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Volume 1 - Number 1 May - September 1997

Volume 15 - Number 1 January 2011

The PDF version of the Atlas of Genetics and Cytogenetics in Oncology and Haematology is a reissue of the original articles published in collaboration with

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Atlas of Genetics and Cytogenetics in Oncology and Haematology

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Scope

The Atlas of Genetics and Cytogenetics in Oncology and Haematology is a peer reviewed on-line journal in

open access, devoted to genes, cytogenetics, and clinical entities in cancer, and cancer-prone diseases.

It presents structured review articles ("cards") on genes, leukaemias, solid tumours, cancer-prone diseases, more

traditional review articles on these and also on surrounding topics ("deep insights"), case reports in hematology,

and educational items in the various related topics for students in Medicine and in Sciences.

Editorial correspondance

Jean-Loup Huret Genetics, Department of Medical Information,

University Hospital

F-86021 Poitiers, France

tel +33 5 49 44 45 46 or +33 5 49 45 47 67

[email protected] or [email protected]

Staff Mohammad Ahmad, Mélanie Arsaban, Houa Delabrousse, Marie-Christine Jacquemot-Perbal, Maureen

Labarussias, Vanessa Le Berre, Anne Malo, Catherine Morel-Pair, Laurent Rassinoux, Sylvie Yau Chun Wan -

Senon, Alain Zasadzinski.

Philippe Dessen is the Database Director, and Alain Bernheim the Chairman of the on-line version (Gustave

Roussy Institute – Villejuif – France).

The Atlas of Genetics and Cytogenetics in Oncology and Haematology (ISSN 1768-3262) is published 12 times

a year by ARMGHM, a non profit organisation, and by the INstitute for Scientific and Technical Information of

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Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1)







Editor

Jean-Loup Huret

(Poitiers, France)

Editorial Board

Sreeparna Banerjee (Ankara, Turkey) Solid Tumours Section

Alessandro Beghini (Milan, Italy) Genes Section

Anne von Bergh (Rotterdam, The Netherlands) Genes / Leukaemia Sections

Judith Bovée (Leiden, The Netherlands) Solid Tumours Section

Vasantha Brito-Babapulle (London, UK) Leukaemia Section

Charles Buys (Groningen, The Netherlands) Deep Insights Section

Anne Marie Capodano (Marseille, France) Solid Tumours Section

Fei Chen (Morgantown, West Virginia) Genes / Deep Insights Sections

Antonio Cuneo (Ferrara, Italy) Leukaemia Section

Paola Dal Cin (Boston, Massachussetts) Genes / Solid Tumours Section

Louis Dallaire (Montreal, Canada) Education Section

Brigitte Debuire (Villejuif, France) Deep Insights Section

François Desangles (Paris, France) Leukaemia / Solid Tumours Sections

Enric Domingo-Villanueva (London, UK) Solid Tumours Section

Ayse Erson (Ankara, Turkey) Solid Tumours Section

Richard Gatti (Los Angeles, California) Cancer-Prone Diseases / Deep Insights Sections

Ad Geurts van Kessel (Nijmegen, The Netherlands) Cancer-Prone Diseases Section

Oskar Haas (Vienna, Austria) Genes / Leukaemia Sections

Anne Hagemeijer (Leuven, Belgium) Deep Insights Section

Nyla Heerema (Colombus, Ohio) Leukaemia Section

Jim Heighway (Liverpool, UK) Genes / Deep Insights Sections

Sakari Knuutila (Helsinki, Finland) Deep Insights Section

Lidia Larizza (Milano, Italy) Solid Tumours Section

Lisa Lee-Jones (Newcastle, UK) Solid Tumours Section

Edmond Ma (Hong Kong, China) Leukaemia Section

Roderick McLeod (Braunschweig, Germany) Deep Insights / Education Sections

Cristina Mecucci (Perugia, Italy) Genes / Leukaemia Sections

Yasmin Mehraein (Homburg, Germany) Cancer-Prone Diseases Section

Fredrik Mertens (Lund, Sweden) Solid Tumours Section

Konstantin Miller (Hannover, Germany) Education Section

Felix Mitelman (Lund, Sweden) Deep Insights Section

Hossain Mossafa (Cergy Pontoise, France) Leukaemia Section

Stefan Nagel (Braunschweig, Germany) Deep Insights / Education Sections

Florence Pedeutour (Nice, France) Genes / Solid Tumours Sections

Elizabeth Petty (Ann Harbor, Michigan) Deep Insights Section

Susana Raimondi (Memphis, Tennesse) Genes / Leukaemia Section

Mariano Rocchi (Bari, Italy) Genes Section

Alain Sarasin (Villejuif, France) Cancer-Prone Diseases Section

Albert Schinzel (Schwerzenbach, Switzerland) Education Section

Clelia Storlazzi (Bari, Italy) Genes Section

Sabine Strehl (Vienna, Austria) Genes / Leukaemia Sections

Nancy Uhrhammer (Clermont Ferrand, France) Genes / Cancer-Prone Diseases Sections

Dan Van Dyke (Rochester, Minnesota) Education Section

Roberta Vanni (Montserrato, Italy) Solid Tumours Section

Franck Viguié (Paris, France) Leukaemia Section

José Luis Vizmanos (Pamplona, Spain) Leukaemia Section

Thomas Wan (Hong Kong, China) Genes / Leukaemia Sections




Volume 15, Number 1, January 2011

Table of contents

Gene Section

CCR1 (chemokine (C-C motif) receptor 1) 1 Qiang Gao, Jia Fan

GSK3B (glycogen synthase kinase 3 beta) 7 Dinesh Kumar Thotala, Eugenia M Yazlovitskaya

MAPKAPK2 (mitogen-activated protein kinase-activated protein kinase 2) 11 Roberta Felix, Veruska Alves, Andre Vettore, Gisele Colleoni

MINA (MYC induced nuclear antigen) 15 Makoto Tsuneoka, Kengo Okamoto, Yuji Tanaka

NKX2-1 (NK2 homeobox 1) 19 Theresia Wilbertz, Sebastian Maier, Sven Perner

NUDT6 (nudix (nucleoside diphosphate linked moiety X)-type motif 6) 29 Leigh-Ann MacFarlane, Paul Murphy

PARVB (parvin, beta) 34 Cameron N Johnstone

PIAS3 (protein inhibitor of activated STAT, 3) 38 Gilles Spoden, Werner Zwerschke

PSEN2 (presenilin 2 (Alzheimer disease 4)) 42 Morgan Newman

RASSF6 (Ras association (RalGDS/AF-6) domain family member 6) 46 Luke B Hesson, Farida Latif

RPA2 (replication protein A2, 32kDa) 54 Anar KZ Murphy, James A Borowiec

S100A7 (S100 calcium binding protein A7) 58 Jill I Murray, Martin J Boulanger

SOX10 (SRY (sex determining region Y)-box 10) 64 Michael Wegner

TNFSF18 (tumor necrosis factor (ligand) superfamily, member 18) 67 Theresa Placke, Hans-Georg Kopp, Benjamin Joachim Schmiedel, Helmut Rainer Salih

USF1 (upstream transcription factor 1) 72 Adrie JM Verhoeven

WNK2 (WNK lysine deficient protein kinase 2) 76 Peter Jordan

Leukaemia Section

t(1;2)(p36;p21) 79 Jean-Loup Huret

t(11;14)(q13;q32) in multiple myeloma Huret JL, Laï JL




t(2;18)(q11;q21) 81 Jean-Loup Huret

t(2;21)(q11;q22) 83 Jean-Loup Huret

Solid Tumour Section

Liver: Nested stromal epithelial tumor 85 Y Albert Yeh

Deep Insight Section

Inhibitors of the heat shock protein 90: from cancer clinical trials to neurodegenerative diseases 88 Jean François Peyrat, Samir Messaoudi, Jean Daniel Brion, Mouad Alami

LDI-PCR in Cancer Translocation Mapping 105 Björn Schneider, Hans G Drexler, Roderick AF MacLeod

Case Report Section

Unbalanced rearrangement der(9;18)(p10;q10) in a patient with polycythemia vera 114 Xinjie Xu, Xueyan Chen, Elizabeth A Rauch, Eric B Johnson, Kate J Thompson, Jennifer JS Laffin,

Gordana Raca, Daniel F Kurtycz

Gene Section Review

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1) 1



CCR1 (chemokine (C-C motif) receptor 1) Qiang Gao, Jia Fan

Liver Cancer Institute, Zhong Shan Hospital and Shanghai Medical School, Fudan University,

Shanghai, P R China (QG, JF)

Published in Atlas Database: April 2010

Online updated version : http://AtlasGeneticsOncology.org/Genes/CCR1ID44379ch3p21.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI CCR1ID44379ch3p21.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Other names: CD191, CKR-1, CKR1, CMKBR1,

HM145, MIP1aR, SCYAR1

HGNC (Hugo): CCR1

Location: 3p21.31

DNA/RNA

Note

CCR1, a member of the beta chemokine receptor

family, is a seven transmembrane protein similar to

G protein-coupled receptors. CCR1 is the first

human CC chemokine receptor to be identified at

the cDNA level. It has a functional viral homolog,

US28, which is a human cytomegalovirus.The

ligands of this receptor include macrophage

inflammatory protein 1 alpha (MIP-1 alpha),

regulated on activation normal T expressed and

secreted protein (RANTES), monocyte

chemoattractant protein 3 (MCP-3), and myeloid

progenitor inhibitory factor-1 (MPIF-1). This gene

and other chemokine receptor genes, including

CCR2, CCRL2, CCR3, CCR5 and CCXCR1, form

a gene cluster on chromosome 3p.

Description

Sequence length: 6633 bp; coding sequence: CDS

72-1139. 2 exons; number of SNPs: 97.

Transcription

2690 bp mRNA, no alternative splicing.

Pseudogene

No pseudogenes have been reported for CCR1.

Protein

Note

Chemokine receptors are cytokine receptors found

on the surface of certain cells, which interact with a

type of cytokine called a chemokine. They each

have a 7 transmembrane structure and couple to G-

protein for signal transduction within a cell, making

them members of a large protein family of G

protein-coupled receptors. Following interaction

with their specific chemokine ligands, chemokine

receptors trigger a flux in intracellular calcium

(Ca2+

) ions (calcium signaling). This causes cell

responses, including the onset of a process known

as chemotaxis that traffics the cell to a desired

location within the organism.

CCR1 (chemokine (C-C motif) receptor 1) Gao Q, Fan J


Predicted structure and amino acid sequence of CCR1. The typical serpentine structure is depicted with three extracellular

(top) and three intracellular (bottom) loops and seven transmembrane domains. The shaded horizontal band represents the cell membrane. Amino acids are listed with a single letter code.

Chemokine receptors share many common

structural features; they are composed of about 350

amino acids that are divided into a short and acidic

N-terminal end, seven helical transmembrane

domains with three intracellular and three

extracellular hydrophilic loops, and an intracellular

C-terminus containing serine and threonine residues

that act as phosphorylation sites during receptor

regulation. The first two extracellular loops of

chemokine receptors are linked together by

disulfide bonding between two conserved cysteine

residues. The N-terminal end of a chemokine

receptor binds to chemokine(s) and is important for

ligand specificity. G-proteins couple to the C-

terminal end, which is important for receptor

signaling following ligand binding.

Description

355 amino acids; 41173 Da.

Expression

Monocyte/macrophages; T cells; platelets; tonsil B

lymphocytes; blood derived mast cells, dendritic

cells, basophils and eosinophils; bone marrow

stromal cells; microvascular endothelial cells;

vascular smooth muscle cells.

Localisation

Cell membrane; multi-pass membrane protein.

Function

Receptor for a C-C type chemokine. Binds to CCL3

(MIP-1-alpha), CCL5 (RANTES), CCL7 (MCP-3),

CCL9 (MIP-1-gamma), CCL14 (HCC-1), CCL15

(MIP-1-delta), CCL16 (HCC-4) and CCL23 (MIP-

3), and, less efficiently, to MIP-1-beta or MCP-1

and subsequently transduces a signal by increasing

the intracellular calcium ions level. The major

function of CCR1 is to regulate leukocyte

trafficking in hematopoiesis and in innate and

adaptive immunity. Other functions include

angiogenic activity, ischemia/reperfusion injury,

immune-cell differentiation, phagocyte activation,

and affecting stem cell proliferation.

Homology

CCR1 protein contains considerable amino acid

sequence homology to other C-C chemokines:

CCR2B (56%), CCR3 (54%), CCR4 (49%), CCR5

(55%).

Implicated in

Hematolymphoid neoplasia

Prognosis

CCR1 expression correlates with overall survival in

the non-germinal center subtype of diffuse large B-



cell lymphoma. In follicular lymphoma, high levels

of CCR1 are associated with a shorter survival

interval, and CCR1 is a marker of an immune

switch between macrophages and a T cell-dominant

response.

Oncogenesis

CCR1 is expressed in intraepithelial B cells of

human tonsil and granulocytic/monocytic cells in

the bone marrow. Immunohistochemical analysis of

944 cases of hematolymphoid neoplasia identified

CCR1 expression in a subset of B- and T-cell

lymphomas, plasma cell myeloma, acute myeloid

leukemia, and classical Hodgkin lymphoma. In 13

patients with chronic lymphocytic leukemia (CLL),

9 with hairy cell leukemia (HCL), 5 with mantle

cell lymphoma (MCL), 5 with marginal zone B-cell

lymphoma (MZL), 6 with small lymphocytic

lymphoma (SLL), and 5 with follicular cell

lymphoma (FCL), flow cytometry analysis

demonstrated that CCR1 was expressed in 70% of

patients with CLL and 40% of those with HCL but

was lacking in patients with MCL, MZL, SLL, and

circulating normal B cells.

Circulating CD3+ T cells derived from healthy

individuals and acute myelogenous leukemia

patients with therapy-induced cytopenia after

conventional chemotherapy or allogeneic stem cell

transplantation showed no qualitative differences in

CCR1 expression, that is, low expression for all the

three groups.

Multiple myeloma

Prognosis

In 80 multiple myeloma (MM) patients with bone

marrow samples, patients with active disease

showed a significantly lower expression of CCR1,

CCR2, as well as CXCR4 than patients with non-

active disease. This chemokine receptor expression

profile correlated with serum beta2-microglobulin,

C-reactive protein and hemoglobin. Multivariate

analysis identified the chemokine receptor

expression profile as an independent prognostic

factor.

Oncogenesis

Human MM cells express at least three different

chemokine receptors that are functionally involved

in MM cell migration, i.e. CCR1, CCR2 and

CXCR4, some also CCR6 and CXCR3. cDNA

arrays identified CCR1 and CCR2 are

overexpressed in myeloma cells compared to

autologous B-lymphoblastoid cell lines. The

expression of CCR1 and the migration to their

ligands, RANTES and MIP-1alpha, respectively,

were demonstrated in MM cell lines and primary

MM cells.

Osteoclasts (OCL) secrete high levels of CCL3 and

MM cells the express CCR1, the interaction

between which plays a key role in the pathogenesis

of MM-related osteolytic bone disease. Through

CCL3-CCR1 axis OCL cells promote OCL

formation and, in turn, OCL enhance MM cell

proliferation.

In murine models of MM, MIP-1alpha, an OCL

stimulatory factor produced by primary MM cells,

increases bone destruction and tumor burden, by

interacting with chemokine receptors CCR1 and

CCR5 that widely expressed in human OCL

precursors, myeloma cell lines, and purified

marrow plasma cells from MM patients.

Neutralizing antibodies to CCR1 or CCR5 inhibited

MIP-1alpha-induced OCL formation. Furthermore,

MCP-3, which binds CCR1 but not CCR5, and the

CCR1-specific antagonist, BX471, markedly

inhibited OCL formation stimulated with MIP-

1alpha. Anti-CCR1, anti-CCR5, or BX471 also

inhibited the upregulation of beta1 integrin

myeloma cells induced by MIP-1alpha, as well as

the adherence of myeloma cells to stromal cells and

IL-6 production by stromal cells in response to

myeloma cells.

The oncogene c-maf is translocated in

approximately 5%-10% of MM. By gene

expression profiling, three c-maf target genes,

cyclin D2, integrin beta7 and CCR1, were

identified.

Hepatocellular carcinoma

Oncogenesis

Hepatic myofibroblast LI90 cells express and

secrete MCP-1/CCL2. Through its receptors CCR1

and CCR2 as well, LI90 induces human

hepatocellular carcinoma (HCC) Huh7 cell

migration and invasion, which are strongly

inhibited by heparin, beta-D-xyloside and anti-

syndecan-1 and -4 antibodies. RANTES/CCL5

strongly stimulates the migration and the invasion

of Huh7 cells by stimulating the tyrosine

phosphorylation of focal adhesion kinase as well as

activating matrix metalloproteinase-9, and to a

lesser extent that of Hep3B cells. The RANTES-

induced migration and invasion of Huh7 cells are

also strongly inhibited by anti-CCR1 antibodies and

heparin, as well as by beta-d-xyloside treatment of

the cells, suggesting that CCR1 and

glycosaminoglycans are involved in these events.

We found that the miRNA-mediated knockdown

expression of CCR1 significantly inhibited the

invasive ability of and reduced the secretion of

MMP-2 in hepatocellular carcinoma HCCLM3

cells, but only had a minor effect on the cellular

proliferation. CCR1 expression was also detected

on primary HCC cells and to a lesser degree, on

endothelial cells in HCC tissues but not in normal

liver tissues. Similarly, CCL3 expression was

detected in HCC cells, endothelial cells, and to a

lesser degree, fibroblast-like cells in HCC tissue,

whereas only occasional vascular endothelial cells

and inflammatory cells in normal liver tissues were

weakly positive for CCL3. IL-1 enhances the local

production of CCL3, which interact with CCR1

expressed on HCC cells, in an autocrine and/or



paracrine manner. In a murine HCC model, injected

tumor cells were transfected with HSV-thymidine

kinase gene and then treated with ganciclovir

(GCV). GCV treatment induced massive tumor cell

apoptosis accompanied with intratumoral

CCR1+CCR5+ dendritic cell infiltration. Tumor-

infiltrating T cells and macrophages expressed

CCL3, suggesting CCR1-CCL3 play a crucial role

in the regulation of intratumoral dendritic cell

accumulation and the subsequent establishment of

tumor immunity following induction of tumor

apoptosis by suicide genes. CCL3 and CCR1 are

also expressed in 2 different models of HCC, i.e.,

N-nitrosodiethylamine (DEN)-induced HCC and

HCC induced by hepatitis B virus. After DEN

treatment, tumor foci number and sizes were

remarkably reduced in CCR1- and CCL3-deficient

mice, comparing with wild-type (WT) mice. Also,

tumor angiogenesis markedly diminished,

intratumoral Kupffer cells number reduced, MMP9

gene expression attenuated and MMP9+ cell

numbers decreased in CCL3- and CCR1-deficient

mice, as compared with WT mice. These

observations suggest the contribution of the CCR1-

CCL3 axis to HCC progression.

Colorectal cancer

Prognosis

The expression of CCR1 is higher in colorectal

carcinoma than normal tissues, and correlates with

lymph node metastasis, deep invasion, poor

differentiation and advanced Dukes' stage.

Oncogenesis

Inactivation of TGF-beta family signaling within

colon cancer increases CCL9 and promotes

recruitment of the matrix metalloproteinase-

expressing stromal cells that carry CCR1. Lack of

CCR1 prevents the accumulation of MMP-

expressing cells at the invasive front and suppresses

tumor invasion. In a murine model of invasive

colorectal cancer in which TGF-beta family

signaling is blocked, CD34+ CCR1+ immature

myeloid cell is recruited from the bone marrow to

the tumor invasive front where expression of CCL9

is increased. These immature myeloid cells express

MMP9, MMP2 and CCR1 and migrate toward the

ligand CCL9. Lack of CCR1 prevents accumulation

of CD34+ immature myeloid cell at the invasive

front and suppresses tumor invasion.

Non-small cell lung cancer

Oncogenesis

CCR1 expression correlated with the aggressive

phenotype of the non-small cell lung cancer

(NSCLC) cells. CCR1 knockdown significantly

suppressed the invasiveness of NSCLC cells and

significantly reduced the expression of matrix

metalloproteinase-9, but had only a minor effect on

cell proliferation.

Oral squamous cell carcinoma

Oncogenesis

Expression of CCL3 and CCR1 is significant higher

in oral squamous cell carcinoma compared with the

normal controls. The percentages of CCL3+ and

CCR1+ cells were observed to be similar in

parenchyma and stroma in cases without lymph

node metastasis when compared with lymph node

metastasis positive cases.

Ovarian cancer

Oncogenesis

mRNA for CCR1, -2a, -2b, -3, -4, -5, and -8 was

detected in cells from human ovarian cancer ascites.

Further, flowcytometry showed CD14+

macrophages within ascites consistently expressed

CCR1, -2, and -5, and >60% of all T cells

expressed CCR1. Although ovarian cancer ascitic

and blood monocyte/macrophages express CCR1,

they failed to migrate in response to the RANTES.

Compared with that of normal blood, cell surface

expression level for CCR1 was higher in ascites. In

a monocytic cell line in vitro, CCR1 mRNA

expression was increased 5-fold by hypoxia. In 25

patients with ovarian cancer, CCR1 was detected in

samples from 75% of patients, where CCR1

localised to macrophages and lymphocytes, and

there was a correlation between numbers of CD8+

cells and CCR1+ cells.

Prostate cancer

Oncogenesis

Androgen receptor negative human prostate cancer

cell line DU-145 cells selectively expressed

CXCR4 and CCR1 at high levels compared with

DU-145/AR cells that express androgen receptor.

DU-145 showed vigorous migratory responses to

CXCL12 and CCL3. In contrast, neither CXCL12

nor CCL3 affected the migration of DU-145/AR

cells.

Breast cancer

Oncogenesis

The expression of CCR5 was higher than that of

CCR1 in the peripheral blood mononuclear cells

(PBMC) of healthy women, while the PBMC of the

breast cancer patients showed overexpression of

CCR1 and downregulation of CCR5. The

differential effects of MIP-1alpha and MIP-1beta

on the PBMC of healthy women and breast cancer

patients correlated with the expression levels of

CCR1 and CCR5 in these monocytes. In murine

model of breast cancer, CCL5 (RANTES) was

produced by the tumor cells, and its receptors,

CCR1 and CCR5, were expressed by the infiltrating

leukocytes. In mice treatment with Met-CCL5, an

antagonist of CCR1 and CCR5, the volume and

weight of tumors were significantly decreased

compared with control-treated tumors.



The total cell number obtained after collagenase

digestion was decreased in Met-CCL5-treated

tumors as was the proportion of infiltrating

macrophages. Furthermore, chemokine antagonist

treatment increased stromal development and

necrosis.

Glioma

Oncogenesis

Co-cultured human glioma U87 cells induced an

activated phenotype in HUVECs. These tumour-

activated endothelial cells coordinately expressed

matching pairs of receptors/ligands were found to

be, including CCR1-RANTES axis.

Osteogenic sarcoma

Oncogenesis

The activities of phospholipase C (PLC), protein

kinase C delta (PKCdelta) and NF-kappaB were

enhanced by Lkn-1 (CCL15) stimulation on CCR1+

human osteogenic sarcoma cells. Inhibitors of G

protein, PLC, PKCdelta and NF-kappaB inhibited

the chemotactic activity of Lkn-1 on CCR1+

osteogenic sarcoma cells indicating that Lkn-1-

induced chemotaxis involving these signaling

pathways.

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Silva TA, Ribeiro FL, Oliveira-Neto HH, Watanabe S, Alencar Rde C, Fukada SY, Cunha FQ, Leles CR, Mendonça EF, Batista AC. Dual role of CCL3/CCR1 in oral squamous cell carcinoma: implications in tumor metastasis and local host defense. Oncol Rep. 2007 Nov;18(5):1107-13

Sutton A, Friand V, Papy-Garcia D, Dagouassat M, Martin L, Vassy R, Haddad O, Sainte-Catherine O, Kraemer M, Saffar L, Perret GY, Courty J, Gattegno L, Charnaux N. Glycosaminoglycans and their synthetic mimetics inhibit RANTES-induced migration and invasion of human hepatoma cells. Mol Cancer Ther. 2007 Nov;6(11):2948-58

Vallet S, Raje N, Ishitsuka K, Hideshima T, Podar K, Chhetri S, Pozzi S, Breitkreutz I, Kiziltepe T, Yasui H, Ocio EM, Shiraishi N, Jin J, Okawa Y, Ikeda H, Mukherjee S, Vaghela N, Cirstea D, Ladetto M, Boccadoro M, Anderson KC. MLN3897, a novel CCR1 inhibitor, impairs osteoclastogenesis and inhibits the interaction of multiple myeloma cells and osteoclasts. Blood. 2007 Nov 15;110(10):3744-52

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cancer. Cancer Immunol Immunother. 2008 May;57(5):635-45

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This article should be referenced as such:

Gao Q, Fan J. CCR1 (chemokine (C-C motif) receptor 1). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):1-6.

Gene Section Review




GSK3B (glycogen synthase kinase 3 beta) Dinesh Kumar Thotala, Eugenia M Yazlovitskaya

Department of Radiation Oncology, Vanderbilt Ingram Cancer Center, Vanderbilt University, SS1411

Medical Center North, 1161 21 Avenue S, Nashville, TN 37232, USA (DKT, EMY)


Online updated version : http://AtlasGeneticsOncology.org/Genes/GSK3BID40761ch3q13.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI GSK3BID40761ch3q13.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity

Other names: EC 2.7.11.26

HGNC (Hugo): GS3KB

Location: 3q13.33

Local order:

Human: Nuclear receptor subfamily 1, group I,

member 2 (NR1I2); GSK3B; G-protein coupled

receptor 156 (GPR156).

Mouse: G-protein coupled receptor 156 (Gpr156);

Gsk3b; Nuclear receptor subfamily 1, group I,

member 2 (Nr1i2).

DNA/RNA

Description

According to Entrez-Gene, human GSK3B maps to

locus NC_000003.11.

This gene contains 12 exons that encompass

266971 bp of genomic DNA. In mice, GSK3B

maps to NC_000082.5 and contains 11 exons that

span 157079 bp of DNA within the mouse genome.

Transcription

Human GSK3B mRNA (NM_002193.3) consists of

7134 bp, and murine GSK3B mRNA (NM_019827)

contains 8298 bp. Alternatively spliced transcript

variants encoding different isoforms (1 and 2) have

been found for human gene. Transcript variant 2 is

missing an in-frame coding exon (9) compared to

variant 1, resulting in a shorter isoform 2 lacking a

13 aa segment compared to isoform 1.

Pseudogene

No pseudogene has been identified for GSK3B.

A) Human GSK3B gene, isoform 1. B) Mouse Gsk3b gene. GSK3B is comprised of 12 exons in human and 11 exons in

mouse. The ATG start codon is located within exon 1 and the TAG stop codon is found in exon 12 (Human) and 11 (Mouse). The sizes of exons for human gene 1-12 are 1071 bp, 191 bp, 85 bp, 110 bp, 130 bp, 106 bp, 97 bp, 95 bp, 38 bp, 186 bp, 98 bp and 604 bp, respectively. The sizes of exons for mouse gene 1-11 are 1613 bp, 193 bp, 83 bp, 110 bp, 130 bp, 106 bp, 97 bp, 95 bp,

186 bp, 98 bp and 5577 bp, respectively.

GSK3B (glycogen synthase kinase 3 beta) Thotala DK, Yazlovitskaya EM


GSK3B structure. GSK3B is a 46-47 kDa protein consisting of 433 and 420 amino acids in human and mouse respectively. The

protein contains an N-terminal domain, a kinase domain and a C-terminal domain. Phosphorylation of Tyr216 located in the T-loop (activation site) facilitates substrate phosphorylation by GSK3B but is not strictly required for its kinase activity.

Phosphorylation of GSK3B at Ser9 in N-terminal region leads to inhibition of its kinase activity. Binding domain (BD) includes GSK3B specific binding sites for substrates and protein complexes (e.g., p53).

Protein

Description

Glycogen synthase kinase-3 beta (GSK3B) was

named due to its ability to phosphorylate and

inactivate glycogen synthase. GSK3B is a

multifunctional serine/threonine kinase which has

been implicated in multiple biological processes

including embryonic development, cell

differentiation, apoptosis and insulin response.

GSK3B is a key component in neuronal functions

and has been implicated in major diseases involving

the central nervous system.

Expression

GSK3B was originally isolated from the skeletal

muscle but it is ubiquitously expressed in almost all

the tissues. However, abundant expression is

detected in brain tissue, especially in the neurons

when compared to the astrocytes. The high level of

expression in the brain is due to its vital role in the

neuronal signaling. Dysregulation of GSK3B

expression leads to various pathological conditions

such as diabetes or insulin resistance, neuronal

dysfunction and neuronal diseases.

Localisation

GSK3B is generally considered a cytosolic protein;

however, it is reported to be present in the nucleus

and mitochondria. Nuclear and mitochondrial

localization of GSK3B correlates with its higher

kinase activity compared to cytosolic protein.

Translocation and specific cellular localization of

GSK3B determine its involvement in signaling

pathways, regulate its interaction with substrates

and participation in protein complex formation, and

influence gene expression and transcription.

Function

GSK3B is a multifunctional protein kinase which is

implicated in a large number of cellular processes

and diseases. GSK3B is regulated by serine

(inhibitory) and tyrosine (activating)

phosphorylation. More than 40 proteins have been

reported to be phosphorylated by GSK3B. GSK3B

substrates include metabolic and signaling proteins

like glycogen synthase, Acetyl CoA carboxylase,

Axin, Cyclin D1; structural proteins like Tau,

neural cell adhesion protein (NCAM); transcription

factors like beta-catenin, p53, Myc, NFkappaB,

CREB and AP-1; apoptotic-related proteins like

Bax and p53. GSK3B also regulates various cellular

processes by binding to protein complexes.

Homology

The GSK3B gene is conserved in human,

chimpanzee, dog, cow, rat, chicken, zebrafish, fruit

fly, mosquito, C. elegans, A. thaliana, rice, and P.

falciparum.

Mutations

Germinal

1. Several rare sequence variants in GSK3B were

identified in the case-control study of patients with

probable Alzheimer disease (AD), familial

frontotemporal dementia (FTD), primary

progressive aphasia, and aged healthy subjects. An

intronic polymorphism (IVS2-68G>A) occurred at

more than twice the frequency among patients with

FTD (10.8%) and patients with AD (14.6%) than in

aged healthy subjects (4.1%).

2. GSK3beta promoter single-nucleotide

polymorphism (rs334558) influences transcriptional

strength, and the less active form was associated

with less detrimental clinical features of mood

disorders. Effect of rs334558 was studied on grey

matter volumes of patients affected by chronic

schizophrenia. Carriers of the less active C allele

variant showed significantly higher brain volumes

in an area encompassing posterior regions of right

middle and superior temporal gyrus, within the

boundaries of Brodmann area 21. The temporal

lobe is the brain parenchymal region with the most

consistently documented morphometric

abnormalities in schizophrenia, and

neuropathological processes in these regions

develop soon at the beginning of the illness.

Implicated in

Ovarian cancer

Note

Ovarian cancer is a leading cause of death from

gynecological malignancies. GSK3B promotes

ovarian cancer cell proliferation by regulating



Cyclin D1. GSK3B-dependent increased Cyclin D1

expression in ovarian cancer cells supports a

possibility that GSK3B is involved in ovarian

tumor chemotherapy resistance. Therefore, it is

possible that combination of traditional

chemotherapy and GSK3B inhibitors would benefit

ovarian cancer patient response.

Prostate cancer

Note

Androgen receptor (AR) regulates growth of

normal and cancer prostate cells. AR

phosphorylation status is associated with its

transcriptional activation. GSK3B interacts directly

with the AR, modulates AR signaling and plays

important role in the control of the proliferation of

normal and malignant androgen-regulated tissues.

Therefore, pharmacological inhibitors designed to

increase GSK3B activity could be useful in prostate

cancer therapy.

Pancreatic cancer

Note

It was shown that pancreatic cancer cells contain a

pool of active GSK3B, and that pharmacological

inhibition of GSK3B kinase activity using small

molecule inhibitors or genetic depletion of GSK3B

by RNA interference leads to decreased cancer cell

proliferation and survival. Hence GSK3B has

potential as an important new target in the treatment

of pancreatic cancer.

Colorectal cancer

Note

Colon cancer cell lines and colon cells from

colorectal cancer patients have higher levels of

GSK3B expression than their normal counterparts.

Inhibition of GSK3B activity either by chemical

inhibitors or by expression by RNA interference

targeting GSK3B induced apoptosis and attenuation

of proliferation of colon cancer cells in vitro. Hence

GSK3B has a potential as therapeutic target in

colorectal cancer.

Neuroblastoma

Note

Treatment of B65 neuroblastoma cell line with

GSK3B inhibitors Lithium or SB415286 caused a

decrease in cell proliferation that was associated

with G2/M cell cycle arrest due to regulating the

phosphorylation of Cdc2. Therefore, GSK3B and

Cdc2 could be potential pharmacological targets in

neuroblastoma.

Glioblastoma

Note

Glioblastoma is the most frequent malignant tumor

of the brain and represents a subset of cancers that

is mostly nonresponsive to currently available

anticancer treatments.

The current standard therapy for newly diagnosed

glioblastoma consists of surgical resection of the

tumor to the extent that is safe and feasible,

followed by chemotherapy and irradiation. There

has been an emerging paradigm for the combination

of chemotherapy and molecular targeted therapy to

improve therapeutic efficiency. Glioblastoma cells

depend on deregulated GSK3B to survive,

proliferate, and resist chemotherapy and radiation.

Pretreatment with low-dose GSK3B inhibitor

enhanced the cytocidal effect of ionizing radiation

in glioblastoma cells. At the same time, GSK3B

inhibitors have been shown to protect normal

hippocampal neurons from radiation-induced

apoptosis. Therefore, GSK3B inhibition provides

dual benefits for the glioblastoma patients treated

with radiation: by attenuating tumor proliferation

and by protecting host brain tissue from

degradation and allowing its repair.

Insulin resistance and diabetes

Note

Insulin resistance is caused by the inability of

insulin sensitive tissues to respond to insulin and

efficiently clear blood glucose. Insulin signaling

involves autophosphorylation of the insulin

receptor leading to the activation of PI3K which

activates PKB (Akt). The activated PKB

phosphorylates and inactivates GSK3B.

Dysregulation of GSK3B results in impaired insulin

signaling leading to diabetes. Inhibitors of GSK3B

improve insulin signaling and maintain proper

glucose levels.

Alzheimer's disease

Note

Alzheimer's disease (AD) is a chronic disorder that

slowly destroys neurons and causes serious

cognitive disability. The two neuropathologiocal

features of Alzheimer's disease are neurofibrillary

tangles and amyloid plaques. GSK3B has been

implicated in both neuropathologies. In addition,

presenilin 1 (PS1) have been linked to Alzheimer's

disease. Presenilin 1 binds to and regulates GSK3B

activity. Presenilin 1 mutations might compromise

neuronal function by increasing GSK3B activity.

Schizophrenia

Note

Schizophrenia is a severe brain illness in which the

disrupted in schizophrenia 1 (DISC1) gene is

disrupted by a balanced chromosomal translocation.

DISC1 is highly expressed in neural progenitor

cells and required for embryonic brain

development. DISC1 regulates beta-catenin

turnover by inhibiting GSK3B activity. GSK3B

inhibitors are able to normalize progenitor

proliferation and behavioral defects caused by

DISC1 loss of function.



Bipolar affective disorder

Note

Patients with bipolar affective disorder have a

history of experiencing manic episodes that are

often interspersed with depression, and major

depression is commonly referred to as mood

disorders. Lithium, a known GSK3B inhibitor, is

one of the most widely used mood-stabilizing

agents for the treatment of bipolar disorder.

References Lau KF, Miller CC, Anderton BH, Shaw PC. Molecular cloning and characterization of the human glycogen synthase kinase-3beta promoter. Genomics. 1999 Sep 1;60(2):121-8

Dajani R, Fraser E, Roe SM, Young N, Good V, Dale TC, Pearl LH. Crystal structure of glycogen synthase kinase 3 beta: structural basis for phosphate-primed substrate specificity and autoinhibition. Cell. 2001 Jun 15;105(6):721-32

Martinez A, Castro A, Dorronsoro I, Alonso M. Glycogen synthase kinase 3 (GSK-3) inhibitors as new promising drugs for diabetes, neurodegeneration, cancer, and inflammation. Med Res Rev. 2002 Jul;22(4):373-84

Bhat RV, Budd Haeberlein SL, Avila J. Glycogen synthase kinase 3: a drug target for CNS therapies. J Neurochem. 2004 Jun;89(6):1313-7

Cohen P, Goedert M. GSK3 inhibitors: development and therapeutic potential. Nat Rev Drug Discov. 2004 Jun;3(6):479-87

Jope RS, Johnson GV. The glamour and gloom of glycogen synthase kinase-3. Trends Biochem Sci. 2004 Feb;29(2):95-102

Meijer L, Flajolet M, Greengard P. Pharmacological inhibitors of glycogen synthase kinase 3. Trends Pharmacol Sci. 2004 Sep;25(9):471-80

Wang L, Lin HK, Hu YC, Xie S, Yang L, Chang C. Suppression of androgen receptor-mediated transactivation and cell growth by the glycogen synthase kinase 3 beta in prostate cells. J Biol Chem. 2004 Jul 30;279(31):32444-52

Ougolkov AV, Fernandez-Zapico ME, Savoy DN, Urrutia RA, Billadeau DD. Glycogen synthase kinase-3beta participates in nuclear factor kappaB-mediated gene transcription and cell survival in pancreatic cancer cells. Cancer Res. 2005 Mar 15;65(6):2076-81

Shakoori A, Ougolkov A, Yu ZW, Zhang B, Modarressi MH, Billadeau DD, Mai M, Takahashi Y, Minamoto T. Deregulated GSK3beta activity in colorectal cancer: its association with tumor cell survival and proliferation. Biochem Biophys Res Commun. 2005 Sep 9;334(4):1365-73

Cao Q, Lu X, Feng YJ. Glycogen synthase kinase-3beta positively regulates the proliferation of human ovarian cancer cells. Cell Res. 2006 Jul;16(7):671-7

Yazlovitskaya EM, Edwards E, Thotala D, Fu A, Osusky KL, Whetsell WO Jr, Boone B, Shinohara ET, Hallahan DE. Lithium treatment prevents neurocognitive deficit

resulting from cranial irradiation. Cancer Res. 2006 Dec 1;66(23):11179-86

Garcea G, Manson MM, Neal CP, Pattenden CJ, Sutton CD, Dennison AR, Berry DP. Glycogen synthase kinase-3 beta; a new target in pancreatic cancer? Curr Cancer Drug Targets. 2007 May;7(3):209-15

Schaffer BA, Bertram L, Miller BL, Mullin K, Weintraub S, Johnson N, Bigio EH, Mesulam M, Wiedau-Pazos M, Jackson GR, Cummings JL, Cantor RM, Levey AI, Tanzi RE, Geschwind DH. Association of GSK3B with Alzheimer disease and frontotemporal dementia. Arch Neurol. 2008 Oct;65(10):1368-74

Thotala DK, Hallahan DE, Yazlovitskaya EM. Inhibition of glycogen synthase kinase 3 beta attenuates neurocognitive dysfunction resulting from cranial irradiation. Cancer Res. 2008 Jul 15;68(14):5859-68

Eom TY, Jope RS. GSK3 beta N-terminus binding to p53 promotes its acetylation. Mol Cancer. 2009 Mar 5;8:14

Machado-Vieira R, Manji HK, Zarate CA Jr. The role of lithium in the treatment of bipolar disorder: convergent evidence for neurotrophic effects as a unifying hypothesis. Bipolar Disord. 2009 Jun;11 Suppl 2:92-109

Mao Y, Ge X, Frank CL, Madison JM, Koehler AN, Doud MK, Tassa C, Berry EM, Soda T, Singh KK, Biechele T, Petryshen TL, Moon RT, Haggarty SJ, Tsai LH. Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3beta/beta-catenin signaling. Cell. 2009 Mar 20;136(6):1017-31

Miyashita K, Kawakami K, Nakada M, Mai W, Shakoori A, Fujisawa H, Hayashi Y, Hamada J, Minamoto T. Potential therapeutic effect of glycogen synthase kinase 3beta inhibition against human glioblastoma. Clin Cancer Res. 2009 Feb 1;15(3):887-97

Pizarro JG, Folch J, Esparza JL, Jordan J, Pallàs M, Camins A. A molecular study of pathways involved in the inhibition of cell proliferation in neuroblastoma B65 cells by the GSK-3 inhibitors lithium and SB-415286. J Cell Mol Med. 2009 Sep;13(9B):3906-17

Takahashi-Yanaga F, Sasaguri T. Drug development targeting the glycogen synthase kinase-3beta (GSK-3beta)-mediated signal transduction pathway: inhibitors of the Wnt/beta-catenin signaling pathway as novel anticancer drugs. J Pharmacol Sci. 2009 Feb;109(2):179-83

Benedetti F, Poletti S, Radaelli D, Bernasconi A, Cavallaro R, Falini A, Lorenzi C, Pirovano A, Dallaspezia S, Locatelli C, Scotti G, Smeraldi E. Temporal lobe grey matter volume in schizophrenia is associated with a genetic polymorphism influencing glycogen synthase kinase 3-beta activity. Genes Brain Behav. 2010 Jun 1;9(4):365-71

Thotala DK, Geng L, Dickey AK, Hallahan DE, Yazlovitskaya EM. A new class of molecular targeted radioprotectors: GSK-3beta inhibitors. Int J Radiat Oncol Biol Phys. 2010 Feb 1;76(2):557-65


Thotala DK, Yazlovitskaya EM. GSK3B (glycogen synthase kinase 3 beta). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):7-10.

Gene Section Mini Review




MAPKAPK2 (mitogen-activated protein kinase-activated protein kinase 2) Roberta Felix, Veruska Alves, Andre Vettore, Gisele Colleoni

Laboratory of Cancer Molecular Biology, Federal University of Sao Paulo UNIFESP/EPM, Sao

Paulo, Brazil (RF, VA, AV, GC)


Online updated version : http://AtlasGeneticsOncology.org/Genes/MAPKAPK2ID41295ch1q32.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI MAPKAPK2ID41295ch1q32.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity

Other names: MAPKAPK-2, MK2

HGNC (Hugo): MAPKAPK2

Location: 1q32.1

Note: MAPKAPK2 is involved in many cellular

processes including: stress and inflammatory

response, nuclear export, gene expression

regulation and cell proliferation, acting with p38

MAP gene.

DNA/RNA

Note

MAPKAPK2 encodes a member of the Ser/Thr

protein kinase regulated through direct

phosphorylation by p38 MAP kinase. Inhibition of

the p38 MAPK pathway could be a possible target

to inflammatory diseases therapy. Unfortunately,

blocking p38 MAPK activation "in vivo" implies in

high toxicity and it does not have oral

bioavailability. MAPKAPK2/MK2 inhibitors acting

downstream of p38 could be reasonable solutions to

overcome this problem (Duraisamy et al., 2008).

Description

Size 49,338 bases, starts at 204924912 and ends at

204974256 bp from pter with plus strand

orientation.

Transcription

We found some discordant information regarding

MAPKAPK2 splice variants. Kervinen et al. 2006,

described that the human MAPKAPK2 gene

encodes two alternatively spliced transcripts and

also that this gene contains 14 different introns (13

gt-ag, 1 gc-ag). Transcription produces 9 different

mRNAs, 8 alternatively spliced variants and 1

unspliced form. There are 5 probable alternative

promoters and 3 validated alternative

polyadenylation sites. The mRNAs appear to differ

by truncation of the 5' end, presence or absence of 7

cassette exons, overlapping exons with different

boundaries, alternative splicing or retention of 3

introns.

Pseudogene

ATF4C - Cyclic AMP-dependent transcription

factor ATF-4, localized at chromosome 17,

location: 17q25.1.

Protein

Description

MAPKAPK2 has two alternatively spliced

transcripts, encoding 400 and 370 amino acids, with

sequence heterogeneity from Lys-353 to the C-

terminus. Crystal structure shows: 1) an

autoinhibitory domain, consisting of 328-370

residues; 2) a helix-turn-helix structure that

occupies the substrate-binding cleft of the kinase

domain and inhibits kinase function (ter Haar et al.,

2007).

Localisation

The 400-residue MAPKAPK2 (isoform 1) consists

of an N-terminal Pro-rich region, a kinase domain,

an autoinhibitory domains, and C-terminal nuclear

export (NES) and nuclear localization (NLS)

signals. The 1-370 isoform (isoform 2) lacks NES

and NLS, consistent with its presence only in the

MAPKAPK2 (mitogen-activated protein kinase-activated protein kinase 2)

Felix R, et al.


cytoplasm. MAPKAPK2 also phosphorylates

proteins found in both the nucleus (cAMP-response

element-binding protein, or CREB) and cytoplasm

(HSP25/27 and LSP-1) (Kervinen et al., 2006).

Function

MAPKAPK2 is required for both cytokines

production and cell migration (Kotlyarov et al.,

2002).

MAPKAPK2 is activated upon stress by p38

MAPK, which binds C terminus of MAPKAPK2,

leading to subsequently phosphorylation of its

regulatory sites. After activation, MAPKAPK2 is

transferred from nucleus to cytoplasm, and

cotransport p38 to the new localization. In murine

knockout model, MAPKAPK2 blockage leads to a

dramatic reduction of tumor necrosis factor (TNF)

production in response to lipopolysaccharide

(Kotlyarov et al., 2002). One of the major

substrates of MAPKAPK2 is the heat shock protein

HSP27, which stimulates actin polymerization in

order to facilitate recovery from destruction of

cytoskeleton during cellular stresses.

Homology

Isoform 1 and 2 see figure below.


Felix R, et al.


Mutations

Note

There is one described mutation: position 804 of

mRNA, allele change GCC to GGC. At protein

level, residue change A [Ala] to G [Gly].

Implicated in

Multiple myeloma (MM)

Note

Hideshima et al. 2004 have shown that

overexpression of HSP27 confers resistance to

bortezomib, a proteasome inhibitor currently used

as front line MM therapy, combined with

corticosteroids and immunomodulatory drugs, such

as thalidomide and lenalidomide. Therefore,

overexpression of MAPKAPK2 could be related to

MM resistance to chemotherapy. They

hypothesized that inhibition of MAPKAPK2

activity could augment bortezomib cytotoxicity by

down regulating HSP27. Felix et al. 2009, at their

gene expression studies, supported further

exploitation of this pathway as therapeutic target in

MM, although immunohistochemistry did not show

high frequency of protein expression in MM (21%)

(Felix et al., 2009).

Panel showing bone marrow samples of MM cases: A -

plasma cells nuclear and cytoplasmatic positivity for MAPKAPK2; B - sample negative for MAPKAPK2 (400X).

Bladder cancer

Note

Kumar et al. 2010, showed that overexpression of

the matrix metalloproteinases MMP-2 and MMP-9

have prognostic value in transitional cell carcinoma

of bladder. p38 MAPK modulated MMP-2/9

mRNA expression and MMP-2/9 activity as

mediators of tumor cells invasive capacity.

Therefore, p38 MAPK inhibition blocks MMP-2/9

activities mediated by MAPKAPK2 (Kumar et al.,

2010).

Skin tumor

Note

Using the two-stage chemical carcinogenesis

model, Johansen et al. 2009 studied the effect of

MAPKAPK2-deficiency and TNF-alpha-deficiency

on skin tumor development in mice. Their findings

demonstrate a dual role of MAPKAPK2 in the early

stages of tumor promotion through regulation of

both the inflammatory response and apoptosis of

DNA-damaged cells. These results also identify

MAPKAPK2 as a possible target for skin

carcinoma therapy (Johansen et al., 2009).

Prostate cancer

Note

TGFbeta is an important regulator of cell adhesion

and motility in a variety of cell types. p38 MAP

kinase is necessary for TGFbeta -mediated up-

regulation of matrix metalloproteinase type 2

(MMP-2), as well as TGFbeta -dependent increases

in prostate cell invasion. Xu et al. 2006

demonstrated, after transient transfection, that both

MAPKAPK2 and HSP27 are necessary for

TGFbeta -mediated increases in MMP-2 activity in

any cell type, as well as prostate cancer cells (Xu et

al., 2006).

Alzheimer's disease (AD)

Note

Culbert et al. 2006 suggested that MAPKAPK2

plays a role in neuroinflammatory and

neurodegenerative diseases, such as AD. The

MAPKAPK2 activation and expression were

increased in lipopolysaccharide (LPS) + interferon

gamma-stimulated microglial cells, demonstrating

MAPKAPK2 ability in eliciting a pro-inflammatory

response. Again, MAPKAPK2 pathway can be

considered a target for control of this degenerative

brain disease.

Psoriatic skin

Note

Alterations in this specific signal transduction

pathway may be involved in increased expression

of proinflammatory cytokines in inflammatory

diseases (Johansen et al., 2006). The increased

activation of MAPKAPK2 is responsible for the

elevated TNFalpha protein expression in psoriatic


Felix R, et al.


skin, making this pathway a potential target in the

treatment of psoriasis (Johansen et al., 2006).

References Kotlyarov A, Yannoni Y, Fritz S, Laass K, Telliez JB, Pitman D, Lin LL, Gaestel M. Distinct cellular functions of MK2. Mol Cell Biol. 2002 Jul;22(13):4827-35

Hideshima T, Podar K, Chauhan D, Ishitsuka K, Mitsiades C, Tai YT, Hamasaki M, Raje N, Hideshima H, Schreiner G, Nguyen AN, Navas T, Munshi NC, Richardson PG, Higgins LS, Anderson KC. p38 MAPK inhibition enhances PS-341 (bortezomib)-induced cytotoxicity against multiple myeloma cells. Oncogene. 2004 Nov 18;23(54):8766-76

Culbert AA, Skaper SD, Howlett DR, Evans NA, Facci L, Soden PE, Seymour ZM, Guillot F, Gaestel M, Richardson JC. MAPK-activated protein kinase 2 deficiency in microglia inhibits pro-inflammatory mediator release and resultant neurotoxicity. Relevance to neuroinflammation in a transgenic mouse model of Alzheimer disease. J Biol Chem. 2006 Aug 18;281(33):23658-67

Johansen C, Funding AT, Otkjaer K, Kragballe K, Jensen UB, Madsen M, Binderup L, Skak-Nielsen T, Fjording MS, Iversen L. Protein expression of TNF-alpha in psoriatic skin is regulated at a posttranscriptional level by MAPK-activated protein kinase 2. J Immunol. 2006 Feb 1;176(3):1431-8

Kervinen J, Ma H, Bayoumy S, Schubert C, Milligan C, Lewandowski F, Moriarty K, Desjarlais RL, Ramachandren K, Wang H, Harris CA, Grasberger B, Todd M, Springer BA, Deckman I. Effect of construct design on MAPKAP kinase-2 activity, thermodynamic stability and ligand-binding affinity. Arch Biochem Biophys. 2006 May 15;449(1-2):47-56

Xu L, Chen S, Bergan RC. MAPKAPK2 and HSP27 are downstream effectors of p38 MAP kinase-mediated matrix metalloproteinase type 2 activation and cell invasion in human prostate cancer. Oncogene. 2006 May 18;25(21):2987-98

ter Haar E, Prabhakar P, Liu X, Lepre C. Crystal structure of the p38 alpha-MAPKAP kinase 2 heterodimer. J Biol Chem. 2007 Mar 30;282(13):9733-9

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Felix R, Alves V, Vettore A, Colleoni G. MAPKAPK2 (mitogen-activated protein kinase-activated protein kinase 2). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):11-14.

Gene Section Review




MINA (MYC induced nuclear antigen) Makoto Tsuneoka, Kengo Okamoto, Yuji Tanaka

Laboratory of Molecular and Cellular Biology, Department of Molecular Pharmacology, Faculty of

Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-

0033, Japan (MT, KO, YT)


Online updated version : http://AtlasGeneticsOncology.org/Genes/MINAID44409ch3q11.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI MINAID44409ch3q11.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Other names: DKFZp762O1912, FLJ14393,

MDIG, MINA53, NO52

HGNC (Hugo): MINA

Location: 3q11.2

Note: MINA (myc induced nuclear antigen) is a

gene whose expression is directly induced by c-

MYC protein. The MINA gene encodes a protein

with a molecular weight of 53 kDa that is localized

in the nucleoplasm and nucleolus.

DNA/RNA

Description

The human MINA gene consists of twelve exons

spanning a 30 kb. The translation start site locates

in exon 2, which follows two distinct exons, exon

1a and exon 1b.

Thus, there are two transcription initiation sites in

the human MINA gene. The exon 1b exists 0.25 kb

downstream of the exon 1a. The stop codon (TAG)

exists in the last exon, exon 10. The open reading

frame of the coding region is 1398 bp, encoding

465 amino acids. mRNA encoding 464 amino acids

(lacking 297Q) is also generated by alternative

splicing due to the lack of the first three bp of exon

7.

Transcription

The human MINA coding sequence consists of

1398 bp from the start codon to the stop codon. In

addition to the 1395 bp-coding sequence, multiple

alternative spliced transcript variants have been

found for this gene. c-MYC protein stimulates the

transcription of MINA through the E-box near the

transcription start sites (Tsuneoka et al., 2002). The

expression of MINA is also induced by serum

(Tsuneoka et al., 2002).

a. Exon-intron structure of the MINA gene. There are two transcription initiation sites at exon 1a and exon 1b. b. mRNA for

human MINA that encodes MINA protein. The protein is coded from exon 2 to exon 10.

MINA (MYC induced nuclear antigen) Tsuneoka M, et al.


Protein

Structural features of MINA protein. The positions of the

JmjC domain is shown (green).

Description

Structure: MINA is a member of the jumonji C

(JmjC) protein family, and suspected to hydroxylate

some proteins to control gene expression.

Activation: The expression of mRNA is elevated

by c-MYC protein, and frequently increased in

various types of cancers, including human colon

cancer, esophageal squamous cell carcinoma

(ESCC). In some types of cancers such as ESCC,

patients with high expression of MINA53 had

shorter survival periods. MINA expression is also

activated not only MYC but also by other factors,

because there are lymphoma and lung cancer tissues

where the expression of MYC is downregulated but

the expression of MINA is elevated (Teye et al.,

2007; Komiya et al., 2010). The expression of

MINA is also activated and mineral dust in human

alveolar macrophage and human lung cancer cell

line, A549 (Zhange et al., 2005).

Expression

MINA is ubiquitously expressed. The expression of

MINA is frequently increased in various types of

human cancers. In mice, MINA expression is high

in some non-neoplastic tissues, including spleen,

thymus, colon and testis, but low in skeletal muscle,

cerebellum, and seminal vesicle. In testis the

expression of MINA is high in spermatogonia and

mitotic prophase cells and weakly in early

pachytene spermatocyte but absent in late

pachytene spermatocytes (Tsuneoka et al., 2006).

Localisation

MINA is diffusely nucleoplasmic and some portion

is accumulated in nucleolus (Tsuneoka et al., 2002).

Function

Specific inhibition of MINA expression suppressed

cell proliferation in some cultured cell lines

(Tsuneoka et al., 2002). Forced expression of

MINA in NIH/3T3 cells induces cell

transformation, and MINA-transfected NIH/3T3

clones produce tumor in nude mice (Komiya et al.,

2010). Therefore, MINA has oncogenic potential.

MINA is a nuclear protein and a member of the

jumonji C (JmjC) protein family. Thus, MINA is

suspected to hydroxylate some proteins to control

gene expression, but its substrate is not clear.

Gene activation: MINA regulates several genes

which are also regulated by MYC. Genes regulated

by MINA but not by MYC include HGF, EGFR,

and IL6 (Komiya et al., 2010).

Gene suppression: Recently, MINA was identified

as a genetic determinant of T(H)2 bias. MINA

specifically binds to and represses the IL4

promoter. MINA overexpression in transgenic mice

impaired IL4 expression, whereas its knockdown in

primary CD4(+) T cells led to IL4 de-repression.

Therefore MINA controls helper T cell

differentiation through an IL4-regulatory pathway

(Okamoto et al,. 2009). These findings suggest that

MINA may play a role on carcinogenesis also in the

field of cancer immunology.

Ribosome biogenesis: MINA is accumulated in

nucleolus (Tsuneoka et al., 2002).

Immunolocalization studies revealed that MINA is

highly concentrated in the granular component of

nucleoli (Eilbracht et al., 2005). MINA is a

constituent of free preribosomal particles but is

absent from cytoplasmic ribosomes. MINA

interacts with various ribosomal proteins as well as

with a distinct set of non-ribosomal nucleolar

proteins. These results suggest that MINA is

directly involved in ribosome biogenesis, most

likely during the assembly process of preribosomal

particles (Eilbracht et al., 2005). MINA was also

suggested to be involved in ribosomal RNA

transcription (Lu et al., 2009).

Homology

The primary sequence of MINA has similarity to

nucleolar protein NO66, which also has a JmjC

domain. The JmjC domain of MINA has 50%

identity to that of NO66. In 2010, it was report that

NO66 directly interacts with Osterix (Osx), which

is an osteoblast-specific transcription factor

required for osteoblast differentiation and bone

formation. NO66 exhibits a JmjC-dependent

histone demethylase activity, which is specific for

both H3K4me and H3K36me in vitro and in vivo. It

was suggested that interactions between NO66 and

Osx regulate Osx-target genes in osteoblasts by

modulating histone methylation states (Sinha et al.,

2010).

Implicated in

Neoplasm diseases

Note

Colon cancer, esophageal squamous cell carcinoma,

gingival squamous cell carcinoma, subtypes of

human lymphoma, renal cell carcinoma,

neuroblastoma, gastric carcinoma, lung cancer and

hepatoma.

Disease

MINA expression is elevated in several types of

carcinomas.

Prognosis

MINA is preferentially expressed in some types of

cancers with a poor prognosis, including

esophageal squamous cell carcinoma, advanced



renal cell carcinoma, neuroblastoma. The

expression levels of MINA may be used as a

prognositic marker in these cancers. On the other

hand, elevated expression of MINA in lung cancer

patients is associated with favorable prognosis.

Colon cancer

Note

The expression of MINA is elevated in all the

adenocarcinomas compared to adjacent non-

neoplastic tissues, which shows little staining.

MINA is expressed in all pathological grades of

cancer as well as in the adenoma. Staining patterns

of Ki-67, a biomarker for cell proliferation, are

similar to those of MINA in most cases. While anti-

Ki-67 antibody strongly stains some well-

proliferating non-neoplastic cells including cells in

the deeper part of the crypts and in lymphoid

germinal centers, antibody to MINA rarely stained

those cells. These results indicate that the elevated

expression of MINA is a characteristic feature in

colon cancer (Teye et al., 2004).

Esophageal cancer

Note

The expression of MINA in tumors is increased

compared with that in adjacent non-neoplastic

tissues. MINA was highly expressed in more than

80% of specimens. Anti-MINA antibody stained

tumors more efficiently than antibody against Ki-

67, a cell proliferation biomarker, in some cancer

specimens. Patients with high expression of MINA

has shorter survival periods, whereas the expression

level of Ki-67 in ESCC shows no relationship to

patient outcome (Tsuneoka et al., 2004).

Primary gingival squamous cell carcinoma

Note

A significant correlation was found between the

expression of MINA and that of Ki-67 in patients

with gingival squamous cell carcinoma or

dysplastic gingiva. No significant correlation was

noted between the expression of MINA or Ki-67

and prognostic factors such as the degree of

differentiation, lymph node metastasis, stage, and

tumor diameter (Kuratomi et al., 2006).

Lymphoma

Note

Although MINA expression is not prominent in

lymphoma in general, it is related to tumor

progression of B cell lymphoma (Teye et al., 2007).

Renal cell carcinoma (RCC)

Note

MINA is expressed in the nuclei of tumor cells and

tubular nuclei of normal renal tissue.

The expression level of MINA is significantly

higher in patients with poor prognostic factors

(stage IV, MVI-positive, and sarcomatoid RCC,

and high Ki-67 LI). The prognosis of high MINA-

expressing tumors was significantly poorer than

that of non-MINA-high tumors (Ishizaki et al.,

2007).

Neuroblastoma

Note

Surgically obtained neuroblastoma specimens were

immunohistochemically stained to determine the

MINA and Cap43 expression levels. A significant

relationship was found between MINA and Ki-67,

between MINA and neurotrophic tyrosine kinase,

receptor, type 1 (TrkA), and between Cap43 and

TrkA. The prognosis is significantly favorable in

the Cap43 high-expression cases, whereas it is

significantly poor in the MINA high-expression

cases (Fukahori et al., 2007).

Gastric carcinomas

Note

Elevated expression of MINA was observed in

91.1% of the gastric carcinomas. No significant

associations were found between MINA and

clinicopathological characteristics such as sex, age,

histological differentiation, distant metastasis and

lymph node metastasis. However, there was a

significant association with depth of invasion and

TMN stage. MINA expression was positively

associated with a proliferation marker, PCNA, level

(Zhang et al., 2008).

Lung cancers

Note

The expression of MINA is elevated in lung cancer

tissues (Lu et al., 2009; Komiya et al., 2010). The

overexpression of MINA is an early event in lung

cancer occurence (Lu et al., 2009; Komiya et al.,

2010). Further, patients with negative staining for

MINA has shorter survival than patients with

positive staining for MINA, especially in stage I or

with squamous cell carcinoma. These results

suggest that overexpression of MINA in lung

cancer patients is associated with favorable

prognosis (Komiya et al., 2009). MINA may inhibit

lung cancer cell invasion (Komiya et al., 2009).

Hepatoma

Note

MINA is diffusely expressed in the nuclei of cancer

cells in the tumor nodule, and is often strong at the

periphery of tumor nodules. MINA expression is

higher in poorly differentiated hepatocellular

carcinoma (HCC) than in well-differentiated HCC,

and there is significant relationship between MINA

expression and histological grade. The high MINA

expression is associated with high expression of a

proliferation marker, antibody to Ki-67. MINA

expression is high in the tumors of > 2 cm of

diameter than in ≤ 2 cm (Ogasawara et al., 2010 in

press).



References Tsuneoka M, Koda Y, Soejima M, Teye K, Kimura H. A novel myc target gene, mina53, that is involved in cell proliferation. J Biol Chem. 2002 Sep 20;277(38):35450-9

Teye K, Tsuneoka M, Arima N, Koda Y, Nakamura Y, Ueta Y, Shirouzu K, Kimura H. Increased expression of a Myc target gene Mina53 in human colon cancer. Am J Pathol. 2004 Jan;164(1):205-16

Tsuneoka M, Fujita H, Arima N, Teye K, Okamura T, Inutsuka H, Koda Y, Shirouzu K, Kimura H. Mina53 as a potential prognostic factor for esophageal squamous cell carcinoma. Clin Cancer Res. 2004 Nov 1;10(21):7347-56

Eilbracht J, Kneissel S, Hofmann A, Schmidt-Zachmann MS. Protein NO52--a constitutive nucleolar component sharing high sequence homologies to protein NO66. Eur J Cell Biol. 2005 Mar;84(2-3):279-94

Zhang Y, Lu Y, Yuan BZ, Castranova V, Shi X, Stauffer JL, Demers LM, Chen F. The Human mineral dust-induced gene, mdig, is a cell growth regulating gene associated with lung cancer. Oncogene. 2005 Jul 21;24(31):4873-82

Kuratomi K, Yano H, Tsuneoka M, Sakamoto K, Kusukawa J, Kojiro M. Immunohistochemical expression of Mina53 and Ki67 proteins in human primary gingival squamous cell carcinoma. Kurume Med J. 2006;53(3-4):71-8

Tsuneoka M, Nishimune Y, Ohta K, Teye K, Tanaka H, Soejima M, Iida H, Inokuchi T, Kimura H, Koda Y. Expression of Mina53, a product of a Myc target gene in mouse testis. Int J Androl. 2006 Apr;29(2):323-30

Fukahori S, Yano H, Tsuneoka M, Tanaka Y, Yagi M, Kuwano M, Tajiri T, Taguchi T, Tsuneyoshi M, Kojiro M. Immunohistochemical expressions of Cap43 and Mina53 proteins in neuroblastoma. J Pediatr Surg. 2007 Nov;42(11):1831-40

Ishizaki H, Yano H, Tsuneoka M, Ogasawara S, Akiba J, Nishida N, Kojiro S, Fukahori S, Moriya F, Matsuoka K,

Kojiro M. Overexpression of the myc target gene Mina53 in advanced renal cell carcinoma. Pathol Int. 2007 Oct;57(10):672-80

Teye K, Arima N, Nakamura Y, Sakamoto K, Sueoka E, Kimura H, Tsuneoka M. Expression of Myc target gene mina53 in subtypes of human lymphoma. Oncol Rep. 2007 Oct;18(4):841-8

Zhang Q, Hu CM, Yuan YS, He CH, Zhao Q, Liu NZ. Expression of Mina53 and its significance in gastric carcinoma. Int J Biol Markers. 2008 Apr-Jun;23(2):83-8

Hemmers S, Mowen KA. T(H)2 bias: Mina tips the balance. Nat Immunol. 2009 Aug;10(8):806-8

Okamoto M, Van Stry M, Chung L, Koyanagi M, Sun X, Suzuki Y, Ohara O, Kitamura H, Hijikata A, Kubo M, Bix M. Mina, an Il4 repressor, controls T helper type 2 bias. Nat Immunol. 2009 Aug;10(8):872-9

Komiya K, Sueoka-Aragane N, Sato A, Hisatomi T, Sakuragi T, Mitsuoka M, Sato T, Hayashi S, Izumi H, Tsuneoka M, Sueoka E. Mina53, a novel c-Myc target gene, is frequently expressed in lung cancers and exerts oncogenic property in NIH/3T3 cells. J Cancer Res Clin Oncol. 2010 Mar;136(3):465-73

Komiya K, Sueoka-Aragane N, Sato A, Hisatomi T, Sakuragi T, Mitsuoka M, Sato T, Hayashi S, Izumi H, Tsuneoka M, Sueoka E. Expression of Mina53, a novel c-Myc target gene, is a favorable prognostic marker in early stage lung cancer. Lung Cancer. 2010 Aug;69(2):232-8

Sinha KM, Yasuda H, Coombes MM, Dent SY, de Crombrugghe B. Regulation of the osteoblast-specific transcription factor Osterix by NO66, a Jumonji family histone demethylase. EMBO J. 2010 Jan 6;29(1):68-79


Tsuneoka M, Okamoto K, Tanaka Y. MINA (MYC induced nuclear antigen). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):15-18.

Gene Section Review




NKX2-1 (NK2 homeobox 1) Theresia Wilbertz, Sebastian Maier, Sven Perner

Institute of Pathology, University Hospital Tubingen, Germany (TW, SM, SP)


Online updated version : http://AtlasGeneticsOncology.org/Genes/NKX2-1ID44015ch14q13.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI NKX2-1ID44015ch14q13.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Other names: BCH, BHC, NK-2, NKX2.1,

NKX2A, TEBP, TITF1, TTF-1, TTF1

HGNC (Hugo): NKX2-1

Location: 14q13.3

DNA/RNA

Description

NKX2-1 is regulated by two promoter regions: the

first one is located in intron 1 (5' of exon 1,

regulation of NKX2-1 in lung and thyroid cells).

The second one is situated in the 5' flanking region

of exon 1, it is a 330 bp TATA-less region

containing multiple palindromes and G/C-rich

elements. It regulates NKX2-1 in lung epithelial

cells responding to transcription factors sp1 and

sp3.

Transcription

NKX2-1 is transcribed in two highly conserved

forms: mRNA-isoform 1 contains exon 1, exon 2,

and exon 3, it is translated into a 401 amino acid

protein and represents the minor transcript. mRNA-

isoform 2 is the predominant transcript containing

exon 2 and exon 3. It is translated into a 371 aa

protein.

Figure 1. NKX2-1 gene and NKX2-1 mRNA.

NKX2-1 (NK2 homeobox 1) Wilbertz T, et al.


Figure 2. Upstream and downstream targets of NKX2-1.

Protein

Description

The NKX2-1 protein includes three functional

domains: an N-terminal transactivation domain, a

DNA-binding transactivation domain and a C-

terminal transactivation domain.

Expression

In the lung, expression of NKX2-1 is consistent

throughout all life stages from fetal to adult tissue.

It is expressed in conducting airways type II

alveolar epithelial cells and Clara cells and

uniformly in the terminal respiratory unit.

NKX2-1 expression is also found in thyroid

follicular cells and both normal and hyperplastic C

cells where it activates calcitonin gene expression.

NKX2-1 is not expressed in adult neurons of the

basal ganglia.

During embryonic and fetal development, NKX2-1

expression is found in various tissues (e.g. brain,

lung, thyroid), for details see "function" →

"Embryonic and fetal development".

Localisation

NKX2-1 is a nuclear transcription factor.

Function

In the lung, NKX2-1 regulates the expression of the

lung-specific genes: surfactant protein SP-A, SP-B,

SP-C and Clara cell secretory protein (CCSP).

It cooperates with C/EBPalpha in transactivating

CCSP.

In the transcription of SP-C, NKX2-1 interacts with

nuclear factor I to differentially regulate the

transcription. The longer NKX2-1 isoform reduces

transactivation of SP-C, probably due to some kind

of interference.

NKX2-1 is a key activator of SP-B gene expression

having at least two binding sites at the SP-B

promoter and enhancer. The transactivation

capacity of NKX2-1 regarding the expression of

SP-B is controlled by the sphingolipid ceramide

which is produced in inflammation and reduces

NKX2-1 binding capacity to the SP-B promoter.

SP-B transcription is also inhibited by TGFbeta1-

mediated interaction of smad3 with NKX2-1.

Moreover, NKX2-1 interacts with retinoic acid

receptor (RAR), nuclear receptor coactivators

(p160, CBP/p300) and signal transducers and

activators of transcription 3 (STAT3) in regulation

of SP-B expression.



Furthermore, NKX2-1 regulates the expression of

the secretoglobulin 3A2 gene (SCGB3A2) in mouse

airways in cooperation with CAATT/enhancer

binding proteins alpha and delta as well as the

expression of ABCA3 which encodes for a lipid

transporter critical for surfactant function at birth

and formation of lamellar bodies.

NKX2-1 also plays an important role in the

endocrine system: it regulates the expression of the

thyroid-specific genes thyroglobulin, thyroid

peroxidase, thyrotropin receptor and sodium-

iodide-symporter, therefore being crucial for proper

thyroid hormone synthesis.

Deletion of NKX2-1 in differentiated neurons of

the hypothalamus in mice causes delayed puberty,

reduced reproductive capacity and a shorter

reproductive span in female mice, suggesting that

NKX2-1 plays an important role in juvenile and

adult endocrine function.

During embryonic and fetal development, NKX2-1

is active in various organs, especially lung, thyroid

and brain.

As a crucial factor for lung development, NKX2-1

is expressed in the ventral foregut endoderm at a

very early stage functioning as a signal which is

essential for specification of a pulmonary cell fate

instead of a liver cell fate. At a later stage, NKX2-1

is critical to the formation of distal pulmonary

structures (whereas proximal lung differentiation is

NKX2-1-independent), a function in which it is

inhibited by TGF-beta.

In addition to that NKX2-1 regulates surfactant

protein genes that are important for the

development of alveolar stability at birth. It induces

SP-A gene expression in fetal lung type II cells

through increased binding of NKX2-1 (mediated by

cAMP) and the NFkappa-B proteins p50 and p65.

Supporting the notion of NKX2-1-dependent SP-

expression, lung and associated respiratory

dysfunction in neonates caused by SP-B-deficiency

are partly induced by down-regulation of NKX2-1.

The main therapeutical option, prenatal

glucocorticoid treatment, induces the expression of

NKX2-1. NKX2-1 regulates expression of

uteroglobin-related protein-1 and claudin-18 during

lung development.

During thyroid gland organogenesis NKX2-1 is

expressed in the ultimobranchial body (UBB) and

in the thyroid diverticulum. It is important for the

survival of UBB-cells and eventually their

dissemination into the thyroid diverticulum and for

the formation of the UBB-derived vesicular

structure. Pendrin and thyroglobulin are

downstream targets of NKX2-1 during thyroid

differentiation. The transactivational activity of

NKX2-1 during thyroid development can be

inhibited by NKX2-5.

In the course of brain development, NKX2-1

expression is found in both telencephalic and

diencephalic domains. It cooperates with Gsh2 to

pattern the ventral telencephalon. Lack of

functional NKX2-1 protein in neurons impairs

developmental differentiation and organization of

basal ganglia and basal forebrain. NKX2-1

upregulates the transcription of nestin, an

intermediate filament protein expressed in

multipotent neuroepithelial cells, by direct binding

to a HRE-CRE-like site (NestBS) within a CNS-

specific enhancer, which indicates that nestin might

be at least one of the effectors of NKX2-1 during

forebrain development.

NKX2-1 expression occurs in neurons of the

arcuate nucleus of the hypothalamus and in glia

cells (tanycytes) in neonatal and adult mice, as well

as in fetal and adult pituicytes suggesting that

NKX2-1 is essential for proper development of the

hypothalamus. Lack of NKX2-1 causes aberrant

trajectory of the dopaminergic pathway in the

developing hypothalamus (mouse-model),

development of GABAergic and cholinergic

neurons is also impaired in NKX2-1 defective mice.

Furthermore, NKX2-1 regulates the specification of

oligodendrocytes and controls the postmitotic

migration of interneurons originating in the medial

ganglionic eminence to either the cortex

(downregulation of NKX2-1) or the striatum

(maintenance of NKX2-1 expression and thus direct

transcriptional activation of neuropilin-2, a

guidance receptor in postmitotic cells). By directly

activating Lhx6 during embryonic development

NKX2-1 plays an essential role for the specification

of cortical interneurons which express parvalbumin

or somatostatin.

In accordance with the findings concerning the role

of NKX2-1 in the development of the above-

mentioned organs, NKX2-1-defective mice die at

birth due to a characteristic set of malformations

and functional impairments: hypoplastic lungs and

insufficient surfactant production, defective

hypothalamus, absence of thyroid and pituitary

gland, delayed development of dopaminergic,

GABAergic and cholinergic neurons.

Mutations

Note

Germinal

Mutations in NKX2-1 (for details see table 1) can

cause benign hereditary chorea (BHC, a dyskinesia,

i.e. a neurological disorder characterized by

abnormal involuntary movements) and brain-lung-

thyroid syndrome (in addition to BHC, patients

suffer from congenital hypothyroidism and infant

respiratory distress syndrome).

A heterozygous substitution at position 1016 in the

coding sequence (C → T) leads to a mutant NKX2-

1 protein (A339V) and can contribute to a

predisposition for multinodular goiter and papillary

thyroid carcinoma.



Brain-lung-thyroid syndrome congenital hypothyreoidism, infant respiratory distress syndrome, benign hereditary chorea

SNP bp 523 G → T premature stop codon at postition 175

SNP bp 609 C → A premature stop codon at position 145

SNP bp 1320 C → A premature stop codon at position 75

SNP bp 2626 G → T missense mutation: valine → phenylalanine at position 14 of DNA-

binding-domain

SNP splice acceptor

site of intron 2 A → T altered mRNA structure => incorrect removal of introns

Deletion 14q11.2-q13.3

Insertion bp 2595

insertion of GG frameshift mutation: causes truncated protein lacking

the entire third helix of the homeodomain

Cancer predisposition can contribute to predisposition for multinodular goiter and papillary thyroid carcinoma.

SNP bp 1016 C → T missense mutation: A339V

Table 1. Mutations in NKX2-1 gene.

For other heterozygous NKX2-1 mutations in

humans, phenotypes vary widely.

Thyroid dysfunction ranges from mild

hypothyrotrophinaemia to severe congenital

hypothyroidism due to thyroid hypoplasia or even

agenesis. Implication of the lung ranges from a

slight increase in pulmonary infections to severe

neonatal respiratory distress syndrome.

Homozygous NKX2-1 mutations in humans are

probably not viable.

Implicated in

Various cancers

Note

NKX2-1 expression has been found in a variety of

tumor entities, especially lung and thyroid tumors

(for details see table 2).

Lung neoplasms

Disease

NKX2-1 is strongly expressed in 75-90% of

primary lung adenocarcinomas, whereas only 1/4 of

bronchioloalveolar carcinomas show NKX2-1

positivity. Among non-small cell lung cancers,

NKX2-1 is not expressed in squamous cell lung

cancer.

Small cell lung cancer, as well as pulmonary

carcinoids and non-neuroendocrine large-cell

carcinomas partly exhibit NKX2-1 protein

expression.

Prognosis

Overall, NKX2-1 expression is a predictor for

better survival in adenocarcinomas of the lung (just

one smaller study suggested that NKX2-1

expression is associated with poor prognosis).

Controversially, NKX2-1 pathway activation in

lung cancers is associated with poor survival and

cisplatin resistance if PAX9 or Nkx2-8 pathways

are activated at the same time.

Oncogenesis

NKX2-1 is highly amplified in 5-15% of primary

lung adenocarcinomas. In cells harbouring NKX2-1

amplification, this recurrent gene amplification

seems to be a mechanism of high-level NKX2-1

expression.

For a subset of lung adenocarcinomas (especially

those which are derived from the terminal

respiratory unit) sustained expression of NKX2-1

has been shown to be crucial for the survival of

tumor cells. In these tumors RNAi inhibition of

NKX2-1 induces proliferation inhibition, growth

inhibition and apoptosis (lineage-specific

dependency model).

Interestingly, NKX2-1 is also an activator of HOP

(Hsp70/Hsp90 Organizing Protein), a potential

tumor suppressor gene in lung cancer, and it

inhibits EMT (epithelial to mesenchymal

transition). NKX2-1 restores epithelial phenotypes

in lung adenocarcinomas, acting as an adversary of

the EMT-stimulating TGF-beta and a suppressor of

tumor progression and invasiveness. TGF-beta

inhibits the expression of NKX2-1 and thus lung

morphogenesis.

Moreover, NKX2-1 is expressed in most metastatic

lung adenocarcinomas.

Thyroid neoplasms

Disease

Well-differentiated thyroid follicular cell tumors,

such as follicular adenomas, follicular carcinomas

and papillary carcinomas exhibit strong nuclear

positivity for NKX2-1 staining. In contrast,

undifferentiated thyroid carcinomas show low or no

immunoreaction.



Consistently expressed

Occasionally

expressed Not expressed

Thyroid

- Papillary carcinoma

- Follicular carcinoma

- Medullary carcinoma

- Hurthle cell carcinoma

- Follicular adenoma

- Hyperplastic follicular

cells

- Undifferentiated thyroid

carcinomas

Lung

- Adenocarcinoma

- Small cell lung cancer

(SCLC)

- Pleural effusions of SCLC

- Pulmonary sclerosing

hemangioma

- Bronchioloalveolar

carcinoma (except for

mucinous parts)

- Non-neuroendocrine

large-cell carcinoma

- Signet-ring cell

carcinomas of lung origin

- Pulmonary carcinoids

(50%) - Squamous cell lung cancer

- Pleural mesothelioma

- Bronchioloalveolar

carcinomas (just mucinous

parts)

- Basaloid carcinoma of the

lung

Gastrointestinal system - Small cell cancer of the

esophagus - Colorectal carcinoma

Genitourinary system

- Small cell carcinoma

of the urine bladder

- Nephroblastoma

- Endometrial

carcinoma

- Endocervical

carcinoma

Thymus

- Thymic carcinoma

- Thymoma

Skin

- Merkel cell carcinoma

Neuroectodermal

- Ependymoma

- Glioblastoma - Astrocytoma

- Oligodendroglioma

- Medulloblastoma

- Paraganglioma

- Ganglioglioma

Neuroendocrine

(carcinoid tumorlets,

neuroendocrine cell

hyperplasia, typical

carcinoids, atypical

carcinoids, large cell

neuroendocrine

carcinomas)

- Thyroid origin

- Pulmonary origin - Thymic origin

- Gastrointestinal origin

- Pancreatic origin

- Ovarian origin

- Parathyroid adenoma

- Pituitary adenoma

- Pheochromocytoma

Body cavity fluids

- Lung origin

(adenocarcinoma)

- Genitourinary origin

- Gastrointestinal origin

- Breast origin

- Ovarian origin

Table 2. Expression of NKX2-1 in different tumor entities.

http://atlasgeneticsoncology.org/Tumors/LungSmallCellID5142.html

http://atlasgeneticsoncology.org/Tumors/colon5006.html

http://atlasgeneticsoncology.org/Tumors/PituitAdenomID5051.html

http://atlasgeneticsoncology.org/Tumors/pheochromocytomaID5026.html



Concerning parafollicular cells, NKX2-1

expression can be found in normal and hyperplastic

c-cells, as well as in medullary thyroid carcinomas.

However, the signal intensity is much weaker and

less homogenous than observed in tumors

originating from follicular thyroid cells.

Non-malignant branchiogenic cysts can easily be

confounded with papillary thyroid carcinomas.

Since positive immunostaining for NKX2-1 has

been found in a subset of these non-malignant

cervical cysts, NKX2-1 cannot serve to distinguish

between both entities.

Oncogenesis

NKX2-1 is expressed in most differentiated thyroid

neoplasms, but not in undifferentiated tumors of

thyroid origin. On DNA-level, normal thyroids and

papillary carcinomas do not exhibit DNA

methylation in the CpG of NKX2-1 promoter,

whereas undifferenciated thyroid carcinomas show

DNA methylation in this region in about 60%. Most

metastases of thyroid origin are positive for NKX2-

1 expression.

A heterozygous germline mutation, which leads to a

mutant NKX2-1 protein has been shown to be

associated with increased cell proliferation.

Consequently, it might contribute to a

predisposition for multinodular goiter and papillary

thyroid carcinoma (for details see section

mutations).

Neoplasms of the gastrointestinal tract

Disease

Small cell esophageal cancers exhibit NKX2-1

expression in the majority of cases. In contrast,

carcinoids originating from the gastrointestinal

tract, such as ileal, appendical, duodenal,

ampullary, rectal, pancreatic and gastric carcinoids

are negative for NKX2-1 immunohistochemical

staining.

Neoplasms of the genitourinary tract

Disease

NKX2-1 seems to be implicated in neoplasms

arising from the urinary system. Small cell

carcinomas of the urinary bladder are positive for

NKX2-1 staining in 25-40% of cases. Likewise,

large cell neuroendocrine bladder carcinomas

exhibit NKX2-1 expression. In one study, 1/6 of a

set of nephroblastomas showed nuclear positivity

for NKX2-1, whereas metanephric adenomas and

cystic nephromas were NKX2-1 negative.

NKX2-1 expression can be found in benign tubal

and endometrial epithelia, as well as in benign

tumors originating from these tissues. In addition,

malignant tumors of the female genital tract, such

as endocervical adenocarcinomas, small cell

carcinomas of the uterine cervix, endometrioid

adenocarcinomas, serous carcinomas, clear cell

carcinomas, and uterine malignant mixed Mullerian

tumors show positivity for NKX2-1. Staining

morphology in these tumors differs from rare

positive cells to a diffusely positive staining pattern.

Prognosis

No correlation could be detected between positive

NKX2-1 immunostaining in small cell carcinomas

of the urinary bladder and clinicopathologic

features (including outcome, age, sex, smoking

history, stage and metastatic status).

Neuroendocrine neoplasms

Disease

Among well-differentiated neuroendocrine tumors,

only those tumors originating from the lung or

thyroid are positive for NKX2-1 expression.

Neither gastrointestinal typical or atypical

carcinoids, nor neuroendocrine tumors from other

sites (e.g. Merkel cell carcinomas, thymic

carcinoids, ovarian large cell neuroendocrine

carcinomas) show NKX2-1 expression.

Concerning small cell carcinomas, NKX2-1

expression is not specific for small cell lung cancer,

as NKX2-1 expression can also be found in small

cell carcinomas originating from the esophagus,

prostate, bladder or uterine cervix.

Neoplasms of neuroectodermal origin

Disease

NKX2-1 occasionally has been detected in

glioblastoma multiforme and in ependymomas of

the third ventricle. Other primary brain tumors,

such as astrocytomas, oligodendrogliomas,

medulloblastomas and gangliomas from various

sites do not exhibit NKX2-1 expression.

Sellar tumors, including pituicytomas, atypical

pituicytomas, granular cell tumors and spindle cell

oncocytomas can show positive immunostaining for

NKX2-1.

To be noted

Note

NKX2-1 has been well studied in neoplasms of the

lung and thyroid, but lacks a sufficient level of

evidence in other tumor entities.

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Kwei KA, Kim YH, Girard L, Kao J, Pacyna-Gengelbach M, Salari K, Lee J, Choi YL, Sato M, Wang P, Hernandez-Boussard T, Gazdar AF, Petersen I, Minna JD, Pollack JR. Genomic profiling identifies TITF1 as a lineage-specific oncogene amplified in lung cancer. Oncogene. 2008 Jun 5;27(25):3635-40

Lin JD. Thyroglobulin and human thyroid cancer. Clin Chim Acta. 2008 Feb;388(1-2):15-21

Maeshima AM, Omatsu M, Tsuta K, Asamura H, Matsuno Y. Immunohistochemical expression of TTF-1 in various cytological subtypes of primary lung adenocarcinoma, with special reference to intratumoral heterogeneity. Pathol Int. 2008 Jan;58(1):31-7

Nóbrega-Pereira S, Kessaris N, Du T, Kimura S, Anderson SA, Marín O. Postmitotic Nkx2-1 controls the migration of telencephalic interneurons by direct repression of guidance receptors. Neuron. 2008 Sep 11;59(5):733-45

Pelizzoli R, Tacchetti C, Luzzi P, Strangio A, Bellese G, Zappia E, Guazzi S. TTF-1/NKX2.1 up-regulates the in vivo transcription of nestin. Int J Dev Biol. 2008;52(1):55-62

Perner S, Wagner PL, Demichelis F, Mehra R, Lafargue CJ, Moss BJ, Arbogast S, Soltermann A, Weder W, Giordano TJ, Beer DG, Rickman DS, Chinnaiyan AM, Moch H, Rubin MA. EML4-ALK fusion lung cancer: a rare acquired event. Neoplasia. 2008 Mar;10(3):298-302

Tomita T, Kido T, Kurotani R, Iemura S, Sterneck E, Natsume T, Vinson C, Kimura S. CAATT/enhancer-binding proteins alpha and delta interact with NKX2-1 to



synergistically activate mouse secretoglobin 3A2 gene expression. J Biol Chem. 2008 Sep 12;283(37):25617-27

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Xing Y, Li C, Hu L, Tiozzo C, Li M, Chai Y, Bellusci S, Anderson S, Minoo P. Mechanisms of TGFbeta inhibition of LUNG endodermal morphogenesis: the role of TbetaRII, Smads, Nkx2.1 and Pten. Dev Biol. 2008 Aug 15;320(2):340-50

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Wilbertz T, Maier S, Perner S. NKX2-1 (NK2 homeobox 1). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):19-28.

Gene Section Review




NUDT6 (nudix (nucleoside diphosphate linked moiety X)-type motif 6) Leigh-Ann MacFarlane, Paul Murphy

Dalhousie University, Department of Physiology and Biophysics, Faculty of Medicine, 5850 College

Street Sir Charles Tupper Medical Building, Halifax, Nova Scotia B3H 1X5, Canada (LAM, PM)


Online updated version : http://AtlasGeneticsOncology.org/Genes/NUDT6ID41593ch4q28.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI NUDT6ID41593ch4q28.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Other names: ASFGF2, bFGF, FGF-2, FGF-AS,

FGF2AS, gfg, gfg-1

HGNC (Hugo): NUDT6

Location: 4q28.1

Note: NUDT6 is a novel nudix protein with

unknown function.

DNA/RNA

Note

Human NUDT6 is located on chromosome 4 in the

region of q28 on the reverse strand, opposite to

FGF-2 gene locus. FGF-2 and NUDT6 genes

overlap at 3' ends, and the mRNAs form a sense-

antisense pair. The NUDT6 mRNA (referred to as

FGF-AS) has been implicated in the regulation of

FGF2 mRNA stability.

Figure A. The schematic representation of the overlap between human NUDT6 (FGF-AS) and FGF2 gene transcripts (colored boxes, coding region; connecting vertical lines, complementary regions between transcripts). Adapted from: MacFarlane LA, et

al., 2010. Molecular Endocrinology 24.

NUDT6 (nudix (nucleoside diphosphate linked moiety X)-type motif 6)

MacFarlane LA, Murphy P


Figure B. The schematic representation of the human NUDT6 gene transcripts, variants a-g (red boxes, coding region; yellow

boxes, untranslated region).

Description

The human NUDT6 gene is 34271 bp in length,

composed of a 5'UTR, 16 exons, 6 introns and a

3'UTR. The 5' and 3'UTR contain a variety of

regulatory elements that regulate NUDT6

expression. NUDT6 gene transcription is regulated

by a core promoter mapping from -1871 to +181

(relative to the transcription start site +1, up-stream

-), which is 44 kb downstream from the FGF-2

promoter, however the proximal -151/+181 region

confers almost full transcription activity.

The promoter lacks a consensus TATA box or

CCAAT element. The region between the first two

exons contains two Sp1 transcription factor binding

sites (-372/-58 relative to the first exon start site).

The common upstream region (all subsequent

positions relative to first exon start site) from these

start sites contains a multitude of tissue specific

transcription factor binding sites, which include

lymphocyte specific factors Ets at -229 and -83,

GATA at -662 and +56, Lyf-1 at -981; skeletal

muscle consensus E-boxes at -901 and +30; cardiac

factor Nkx-2.5 at -1501 and -582; liver and adipose

C/EBP factor at -624; and testis specific factors

SRY at -1740, -671, +163, +171 and Sox-5 at -

1472, -632.

Two negative regulatory elements also reside in this

shared upstream region, at -1871 and -1315. The

NUDT6 3'UTR contains a singe AU-rich element

(ARE) and seven AU-rich-like sequences which

negatively regulate mRNA stability. A portion of

the NUDT6 coding region and 3'UTR

(+531/+1167) is fully complementary to the 3'UTR

of FGF2 and interaction through this region leads to

the formation of a sense-antisense pair.

Transcription

The primary transcript can be alternatively spliced

to produce at least 7 splice variants, a-d. Full length

variants a and b only differ in the use of an

alternative first exon, designated 1A or 1B. The 3'

ends of the variants share sequence similarity. Two

transcriptional start sites have been identified, one

15 bp upstream of the 1A exon (designated +1) and

the other 84 bp upstream of the 1B exon (+312). It

is unclear whether another transcription start site

specific for other variants are located further

downstream.

NUDT6 transcripts are often designated FGF-AS

(FGF antisense). The two longest transcripts, a and

b, are classified as cis-antisense because they are

transcribed from the same gene locus, on the

opposite DNA strand and their 3'UTR is fully

complementary to the 3'UTR of FGF-2 over two

regions 583 bp and 56 bp in length.

Pseudogene

NA.

Protein

Note

Human NUDT6 encodes 3 novel nudix proteins

with unknown function.

Description

NUDT6 splice variants a-c contain open reading

frames (ORF) that predict isoforms of a novel nudix

motif protein, originally designated GFG. The

nudix box motif is defined by the consensus

signature amino acid sequence

GX5EX7REUXEEXGU,




Schematic representing predicted NUDT6 isoforms encoded by alternate splice RNA transcripts (deep blue boxes, nudix motif;

light blue box, MTSP-mitochondrial targeting signal peptide).

where X is any amino acid and U is a bulky

hydrophobic amino acid, usually isoleucine, leucine

or valine. To date, three different molecular weight

isoforms have been identified in human, of 35, 28

and 17 kDa, which are presumably generated by

alternative translation initiation. Isoforms are

designated as a, b or c however, this does not

necessarily indicate that the isoform was

synthesized from the corresponding transcript

variant. The 35 kDa isoform is synthesized from the

full length FGF-ASa, by translation initiation from

the in-frame AUG codon located in exon 1A. The

origin of the 28 kDa isoform is unclear. It is

suspected that it is synthesized from an in-frame

CUG codon in exon 2 of either FGF-AS a or b.

However, it is possible that the 28 kDa product is a

proteolytic fragment of the 35 kDa isoform. The 17

kDa isoform may arise from translation initiation at

an in-frame CUG codon in exon 3 of FGF-ASb or

AUG codon in the first exon of FGF-ASc. The

NUDT6 isoforms are detected as stable homo- and

hetero-dimers by western blotting, which can be

disrupted by dithiothreitol (DTT) and boiling.

Potential dimerization domains have been mapped

to both the N-terminus and COOH-terminus of

NUDT6.

Expression

NUDT6 is expressed in a tissue and developmental

stage specific manner. RNA transcripts are detected

in most human tissues including liver, thymus,

spleen, peripheral blood leukocytes, heart skeletal

muscle, testis, colon and kidney. However, which

transcript variants are expressed appears to be tissue

specific. The full length FGF-ASb is thought to be

the predominant variant in most tissues however

variant FGF-ASa is the major variant in normal

hematopoietic tissues. Furthermore, some tissues

co-express FGF-2 and the ratio between FGF-2 and

FGF-AS transcripts varies with tissue and

development stage. FGF-AS levels are relatively

low in many embryonic tissues, with expression

increasing dramatically in a tissue specific manner

postnatally. FGF-2 and FGF-AS exhibit an inverse

relationship in normal tissues, tumor cell lines,

embryonic development and throughout cell cycle

progression.

The level of NUDT6 expression and its ratio with

FGF-2 expression is frequently altered in tumors.

Normal pituitary expresses moderate levels of

NUDT6 and no FGF-2 while pituitary tumors have

reduced NUDT6 expression and high levels of

FGF-2. The NUDT6/FGF-2 expression ratio

decreases dramatically in tumors compared to

normal tissue. Varying NUDT6/FGF-2 ratios have

also been observed in esophageal adenocarcinomas.

Additionally, transient increase in NUDT6

expression occurs in response to treatment with

interleukin-2 and prolactin.

Localisation

NUDT6 can reside in the mitochondria, cytoplasm

and nucleus, however its subcellular localization

varies with isoform, cell type, disease state and

extracellular stimulus. NUDT6a predominantly

localizes to mitochondria whereas NUDT6b and

NUDT6c primarily reside in the cytoplasm and

nucleus. NUDT6 is only found in the cytoplasm of

normal esophageal squamous epithelial cells

whereas in normal lymphocytes it is exclusively

nuclear. However, transformation of these cells

results in the redistribution of NUDT6. Cells from

esophageal adenocarcinoma tumors and lymph

nodes of patient with immunoblastic lymphoma

localize NUDT6 to the nucleus and cytoplasm.

Function

The RNA and protein products appear to have

distinct biological functions. NUDT6 mRNA (FGF-

AS) plays a role in FGF-2 regulation, proliferation,

and cell survival. Additionally, the NUDT6 protein

has been implicated in the control of hormone

production in the pituitary, and possibly in the

removal of potentially hazardous compounds and

metabolites by virtue of its conserved nudix

domain. However, it is not always clear whether a

specific action is a result of the RNA or protein

function and this is further complicated by multiple

antisense splice variants and protein isoforms.

FGF-AS regulates FGF-2 transcript stability.

Although the details of the mechanism involved are

unclear, evidence suggest involvement of RNA

interference and/or a dsRNA duplex formed

between the 3'UTRs of FGF-AS and FGF-2. In

addition to regulating FGF-2 abundance it has been

suggested that it also controls FGF-2 isoform

translation and localization. The regulatory role of

FGF-AS over FGF-2 is thought to account for

observed effects on cell proliferation and survival.

NUDT6 protein is a nudix hydrolase which is a

class of "house cleaning" enzymes capable of

hydrolyzing a broad range of substrates, all defined

as nucleoside diphosphates linked to some other

moiety, that include nucleoside di- and




triphosphates, dinucleoside and diphosphoinositol

polyphosphates, nucleotide sugars and RNA caps.

The substrate of human NUDT6 has yet to be

elucidated and therefore its physiological function

remains unknown. NUDT6 has observed effects on

cell proliferation independent of those associated

with FGF-AS. NUDT6 overexpression in human

colorectal cancer cells increases proliferation.

Perhaps NUDT6's effects on proliferation are

dependent on expression level, isoform and/or cell

type, as is the case with FGF-2. Furthermore,

NUDT6 is involved in hormone production. GFG

expression can increase levels of prolactin.

However it is unclear if these effects are mediated

through the same MAPK pathway utilized by FGF-

2 to increase prolactin expression.

Additionally, NUDT6 alters the isoform ratio of

growth hormone, by increasing synthesis of the 22

kDa isoform and not the 20 kDa.

Homology

NUDT6 contains a conserved nudix motif common

to other members of the Nudix family of

phosphohydrolases. The Nudix motif is

GXXXXXEXXXXXXXREUXEEXGU where U is

Isoleucine, Leucine, or Valine and X is any amino

acid. NUDT6 is highly conserved among man, cow,

mouse, worm, and fruit fly, and GFG homologs

across species are more evolutionarily related to

each other than to other nudix proteins from the

same species.

Implicated in

Esophageal adenocarcinoma

Note

Esophageal adenocarcinoma refers to uncontrolled

growth of glandular cells in the esophagus and the

junction between the esophagus and the stomach.

Prognosis

Elevated expression of FGF-AS in FGF-2

expressing esophageal adenocarcinomas is

associated with reduced tumor reoccurrence

following surgical resection of tumors and

increased survival rates, suggesting that it may be

used as a prognostic indicator.

Oncogenesis

Esophageal adendocarcinoma tumors overexpressed

FGF-AS and cytoplasmic GFG in comparison to

normal match esophageal tissue. However, the

reduced tumor reoccurrence and improved survival

rates specifically correlated to FGF-AS levels, not

GFG levels. Evidence suggests FGF-AS tumor

suppressive role is a result of its post-transcription

control over FGF-2.

Melanoma

Note

Melanoma is a malignant tumor of melanocytes,

which are found primarily in the skin, however they

can develop in melanocytes found in the eye and

bowel. A characteristic of aggressive melanomas is

their ability to form fluid-conducting vasculogenic-

like networks.

Oncogenesis

A preliminary study investigating these 3D tubular

networks within tumors identified NUDT6 as one

of the many genes overexpressed in aggressive

melanomas and speculated it is involved in

promoting self-renewal and tumor cell plasticity in

melanoma cancer networks. Additionally, they

suggest FGF-AS could play a role in the

development of the endothelia-lined vasculature

networks in melanomas indirectly through its

regulatory control over FGF-2 expression, which is

associated with angiogenesis, proliferation and

survival.

Colorectal cancer

Note

Colorectal cancer refers to uncontrolled growth of

cells that line the colon, rectum and appendix,

collectively the large intestine.

Oncogenesis

Induced overexpression of NUDT6 in a variety of

human colorectal cells significantly increases

cancer cell proliferation and their clonogenic

capacity. NUDT6 is described as having tumor

promoting functions in this cellular environment

and it is suggested that it plays a role in colorectal

cancer development and progression.

Endometriosis

Note

Endometriosis is a medical condition affecting the

endometrium lining of the uterus. The endometrium

is comprised of hormonally responsive cells that

proliferate and secrete under the influence of

estrogen and progesterone. Upon menstruation the

endometrium lining is shed as a part of the

menstrual flow. Endometriosis describes the

presence of endometrial cells outside of the uterus,

such as the ovaries, fallopian tubes, bladder and

interstitial space in the abdominal cavity.

Patients with endometriosis lesions have reduced

FGF-AS-b mRNA levels and elevated FGF-2

mRNA levels during the late proliferative phase of

the menstrual cycle, compared to control patients.

This increased FGF/FGF-AS ratio is thought to

contribute to the development of endometriosis.

References Knee RS, Pitcher SE, Murphy PR. Basic fibroblast growth factor sense (FGF) and antisense (gfg) RNA transcripts are expressed in unfertilized human oocytes and in differentiated adult tissues. Biochem Biophys Res Commun. 1994 Nov 30;205(1):577-83

Murphy PR, Knee RS. Identification and characterization of an antisense RNA transcript (gfg) from the human basic fibroblast growth factor gene. Mol Endocrinol. 1994 Jul;8(7):852-9




Bessman MJ, Frick DN, O'Handley SF. The MutT proteins or "Nudix" hydrolases, a family of versatile, widely distributed, "housecleaning" enzymes. J Biol Chem. 1996 Oct 11;271(41):25059-62

Knee R, Murphy PR. Regulation of gene expression by natural antisense RNA transcripts. Neurochem Int. 1997 Sep;31(3):379-92

Gagnon ML, Moy GK, Klagsbrun M. Characterization of the promoter for the human antisense fibroblast growth factor-2 gene; regulation by Ets in Jurkat T cells. J Cell Biochem. 1999 Mar 15;72(4):492-506

Asa SL, Ramyar L, Murphy PR, Li AW, Ezzat S. The endogenous fibroblast growth factor-2 antisense gene product regulates pituitary cell growth and hormone production. Mol Endocrinol. 2001 Apr;15(4):589-99

Mihalich A, Reina M, Mangioni S, Ponti E, Alberti L, Viganò P, Vignali M, Di Blasio AM. Different basic fibroblast growth factor and fibroblast growth factor-antisense expression in eutopic endometrial stromal cells derived from women with and without endometriosis. J Clin Endocrinol Metab. 2003 Jun;88(6):2853-9

Baguma-Nibasheka M, Li AW, Osman MS, Geldenhuys L, Casson AG, Too CK, Murphy PR. Coexpression and regulation of the FGF-2 and FGF antisense genes in leukemic cells. Leuk Res. 2005 Apr;29(4):423-33

Barclay C, Li AW, Geldenhuys L, Baguma-Nibasheka M, Porter GA, Veugelers PJ, Murphy PR, Casson AG. Basic fibroblast growth factor (FGF-2) overexpression is a risk

factor for esophageal cancer recurrence and reduced survival, which is ameliorated by coexpression of the FGF-2 antisense gene. Clin Cancer Res. 2005 Nov 1;11(21):7683-91

Zhang SC, Barclay C, Alexander LA, Geldenhuys L, Porter GA, Casson AG, Murphy PR. Alternative splicing of the FGF antisense gene: differential subcellular localization in human tissues and esophageal adenocarcinoma. J Mol Med. 2007 Nov;85(11):1215-28

Demou ZN, Hendrix MJ. Microgenomics profile the endogenous angiogenic phenotype in subpopulations of aggressive melanoma. J Cell Biochem. 2008 Oct 1;105(2):562-73

MacFarlane LA, Gu Y, Casson AG, Murphy PR. Regulation of fibroblast growth factor-2 by an endogenous antisense RNA and by argonaute-2. Mol Endocrinol. 2010 Apr;24(4):800-12

Sukhthankar M, Choi CK, English A, Kim JS, Baek SJ. A potential proliferative gene, NUDT6, is down-regulated by green tea catechins at the posttranscriptional level. J Nutr Biochem. 2010 Feb;21(2):98-106


MacFarlane LA, Murphy P. NUDT6 (nudix (nucleoside diphosphate linked moiety X)-type motif 6). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):29-33.

Gene Section Review




PARVB (parvin, beta) Cameron N Johnstone

Cancer Metastasis Laboratory, Research Division, Peter MacCallum Cancer Centre, 2 St Andrew's

Place, East Melbourne, 3002, Victoria, Australia (CNJ)


Online updated version : http://AtlasGeneticsOncology.org/Genes/PARVBID46486ch22q13.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI PARVBID46486ch22q13.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity

Other names: CGI-56, affixin, beta-parvin

HGNC (Hugo): PARVB

Location: 22q13.31

Local order: PARVB is located telomeric to the

SAMM50 gene and centromeric to the PARVG

gene at 22q13.31.

DNA/RNA

Note

Genethon marker D22S1171 is located at the 5' end

of the gene (Mongroo et al., 2004). Genethon

marker D22S1171 is located between exon 2 and

exon 1A of the PARVB gene.

The PARVA gene is located at 11p15.3.

Figure A. Generation of transcript diversity by alternative promoter usage. Horizontal lines above the gene structure indicate

human genomic DNA BAC clones. The NCBI accession numbers of the clones, and clone names (in brackets) are shown. Figure adapted from Mongroo et al., 2004.

PARVB (parvin, beta) Johnstone CN


Figure B. Human polyA

+ RNA Multiple Tissue Northern blot (Origene) probed with full-length PARVB1 cDNA probe radiolabeled

to a specific activity of > 5 x 108 cpm / mg (Johnstone C.N., unpublished). The two PARVB mRNA transcripts are indicated. The

higher M.W. band most likely corresponds to non-specific hybridization (n/s).

Description

PARVB3/CLINT, which encodes the longer

Parvin-beta protein isoform is transcribed from

promoter 1 and contains two additional 5' exons

(exons 1 and 2) not present in PARVB1, and

comprises 14 exons in total. PARVB1 encodes the

shorter Parvin-beta protein isoform, is transcribed

from promoter 1A, and comprises 13 exons in total.

Both promoters contain CpG islands that span the

transcription start sites. PARVB3/CLINT contains

70 unique N-terminal amino acids not present in the

short isoform. (See figure A).

Transcription

As with PARVA, human PARVB mRNA

expression is highest in heart, followed by skeletal

muscle, where it localises to the sarcolemma

(Yamaji et al., 2001; Matsuda et al., 2005). Both

PARVB mRNA transcripts are essentially

ubiquitously expressed (Korenbaum et al., 2001),

but with lower expression in gastrointestinal tissues

(stomach, small intestine, colon). (See figure B).

Protein

Description

The major functional domains of Parvin-beta are

two 'atypical' calponin homology (CH) domains,

termed CH1 (106 amino acids) and CH2 (107

amino acids). Each CH domain contains two actin

binding sequences (ABS), although Parvin-beta has

not been shown to bind actin directly (Korenbaum

et al., 2001; Sepulveda and Wu, 2006). Parvin-beta

physically interacts with Dysferlin and ARHGEF6

(alpha-PIX) through the CH1 domain (Matsuda et

al., 2005; Rosenberger et al., 2003) and with ILK

and alpha-actinin through the CH2 domain (Yamaji

et al., 2001; Yamaji et al., 2004). Parvin-beta was

also recently reported to directly interact with AKT

(Kimura et al., 2010).

Expression

PARVB is essentially ubiquitously expressed.

Localisation

Parvin-beta localises to focal adhesions but also to

the nucleus, which is most likely due to NLS motifs

in the N-terminal region (Mongroo et al., 2004;

Johnstone et al., 2008). Parvin-beta is incorporated

into focal adhesions as part of the heterotrimeric

'IPP complex'. The ternary complex contains 1

molecule of integrin linked kinase (ILK), 1 Parvin

isoform, and 1 PINCH (LIMS) isoform, (Legate et

al., 2006). Binding of Parvin-alpha and Parvin-beta

to the kinase domain of ILK is mutually exclusive

(Zhang et al., 2004). Formation of the IPP complex

also dictates total protein levels of each component,

as any excess ILK, Parvin, or PINCH not

incorporated into IPP is degraded in a proteasome-

dependent manner (Fukuda et al., 2003).

Depiction of functional domains of Parvin-beta(long) and Parvin-beta(short). NLS, nuclear localization sequence; ABS,

actin binding sequence; CH, calponin homology. Adapted from Sepulveda and Wu, 2006.



Function

Parvin-beta participates in focal adhesion dynamics

through involvement in the IPP complex. The high

expression levels in cardiac and skeletal muscle

suggest important function(s) in these organs. In

skeletal muscle, it binds dysferlin at the

sarcolemma and thus may be involved with

membrane repair (Yamaji et al., 2001; Matsuda et

al., 2005; Legate et al., 2006). Parvin-beta and

Parvin-alpha appear to negatively regulate the

expression of each other (Zhang et al., 2004;

Johnstone et al., 2008). Parvin-beta may modulate

signalling through ILK as overexpression of Parvin-

beta reduced AKT (S473) and GSK3beta (S9)

phosphorylation in response to EGF stimulation

(Mongroo et al., 2004). Parvin-beta was recently

reported to directly interact with AKT (Kimura et

al., 2010), which may explain its effects on AKT

phosphorylation. Parvin-beta interacts with

ARHGEF6 (alpha-PIX), an exchange factor for

RAC1, thus implicating Parvin-beta in regulation of

RAC signalling downstream of integrin

engagement (Rosenberger et al., 2003). Finally,

Parvin-beta may affect metabolic pathways through

promotion of CDK9-mediated phosphorylation and

activation of PPARgamma transcriptional activity

in the nucleus (Johnstone et al., 2008).

Interestingly, Parvb knockout mice were recently

generated. Whilst constitutive Parva null mice

feature kidney and cardiovascular defects and die

between E10.5 and E14.5 (Lange et al., 2009;

Montanez et al., 2009), constitutive Parvb null mice

are viable (Wickström et al., 2010), although a

detailed phenotypic analysis has not yet been

described.

Homology

Human Parvin-beta is most closely related to

Parvin-alpha [75% identity with Parvin-beta(short)

and 67% identity with Parvin-beta(long)] and more

distantly to Parvin-gamma [41% identity with both

Parvin-beta(short) and Parvin-beta(long)].

Mutations

Note

No mutations reported to date.

Germinal

Germline SNPs are identified in the PARVB gene

by direct sequencing of PCR products amplified

from cDNA prepared from 16 primary ductal

adenocarcinomasand adjacent normal mammary

gland from the same patient. Two non-synonymous

SNPs were identified, W37R, and E175K

(Johnstone et al., manuscript in preparation).

Somatic

No somatic mutations were found in an analysis of

16 breast adenocarcinomas as presented above

(Johnstone et al., manuscript in preparation).

According to the C.O.S.M.I.C. online database

(Forbes et al., 2008), 171 unique cancer samples

have been analysed for alterations in the PARVB

gene, with no somatic changes found. A breakdown

of the samples analysed is given below.

Name Alleles Location Base Position† Amino Acid

Position‡

Amino Acid

change

No. of

Alleles

A98C A/C Intron 1 98** n/a n/a 3/8

W37R T/C Exon 2 252 37 W>R 3/8

D150D C/T Exon 5 593 150 D>D 2/32

E175K G/A Exon 6 666 175 E>K 2/32

A223A C/T Exon 7 812 223 A>A 2/32

G316G^ C/T Exon 12 1097 318 G>G 2/32

F354F^ C/T Exon 13 1205 354 F>F 2/32

† Relative to transcription start site

‡ Relative to translation start site

^ Occur as a haplotype

** Relative to splice site

Cancer Type No. of Specimens Reference

Breast 11 Sjöblom et al., 2006

Glioma 23 Parsons et al., 2008

Clear Cell Renal 101 Dalgliesh et al., 2010

Colon 12 Sjöblom et al., 2006

Lung (cell lines) 11 N/A

http://atlasgeneticsoncology.org/Tumors/GliomaOverviewID5763.html

http://atlasgeneticsoncology.org/Tumors/ClearCellRenalCC5020.html

http://atlasgeneticsoncology.org/Tumors/colon5006.html

http://atlasgeneticsoncology.org/Tumors/LungTumOverviewID5030.html



Pancreas (cell lines) 1 N/A

Mesothelioma (cell lines) 1 N/A

Melanoma (cell lines) 6 N/A

Urinary tract (cell lines) 2 N/A

HNSCC (cell lines) 3 N/A

Implicated in

Breast cancer

Note

Parvin-beta mRNA levels are reduced in primary

human ductal adenocarcinoma compared with

adjacent normal mammary gland. PARVB mRNA

levels are also reduced in MDA-MB-231 and

MDA-MB-453 cell lines. Post-transcriptional

downregulation of protein expression may also

occur in cancer cells such as MCF7 (Mongroo et

al., 2004). Ectopic Parvin-beta expression in MDA-

MB-231 metastatic breast cancer cells increased

adhesion and reduced invasion. Ectopic expression

also reduced tumorigenicity of the same cell line in

nude mice in vivo. Parvin-beta expression did not

affect proliferation of the cells in vitro, but reduced

Ki-67 staining was observed in Parvin-beta

transfectants in vivo (Johnstone et al., 2008).

Parvin-beta overexpression was also reported to

promote apoptosis in HeLa cervical cancer cells

(Zhang et al., 2004).

Prognosis

Association with prognosis has not been studied to

date.

References Korenbaum E, Olski TM, Noegel AA. Genomic organization and expression profile of the parvin family of focal adhesion proteins in mice and humans. Gene. 2001 Nov 14;279(1):69-79

Yamaji S, Suzuki A, Sugiyama Y, Koide Y, Yoshida M, Kanamori H, Mohri H, Ohno S, Ishigatsubo Y. A novel integrin-linked kinase-binding protein, affixin, is involved in the early stage of cell-substrate interaction. J Cell Biol. 2001 Jun 11;153(6):1251-64

Fukuda T, Chen K, Shi X, Wu C. PINCH-1 is an obligate partner of integrin-linked kinase (ILK) functioning in cell shape modulation, motility, and survival. J Biol Chem. 2003 Dec 19;278(51):51324-33

Rosenberger G, Jantke I, Gal A, Kutsche K. Interaction of alphaPIX (ARHGEF6) with beta-parvin (PARVB) suggests an involvement of alphaPIX in integrin-mediated signaling. Hum Mol Genet. 2003 Jan 15;12(2):155-67

Mongroo PS, Johnstone CN, Naruszewicz I, Leung-Hagesteijn C, Sung RK, Carnio L, Rustgi AK, Hannigan GE. Beta-parvin inhibits integrin-linked kinase signaling and is downregulated in breast cancer. Oncogene. 2004 Nov 25;23(55):8959-70

Yamaji S, Suzuki A, Kanamori H, Mishima W, Yoshimi R, et al. Affixin interacts with alpha-actinin and mediates integrin signaling for reorganization of F-actin induced by

initial cell-substrate interaction. J Cell Biol. 2004 May 24;165(4):539-51

Zhang Y, Chen K, Tu Y, Wu C. Distinct roles of two structurally closely related focal adhesion proteins, alpha-parvins and beta-parvins, in regulation of cell morphology and survival. J Biol Chem. 2004 Oct 1;279(40):41695-705

Matsuda C, Kameyama K, Tagawa K, Ogawa M, Suzuki A, et al. Dysferlin interacts with affixin (beta-parvin) at the sarcolemma. J Neuropathol Exp Neurol. 2005 Apr;64(4):334-40

Legate KR, Montañez E, Kudlacek O, Fässler R. ILK, PINCH and parvin: the tIPP of integrin signalling. Nat Rev Mol Cell Biol. 2006 Jan;7(1):20-31

Sepulveda JL, Wu C. The parvins. Cell Mol Life Sci. 2006 Jan;63(1):25-35

Sjöblom T, Jones S, Wood LD, Parsons DW, Lin J, et al. The consensus coding sequences of human breast and colorectal cancers. Science. 2006 Oct 13;314(5797):268-74

Forbes SA, Bhamra G, Bamford S, Dawson E, Kok C, Clements J, Menzies A, Teague JW, Futreal PA, Stratton MR. The Catalogue of Somatic Mutations in Cancer (COSMIC). Curr Protoc Hum Genet. 2008 Apr;Chapter 10:Unit 10.11

Johnstone CN, Mongroo PS, Rich AS, Schupp M, et al. Parvin-beta inhibits breast cancer tumorigenicity and promotes CDK9-mediated peroxisome proliferator-activated receptor gamma 1 phosphorylation. Mol Cell Biol. 2008 Jan;28(2):687-704

Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, et al. An integrated genomic analysis of human glioblastoma multiforme. Science. 2008 Sep 26;321(5897):1807-12

Lange A, Wickström SA, Jakobson M, Zent R, Sainio K, Fässler R. Integrin-linked kinase is an adaptor with essential functions during mouse development. Nature. 2009 Oct 15;461(7266):1002-6

Montanez E, Wickström SA, Altstätter J, Chu H, Fässler R. Alpha-parvin controls vascular mural cell recruitment to vessel wall by regulating RhoA/ROCK signalling. EMBO J. 2009 Oct 21;28(20):3132-44

Dalgliesh GL, Furge K, Greenman C, Chen L, Bignell G, et al. Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature. 2010 Jan 21;463(7279):360-3

Kimura M, Murakami T, Kizaka-Kondoh S, Itoh M, et al. Functional molecular imaging of ILK-mediated Akt/PKB signaling cascades and the associated role of beta-parvin. J Cell Sci. 2010 Mar 1;123(Pt 5):747-55

Wickström SA, Lange A, Montanez E, Fässler R. The ILK/PINCH/parvin complex: the kinase is dead, long live the pseudokinase! EMBO J. 2010 Jan 20;29(2):281-91


Johnstone CN. PARVB (parvin, beta). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):34-37.

http://atlasgeneticsoncology.org/Tumors/SkinMelanomID5416.html

http://atlasgeneticsoncology.org/Tumors/HeadNeckSquamID5090.html

Gene Section Review




PIAS3 (protein inhibitor of activated STAT, 3) Gilles Spoden, Werner Zwerschke

Institute for Medical Microbiology and Hygiene, University Medical Center of the Johannes

Gutenberg University Mainz, Hochhaus am Augustus-platz, 55131 Mainz, Germany (GS); Cell

Metabolism and Differentiation Research Group, Institute for Biomedical Aging Research, Rennweg

10, 6020 Innsbruck, Austria (ZW); Tumorvirology Research Group, Tyrolean Cancer Research

Institute at Medical University Innsbruck, Innrain 66, 6020 Innsbruck, Austria (ZW)


Online updated version : http://AtlasGeneticsOncology.org/Genes/PIAS3ID41709ch1q21.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI PIAS3ID41709ch1q21.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity

Other names: FLJ14651, KChAP, ZMIZ5

HGNC (Hugo): PIAS3

Location: 1q21.1

DNA/RNA

Note

The gene codes for a protein of the PIAS family

(protein inhibitor of activated STAT (signal

transducer and activator of transcription)). PIAS3

regulates the activity of several transcription factors

by direct protein-protein interaction. Further,

PIAS3 is a SUMO (small ubiquitin-like modifier)-

E3 ligase, catalyzing the covalent, post-translational

modification of specific target proteins with

SUMO. Different splice variants of PIAS3 have

been identified but the full-length sequence of some

of these variants has not been described.

Description

The human PIAS3 gene is 10559 bp long and

consists of 14 exons and 13 introns.

Transcription

Transcript length 2902 bp (CDS 1887 bp ; residues

628 aa).

Pseudogene

No pseudogene reported.

Figure 1. PIAS3 gene 10559 bp. Exons 1 to 14 (UTR in white, coding sequence in red) Exon 1: 1-115 (5'UTR: 1-91); Exon 2:

2075-2492; Exon 3: 2650-2734; Exon 4: 2963-3013; Exon 5: 3255-3345; Exon 6: 4201-4335; Exon 7: 4518-4623; Exon 8: 5195-5268; Exon 9: 5417-5577; Exon 10: 7928-8061; Exon 11: 8142-8310; Exon 12: 8495-8628; Exon 13: 8812-8849; Exon 14: 9369-

10559 (3'UTR: 9636-10559).

Figure 2. The schematic domain structure of human PIAS3 protein is shown. SAP domain: nuclear localization and binding to

DNA, transcription factors, coregulators. PINIT: nuclear retention, transcriptional repression. SP-RING: protein-protein interactions, interacts with the SUMO conjugase Ubc9, sumoylation. SIM: binding to SUMO. S/T: variable region, binding to

coactivators.

PIAS3 (protein inhibitor of activated STAT, 3) Spoden G, Zwerschke W


Protein

Description

The human PIAS3 protein is a E3 SUMO-protein

ligase consisting of 628 amino acids. It contains 5

conserved regions, the SAP, PINIT, SP-RING, SIM

and S/T domains (figure 2).

Expression

PIAS3 is ubiquitously expressed.

Localisation

Nuclear as well as cytoplasmic localization.

Function

PIAS3 belongs to the mammalian protein inhibitor

of activated STAT (PIAS) protein family, originally

identified as cytokine-induced inhibitors of the

STAT family of transcription factors. This protein

class, referred to as SUMO-E3 ligases, increases the

efficiency of SUMO conjugation. SUMO, a small

ubiquitin-like modifier protein, is conjugated to a

large number of cellular target proteins. Similar to

enzymatic ubiquitination, the conjugation of

specific SUMO proteins (SUMO-1-SUMO-3) to

target proteins requires an E1-activating enzyme

(Aos1/Uba2) as well as an E2-type SUMO-1-

conjugating enzyme (Ubc9). Similar to many

ubiquitin E3 ligases, these proteins contain a

putative RING finger-like structure (SP-RING,

figure 2), which is essential for their SUMO-E3

ligase activities toward various target proteins.

Sumoylation is a dynamic process with highly

diverse outcomes, ranging from changes in

subcellular localization, signal transduction,

transcriptional regulation to altered activity and

stability of the modified protein. PIAS3 do,

however, not only operate as SUMO-E3, since its

coregulator effects are often independent of its

RING-finger like domain but dependent on its

capability to interact with sumoylated proteins via

its conserved SIM (SUMO-interacting motif) or

SAP (scaffold attachment factor-A/B/acinus/PIAS)

domain (figure 2). Beside the N-terminal SAP, the

SIM and the RING-type zinc-binding domain, a

PINIT motif, and a serine/ threonine-rich C-

terminal region (S/T) is conserved in PIAS3 (figure

2).

PIAS3 is involved in cytoplasmic regulation, such

as functional interaction of PIAS3 with

metabotropic glutamate receptor-8, voltage-gated

potassium channel Kv1.5 and pyruvate kinase

subtype M2, but the majority of so far reported

interactions of the PIAS3 protein occurred with

transcription factors or other proteins linked to

nuclear regulation. PIAS3 can act in both

transcriptional repression and activation. PIAS3 has

been shown to repress STAT3 and Stat5 dependent

transcriptional activation by blocking the DNA-

binding of the factor without influencing its

sumoylation. It interacts with and promotes

sumoylation of the photoreceptor-specific

transcription factor Nr2e3 when bound to specific

promoters, which converts the factor to a

transcriptional repressor. Moreover, PIAS3 was

described as a repressor of microphthalmia

transcription factor (MITF) and it was shown that

PIAS3 blocks NF-kB mediated transcriptional

activation by interacting with the p65/RelA subunit.

Repression of IRF1-mediated transcription by

PIAS3 has also been shown. PIAS3 has been shown

to activate transcription mediated by Smad proteins

through forming a complex with Smads and

coactivator p300/CBP; moreover, PIAS proteins

enhance steroid receptor-dependent transcription

through an SP-RING-mediated interaction and

sumoylation of the coactivator protein

GRIP1/SCR2. Finally, PIAS3 was shown to

modulate the ability of TIF2 to mediate ligand-

enhanced transcription activation positively or

negatively, for different steroid receptors.

Homology

The mammalian PIAS family consists of seven

structurally related proteins (PIAS1, PIAS3,

PIAS3b, PIASxa, PIASxb, PIASy, and PIASyE6)

encoded by four genes. PIAS orthologs are found in

nonvertebrate animal species, plants and yeasts.

Implicated in

Prostate Cancer

Oncogenesis

PIAS3 is expressed in normal prostate and in

prostate cancer cells and has been shown to

modulate the transcriptional activity of androgen

receptor in prostate cancer cells. Moreover, PIAS3

(KChAP) induces increased K+ efflux and

apoptosis in prostate cancer lines.

Glioblastoma multiforme (GBM)

Oncogenesis

The activation of STATs and loss of their natural

inhibitors SOCS and PIAS is common in various

human cancers. STAT3, a cytoplasmic transcription

factor that becomes activated in response to a

variety of cytokines and growth factors is aberrantly

activated in GBM tumors. STAT3 activation

correlates with strongly reduced PIAS3 protein

expression in GBM tissues. Inhibition of PIAS3

resulted in enhanced glioblastoma cellular

proliferation, and, conversely, PIAS3

overexpression inhibits STAT3 transcriptional

activity, expression of STAT3-regulated genes, and

cell proliferation. This suggests that the loss of

PIAS3 in GBM contributes to enhanced STAT3

transcriptional activity and subsequent cell

proliferation.



Melanoma

Oncogenesis

PIAS3 functions as a key molecule in suppressing

the transcriptional activity of both MITF and

STAT3, two transcription factors that play a major

role in the development, proliferation and survival

of mast cells and melanocytes. In addition to its role

in normal cell signaling, constitutively activated

STAT3 signaling directly contributes to

oncogenesis in many human cancers. STAT3

cooperates with MITF in the induction of cellular

transformation. Evidence was provided suggesting

that PIAS3 halt proliferation and induce apoptotic

cell death in mast cells and in melanoma cells by

inhibiting the transcriptional activity of the two

oncogenic factors MITF and STAT3. Therefore

PIAS3 may play a role in tumor suppression by

inhibiting oncogenic processes induced by STAT3

and MITF.

Non-small cell lung cancer (NSCLC)

Disease

The Epidermal Growth Factor Receptor (EGFR)-

STAT3 axis plays an important role in oncogenic

signaling of non-small cell lung cancer (NSCLC).

The negative regulator of STAT3-mediated

transcriptional activation, PIAS3, was shown to

modulate oncogenic EGFR-STAT3 signaling in

lung cancer. Overexpression of PIAS3 decreases

STAT3 transcriptional activity and proliferation of

NSCLC cells and when used in conjunction with

EGFR inhibitors, further increased the anti-

proliferative effects. This suggests that PIAS3 acts

as an inhibitor of EGFR-STAT3 induced oncogenic

action.

To be noted

Note

TAR syndrome (Thrombocytopenia-absent radius)

is a rare genetic disorder characterized by low

platelet counts and bilateral radial aplasia. TAR is

also frequently associated with cardiac

abnormalities and cow's milk intolerance. In 2007 a

research article described a common microdeletion

of 200 kb on chromosome 1q21.1 in patients with

TAR syndrome. PIAS3 is one of 11 genes

encompassed by this microdeletion.

References Chung CD, Liao J, Liu B, Rao X, Jay P, Berta P, Shuai K. Specific inhibition of Stat3 signal transduction by PIAS3. Science. 1997 Dec 5;278(5344):1803-5

Wible BA, Yang Q, Kuryshev YA, Accili EA, Brown AM. Cloning and expression of a novel K+ channel regulatory protein, KChAP. J Biol Chem. 1998 May 8;273(19):11745-51

Ueki N, Seki N, Yano K, Saito T, Masuho Y, Muramatsu M. Isolation and chromosomal assignment of a human gene encoding protein inhibitor of activated STAT3 (PIAS3). J Hum Genet. 1999;44(3):193-6

Bowman T, Garcia R, Turkson J, Jove R. STATs in oncogenesis. Oncogene. 2000 May 15;19(21):2474-88

Bromberg J, Darnell JE Jr. The role of STATs in transcriptional control and their impact on cellular function. Oncogene. 2000 May 15;19(21):2468-73

Junicho A, Matsuda T, Yamamoto T, Kishi H, Korkmaz K, Saatcioglu F, Fuse H, Muraguchi A. Protein inhibitor of activated STAT3 regulates androgen receptor signaling in prostate carcinoma cells. Biochem Biophys Res Commun. 2000 Nov 11;278(1):9-13

Minty A, Dumont X, Kaghad M, Caput D. Covalent modification of p73alpha by SUMO-1. Two-hybrid screening with p73 identifies novel SUMO-1-interacting proteins and a SUMO-1 interaction motif. J Biol Chem. 2000 Nov 17;275(46):36316-23

Gross M, Liu B, Tan J, French FS, Carey M, Shuai K. Distinct effects of PIAS proteins on androgen-mediated gene activation in prostate cancer cells. Oncogene. 2001 Jun 28;20(29):3880-7

Hari KL, Cook KR, Karpen GH. The Drosophila Su(var)2-10 locus regulates chromosome structure and function and encodes a member of the PIAS protein family. Genes Dev. 2001 Jun 1;15(11):1334-48

Hochstrasser M. SP-RING for SUMO: new functions bloom for a ubiquitin-like protein. Cell. 2001 Oct 5;107(1):5-8

Jiménez-Lara AM, Heine MJ, Gronemeyer H. PIAS3 (protein inhibitor of activated STAT-3) modulates the transcriptional activation mediated by the nuclear receptor coactivator TIF2. FEBS Lett. 2002 Aug 28;526(1-3):142-6

Kotaja N, Vihinen M, Palvimo JJ, Jänne OA. Androgen receptor-interacting protein 3 and other PIAS proteins cooperate with glucocorticoid receptor-interacting protein 1 in steroid receptor-dependent signaling. J Biol Chem. 2002 May 17;277(20):17781-8

Levy C, Nechushtan H, Razin E. A new role for the STAT3 inhibitor, PIAS3: a repressor of microphthalmia transcription factor. J Biol Chem. 2002 Jan 18;277(3):1962-6

Nakagawa K, Yokosawa H. PIAS3 induces SUMO-1 modification and transcriptional repression of IRF-1. FEBS Lett. 2002 Oct 23;530(1-3):204-8

Nishida T, Yasuda H. PIAS1 and PIASxalpha function as SUMO-E3 ligases toward androgen receptor and repress androgen receptor-dependent transcription. J Biol Chem. 2002 Nov 1;277(44):41311-7

Rycyzyn MA, Clevenger CV. The intranuclear prolactin/cyclophilin B complex as a transcriptional inducer. Proc Natl Acad Sci U S A. 2002 May 14;99(10):6790-5

Tan JA, Hall SH, Hamil KG, Grossman G, Petrusz P, French FS. Protein inhibitors of activated STAT resemble scaffold attachment factors and function as interacting nuclear receptor coregulators. J Biol Chem. 2002 May 10;277(19):16993-7001

Wible BA, Wang L, Kuryshev YA, Basu A, Haldar S, Brown AM. Increased K+ efflux and apoptosis induced by the potassium channel modulatory protein KChAP/PIAS3beta in prostate cancer cells. J Biol Chem. 2002 May 17;277(20):17852-62

Duval D, Duval G, Kedinger C, Poch O, Boeuf H. The 'PINIT' motif, of a newly identified conserved domain of the PIAS protein family, is essential for nuclear retention of PIAS3L. FEBS Lett. 2003 Nov 6;554(1-2):111-8

Levy C, Sonnenblick A, Razin E. Role played by microphthalmia transcription factor phosphorylation and its



Zip domain in its transcriptional inhibition by PIAS3. Mol Cell Biol. 2003 Dec;23(24):9073-80

Schmidt D, Müller S. PIAS/SUMO: new partners in transcriptional regulation. Cell Mol Life Sci. 2003 Dec;60(12):2561-74

Jang HD, Yoon K, Shin YJ, Kim J, Lee SY. PIAS3 suppresses NF-kappaB-mediated transcription by interacting with the p65/RelA subunit. J Biol Chem. 2004 Jun 4;279(23):24873-80

Johnson ES. Protein modification by SUMO. Annu Rev Biochem. 2004;73:355-82

Joo A, Aburatani H, Morii E, Iba H, Yoshimura A. STAT3 and MITF cooperatively induce cellular transformation through upregulation of c-fos expression. Oncogene. 2004 Jan 22;23(3):726-34

Long J, Wang G, Matsuura I, He D, Liu F. Activation of Smad transcriptional activity by protein inhibitor of activated STAT3 (PIAS3). Proc Natl Acad Sci U S A. 2004 Jan 6;101(1):99-104

Sonnenblick A, Levy C, Razin E. Interplay between MITF, PIAS3, and STAT3 in mast cells and melanocytes. Mol Cell Biol. 2004 Dec;24(24):10584-92

Miura K, Rus A, Sharkhuu A, Yokoi S, Karthikeyan AS, Raghothama KG, Baek D, Koo YD, Jin JB, Bressan RA, Yun DJ, Hasegawa PM. The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses. Proc Natl Acad Sci U S A. 2005 May 24;102(21):7760-5

Sentis S, Le Romancer M, Bianchin C, Rostan MC, Corbo L. Sumoylation of the estrogen receptor alpha hinge region regulates its transcriptional activity. Mol Endocrinol. 2005 Nov;19(11):2671-84

Shuai K, Liu B. Regulation of gene-activation pathways by PIAS proteins in the immune system. Nat Rev Immunol. 2005 Aug;5(8):593-605

Levy C, Lee YN, Nechushtan H, Schueler-Furman O, Sonnenblick A, Hacohen S, Razin E. Identifying a common molecular mechanism for inhibition of MITF and STAT3 by PIAS3. Blood. 2006 Apr 1;107(7):2839-45

Ogata Y, Osaki T, Naka T, Iwahori K, Furukawa M, Nagatomo I, Kijima T, Kumagai T, Yoshida M, Tachibana I, Kawase I. Overexpression of PIAS3 suppresses cell growth and restores the drug sensitivity of human lung cancer cells in association with PI3-K/Akt inactivation. Neoplasia. 2006 Oct;8(10):817-25

Geiss-Friedlander R, Melchior F. Concepts in sumoylation: a decade on. Nat Rev Mol Cell Biol. 2007 Dec;8(12):947-56

Klopocki E, Schulze H, Strauss G, Ott CE, Hall J, Trotier F, Fleischhauer S, Greenhalgh L, Newbury-Ecob RA, Neumann LM, Habenicht R, König R, Seemanova E,

Megarbane A, Ropers HH, Ullmann R, Horn D, Mundlos S. Complex inheritance pattern resembling autosomal recessive inheritance involving a microdeletion in thrombocytopenia-absent radius syndrome. Am J Hum Genet. 2007 Feb;80(2):232-40

Martin S, Nishimune A, Mellor JR, Henley JM. SUMOylation regulates kainate-receptor-mediated synaptic transmission. Nature. 2007 May 17;447(7142):321-5

Palvimo JJ. PIAS proteins as regulators of small ubiquitin-related modifier (SUMO) modifications and transcription. Biochem Soc Trans. 2007 Dec;35(Pt 6):1405-8

Uzunova K, Göttsche K, Miteva M, Weisshaar SR, Glanemann C, Schnellhardt M, Niessen M, Scheel H, Hofmann K, Johnson ES, Praefcke GJ, Dohmen RJ. Ubiquitin-dependent proteolytic control of SUMO conjugates. J Biol Chem. 2007 Nov 23;282(47):34167-75

Brantley EC, Nabors LB, Gillespie GY, Choi YH, Palmer CA, Harrison K, Roarty K, Benveniste EN. Loss of protein inhibitors of activated STAT-3 expression in glioblastoma multiforme tumors: implications for STAT-3 activation and gene expression. Clin Cancer Res. 2008 Aug 1;14(15):4694-704

Kluge A, Dabir S, Kern J, Nethery D, Halmos B, Ma P, Dowlati A. Cooperative interaction between protein inhibitor of activated signal transducer and activator of transcription-3 with epidermal growth factor receptor blockade in lung cancer. Int J Cancer. 2009 Oct 1;125(7):1728-34

Onishi A, Peng GH, Hsu C, Alexis U, Chen S, Blackshaw S. Pias3-dependent SUMOylation directs rod photoreceptor development. Neuron. 2009 Jan 29;61(2):234-46

Rytinki MM, Kaikkonen S, Pehkonen P, Jääskeläinen T, Palvimo JJ. PIAS proteins: pleiotropic interactors associated with SUMO. Cell Mol Life Sci. 2009 Sep;66(18):3029-41

Spoden GA, Morandell D, Ehehalt D, Fiedler M, Jansen-Dürr P, Hermann M, Zwerschke W. The SUMO-E3 ligase PIAS3 targets pyruvate kinase M2. J Cell Biochem. 2009 May 15;107(2):293-302

Yagil Z, Kay G, Nechushtan H, Razin E. A specific epitope of protein inhibitor of activated STAT3 is responsible for the induction of apoptosis in rat transformed mast cells. J Immunol. 2009 Feb 15;182(4):2168-75


Spoden G, Zwerschke W. PIAS3 (protein inhibitor of activated STAT, 3). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):38-41.

Gene Section Review




PSEN2 (presenilin 2 (Alzheimer disease 4)) Morgan Newman

School of Molecular and Biomedical Science, The University of Adelaide, Australia (MN)


Online updated version : http://AtlasGeneticsOncology.org/Genes/PSEN2ID41883ch1q42.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI PSEN2ID41883ch1q42.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity

Other names: AD3L, AD4, PS2, STM2

HGNC (Hugo): PSEN2

Location: 1q42.13

DNA/RNA

Description

Twelve exons, spans approximately 26.7 kb of

genomic DNA in the centromere to telomere

orientation, the translation initation codon is in

exon 4 and the stop codon in exon 12.

Transcription

mRNA of approximately 2.3 kb. Two alternatively

spliced transcript variants encoding different

isoforms of PSEN2 have been identified.

Pseudogene

Not known.

Protein

Description

The open reading frame encodes a 448 amino acid

protein, with an estimated molecular weight of 50

kDa.

It is a multi-spanning transmembrane protein with a

predicted 9 transmembrane domains.

Heterogeneous proteolytic processing generates N-

terminal and C-terminal fragments.

Expression

Neuronal (higher levels in hippocampus and

cerebellum). Isoform 1 is seen in the placenta,

skeletal muscle and heart while isoform 2 is seen in

the heart, brain, placenta, liver, skeletal muscle and

kidney. (In isoform 2 amino-acids 263-296 are

missing).

Localisation

Endoplasmic reticulum, plasma membrane, golgi

apparatus.

Function

Catalytic core of the gamma-secretase complex.

This complex catalyses the intramembrane cleavage

of single-pass membrane proteins such as Notch

and the Amyloid Precursor Protein (APP) to give

intracellular signaling. The released intracellular

domains of Notch or APP form complexes with

other proteins to regulate gene transcription.

Homology

The PSEN2 gene is conserved in chimpanzee, dog,

cow, mouse, rat, chicken, and zebrafish.

Presenilin 2 transcript, lines indicate introns and boxes exons. Untranslated regions are represented as yellow boxes and coding

regions as red boxes.

Presenilin 2 protein domains, bright blue boxes are transmembrane domains (TM).

PSEN2 (presenilin 2 (Alzheimer disease 4)) Newman M


Mutations

Somatic

23 mutations.

Nucleotide

change Disease Reference

Arg62His AD Cruts et al., 1998; Guerreiro et

al., 2008

Arg71Trp AD Guerreiro et al., 2008

Thr122Pro AD Finckh et al., 2000; 2005

Ser130Leu AD Sorbi et al., 2002; Tedde et al.,

2003; Tomaino et al., 2007

Val139Met AD Bernardi et al., 2008

Asn141Ile AD Levy-Lahad et al., 1995; Rogaev

et al., 1995

Met174Val AD Guerreiro et al., 2008

Ser175Cys AD Piscopo et al., 2008

Gln228Leu AD Zekanowski et al., 2003

Met239Ile AD Finckh et al., 2000

Met239Val AD Rogaev et al., 1995; Marcon et

al., 2004

Val393Met AD Lindquist et al., 2008; 2009

Thr430Met AD Lleo et al., 2002; Ezquerra et al.,

2003

Asp439Ala AD Lleo et al., 2001; 2002

Arg62His Breast

Cancer To et al., 2006

Arg71Trp Breast

Cancer To et al., 2006

Tyr231Cys FTD Marcon et al., 2008; 2009

Ala85Val LBD Piscopo et al., 2008

Thr122Arg Atypical

Dementia Binetti et al., 2003

Table. Mutations identified through genetic screening. AD: Alzheimer's Disease, FTD: Frontotemporal Dementia,

LBD: Lewy Body Dementia.

Implicated in

Breast cancer

Disease

Breast cancer is the most common form of cancer

for women. The cancer originates from the breast

tissue where it can be a ductal carcinoma or lobular

carcinoma. They can be further defined as in situ or

invasive cancers.

Oncogenesis

Mutations (see above).

Alzheimer's disease

Note

Mutations (see above) taken from the Alzheimer's

Disease and Frontotemporal Dementia Mutation

Database. Only pathogenic mutations are included.

Disease

Alzheimer's disease is the most prevalent form of

dementia. In affected individuals the disease causes

a progressive and permanent decline in memory and

cognitive abilities. Neuropathogenesis is proposed

to be a result of the accumulation of amyloid-beta

peptides in the brain together with increased

oxidative stress and neuroinflammation. The

presenilin proteins are central to the gamma-

secretase cleavage of the amyloid precursor protein

(APP), releasing the amyloid-beta peptide. Point

mutations in the presenilin genes lead to cases of

familial Alzheimer's disease (and some sporadic

cases) by altering APP cleavage resulting in excess

amyloid-beta formation.

Frontotemporal Dementia (FTD)

Note

Mutation (see above).

Disease

Frontotemporal dementia is a group of related

conditions resulting from the progressive

degeneration of the temporal and frontal lobes of

the brain (frontotemporal lobar degeneration,

FTLD), usually with the presence of abnormal

intracellular protein accumulations. These areas of

the brain play a significant role in decision-making,

behavioral control, emotion and language. The

disorder is often sporadic, familial FTD has been

linked to mutations in several genes, including

those encoding the microtubule-associated protein

tau (MAPT), progranulin (GRN), valosin-

containing protein (VCP) and charged

multivescicular body protein 2B (CHMP2B).

Lewy body Dementia (DLB)

Note

Mutation (see above).

Disease

Dementia with Lewy bodies is a neurodegenerative

disorder associated with abnormal structures (Lewy

bodies) which are clumps of alpha-synuclein and

ubiquitin protein in neurons found in certain areas

of the brain. In addition to dementia, patients with

dementia with Lewy bodies experience

hallucinations, motor impairment, and fluctuating

alertness.

http://www.molgen.ua.ac.be/ADMutations





Diagram taken from http://www.molgen.ua.ac.be/ADMutations. Coloured circles indicate mutation sites. Red: pathogenic,

orange: pathogenic nature unclear, green: not pathogenic.

To be noted

Note

Truncated variant PSEN2 protein (PS2V). Variant

transcript lacks exon 5 due to alternative splicing.

Encodes the first 119 codons of PSEN2 plus a

newly generated five amino acids SSMAG. PS2V is

detected in sporadic Alzheimer's disease, bi-polar

and schizophrenia cases (Sato et al., 1999; Smith et

al., 2004). Cell-culture experiments indicate that

this variant is upregulated under hypoxic conditions

(Sato et al., 1999).

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Rogaev EI, Sherrington R, Rogaeva EA, Levesque G, Ikeda M, Liang Y, Chi H, Lin C, Holman K, Tsuda T. Familial Alzheimer's disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer's disease type 3 gene. Nature. 1995 Aug 31;376(6543):775-8

Lee MK, Slunt HH, Martin LJ, Thinakaran G, Kim G, Gandy SE, Seeger M, Koo E, Price DL, Sisodia SS. Expression of presenilin 1 and 2 (PS1 and PS2) in human and murine tissues. J Neurosci. 1996 Dec 1;16(23):7513-25

Levy-Lahad E, Poorkaj P, Wang K, Fu YH, Oshima J, Mulligan J, Schellenberg GD. Genomic structure and expression of STM2, the chromosome 1 familial Alzheimer disease gene. Genomics. 1996 Jun 1;34(2):198-204

McMillan PJ, Leverenz JB, Poorkaj P, Schellenberg GD, Dorsa DM. Neuronal expression of STM2 mRNA in human brain is reduced in Alzheimer's disease. J Histochem Cytochem. 1996 Nov;44(11):1215-22

Prihar G, Fuldner RA, Perez-Tur J, Lincoln S, Duff K, Crook R, Hardy J, Philips CA, Venter C, Talbot C, Clark RF, Goate A, Li J, Potter H, Karran E, Roberts GW, Hutton M, Adams MD. Structure and alternative splicing of the presenilin-2 gene. Neuroreport. 1996 Jul 8;7(10):1680-4

Wolozin B, Iwasaki K, Vito P, Ganjei JK, Lacanà E, Sunderland T, Zhao B, Kusiak JW, Wasco W, D'Adamio L. Participation of presenilin 2 in apoptosis: enhanced basal activity conferred by an Alzheimer mutation. Science. 1996 Dec 6;274(5293):1710-3

Cruts M, Van Broeckhoven C. Presenilin mutations in Alzheimer's disease. Hum Mutat. 1998;11(3):183-90

Cruts M, Van Broeckhoven C. Molecular genetics of Alzheimer's disease. Ann Med. 1998 Dec;30(6):560-5

Cruts M, van Duijn CM, Backhovens H, Van den Broeck M, et al. Estimation of the genetic contribution of presenilin-1 and -2 mutations in a population-based study of presenile Alzheimer disease. Hum Mol Genet. 1998 Jan;7(1):43-51

Lao JI, Beyer K, Fernández-Novoa L, Cacabelos R. A novel mutation in the predicted TM2 domain of the presenilin 2 gene in a Spanish patient with late-onset Alzheimer's disease. Neurogenetics. 1998 Aug;1(4):293-6




Sato N, Hori O, Yamaguchi A, Lambert JC, Chartier-Harlin MC, Robinson PA, Delacourte A, Schmidt AM, Furuyama T, Imaizumi K, Tohyama M, Takagi T. A novel presenilin-2 splice variant in human Alzheimer's disease brain tissue. J Neurochem. 1999 Jun;72(6):2498-505

Finckh U, Alberici A, Antoniazzi M, Benussi L, Fedi V, Giannini C, Gal A, Nitsch RM, Binetti G. Variable expression of familial Alzheimer disease associated with presenilin 2 mutation M239I. Neurology. 2000 May 23;54(10):2006-8

Finckh U, Müller-Thomsen T, Mann U, Eggers C, Marksteiner J, Meins W, Binetti G, Alberici A, Hock C, Nitsch RM, Gal A. High prevalence of pathogenic mutations in patients with early-onset dementia detected by sequence analyses of four different genes. Am J Hum Genet. 2000 Jan;66(1):110-7

Lleó A, Blesa R, Gendre J, Castellví M, Pastor P, Queralt R, Oliva R. A novel presenilin 2 gene mutation (D439A) in a patient with early-onset Alzheimer's disease. Neurology. 2001 Nov 27;57(10):1926-8

Groth C, Nornes S, McCarty R, Tamme R, Lardelli M. Identification of a second presenilin gene in zebrafish with similarity to the human Alzheimer's disease gene presenilin2. Dev Genes Evol. 2002 Nov;212(10):486-90

Lleó A, Blesa R, Queralt R, Ezquerra M, Molinuevo JL, Peña-Casanova J, Rojo A, Oliva R. Frequency of mutations in the presenilin and amyloid precursor protein genes in early-onset Alzheimer disease in Spain. Arch Neurol. 2002 Nov;59(11):1759-63

Sorbi S, Tedde A, Nacmias B, Ciantelli M, Caffarra P, Ghidoni E, Bracco L, Piccini C.. Novel presenilin 1 and presenilin 2 mutations in early-onset Alzheimer's disease families. Neurobiology of Aging. 2002; 23(1S):S312.

Binetti G, Signorini S, Squitti R, Alberici A, Benussi L, Cassetta E, Frisoni GB, Barbiero L, Feudatari E, Nicosia F, Testa C, Zanetti O, Gennarelli M, Perani D, Anchisi D, Ghidoni R, Rossini PM. Atypical dementia associated with a novel presenilin-2 mutation. Ann Neurol. 2003 Dec;54(6):832-6

Ezquerra M, Lleó A, Castellví M, Queralt R, Santacruz P, Pastor P, Molinuevo JL, Blesa R, Oliva R. A novel mutation in the PSEN2 gene (T430M) associated with variable expression in a family with early-onset Alzheimer disease. Arch Neurol. 2003 Aug;60(8):1149-51

Nornes S, Groth C, Camp E, Ey P, Lardelli M. Developmental control of Presenilin1 expression, endoproteolysis, and interaction in zebrafish embryos. Exp Cell Res. 2003 Sep 10;289(1):124-32

Tedde A, Nacmias B, Ciantelli M, Forleo P, Cellini E, Bagnoli S, Piccini C, Caffarra P, Ghidoni E, Paganini M, Bracco L, Sorbi S. Identification of new presenilin gene mutations in early-onset familial Alzheimer disease. Arch Neurol. 2003 Nov;60(11):1541-4

Zekanowski C, Styczyńska M, Pepłońska B, Gabryelewicz T, Religa D, Ilkowski J, Kijanowska-Haładyna B, et al. Mutations in presenilin 1, presenilin 2 and amyloid precursor protein genes in patients with early-onset Alzheimer's disease in Poland. Exp Neurol. 2003 Dec;184(2):991-6

Marcon G, Giaccone G, Cupidi C, Balestrieri M, Beltrami CA, Finato N, Bergonzi P, Sorbi S, Bugiani O, Tagliavini F. Neuropathological and clinical phenotype of an Italian Alzheimer family with M239V mutation of presenilin 2 gene. J Neuropathol Exp Neurol. 2004 Mar;63(3):199-209

Smith MJ, Sharples RA, Evin G, McLean CA, Dean B, Pavey G, Fantino E, Cotton RG, Imaizumi K, Masters CL, Cappai R, Culvenor JG. Expression of truncated presenilin

2 splice variant in Alzheimer's disease, bipolar disorder, and schizophrenia brain cortex. Brain Res Mol Brain Res. 2004 Aug 23;127(1-2):128-35

Finckh U, Kuschel C, Anagnostouli M, Patsouris E, Pantes GV, Gatzonis S, Kapaki E, Davaki P, Lamszus K, Stavrou D, Gal A. Novel mutations and repeated findings of mutations in familial Alzheimer disease. Neurogenetics. 2005 May;6(2):85-9

To MD, Gokgoz N, Doyle TG, Donoviel DB, Knight JA, Hyslop PS, Bernstein A, Andrulis IL. Functional characterization of novel presenilin-2 variants identified in human breast cancers. Oncogene. 2006 Jun 15;25(25):3557-64

Tomaino C, Bernardi L, Anfossi M, Costanzo A, Ferrise F, Gallo M, Geracitano S, Maletta R, Curcio SA, Mirabelli M, Colao R, Frangipane F, Puccio G, Calignano C, Muraca MG, Paonessa A, Smirne N, Leotta A, Bruni AC. Presenilin 2 Ser130Leu mutation in a case of late-onset "sporadic" Alzheimer's disease. J Neurol. 2007 Mar;254(3):391-3

Bernardi L, Tomaino C, Anfossi M, Gallo M, Geracitano S, Puccio G, Colao R, Frangipane F, Mirabelli M, Smirne N, Giovanni Maletta R, Bruni AC. Late onset familial Alzheimer's disease: novel presenilin 2 mutation and PS1 E318G polymorphism. J Neurol. 2008 Apr;255(4):604-6

Brouwers N, Sleegers K, Van Broeckhoven C. Molecular genetics of Alzheimer's disease: an update. Ann Med. 2008;40(8):562-83

Lindquist SG, Hasholt L, Bahl JM, Heegaard NH, Andersen BB, Nørremølle A, Stokholm J, Schwartz M, Batbayli M, Laursen H, Pardossi-Piquard R, Chen F, St George-Hyslop P, Waldemar G, Nielsen JE. A novel presenilin 2 mutation (V393M) in early-onset dementia with profound language impairment. Eur J Neurol. 2008 Oct;15(10):1135-9

Nornes S, Newman M, Verdile G, Wells S, Stoick-Cooper CL, Tucker B, Frederich-Sleptsova I, Martins R, Lardelli M. Interference with splicing of Presenilin transcripts has potent dominant negative effects on Presenilin activity. Hum Mol Genet. 2008 Feb 1;17(3):402-12

Piscopo P, Marcon G, Piras MR, Crestini A, Campeggi LM, Deiana E, Cherchi R, Tanda F, Deplano A, Vanacore N, Tagliavini F, Pocchiari M, Giaccone G, Confaloni A. A novel PSEN2 mutation associated with a peculiar phenotype. Neurology. 2008 Apr 22;70(17):1549-54

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Guerreiro RJ, Baquero M, Blesa R, Boada M, Brás JM, et al. Genetic screening of Alzheimer's disease genes in Iberian and African samples yields novel mutations in presenilins and APP. Neurobiol Aging. 2010 May;31(5):725-31


Newman M. PSEN2 (presenilin 2 (Alzheimer disease 4)). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):42-45.

Gene Section Review




RASSF6 (Ras association (RalGDS/AF-6) domain family member 6) Luke B Hesson, Farida Latif

Lowy Cancer Centre and Prince of Wales Clinical School, Faculty of Medicine, University of New

South Wales, NSW2052, Australia (LBH), School of Clinical and Experimental Medicine, College of

Medical and Dental Sciences, Department of Medical and Molecular Genetics, University of

Birmingham, Birmingham B15 2TT, UK (FL)


Online updated version : http://AtlasGeneticsOncology.org/Genes/RASSF6ID43462ch4q13.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI RASSF6ID43462ch4q13.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity

Other names: DKFZp686K23225

HGNC (Hugo): RASSF6

Location: 4q13.3

Local order: Centromere-AFP-AFM-RASSF6-

IL8-Telomere.

Note

Brief overview : The RASSF family of tumour

suppressor genes (TSG) encode Ras superfamily

effector proteins that, amongst other functions,

mediate some of the growth inhibitory functions of

Ras proteins. Several members of this family are

inactivated by promoter DNA hypermethylation in

a broad range of cancers, and the RASSF6 gene

may be a frequent target of epigenetic inactivation

in leukaemias. The RASSF6 protein is involved in

the regulation of apoptosis partly by controlling the

function of the proapoptotic mammalian

serine/threonine kinases 1 and 2 (MST1 and MST2)

and modulator of apoptosis 1 (MOAP-1).

Figure 1: RASSF6 gene structure. The RASSF6 gene is composed of at least two isoforms that are transcribed from

immediately upstream (RASSF6A) or from within (RASSF6B) a small CpG island.

RASSF6 (Ras association (RalGDS/AF-6) domain family member 6)

Hesson LB, Latif F


DNA/RNA

Description

The RASSF6 gene occupies 47478 bp of genomic

DNA. RASSF6A [GenBank:NM_201431] contains

11 exons and is transcribed 82 bp upstream from a

small (214 bp) 5' CpG island (at chr4:74,486,045-

74,486,258). RASSF6B [GenBank:NM_177532]

contains 11 exons and is transcribed from within

the same CpG island. There is evidence of

additional splice variants and transcription initiation

sites for the RASSF6 gene, however these have not

been validated.

Protein

Description

RASSF6A [GenBank:NP_958834] is a 369 amino

acid protein whereas RASSF6B

[GenBank:NP_803876] is a 337 amino acid protein.

The RASSF6A protein (figure 2) contains C-

terminal Ras-association (RA) and

Sav/RASSF/Hpo (SARAH) domains that define the

'classical' RASSF family (RASSF1, RASSF2,

RASSF3, RASSF4, RASSF5, RASSF6). The

RASSF6B protein lacks the N-terminal 32 amino

acids present in RASSF6A but is otherwise

identical. There are some inconsistencies in the

literature regarding which may be the major

isoform, however all functional work to date has

studied the larger 369 amino acid protein, which is

hereafter referred to as RASSF6. Using an in-house

antibody Ikeda et al., (2007) were unable to detect

endogenous RASSF6 protein. All functional work

has been performed using overexpressed RASSF6

protein. A commercially available antibody towards

RASSF6 (ProteinTech Group) has been used to

demonstrate re-expression following treatment with

the DNA demethylating agent 5-aza-

2'deoxycytidine (5azaDC) and Trichostatin A

(TSA) in leukaemia cell lines in which RASSF6 is

epigenetically inactivated (Hesson et al., 2009).

Expression

Northern blotting of a normal tissue RNA panel

shows RASSF6 mRNA is highly expressed in

thymus, kidney and placenta, with lower levels of

expression in colon, small intestine and lung (Allen

et al., 2007). In the same study the matched normal

tissue from a primary tumour cDNA panel showed

readily detectable levels of RASSF6 expression in

rectal, pancreatic, liver, breast and stomach tissues.

There has been no systematic analysis of the

expression patterns of the different RASSF6

isoforms. RT-PCR analysis of a range of tumour

cell lines showed that RASSF6 is highly expressed

in HeLa and A549 cells with lower levels observed

in MCF-7, U373, H1299 and HepG2 (Ikeda et al.,

2007). It appears from these studies that the tissue

distribution of RASSF6 expression is much more

restricted than that of the RASSF members

RASSF1, RASSF2 and RASSF5.

Expression of the RASSF6 gene is lost or

downregulated in a variety of solid tumours by

unknown mechanisms, whereas in childhood

leukaemias RASSF6 is inactivated by CpG island

promoter region DNA methylation (see below).

Function

dRASSF antagonises the Hippo pathway

In Drosophila, dRASSF represents the orthologue

of mammalian RASSF1-6. dRASSF protein

competes with Salvador (the Drosophila orthologue

of the mammalian WW45 protein) for binding to

Hippo (the Drosophila orthologue of the

mammalian MST kinases). The dRASSF-Hippo

and Salvador-Hippo interactions appear mutually

exclusive and control Hippo function in very

different ways.

Figure 2: RASSF6 transcript and protein structure. The RASSF6A mRNA (red bar) encodes a 369 amino acid protein

[GenBank:NP_958834] containing a C-terminal Ras-association (RA) domain of the RalGDS/AF-6 variety and acidic coiled-coil Sav/RASSF/Hpo (SARAH) domain. RASSF6B [GenBank:NP_803876] is a predicted 337 amino acid protein lacking the N-

terminal 32 amino acids present in RASSF6A.


Hesson LB, Latif F


Both Salvador or dRASSF are stabilised following

binding to Hippo, however, probing of

immunoprecipitates of Salvador or dRASSF with a

phospho-specific antibody that recognises active

Hippo demonstrates that Hippo is present in

Drosophila cells in two pools; an active form

associated with Salvador and an inactive form

associated with dRASSF (Polesello et al., 2006).

Furthermore, RNAi-mediated dRASSF depletion

led to a marked increase in Hippo activation

following Staurosporine (STS) treatment, a potent

activator of Hippo in Drosophila cells and MSTs in

mammalian cells. Thus, in Drosophila dRASSF

restricts the activation of Hippo, which may

account for the smaller size of dRASSF mutant flies

(Polesello et al., 2006).

Regulation of apoptosis by mammalian RASSF6

Several studies have demonstrated that

overexpression of RASSF6 induces apoptosis and

inhibits the growth of a variety of tumour cell lines

(Ikeda et al., 2007; Allen et al., 2007; Ikeda et al.,

2009). In HeLa cells RASSF6-induced apoptosis

occurred through both caspase-dependent and

caspase-independent pathways, since

overexpression of RASSF6 results in cleavage and

activation of caspase-3 but apoptosis was not

abrogated by z-VAD-FMK, an inhibitor of caspase-

1, caspase-3, caspase-4 and caspase-7 activation

(Ikeda et al., 2007). RASSF6 also induced Bax

activation and cytochrome C release as well as the

release of apoptosis-inducing factor (AIF) and

endonuclease G (endoG) from the mitochondria.

Following release from the mitochondria AIF and

endoG may result in DNA fragmentation even in

the absence of caspase activation. Early evidence

has suggested that the molecular mechanisms of

RASSF6-induced apoptosis are likely to be

complex and multi-layered. Currently, three

signalling routes are implicated in RASSF6-

induced apoptosis (see below), though at present it

is unclear whether these routes act autonomously or

as part of an extensive apoptotic network.

RASSF6 is an effector molecule of K-Ras-

mediated apoptosis

RASSF6 interacts with K-Ras in a GTP-dependent

manner via its Ras-association domain and the

effector domain of K-Ras. Therefore, RASSF6

exhibits the basic properties of a Ras effector

protein. These data are in contradiction to another

report in which RASSF6 did not interact with K-

Ras, H-Ras, N-Ras, M-Ras or TC21 under the same

conditions that RASSF5 binds to these Ras proteins

(Ikeda et al., 2007). Apart from a strict context-

dependency of these interactions The reasons for

this discrepancy is unclear but may be related to a

strict context-dependency of the interaction or to

the requirement of Ras farnesylation as shown by

Allen et al., (2007). Of particular note however, is

the observation that RASSF6 acts synergistically

with activated K-Ras to induce cell death in 293-T

cells (Allen et al., 2007). Taken together these data

suggests RASSF6 does indeed function within a K-

Ras-regulated pathway, most likely through direct

interaction, to determine cell fate.

RASSF6 negatively regulates the proapoptotic

protein MST2

RASSF6 interacts with MST2 via the

Sav/RASSF/Hpo (SARAH) domains within both

proteins (Ikeda et al., 2009). It seems clear from

many studies that several (if not all) classical

RASSF proteins interact with the MST1 and MST2

kinases and that this interaction is at least partly

involved in RASSF-induced apoptosis (van der

Weyden and Adams, 2007). Ikeda et al., (2009)

showed that the SARAH domain of MST2 can bind

both WW45 (the mammalian orthologue of the

Drosophila protein Salvador) and RASSF6 to form

a trimeric complex. However, RASSF6 and WW45

do not interact. This differs somewhat with the

regulation of the Hippo pathway in Drosophila, in

which the Salvador/Hippo and dRASSF/Hippo

complexes are mutually exclusive (see above).

RASSF6 inhibits MST2 kinase activity, however

activation of MST2 releases RASSF6 in a manner

dependent on WW45. Previous studies have

demonstrated that MST2 can extensively

phosphorylate WW45 (Callus et al., 2006). Ikeda et

al., (2009) demonstrated that activation of MST2 by

the phosphatase inhibitor okadaic acid (OA)

reduced the co-immunoprecipitation of RASSF6

with MST2, whilst the association of MST2 and

WW45 remained unchanged. Taken together this

suggests that the activation of MST2 and

subsequent phosphorylation of WW45 results in the

release of RASSF6 from the

WW45/MST2/RASSF6 complex. The release of

RASSF6 was shown to be WW45-dependent since

RASSF6 remained in complex with MST2

following OA treatment of cells in which WW45

had been depleted by RNAi. Furthermore, the

release of RASSF6 appears to be necessary for

RASSF6-mediated apoptosis. However, apoptosis

was vastly reduced in cells transfected with both

MST2 and RASSF6. This block of RASSF6-

induced apoptosis was dependent on the SARAH

domain of MST2 but not the kinase activity of

MST2 suggesting that MST2 blocks RASSF6-

mediated apoptosis by physical interaction.

Interestingly co-expression of RASSF6, MST2 and

WW45 resulted in potent cell death. This effect was

dependent on the SARAH domain of WW45

suggesting that physical interaction of WW45 with

MST2 was necessary to reinstate RASSF6-induced

apoptosis by allowing the release of RASSF6.

Inhibition of MST2 kinase activity by RASSF6 also

results in the inhibition of NDR1 and LATS2

phosphorylation (Ikeda et al., 2009). NDR1 and

LATS2 are known MST2 substrates that form part

of the MST/Hippo tumour suppressor pathway (see

Hergovich and Hemmings, 2009 and Harvey and


Hesson LB, Latif F


Tapon, 2007 for a more thorough review of the

regulation of apoptosis through the MST/Hippo

pathway). Thus release of RASSF6 from the

MST2-WW45 complex allows RASSF6-mediated

and MST2-mediated apoptosis (figure 3). The

induction of apoptosis through these separate

pathways may explain the caspase-dependent and

caspase-independent nature of RASSF6-mediated

apoptosis, since full activation of MSTs is thought

to require caspase cleavage.

MOAP-1 is involved in RASSF6-mediated

apoptosis

The MOAP-1 protein may also be a RASSF6

effector molecule. To date, two independent studies

have demonstrated that RASSF6 also interacts with

MOAP-1 (Allen et al., 2007; Ikeda et al., 2009).

Though this interaction has not been demonstrated

formally at the endogenous level, co-expression of

the two proteins clearly demonstrates that MOAP-1

is a likely mediator of RASSF6-induced apoptosis

in a manner independent of the MST/Hippo tumour

suppressor pathway. MOAP-1 regulates the

'extrinsic' pathway of apoptosis by acting

downstream of death receptors such as the tumour

necrosis factor a receptor 1 (TNF-R1) and TNFa

apoptosis-inducing related ligand receptor 1

(TRAIL-R1). A role for MOAP-1 in regulating

RASSF1A-induced apoptosis has previously been

demonstrated. Following ligand binding the C-

terminal region of MOAP-1 associates with the

death domain of TNF-R1. Subsequently, the TNF-

R1/MOAP-1 receptor complex is internalised and

recruits RASSF1A through the N-terminal cysteine-

rich (C1) domain within RASSF1A (Baksh et al.,

2005; Vos et al., 2006; Foley et al., 2008). Under

'static' conditions MOAP-1 is held in an inactive

conformation, however binding to RASSF1A

results in a conformational change that allows

MOAP-1 to interact with Bax. This in turn induces

a conformational change within Bax that is required

for its insertion into the mitochondrial membrane

and for the release of inner mitochondrial

membrane proteins that can induce apoptosis.

Interestingly, activated K-Ras, RASSF1A and

MOAP-1 synergise to induce Bax activation and

apoptosis (Vos et al., 2006) indicating that cell

death induced by the RASSF1A/MOAP-1

interaction may be regulated by both K-Ras and

death receptors.

The acidic sequence Glu-Glu-Glu-Glu [312

EEEE] in

the SARAH domain of RASSF1A that binds to

MOAP-1 (Baksh et al., 2005) is also partially

conserved in the RASSF6 SARAH domain

[EEEK]. However, this is unlikely to be the sole

site of interaction since RASSF6 lacking the N-

terminal region, the Ras-association domain, or the

SARAH domain also interacted with MOAP-1

(Ikeda et al., 2009). Depletion of MOAP-1 partially

suppresses RASSF6-mediated apoptosis and co-

expression of MST2 vastly reduces the co-

immunoprecipitation of RASSF6 and MOAP-1; an

effect that is abrogated by the expression of WW45.

Taken together these data suggest that the

MST2/WW45 complex binds RASSF6 and

prevents its interaction with MOAP-1. The MST-

Hippo pathway and the MOAP-1 pathway represent

distinct RASSF6-regulated apoptotic pathways that

are triggered by the activation of MST2 (figure 3).

Homology

RASSF6 is one of 10 members of the Ras-

association domain family (RASSF) comprising

RASSF1-10 (please refer to figure 3 of the RASSF2

gene card. RASSF1-6 are termed the 'classical'

RASSF family and contain C-terminal RA and

SARAH domains. Consequently, RASSF1-6 are

most similar in sequence within their C-termini.

RASSF7, RASSF8, RASSF9 and RASSF10

represent evolutionarily conserved but structurally

distinct RASSF members that lack the SARAH

domains and contain N-terminal RA domains.

RASSF7-10 are termed the 'N-terminal' RASSF

family. Many of these RASSF members are

involved in tumourigenesis and several (RASSF1A,

RASSF2, RASSF4, RASSF5A, RASSF6 and

RASSF10) are inactivated in a variety of human

cancers (Hesson et al., 2007; van der Weyden and

Adams, 2007; Hesson et al., 2009). RASSF6 also

has orthologues in several species (table 1)

including Drosophila melanogaster, which is the

orthologue of the human RASSF1-6 genes.


Hesson LB, Latif F


Figure 3: The molecular basis of RASSF6-induced apoptosis. Multiple lines of evidence suggest that RASSF6-induced

apoptosis involves multiple pathways. K-Ras, MST2 and MOAP-1 have all been implicated in RASSF6-induced apoptosis. To date RASSF1, RASSF2, RASSF4, RASSF5 and RASSF6 have been shown to bind to the MST1 and MST2 kinases. Thus, the

RASSF proteins play a major role in regulating apoptosis through the MST/Hippo tumour suppressor pathway leading to apoptosis. The regulation of apoptosis through activation of the TNF-R1 and TRAIL-R1 death receptor pathways has been

shown to involve the interaction of RASSF1A with MOAP-1. This interaction leads to the release of inner mitochondrial membrane proteins that result in apoptosis. RASSF6 also interacts with MOAP-1, though the events downstream have not been fully elucidated. The release of inner mitochondrial membrane proteins is a major facet of RASSF6-induced apoptosis therefore it

seems likely that RASSF6 regulates MOAP-1 function in a similar way to RASSF1A and may feed into the same pathway. The synergistic effects of activated K-Ras, RASSF6 and MOAP-1 overexpression suggests that apoptosis through the

RASSF6/MOAP-1 complex may be at least partially K-Ras-regulated.


Hesson LB, Latif F


Table 1: RASSF6 orthologues in model species.

Mutations

Note

To date no inactivating mutations to RASSF6 have

been described.

Implicated in

Various cancers

Note

Loss of RASSF6 expression in cancer.

Disease

Northern blotting analysis showed RASSF6

expression is lost or downregulated in 30-60% of

primary tumour tissues of the breast, colon, kidney,

liver, pancreas, stomach and thyroid (Allen et al.,

2007). The mechanisms underlying this reduced

expression are not clear and, at least in solid

tumours, loss of RASSF6 expression is not

associated with promoter DNA methylation (Allen

et al., 2007). Other mechanisms of gene silencing

such as histone modifications or long-range

epigenetic silencing may account for RASSF6

downregulation, but this has not been investigated.

In childhood leukaemias silencing of RASSF6

expression by promoter hypermethylation appears

to be an extremely frequent event and was

identified in 94% (48/51) B-ALL and 41% (12/29)

T-ALL (Hesson et al., 2009). To date this remains

the first and only description of a mechanism

accounting for the loss of RASSF6 expression in

cancer but further suggests that epigenetic

inactivation of RASSF6 may be specific to

leukaemias. Further evidence that the loss of

RASSF6 expression is important in cancer is

provided in a study by Finn et al., (2007), which

demonstrated downregulation of RASSF6

expression in malignant versus benign thyroid

tissue.

Pancreatic endocrine tumour

Note

RASSF6 is downregulated in the pancreatic

endocrine tumour cell line BON1 following siRNAi

of Achaete-scute complex-like 1 (ASCL1), a basic

helix loop helix (bHLH) transcription factor

regulated by the NOTCH signalling pathway


Hesson LB, Latif F


(Johansson et al., 2009). ASCL1 also negatively

regulates expression of Dickkopf homologue 1

(DKK1), an antagonist of the Wnt/beta-catenin-

dependent signalling pathway.

Breast cancer

Note

Using an in vitro model of breast carcinogenesis,

human non-tumourigenic immortalised breast

epithelial cell line MCF-10A cells were selected

following exposure to the mutagen ICR191 to

create transformed cells (Zientek-Targosz et al.,

2008). One of the genes found to contain a

frameshift mutation following this selection was

RASSF6. The authors postulate that this indicates

that inactivation of RASSF6 may be involved in the

transformation of breast epithelial cells thus

indicating the potential importance of RASSF6 in

tumour development.

Childhood B-ALL

Note

RASSF6 is downregulated in mature B-cells in

response to B-cell activating factor (BAFF)

treatment (Saito et al., 2008). BAFF is a tumour

necrosis factor (TNF) superfamily member, which

is thought to be involved in the survival and

maturation of B-cells partly by inhibiting apoptosis.

This finding reiterates the potential importance of

the frequent epigenetic inactivation of RASSF6 in

childhood B-ALL (Hesson et al., 2009), which

would nullify RASSF6-mediated apoptosis.

Viral-induced bronchiolitis

Disease

Genetic mapping studies have linked the RASSF6

locus to viral-induced bronchiolitis, the most

common cause of infant hospital admissions in the

industrialised world (Hull et al., 2004; Smyth and

Openshaw, 2006). The 250 kb interval at 4q13.3

implicated by Hull et al., (2004) includes three

genes (AFM, RASSF6 and IL8), however it is

unclear which gene is the important player. The

predominant cause of acute viral bronchiolitis is

infection with the respiratory syncytial virus (RSV).

A major consequence of RSV infection is

stimulation of the NF-kappaB pathway (Hull et al.,

2004), which is thought to modulate the degree of

inflammation and support viral replication, perhaps

by suppressing apoptosis (Bitko et al., 2004). Using

an NF-kappaB luciferase reporter system in A549

lung tumour cells it has been found that RASSF6

can suppress the serum-induced basal levels of NF-

kappaB reporter expression by approximately

fivefold (Allen et al., 2007). Therefore, RASSF6

remains a promising susceptibility gene candidate

for viral-induced bronchiolitis.

Heavy metal detoxification

Note

Expression of murine Rassf6 was upregulated

following subcutaneous injection of Cadmium,

possibly implicating Rassf6 in the cellular

mechanisms of heavy metal detoxification

(Wimmer et al., 2005). However, it seems equally

likely that the observed upregulation of Rassf6

could be due to increased apoptosis caused by the

toxic effects of heavy metal exposure.

References Bitko V, Garmon NE, Cao T, Estrada B, Oakes JE, Lausch RN, Barik S. Activation of cytokines and NF-kappa B in corneal epithelial cells infected by respiratory syncytial virus: potential relevance in ocular inflammation and respiratory infection. BMC Microbiol. 2004 Jul 15;4:28

Hull J, Rowlands K, Lockhart E, Sharland M, Moore C, Hanchard N, Kwiatkowski DP. Haplotype mapping of the bronchiolitis susceptibility locus near IL8. Hum Genet. 2004 Feb;114(3):272-9

Baksh S, Tommasi S, Fenton S, Yu VC, Martins LM, Pfeifer GP, Latif F, Downward J, Neel BG. The tumor suppressor RASSF1A and MAP-1 link death receptor signaling to Bax conformational change and cell death. Mol Cell. 2005 Jun 10;18(6):637-50

Wimmer U, Wang Y, Georgiev O, Schaffner W. Two major branches of anti-cadmium defense in the mouse: MTF-1/metallothioneins and glutathione. Nucleic Acids Res. 2005;33(18):5715-27

Callus BA, Verhagen AM, Vaux DL. Association of mammalian sterile twenty kinases, Mst1 and Mst2, with hSalvador via C-terminal coiled-coil domains, leads to its stabilization and phosphorylation. FEBS J. 2006 Sep;273(18):4264-76

Polesello C, Huelsmann S, Brown NH, Tapon N. The Drosophila RASSF homolog antagonizes the hippo pathway. Curr Biol. 2006 Dec 19;16(24):2459-65

Smyth RL, Openshaw PJ. Bronchiolitis. Lancet. 2006 Jul 22;368(9532):312-22

Vos MD, Dallol A, Eckfeld K, Allen NP, Donninger H, Hesson LB, Calvisi D, Latif F, Clark GJ. The RASSF1A tumor suppressor activates Bax via MOAP-1. J Biol Chem. 2006 Feb 24;281(8):4557-63

Allen NP, Donninger H, Vos MD, Eckfeld K, Hesson L, Gordon L, Birrer MJ, Latif F, Clark GJ. RASSF6 is a novel member of the RASSF family of tumor suppressors. Oncogene. 2007 Sep 13;26(42):6203-11

Finn SP, Smyth P, Cahill S, Streck C, O'Regan EM, Flavin R, Sherlock J, Howells D, Henfrey R, Cullen M, Toner M, Timon C, O'Leary JJ, Sheils OM. Expression microarray analysis of papillary thyroid carcinoma and benign thyroid tissue: emphasis on the follicular variant and potential markers of malignancy. Virchows Arch. 2007 Mar;450(3):249-60

Harvey K, Tapon N. The Salvador-Warts-Hippo pathway - an emerging tumour-suppressor network. Nat Rev Cancer. 2007 Mar;7(3):182-91

Hesson LB, Cooper WN, Latif F. The role of RASSF1A methylation in cancer. Dis Markers. 2007;23(1-2):73-87


Hesson LB, Latif F


Ikeda M, Hirabayashi S, Fujiwara N, Mori H, Kawata A, Iida J, Bao Y, Sato Y, Iida T, Sugimura H, Hata Y. Ras-association domain family protein 6 induces apoptosis via both caspase-dependent and caspase-independent pathways. Exp Cell Res. 2007 Apr 15;313(7):1484-95

van der Weyden L, Adams DJ. The Ras-association domain family (RASSF) members and their role in human tumourigenesis. Biochim Biophys Acta. 2007 Sep;1776(1):58-85

Foley CJ, Freedman H, Choo SL, Onyskiw C, Fu NY, Yu VC, Tuszynski J, Pratt JC, Baksh S. Dynamics of RASSF1A/MOAP-1 association with death receptors. Mol Cell Biol. 2008 Jul;28(14):4520-35

Saito Y, Miyagawa Y, Onda K, Nakajima H, Sato B, Horiuchi Y, Okita H, Katagiri YU, Saito M, Shimizu T, Fujimoto J, Kiyokawa N. B-cell-activating factor inhibits CD20-mediated and B-cell receptor-mediated apoptosis in human B cells. Immunology. 2008 Dec;125(4):570-90

Zientek-Targosz H, Kunnev D, Hawthorn L, Venkov M, Matsui S, Cheney RT, Ionov Y. Transformation of MCF-10A cells by random mutagenesis with frameshift mutagen ICR191: a model for identifying candidate breast-tumor suppressors. Mol Cancer. 2008 Jun 5;7:51

Hergovich A, Hemmings BA. Mammalian NDR/LATS protein kinases in hippo tumor suppressor signaling. Biofactors. 2009 Jul-Aug;35(4):338-45

Hesson LB, Dunwell TL, Cooper WN, Catchpoole D, Brini AT, Chiaramonte R, Griffiths M, Chalmers AD, Maher ER, Latif F. The novel RASSF6 and RASSF10 candidate tumour suppressor genes are frequently epigenetically inactivated in childhood leukaemias. Mol Cancer. 2009 Jul 1;8:42

Ikeda M, Kawata A, Nishikawa M, Tateishi Y, Yamaguchi M, Nakagawa K, Hirabayashi S, Bao Y, Hidaka S, Hirata Y, Hata Y. Hippo pathway-dependent and -independent roles of RASSF6. Sci Signal. 2009 Sep 29;2(90):ra59

Johansson TA, Westin G, Skogseid B. Identification of Achaete-scute complex-like 1 (ASCL1) target genes and evaluation of DKK1 and TPH1 expression in pancreatic endocrine tumours. BMC Cancer. 2009 Sep 10;9:321


Hesson LB, Latif F. RASSF6 (Ras association (RalGDS/AF-6) domain family member 6). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):46-53.

Gene Section Review




RPA2 (replication protein A2, 32kDa) Anar KZ Murphy, James A Borowiec

Dept of Biochemistry and New York University Cancer Institute, New York University School of

Medicine, New York, New York 10016, USA (AKZM, JAB)


Online updated version : http://AtlasGeneticsOncology.org/Genes/RPA2ID42146ch1p35.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI RPA2ID42146ch1p35.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity

Other names: REPA2, RPA32

HGNC (Hugo): RPA2

Location: 1p35.3

Local order: The human RPA2 gene maps on

1p35.3 between the SMPDL3B (sphingomyelin

phosphodiesterase, acid-like 3B) and C1orf38

(interferon-gamma inducible gene ICB-1 (induced

by contact to basement membrane)).

DNA/RNA

Description

The RPA2 gene is contained within 24.5 kb of

chromosome 1.

The coding sequence is contained within nine

exons. There is no confirmed alternative splicing of

the RPA2 gene, or differential promoter usage.

Transcription

The RPA2 mRNA transcript is 1.5 kb. The RPA2

promoter contains four E2F consensus sequences

within the region about 400 bp upstream of the

mRNA start site, and putative binding sites for

ATF-1 and SP-1 transcription factors. Expression is

upregulated 2 to 3-fold by E2F, with mutation of

the three start site-proximal E2F sites causing a loss

of E2F responsiveness (Kalma et al., 2001).

Pseudogene

RPA2 does not have known pseudogenes.

The sequence numbering corresponds to EMBL locus DQ001128 (26.6 kb). Exon are indicated as boxes (yellow = 5' UTR, blue = CDS, red = 3' UTR), and introns with orange lines. Two lengthy introns have been truncated (indicated with parallel diagonal

lines) to improve viewability.

RPA2 (replication protein A2, 32kDa) Murphy AKZ, Borowiec JA


Protein

Upper panel: Schematic showing the key domains of RPA2.

Lower panel: RPA2 phosphorylation sites are shown in bold, with the primary responsible kinases indicated above each site. Some sites can be phosphorylated by more than

one kinase (e.g., T21 by ATM and DNA-PK).

Description

RPA2 is the middle subunit of the heterotrimeric

Replication Protein A (RPA; (reviewed in Binz et

al., 2004)). The subunit is composed of 270

residues, and has a nominal molecular weight of

29.2 kDa. RPA2 contains an N-terminal

phosphorylation region with 7 phosphorylation

sites, a central DNA-binding domain (termed DBD-

D), and a C-terminal region that can form a three-

helix bundle. One helix of the three helix bundle is

contributed by each RPA subunit, with this

structure responsible for supporting

heterotrimerization of the RPA complex

(Bochkareva et al., 2002). At least in the non-

phosphorylated state, the N-terminal region is

unstructured. DBD-D is constructed from an

oligonucleotide/oligosaccharide binding (OB) fold

(Bochkarev et al., 1999), one of six OB folds found

with the RPA heterotrimer (four OB folds are

located in RPA1, and one within RPA3).

Expression

RPA is an essential factor for DNA replication and

repair, and hence is expressed in all tissues.

Localisation

Nuclear.

Function

General function: RPA is a heterotrimeric single-

stranded DNA (ssDNA) binding protein that is

essential for chromosomal DNA replication,

homologous recombination, and particular DNA

repair reactions (nucleotide excision repair). The

apparent association constant of the RPA: ssDNA

complex is 109 - 10

11 M

-1 (Kim et al., 1992). While

RPA2 contains a central DBD (Philipova et al.,

1996), the major effect of mutating DBD-D is to

decrease the size of the ssDNA occluded by RPA

binding, with only minor effects on RPA: ssDNA

affinity (Bastin-Shanower and Brill, 2001). A key

function of the RPA2 subunit is to regulate RPA

activity in DNA replication and repair reactions,

through the RPA2 phosphorylation state (see

below).

1) RPA2 phosphorylation. The N-terminal 33

residues of RPA2 contain seven phosphorylation

sites. In interphase cells, genotoxic stress (e.g.,

caused by chromosomal double-strand DNA breaks

or DNA replication stress) induces RPA2

phosphorylation by members of the

phosphatidylinositol 3-kinase-like kinase (PIKK;

ATM, ATR, and DNA-PK) and cyclin-dependent

kinases (CDK) families (reviewed in Binz et al.,

2004). Mutation of particular RPA2

phosphorylation sites causes defects in homologous

recombination (Lee et al., 2010), and Rad51

recruitment to nuclear repair foci (Anantha et al.,

2008; Lee et al., 2010). Mutation of these sites also

causes genomic instability in response to DNA

replication stress induced by cellular treatment with

hydroxyurea (Vassin et al., 2009). RPA

phosphorylation also increases cell viability in

response to DNA damage arising during mitosis

(Anantha et al., 2008). Modification of sites in the

phosphorylation region of RPA2 proceeds in a

favored order in response to genotoxic stress

(Anantha et al., 2007). The phosphorylation of

individual RPA2 residues is dependent on the type

of DNA damage or replication stress encountered

(Anantha et al., 2007; Vassin et al., 2009). RPA2 is

a substrate both for PP2A and PP4 phosphatases

(Feng et al., 2009; Lee et al., 2010).

2) Involvement of RPA2 in protein-protein

interactions. RPA2 interacts with the nucleotide

excision repair factor XPA (He et al., 1995), base

excision repair enzyme UNG2 (Mer et al., 2000),

homologous recombination (HR) factor Rad52

(Mer et al., 2000), replication checkpoint protein

Tipin (Unsal-Kacmaz et al., 2007), and the

annealing helicase HARP/SMARCAL1 (Bansbach

et al., 2009; Ciccia et al., 2009; Yuan et al., 2009).

These interactions likely aid the multiple roles of

RPA in facilitating DNA repair.

Homology

A close homolog of RPA2, termed RPA4, is located

on Xq21.33 (Haring et al., 2010).

Mutations

Note

Naturally-occurring mutations of human RPA2

have not yet been described. A small number of

genetic polymorphisms have been described in SNP

datasets (Y14S, G15R, and N203S), but these have

not yet been reported to have any biological effects

(NIEHS SNPs program).



Implicated in

Colorectal adenocarcinoma

Disease

Overexpression of the RPA2 (and RPA1) proteins

have been found to be prognostic indicator of colon

cancer. Strong associations between RPA2

expression and disease stage, lymph node

metastasis, and the histological grade of carcinomas

have been observed.

Prognosis

In addition, RPA2 protein expression correlates

with poor survival of stage II and III patients

(Givalos et al., 2007).

Ductal breast carcinoma

Disease

Levels of anti-RPA2 antibodies was observed to be

significantly higher in sera from breast cancer

patients (10.9%; n = 801) as compared to normal

controls (0.0%; n = 221). Examining individuals

with early stage intraductal in situ carcinomas,

10.3% (n = 39) similarly showed the presence of

high levels of anti-RPA2 antibodies. Even so,

follow-up studies indicated that there were no

apparent differences in mean survival, occurrences

of a second primary tumor, or metastasis frequency

between breast cancer patients that were positive or

negative for anti-RPA2 sera. Although RPA is a

nuclear protein, RPA was seen to be localized to

both nuclei and cytoplasm in the cells of at least

one breast tumor, with RPA also over-expressed

(Tomkiel et al., 2002).

Non-small cell carcinoma

Disease

A fraction of individuals with squamous cell lung

cancer were found to have significant levels of anti-

RPA2 antibodies (9.1%; n = 22) (Tomkiel et al.,

2002).

Laryngeal tumors

Disease

One patient (out of 35; 2.9%) with head and neck

tumors tested positive for the presence of anti-

RPA2 sera (Tomkiel et al., 2002).

Promyelocytic leukemia

Disease

A derivative of the human HL-60 promyelocytic

leukemia cell line (HL-60/P1), selected for its

decreased sensitivity to undergo apoptosis in

response to TNF-related apoptosis-inducing ligand

(TRAIL), was found to have decreased (2-fold)

expression of RPA2 (Petrak et al., 2009).

Sjögren syndrome

Disease

Serum from a patient with Sjögren syndrome was

found to have high levels of anti-RPA2 antibodies.

A higher rate of non-Hodgkin lymphoma, and

lymphoid malignancies, is seen in individuals with

Sjögren syndrome, compared to normal individuals

(Garcia-Lozano et al., 1995).

Systemic lupus erythematosus (SLE)

Disease

One out of 55 individuals with autoimmune

disorders was found to test positive for anti-RPA2

antibodies (1.8%). This individual had SLE, and

secondary Sjögren syndrome (Garcia-Lozano et al.,

1995).

Rheumatoid arthritis (RA)

Note

Fibroblast-like synoviocytes (FLSs) are a cell type

whose invasive properties provide an indicator of

RA severity. Microarray studies from FLSs in DA

rats (arthritis-susceptible inbred model) show a

modest increase in the level of RPA2 mRNA,

compared to back-crossed arthritis-resistant

DA.F344 (Cia5d) congenic strains (Laragione et al.,

2008).

References Kim C, Snyder RO, Wold MS. Binding properties of replication protein A from human and yeast cells. Mol Cell Biol. 1992 Jul;12(7):3050-9

Garcia-Lozano R, Gonzalez-Escribano F, Sanchez-Roman J, Wichmann I, Nuñez-Roldan A. Presence of antibodies to different subunits of replication protein A in autoimmune sera. Proc Natl Acad Sci U S A. 1995 May 23;92(11):5116-20

He Z, Henricksen LA, Wold MS, Ingles CJ. RPA involvement in the damage-recognition and incision steps of nucleotide excision repair. Nature. 1995 Apr 6;374(6522):566-9

Philipova D, Mullen JR, Maniar HS, Lu J, Gu C, Brill SJ. A hierarchy of SSB protomers in replication protein A. Genes Dev. 1996 Sep 1;10(17):2222-33

Bochkarev A, Bochkareva E, Frappier L, Edwards AM. The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding. EMBO J. 1999 Aug 16;18(16):4498-504

Mer G, Bochkarev A, Gupta R, Bochkareva E, Frappier L, Ingles CJ, Edwards AM, Chazin WJ. Structural basis for the recognition of DNA repair proteins UNG2, XPA, and RAD52 by replication factor RPA. Cell. 2000 Oct 27;103(3):449-56

Bastin-Shanower SA, Brill SJ. Functional analysis of the four DNA binding domains of replication protein A. The role of RPA2 in ssDNA binding. J Biol Chem. 2001 Sep 28;276(39):36446-53

Kalma Y, Marash L, Lamed Y, Ginsberg D. Expression analysis using DNA microarrays demonstrates that E2F-1 up-regulates expression of DNA replication genes including replication protein A2. Oncogene. 2001 Mar 15;20(11):1379-87

Bochkareva E, Korolev S, Lees-Miller SP, Bochkarev A. Structure of the RPA trimerization core and its role in the multistep DNA-binding mechanism of RPA. EMBO J. 2002 Apr 2;21(7):1855-63



Tomkiel JE, Alansari H, Tang N, Virgin JB, Yang X, VandeVord P, Karvonen RL, Granda JL, Kraut MJ, Ensley JF, Fernández-Madrid F. Autoimmunity to the M(r) 32,000 subunit of replication protein A in breast cancer. Clin Cancer Res. 2002 Mar;8(3):752-8

Binz SK, Sheehan AM, Wold MS. Replication protein A phosphorylation and the cellular response to DNA damage. DNA Repair (Amst). 2004 Aug-Sep;3(8-9):1015-24

Anantha RW, Vassin VM, Borowiec JA. Sequential and synergistic modification of human RPA stimulates chromosomal DNA repair. J Biol Chem. 2007 Dec 7;282(49):35910-23

Givalos N, Gakiopoulou H, Skliri M, Bousboukea K, Konstantinidou AE, Korkolopoulou P, Lelouda M, Kouraklis G, Patsouris E, Karatzas G. Replication protein A is an independent prognostic indicator with potential therapeutic implications in colon cancer. Mod Pathol. 2007 Feb;20(2):159-66

Unsal-Kaçmaz K, Chastain PD, Qu PP, Minoo P, Cordeiro-Stone M, Sancar A, Kaufmann WK. The human Tim/Tipin complex coordinates an Intra-S checkpoint response to UV that slows replication fork displacement. Mol Cell Biol. 2007 Apr;27(8):3131-42

Anantha RW, Sokolova E, Borowiec JA. RPA phosphorylation facilitates mitotic exit in response to mitotic DNA damage. Proc Natl Acad Sci U S A. 2008 Sep 2;105(35):12903-8

Laragione T, Brenner M, Li W, Gulko PS. Cia5d regulates a new fibroblast-like synoviocyte invasion-associated gene expression signature. Arthritis Res Ther. 2008;10(4):R92

Bansbach CE, Bétous R, Lovejoy CA, Glick GG, Cortez D. The annealing helicase SMARCAL1 maintains genome integrity at stalled replication forks. Genes Dev. 2009 Oct 15;23(20):2405-14

Ciccia A, Bredemeyer AL, Sowa ME, Terret ME, Jallepalli PV, Harper JW, Elledge SJ. The SIOD disorder protein

SMARCAL1 is an RPA-interacting protein involved in replication fork restart. Genes Dev. 2009 Oct 15;23(20):2415-25

Feng J, Wakeman T, Yong S, Wu X, Kornbluth S, Wang XF. Protein phosphatase 2A-dependent dephosphorylation of replication protein A is required for the repair of DNA breaks induced by replication stress. Mol Cell Biol. 2009 Nov;29(21):5696-709

Petrak J, Toman O, Simonova T, Halada P, Cmejla R, Klener P, Zivny J. Identification of molecular targets for selective elimination of TRAIL-resistant leukemia cells. From spots to in vitro assays using TOP15 charts. Proteomics. 2009 Nov;9(22):5006-15

Vassin VM, Anantha RW, Sokolova E, Kanner S, Borowiec JA. Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress. J Cell Sci. 2009 Nov 15;122(Pt 22):4070-80

Yuan J, Ghosal G, Chen J. The annealing helicase HARP protects stalled replication forks. Genes Dev. 2009 Oct 15;23(20):2394-9

Haring SJ, Humphreys TD, Wold MS. A naturally occurring human RPA subunit homolog does not support DNA replication or cell-cycle progression. Nucleic Acids Res. 2010 Jan;38(3):846-58

Lee DH, Pan Y, Kanner S, Sung P, Borowiec JA, Chowdhury D. A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination. Nat Struct Mol Biol. 2010 Mar;17(3):365-72


Murphy AKZ, Borowiec JA. RPA2 (replication protein A2, 32kDa). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):54-57.

Gene Section Review




S100A7 (S100 calcium binding protein A7) Jill I Murray, Martin J Boulanger

Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia

V8W 3P6, Canada (JIM, MJB)


Online updated version : http://AtlasGeneticsOncology.org/Genes/S100A7ID42194ch1q21.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI S100A7ID42194ch1q21.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity

Other names: PSOR1, S100A7c

HGNC (Hugo): S100A7

Location: 1q21.3

Local order: S100A7 is located on chromosome

1cen-q21 between D1Z5 and MUC1 (Borglum et

al., 1995).

Note: S100A7 is also known as psoriasin, psoriasin

1, S100 calcium binding protein A7, S100-A7,

S100A7c, and PSOR1.

S100A7, a member of the S100 family, was first

identified as a protein upregulated in psoriasis

(Madsen et al., 1991).

DNA/RNA

Note

S100A7 is located on chromosome 1q21 within the

epidermal differentiation complex.

Description

The S100A7 gene has 3 exons and 2 introns with a

genomic structure similar to other S100 family

members. Exon 1 encodes the 5' untranslated region

while exons 2 and 3 contain the protein coding

sequence. Exon 2 encodes the start codon and the

non-canonical N-terminal EF-hand while exon 3

encodes the carboxyl-terminal EF-hand.

Transcription

The S100A7 gene encodes for a single

constitutively spliced transcript. An EST has been

reported in which an alternative promoter is used to

produce an identical S100A7 mRNA (See Ensembl,

UCSC genome browser).

Pseudogene

Five copies of S100A7 in the human genome have

been reported including the closely related paralog

S100A15 (also known as S100A7A) (Kulski et al.,

2003; Wolf et al., 2003). Two of the five reported

copies of S100A7, S100A7d (S100A7P1) and

S100A7e (S100A7P2), are proposed to be non-

coding pseudogenes (Kulski et al., 2003; Marenholz

et al., 2006).

The S100A7 genomic organization includes 3 exons and 2 introns with exons 2 and 3 containing the protein encoding sequence

(Semprini et al., 1999). The EF-hand domains are highlighted (Burgisser et al., 1995).

S100A7 (S100 calcium binding protein A7) Murray JI, Boulanger MJ


A. S100A7 primary sequence highlighting the calcium- and zinc-binding residues and the EF-hand domains.

B. The 3D structure of zinc- and calcium-bound S100A7 dimer (2psr).

Protein

Note

S100A7 is a member of the S100 family of

calcium-binding signaling proteins. S100A7 has

both intracellular and extracellular functions.

Description

S100A7 is a small 11.4 kDa protein containing a C-

terminal canonical calcium-binding EF-hand motif

and an N-terminal non-canonical EF-hand motif

which is characteristic of the S100 protein family.

S100A7 forms a homodimer with one Ca2+

ion

bound by the canonical EF-hand motif in each

monomer and two Zn2+

ions located at the dimer

interface (Brodersen et al., 1999). S100A7

monomers and putative higher order multimers

have been observed in both psoriatic and healthy

epidermis (Ruse et al., 2001).

Expression

S100A7 is present at low levels in healthy skin,

however it is highly upregulated in psoriatic

epidermal keratinocytes (Madsen et al., 1991). E.

Coli has been shown to induce S100A7 expression

in keratinocytes (Gläser et al., 2005).

S100A7 expression is upregulated in several

cancers including skin, breast, lung, head, neck,

cervix, bladder and gastric cancer (for review see

Emberley et al., 2004).

S100A7 expression is induced in MCF10 cells by

stresses such as serum deprivation and cell

confluency (Enerback et al., 2002).

S100A7 is repressed by BRCA1 in a c-myc

dependent manner in HCC-BR116 cells (Kennedy

et al., 2005). 17beta-estradiol treatment increased

S100A7 expression in an estrogen receptor beta

dependent manner in MCF-7 cells (Skliris et al.,

2007). Epidermal Growth Factor induces S100A7

expression in MCF-7 and MDA-MB-468 cells

(Paruchuri et al., 2008).

S100A7 expression is induced by proinflammatory

cytokines in skin and breast cancer cells. S100A7

expression is enhanced in human keratinocytes by

stimulation with the cytokine IL-22 in combination

with IL-17 or IL-17F (Liang et al., 2006).

Oncostatin-M was shown to induce S100A7

expression in human epidermal cell skin

equivalents (Gazel et al., 2006). S100A7 expression

is induced by the cytokines oncostatin-M and IL-6

in MCF-7, TD47 and MDA-MB-468 cell lines

(West and Watson, 2010).

Localisation

S100A7 is localized to the cytoplasm, nucleus, cell

periphery and is also secreted from cells.



In keratinocytes, S100A7 is observed in the

cytoplasm when untreated and at the cell periphery

upon stimulation with calcium (Ruse et al., 2003).

S100A7 is expressed at low levels or is not detected

in healthy breast cells. In breast cancer cells,

however, S100A7 is observed in the nucleus and

cytoplasm and is also secreted (Al-Haddad et al.,

1999; Enerback et al., 2002).

Function

S100A7 has been shown to function as a

chemotactic factor for neutrophils and CD4+ T

cells (Jinquan et al., 1996). S100A7 binds RAGE

(receptor for advanced glycation end products) in a

zinc-dependent manner and is proposed to mediate

chemotaxis in a RAGE-dependent manner (Wolf et

al., 2008). S100A7 present in skin functions as a

Zn-dependent antimicrobial towards E.Coli (Glaser

et al., 2005). S100A7 has also been shown to play

an antibacterial role in wound healing (Lee and

Eckert, 2007). S100A7 is a substrate for

transglutaminase (Ruse et al., 2001).

S100A7 interacts, co-purifies and colocalizes in the

cytoplasm with epidermal-type fatty acid-binding

protein (E-FABP), a protein which is also

upregulated in psoriasis (Hagens et al., 1999; Ruse

et al., 2003). S100A7 has been shown to interact

with RanBPM by yeast two-hybrid and co-

immunoprecipitation studies in breast cancer cells

(Emberley et al., 2002). S100A7 has been shown to

interact with the multifunctional signalling protein,

Jab1, yeast two-hybrid and co-immunoprecipitation

studies in breast cancer cells (Emberley et al.,

2003). The Jab1-S100A7 interaction and

downstream effects were disrupted by mutation of a

Jab1-binding site (Emberley et al., 2003; West et

al., 2009).

Homology

S100A7 is a member of the S100 family of

vertebrate proteins. Among the S100 family,

S100A7 is the most divergent (Burgisser et al.,

1995) with the exception of a recently identified

paralog S100A715 (or S100A7A), with which it

shares 93% similarity (Wolf et al., 2003). A bovine

ortholog to S100A7, Bosd3 (Virtanen, 2006) and

equine ortholog (Leeb et al., 2005) have also been

reported. The mouse S100A7, which has 40%

similarity (Webb et al., 2005), has been assigned

the designation mouse S100A15 (Wolf et al., 2006).

Mutations

Note

An allergy associated polymorphism of S100A7

(rs3014837) has been reported (Bryborn et al.,

2008).

Implicated in

Psoriasis and other skin diseases

Note

S100A7 is associated with inflammation in several

skin diseases (Algermissen et al., 1996). S100A7

was originally identified as a protein secreted from

psoriatic skin (Madsen et al., 1991). S100A7 is also

overexpressed in skin lesions of patients with lichen

sclerosus (Gambichler et al., 2009), acne inversa

(Schlapbach et al., 2009), and middle ear

cholesteatoma (Kim et al., 2009).

Non-melanoma skin cancer

Note

S100A7 may play a role in the progression of skin

cancer. S100A7 expression is not observed in

healthy epidermis. When S100A7 levels were

studied by immunohistochemistry in squamous cell

carcinoma skin lesions, higher levels of expression

were found in pre-invasive squamous cell

carcinoma in situ compared to invasive squamous

cell carcinoma (Alowami et al., 2003). In a separate

study, S100A7 mRNA levels, determined by real-

time PCR, were upregulated in pre-cancerous skin

lesions and epithelial skin tumours including basal

cell carcinoma and squamous cell carcinoma

(Moubayed et al., 2007).

Melanoma

Note

S100A7 protein was observed at higher levels in the

urine of melanoma patients compared to healthy

controls (Brouard et al., 2002), although S100A7

was not detected in melanoma cells (Petersson et

al., 2009).

Ductal carcinoma in situ (DCIS) and breast cancer

Note

S100A7 was first associated with primary breast

cancer (Moog-Lutz et al., 1995). Later studies

identified S100A7 as one of the most highly

expressed genes in DCIS, a key stage before the

transition to invasive breast cancer (Leygue et al.,

1996; Enerback et al., 2002). When S100A7 is

expressed in later stages of breast cancer it is

associated with the agressive estrogen-negative

tumors and poor prognosis (Al-Haddad et al., 1999;

Emberley et al., 2004). In vivo mouse model

studies have shown that S100A7 promotes

tumorigenesis (Emberley et al., 2003; Krop et al.,

2005). Several of the tumorigenic effects of

S100A7, including upregulation of NF-kappaB,

PI3K-Akt, and AP-1 as well as promotion of cell

survival, are mediated by the interaction of S100A7

with Jab1 (Emberley et al., 2003; Emberley et al.,

2005).



Epithelial ovarian cancer

Note

S1007 mRNA and protein levels are upregulated in

epithelial ovarian carcinoma tissue compared to

normal and benign ovary tissue (Gagnon et al.,

2008). Autoantibodies to S100A7 were detected at

higher levels in the plasma of early and late-stage

ovarian cancer patients compared to healthy

controls (Gagnon et al., 2008). S100A7

autoantibodies may be useful as a biomarker for

epithelial ovarian cancer (for review see Piura and

Piura, 2009).

Lung squamous cell carcinoma

Note

S100A7 is associated with non-small lung

squamous cell carcinoma metastasis to the brain

(Zhang et al., 2007). Proteomic studies identified

S100A7 as a protein upregulated in a brain

metastasis lung squamous cell carcinoma cell line

and S100A7 overexpression was confirmed in brain

metastasis tissues (Zhang et al., 2007).

Bladder squamous cell carcinoma

Note

S100A7 was detected in bladder squamous cell

carcinoma tumors and also in the urine of patients

with bladder squamous cell carcinoma (Celis et al.,

1996; Ostergaard et al., 1997). As a result, S100A7

has been proposed to be a potential biomarker for

bladder squamous cell carcinoma (Celis et al.,

1996; Ostergaard et al., 1997; Ostergaard et al.,

1999).

Oral squamous cell carcinoma

Note

S100A7 is associated with oral squamous cell

carcinoma (Zhou et al., 2008; Kesting et al., 2009).

RT-PCR and immunofluorescence studies showed

that S100A7 mRNA and protein levels respectively

are up-regulated in oral squamous cell carcinoma

tissues compared to normal oral tissues (Kesting et

al., 2009).

Head-and-neck squamous cell carcinoma

Note

S100A7 is a highly upregulated biomarker in head-

and-neck squamous cell carcinomas (Ralhan et al.,

2008).

Gastric cancer

Note

SAGE (serial analysis of gene expression) studies

identified S100A7 as one of the top twenty genes

upregulated in gastric cancer (El-Rifai et al., 2002).

Further mining of publicly available SAGE, virtual

Northern Blot, and microarray data confirmed the

association of S100 proteins such as S100A7 with

gastric cancer (Liu et al., 2008).

Chronic rhinosinusitis

Note

Chronic rhinosinusitis (CRS) is characterized by a

persistant inflammation of the nasal mucosa. It has

been proposed that the antibacterial function of

S100A7 play a role in protecting against the

environmental factors that contribute to chronic

rinosinusitis (for review see Tieu et al., 2009).

Reduced levels of S100A7 were detected in the

nasal lavage fluid of patients with allergic rhinitis

when compared to controls (Bryborn et al., 2005).

A polymorphism (RS3014837) has been linked

with allergic individuals in Sweden (Bryborn et al.,

2008).

Systemic sclerosis (SSc)

Note

S100A7 is upregulated in the saliva of patients with

systemic sclerosis when compared to healthy

individuals and has been proposed as a potential

biomarker for systemic sclerosis with pulmonary

involvement (Giusti et al., 2007; Baldini et al.,

2008).

Alzheimer's disease

Note

A recent study has suggested that S100A7 is a

potential biomarker for Alzheimer's disease.

Increased levels of S100A7 were detected in the

cerebralspinal fluid and brain of patients with

Alzheimer's disease (Qin et al., 2009).

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Murray JI, Boulanger MJ. S100A7 (S100 calcium binding protein A7). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):58-63.





SOX10 (SRY (sex determining region Y)-box 10) Michael Wegner

Institut fuer Biochemie, Emil-Fischer-Zentrum, Universitaet Erlangen-Nuernberg, 91054 Erlangen,

Germany (MW)


Online updated version : http://AtlasGeneticsOncology.org/Genes/SOX10ID43768ch22q13.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI SOX10ID43768ch22q13.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity

Other names: DOM, MGC15649, WS2E, WS4

HGNC (Hugo): SOX10

Location: 22q13.1

Local order: Flanked by POLR2F (DNA-directed

RNA polymerase II polypeptide F) and PICK1

(protein interacting with PRKCA 1).

DNA/RNA

Note

SOX10 was first identified as a gene mutated in

patients suffering from Waardenburg syndrome

type 4C (WS4C). SOX10 mutations also cause

Waardenburg syndrome type 2E (WS2E) with or

without neurologic involvement, Yemenite deaf-

blind hypopigmentation syndrome and PCWH

syndrome. They usually occur in the heterozygous

state and can be either sporadic or familial.

Description

DNA size: 12.22kb; 5 Exons.

Transcription

mRNA size: 2882 nucleotides.

Protein

Note

The SOX10 protein belongs to subgroup E of the

SOX protein family. All 20 human members of this

protein family possess a high-mobility-group

(HMG) domain with three alpha-helical regions and

close similarity to the one found in the male sex

determining factor SRY. SOX10 functions as

transcription factor and structural protein in

chromatin. SOX9 and SOX8 are its closest relatives

among human SOX proteins.

Description

SOX10 consists of 466 amino acids. The following

domains exist (from amino terminal to carboxy

terminal): DNA-dependent dimerization domain

(amino acids 61-101), DNA-binding HMG-domain

(amino acids 101-180), context-dependent

transactivation domain K2 (amino acids 233-306)

and main transactivation domain TA (amino acids

400-462). SOX10 possesses two nuclear

localization signals (NLS) at the beginning and the

end of the HMG domain and a nuclear export

sequence (NES in the middle).

The SOX10 gene with its 5 exons. The open reading frame (orange) is split between exons 3-5. The 5' untranslated region is

generated from exons 1-3 and the 3' untranslated region corresponds to the hind part of exon 5.

SOX10 (SRY (sex determining region Y)-box 10) Wegner M


Human SOX10 and its domains including the DNA-dependent dimerization domain (Dim), the DNA-binding HMG domain, the

context-dependent transactivation domain K2 and the main transactivation domain (TA). Numbers indicate amino acid positions. The bottom shows the exact amino acid sequence of the HMG domain with its three alpha-helices, the 2 nuclear localization

signals (NLS1, NLS2) and the nuclear export sequence (NES).

Expression

SOX10 expression is first detected during

embryonic development in the emerging neural

crest and continues transiently or permanently in

many non-ectomesenchymal derivates of the neural

crest including melanocytes, adrenal medulla and

the developing peripheral nervous system. Within

the developing central nervous system SOX10

marks cells of the oligodendrocyte lineage. In the

adult, SOX10 is predominantly found in

oligodendrocytes, peripheral glial cells,

melanocytes and adult neural crest stem cell

populations.

Localisation

SOX10 is predominantly found in the nucleus as

expected for a transcription factor but possesses the

ability to shuttle between cytoplasm and nucleus

because of the presence of both NLS and NES in

the protein.

Function

SOX10 has multiple roles during development. In

neural crest stem cells, SOX10 is needed for self-

renewal, survival and maintenance of pluripotency.

SOX10 is furthermore required for specification of

melanocytes and peripheral glia from the neural

crest. After specification, SOX10 continues to be

essential for lineage progression and maintenance

of identity in peripheral glia. Terminal

differentiation of oligodendrocytes also depends on

SOX10. SOX10 exerts these functions through

interactions with different sets of transcription

factors. SOX10 probably shares further roles with

its close relatives SOX9 and SOX8 with which it is

co-expressed in several cell types and functions in a

partly redundant manner.

Homology

SOX10 is highly conserved among vertebrates.

Human SOX10 shares 98% identity with Mus

musculus Sox10, 97% identity with Sox10 from

Rattus norvegicus and Canis lupus familiaris, 96%

identity with Bos taurus Sox10 and 82% identity

with Gallus gallus Sox10.

Mutations

Note

SOX10 mutations have so far primarily been

identified as a cause for neurocristopathies

including WS4C, WS2E with or without neurologic

involvement, PCWH syndrome and Yemenite deaf-

bling hypopigmentation syndrome.

Germinal

Missense mutations: S135T, A157V, Q174P.

Nonsense mutations: R43X, T83X, T173X,

E189X, T207X, Q234X, Q250X, S251X, T313X,

S346X, Q364X, Q372X, S376X, Q377X.

Insertions: (L160 R161) dup.

Carboxy terminal extensions: X467C ext82,

X467L ext86, X467T ext86.

Frameshift mutations: S17C fsX17, E57S fsX57,

A110L fsX2, P169R fsX117, R215P fsX64, R261A

fsX25, G266A fsX20, I271S fsX15, H283L fsX11,

H306T fsX5, G308A fsX3, V350C fsX52, A354P

fsX3, E359D fsX42, Q399V fsX2.

Splice mutations: int3 pos.428 +2T>G, int4

pos.698 -2A>C.

Somatic

Missense mutations: R43Q, Q125X, A361V,

G413S, G413D, H414Y, A424V.

Frameshift mutations: P14P fsX10, S449S fsX66.

Implicated in

Melanoma

Note

SOX10 is expressed homogenously in primary and

metastatic melanoma and was identified as a

melanoma tumor antigen. It is often co-expressed

with its relative SOX9. Somatic SOX10 mutations

occur in early stage melanoma. SOX10 upregulates

MITF, MET and Nestin expression in melanoma

and responds to Wnt signals. Its nuclear localization

is controlled by the Tam tyrosine receptor kinase

Tyro3 and its activity is modulated by the

transcription factor SOX5. In sentinel lymph nodes,

SOX10 is a reliable marker for metastatic

melanoma.

SOX10 (SRY (sex determining region Y)-box 10) Wegner M


Clear cell sarcoma

Note

SOX10 is widely expressed in clear cell sarcoma

where it cooperates with EWS-ATF1 fusions in

MITF activation.

Malignant nerve sheath tumor (MNST)

Note

SOX10 is present in MNST. Expression levels

appear lower than the ones in plexiform

neurofibromas from which MNST arise or in

Schwann cells. SOX10 expression levels are

positively correlated with ErbB3 levels and

inversely correlated with SOX9 levels.

Schwannoma

Note

Homogenous SOX10 expression has been detected

throughout this neoplasm.

Ganglioneuroma

Note

SOX10 expression has been detected. Levels

decrease with increasing grade.

Glioma

Note

SOX10 transcripts and protein were found in

astrocytoma, oligodendroglioma and glioblastoma.

Expression levels and number of expressing cells

within the tumor usually diminish with advancing

grade and malignant progression. SOX10 levels are

particularly high in pilocytic astrocytoma. No

correlation with 1p and 19q deletions has been

detected. In a mouse model, SOX10 has been found

to act synergistically with PDGF during glioma

development, although it was not sufficient to

induce gliomagenesis on its own.

References Pingault V, Bondurand N, Kuhlbrodt K, Goerich DE, Préhu MO, Puliti A, Herbarth B, Hermans-Borgmeyer I, Legius E, Matthijs G, Amiel J, Lyonnet S, Ceccherini I, Romeo G, Smith JC, Read AP, Wegner M, Goossens M. SOX10 mutations in patients with Waardenburg-Hirschsprung disease. Nat Genet. 1998 Feb;18(2):171-3

Khong HT, Rosenberg SA. The Waardenburg syndrome type 4 gene, SOX10, is a novel tumor-associated antigen identified in a patient with a dramatic response to immunotherapy. Cancer Res. 2002 Jun 1;62(11):3020-3

Mollaaghababa R, Pavan WJ. The importance of having your SOX on: role of SOX10 in the development of neural crest-derived melanocytes and glia. Oncogene. 2003 May 19;22(20):3024-34

Inoue K, Khajavi M, Ohyama T, Hirabayashi S, Wilson J, Reggin JD, Mancias P, Butler IJ, Wilkinson MF, Wegner M,

Lupski JR. Molecular mechanism for distinct neurological phenotypes conveyed by allelic truncating mutations. Nat Genet. 2004 Apr;36(4):361-9

Gershon TR, Oppenheimer O, Chin SS, Gerald WL. Temporally regulated neural crest transcription factors distinguish neuroectodermal tumors of varying malignancy and differentiation. Neoplasia. 2005 Jun;7(6):575-84

Wegner M, Stolt CC. From stem cells to neurons and glia: a Soxist's view of neural development. Trends Neurosci. 2005 Nov;28(11):583-8

Addo-Yobo SO, Straessle J, Anwar A, Donson AM, Kleinschmidt-Demasters BK, Foreman NK. Paired overexpression of ErbB3 and Sox10 in pilocytic astrocytoma. J Neuropathol Exp Neurol. 2006 Aug;65(8):769-75

Bannykh SI, Stolt CC, Kim J, Perry A, Wegner M. Oligodendroglial-specific transcriptional factor SOX10 is ubiquitously expressed in human gliomas. J Neurooncol. 2006 Jan;76(2):115-27

Davis IJ, Kim JJ, Ozsolak F, Widlund HR, Rozenblatt-Rosen O, Granter SR, Du J, Fletcher JA, Denny CT, Lessnick SL, Linehan WM, Kung AL, Fisher DE. Oncogenic MITF dysregulation in clear cell sarcoma: defining the MiT family of human cancers. Cancer Cell. 2006 Jun;9(6):473-84

Kelsh RN. Sorting out Sox10 functions in neural crest development. Bioessays. 2006 Aug;28(8):788-98

Ferletta M, Uhrbom L, Olofsson T, Pontén F, Westermark B. Sox10 has a broad expression pattern in gliomas and enhances platelet-derived growth factor-B--induced gliomagenesis. Mol Cancer Res. 2007 Sep;5(9):891-7

Nonaka D, Chiriboga L, Rubin BP. Sox10: a pan-schwannian and melanocytic marker. Am J Surg Pathol. 2008 Sep;32(9):1291-8

Blochin E, Nonaka D. Diagnostic value of Sox10 immunohistochemical staining for the detection of metastatic melanoma in sentinel lymph nodes. Histopathology. 2009 Nov;55(5):626-8

Cronin JC, Wunderlich J, Loftus SK, Prickett TD, Wei X, Ridd K, Vemula S, Burrell AS, Agrawal NS, Lin JC, Banister CE, Buckhaults P, Rosenberg SA, Bastian BC, Pavan WJ, Samuels Y. Frequent mutations in the MITF pathway in melanoma. Pigment Cell Melanoma Res. 2009 Aug;22(4):435-44

Zhu S, Wurdak H, Wang Y, Galkin A, Tao H, Li J, Lyssiotis CA, Yan F, Tu BP, Miraglia L, Walker J, Sun F, Orth A, Schultz PG, Wu X. A genomic screen identifies TYRO3 as a MITF regulator in melanoma. Proc Natl Acad Sci U S A. 2009 Oct 6;106(40):17025-30

Mascarenhas JB, Littlejohn EL, Wolsky RJ, Young KP, Nelson M, Salgia R, Lang D. PAX3 and SOX10 activate MET receptor expression in melanoma. Pigment Cell Melanoma Res. 2010 Apr;23(2):225-37


Wegner M. SOX10 (SRY (sex determining region Y)-box 10). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):64-66.

Gene Section Review




TNFSF18 (tumor necrosis factor (ligand) superfamily, member 18) Theresa Placke, Hans-Georg Kopp, Benjamin Joachim Schmiedel, Helmut Rainer Salih

Eberhard Karls University of Tuebingen, Department of Hematology/Oncology, Otfried-Mueller-Str.

10, 72076 Tuebingen, Germany (TP, HGK, BJS, HRS)


Online updated version : http://AtlasGeneticsOncology.org/Genes/TNFSF18ID42639ch1q25.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI TNFSF18ID42639ch1q25.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity

Other names: AITRL, GITRL, MGC138237, TL6

HGNC (Hugo): TNFSF18

Location: 1q25.1

DNA/RNA

Description

2 transcript versions published:

mRNA 748 bp: 3 exons (1-223, 224-254, 255-748)

-> coding for 199 aa (2-601),

mRNA 610 bp: 3 exons (1-176, 177-207, 208-610)

-> coding for 177 aa (21-554).

Transcription

Accurate start codon is not clearly defined, 2

transcript versions are published (differing start

codons in exon 1).

Pseudogene

Unknown.

Protein

Description

TNFSF18/GITR ligand (GITRL) is a single-pass

type II transmembrane protein and contains 2

potential glycosylation sites (predicted at 129 aa

and 161 aa). TNFSF18 encompasses 177 or 199 aa

and thus has a molecular weight of about 20 kDa.

In the 177 aa long version, amino acids 1-28

constitute the cytoplasmic domain, 29-49 the

transmembrane domain, and 50-177 the

extracellular domain, whereas in the 199 aa long

variant the amino acids 1-50 constitute the

cytoplasmic domain, 51-71 the transmembrane

domain, and 72-199 the extracellular domain.

Expression

TNFSF18 is expressed on DC, monocytes,

macrophages, B cells, activated T cells, endothelial

cells, osteoclasts and various healthy non-lymphoid

tissues (e.g., testis, ...).

Figure 1. Schematic illustration of the gene structure of human TNFSF18 on chromosome 1. Both published transcript variants are shown. Red boxes represent the mRNA transcript within the gene. The smaller boxes at the beginning and the end of the

transcripts indicate untranslated regions, while the larger boxes display the translated parts.

TNFSF18 (tumor necrosis factor (ligand) superfamily, member 18)

Placke T, et al.


Figure 2. Schematic illustration of the structure of TNFSF18 protein versions, according to the two published transcripts.

The fact that TNFSF18 is constitutively expressed

on resting antigen-presenting cells distinguishes it

from most other TNF family members, which are

not detectable in resting state and are upregulated

following activation.

In addition, TNFSF18 is constitutively expressed

and released as soluble form by solid tumors of

different histological origin and various

hematopoietic malignancies.

Localisation

TNFSF18 is a type II transmembrane protein. A

soluble form of the molecule has been shown to be

released by a yet unknown mechanism e.g. by

tumor cells.

Function

TNFSF18 is the only known ligand for GITR

(TNFRSF18, AITR), which is mainly expressed by

lymphatic cells like T lymphocytes and NK cells.

Upon interaction with its receptor, TNFSF18 is,

like many other TNF family members, capable to

transduce bidirectional signals, i.e. in the receptor

and the ligand bearing cell. Transduction of signals

into TNFSF18 bearing cells has been shown to

cause differentiation of osteoclasts, to activate

macrophages and to alter cytokine production of

healthy myeloid cells, but also of carcinoma and

leukemia cells and influences apoptosis. Activation

of macrophages via TNFSF18 results in increased

secretion of inflammatory mediators like MMP-9,

NO and TNF. In healthy macrophages and myeloid

leukemia cells, TNFSF18 signaling has been found

to involve the MAP kinase pathway.

Binding to TNFRSF18 may induce signaling

through this receptor, which, in mice, has been

implicated in the development of autoimmune

diseases, graft versus host disease and in the

immune response against infectious pathogens and

tumors.

Available data suggest that TNFRSF18 may

mediate different effects in mice and men, and most

functional studies regarding the role of TNFRSF18

in tumor immunology have been performed using

agonistic antibodies or injection of adenovirus

expressing recombinant TNFSF18 into tumors,

which might not reflect the consequences of

TNFRSF18 interaction with its natural ligand in

vivo. In line, studies evaluating immune responses

in GITR-/-

mice have so far not led to a clear picture

of the role of TNFRSF18 in normal physiology.

Homology

The TNFSF18 gene is conserved in human,

chimpanzee, dog, mouse, and rat. The homology

among the other TNF family members is highest

with OX40L.

Mutations

Note

No published single nucleotide polymorphisms

(SNPs).

Implicated in

Host-tumor interaction

Note

In mice, it has been shown that application of the

agonistic GITR antibody DTA-1 delays tumor

progression and can even lead to complete tumor

rejection. Similar results were obtained by using

GITRL-Fc fusion protein. Transfection of tumor

cells with GITRL causes rejection of the tumor and

prolonged survival, while parental cells cannot be

rejected. This effect can be reversed by

administration of a blocking GITRL antibody.

There is evidence that expression of GITRL

promotes the development of tumor-specific T

cells. Re-challenge of mice which once successfully

rejected GITRL-positive tumor results in complete

rejection of both transfected and non-transfected

tumors. Several studies showed increased

infiltration of CD8+ cells in GITRL-expressing

tumors. By the use of depletion experiments and

athymic nude mice it has been shown that for

GITR-GITRL dependent rejection of tumors both

CD4+ and CD8+ T cells as well as NK cells are

required.


Placke T, et al.


In humans, controversial data regarding the

function of GITR and GITRL in tumor

immunology were described. Hanabuchi et al.

reported that NK cells are activated by engagement

of GITRL on plasmacytoid dendritic cells, which

can be blocked by anti-GITRL antibody. In

contrast, Baltz et al. and Baessler et al.

demonstrated substantial GITRL expression on

tumor cells and leukemic blasts resulting in

diminished NK cell reactivity. Blockade of GITR-

GITRL interaction by anti-GITR antibody

abrogated the inhibitory effect of GITRL.

Furthermore, stimulation of GITRL substantially

induced the production of TGF-beta and IL-10 by

tumor cells and leukemic blasts. Additionally, they

reported that human GITRL is released by tumor

cells in a soluble form which impairs NK cell

reactivity alike the membrane-bound form. Thus,

GITRL expression seems to affect the interaction of

human tumor cells with the immune system by

influencing tumor cell immunogenity and

metastasis and creating an immunosuppressive

cytokine microenvironment. The inhibitory effect

of GITRL on human NK cells was further

supported by Liu et al., who reported inhibition of

NK cell proliferation and cytokine production and

increased apoptosis after GITR stimulation. These

controversial data regarding the function of GITR

on human NK cells may be due to the usage of

different reagents and different experimental

condition.

The results regarding the role of GITR and GITRL

in tumor immunology are controversial in mice and

humans. Thus, GITR and GITRL may mediate

different effects in mice and men, and in line

suppression of human regulatory T cells, in contrast

to their murine counterparts, is not inhibited by

GITR. Many studies employed agonistic antibodies

or recombinant protein for GITR stimulation and

not constitutively GITRL-expressing cells. Thus,

these studies do not involve possible influences of

reverse signaling mediated by GITRL, which may

change reaction of GITRL-bearing cells and may in

turn alter functions of GITR-bearing cells.

Autoimmune disease

Note

The influence of GITR and GITRL was tested in

different mouse models of autoimmune disease.

Onset of autoimmune diabetes in NOD mice is

accelerated if they are treated with agonistic GITR

mAb, and activation of CD4+ T cells is increased

compared to control treated mice. Likewise,

application of a blocking GITRL antibody protected

from diabetes. In GITR -/- mice, experimental

autoimmune diseases take an attenuated course.

GITR -/- mice with collagen-induced arthritis show

less joint inflammation and bone erosion than

wildtype mice. Furthermore, lower concentrations

of inflammatory mediators were reported. In line

with these findings, GITR triggering antibody

exacerbates collagen-induced arthritis in wildtype

mice compared to control-treated siblings.

However, all these studies regarding the function of

GITR and its ligand in autoimmune disease were

performed in mice. Further investigation is needed

to elucidate the relevance of GITR and GITR ligand

in human autoimmune disease and to clarify the

similarities and differences of these molecules in

mice and men.

References Nocentini G, Giunchi L, Ronchetti S, Krausz LT, Bartoli A, Moraca R, Migliorati G, Riccardi C. A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis. Proc Natl Acad Sci U S A. 1997 Jun 10;94(12):6216-21

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Shimizu J, Yamazaki S, Takahashi T, Ishida Y, Sakaguchi S. Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol. 2002 Feb;3(2):135-42

Shin HH, Lee MH, Kim SG, Lee YH, Kwon BS, Choi HS. Recombinant glucocorticoid induced tumor necrosis factor receptor (rGITR) induces NOS in murine macrophage. FEBS Lett. 2002 Mar 13;514(2-3):275-80

Spinicelli S, Nocentini G, Ronchetti S, Krausz LT, Bianchini R, Riccardi C. GITR interacts with the pro-apoptotic protein Siva and induces apoptosis. Cell Death Differ. 2002 Dec;9(12):1382-4

Lee HS, Shin HH, Kwon BS, Choi HS. Soluble glucocorticoid-induced tumor necrosis factor receptor (sGITR) increased MMP-9 activity in murine macrophage. J Cell Biochem. 2003 Apr 1;88(5):1048-56

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Muriglan SJ, Ramirez-Montagut T, Alpdogan O, Van Huystee TW, Eng JM, Hubbard VM, Kochman AA, Tjoe KH, Riccardi C, Pandolfi PP, Sakaguchi S, Houghton AN, Van Den Brink MR. GITR activation induces an opposite effect on alloreactive CD4(+) and CD8(+) T cells in graft-versus-host disease. J Exp Med. 2004 Jul 19;200(2):149-57


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Ronchetti S, Zollo O, Bruscoli S, Agostini M, Bianchini R, Nocentini G, Ayroldi E, Riccardi C. GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations. Eur J Immunol. 2004 Mar;34(3):613-22

Shin HH, Kim SJ, Lee HS, Choi HS. The soluble glucocorticoid-induced tumor necrosis factor receptor causes cell cycle arrest and apoptosis in murine macrophages. Biochem Biophys Res Commun. 2004 Mar 26;316(1):24-32

Calmels B, Paul S, Futin N, Ledoux C, Stoeckel F, Acres B. Bypassing tumor-associated immune suppression with recombinant adenovirus constructs expressing membrane bound or secreted GITR-L. Cancer Gene Ther. 2005 Feb;12(2):198-205

Esparza EM, Arch RH. Glucocorticoid-induced TNF receptor functions as a costimulatory receptor that promotes survival in early phases of T cell activation. J Immunol. 2005 Jun 15;174(12):7869-74

Esparza EM, Arch RH. Glucocorticoid-induced TNF receptor, a costimulatory receptor on naive and activated T cells, uses TNF receptor-associated factor 2 in a novel fashion as an inhibitor of NF-kappa B activation. J Immunol. 2005 Jun 15;174(12):7875-82

Ko K, Yamazaki S, Nakamura K, Nishioka T, Hirota K, Yamaguchi T, Shimizu J, Nomura T, Chiba T, Sakaguchi S. Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on tumor-infiltrating Foxp3+CD25+CD4+ regulatory T cells. J Exp Med. 2005 Oct 3;202(7):885-91

Patel M, Xu D, Kewin P, Choo-Kang B, McSharry C, Thomson NC, Liew FY. Glucocorticoid-induced TNFR family-related protein (GITR) activation exacerbates murine asthma and collagen-induced arthritis. Eur J Immunol. 2005 Dec;35(12):3581-90

Watts TH. TNF/TNFR family members in costimulation of T cell responses. Annu Rev Immunol. 2005;23:23-68

Baumgartner-Nielsen J, Vestergaard C, Thestrup-Pedersen K, Deleuran M, Deleuran B. Glucocorticoid-induced tumour necrosis factor receptor (GITR) and its ligand (GITRL) in atopic dermatitis. Acta Derm Venereol. 2006;86(5):393-8

Cohen AD, Diab A, Perales MA, Wolchok JD, Rizzuto G, Merghoub T, Huggins D, Liu C, Turk MJ, Restifo NP, Sakaguchi S, Houghton AN. Agonist anti-GITR antibody enhances vaccine-induced CD8(+) T-cell responses and tumor immunity. Cancer Res. 2006 May 1;66(9):4904-12

Cuzzocrea S, Nocentini G, Di Paola R, Agostini M, Mazzon E, Ronchetti S, Crisafulli C, Esposito E, Caputi AP, Riccardi C. Proinflammatory role of glucocorticoid-induced TNF receptor-related gene in acute lung inflammation. J Immunol. 2006 Jul 1;177(1):631-41

Hanabuchi S, Watanabe N, Wang YH, Wang YH, Ito T, Shaw J, Cao W, Qin FX, Liu YJ. Human plasmacytoid predendritic cells activate NK cells through glucocorticoid-induced tumor necrosis factor receptor-ligand (GITRL). Blood. 2006 May 1;107(9):3617-23

Kim J, Choi WS, Kim HJ, Kwon B. Prevention of chronic graft-versus-host disease by stimulation with glucocorticoid-induced TNF receptor. Exp Mol Med. 2006 Feb 28;38(1):94-9

Mahesh SP, Li Z, Liu B, Fariss RN, Nussenblatt RB. Expression of GITR ligand abrogates immunosuppressive function of ocular tissue and differentially modulates inflammatory cytokines and chemokines. Eur J Immunol. 2006 Aug;36(8):2128-38

Ramirez-Montagut T, Chow A, Hirschhorn-Cymerman D, Terwey TH, Kochman AA, Lu S, Miles RC, Sakaguchi S, Houghton AN, van den Brink MR. Glucocorticoid-induced TNF receptor family related gene activation overcomes tolerance/ignorance to melanoma differentiation antigens and enhances antitumor immunity. J Immunol. 2006 Jun 1;176(11):6434-42

Baltz KM, Krusch M, Bringmann A, Brossart P, Mayer F, Kloss M, Baessler T, Kumbier I, Peterfi A, Kupka S, Kroeber S, Menzel D, Radsak MP, Rammensee HG, Salih HR. Cancer immunoediting by GITR (glucocorticoid-induced TNF-related protein) ligand in humans: NK cell/tumor cell interactions. FASEB J. 2007 Aug;21(10):2442-54

Cuzzocrea S, Ronchetti S, Genovese T, Mazzon E, Agostini M, Di Paola R, Esposito E, Muià C, Nocentini G, Riccardi C. Genetic and pharmacological inhibition of GITR-GITRL interaction reduces chronic lung injury induced by bleomycin instillation. FASEB J. 2007 Jan;21(1):117-29

Grohmann U, Volpi C, Fallarino F, Bozza S, Bianchi R, Vacca C, Orabona C, Belladonna ML, Ayroldi E, Nocentini G, Boon L, Bistoni F, Fioretti MC, Romani L, Riccardi C, Puccetti P. Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy. Nat Med. 2007 May;13(5):579-86

Ronchetti S, Nocentini G, Bianchini R, Krausz LT, Migliorati G, Riccardi C. Glucocorticoid-induced TNFR-related protein lowers the threshold of CD28 costimulation in CD8+ T cells. J Immunol. 2007 Nov 1;179(9):5916-26

Zhou P, L'italien L, Hodges D, Schebye XM. Pivotal roles of CD4+ effector T cells in mediating agonistic anti-GITR mAb-induced-immune activation and tumor immunity in CT26 tumors. J Immunol. 2007 Dec 1;179(11):7365-75

Bae EM, Kim WJ, Suk K, Kang YM, Park JE, Kim WY, Choi EM, Choi BK, Kwon BS, Lee WH. Reverse signaling initiated from GITRL induces NF-kappaB activation through ERK in the inflammatory activation of macrophages. Mol Immunol. 2008 Jan;45(2):523-33

Baltz KM, Krusch M, Baessler T, Schmiedel BJ, Bringmann A, Brossart P, Salih HR. Neutralization of tumor-derived soluble glucocorticoid-induced TNFR-related protein ligand increases NK cell anti-tumor reactivity. Blood. 2008 Nov 1;112(9):3735-43

Igarashi H, Cao Y, Iwai H, Piao J, Kamimura Y, Hashiguchi M, Amagasa T, Azuma M. GITR ligand-costimulation activates effector and regulatory functions of CD4+ T cells. Biochem Biophys Res Commun. 2008 May 16;369(4):1134-8

Liu B, Li Z, Mahesh SP, Pantanelli S, Hwang FS, Siu WO, Nussenblatt RB. Glucocorticoid-induced tumor necrosis factor receptor negatively regulates activation of human primary natural killer (NK) cells by blocking proliferative signals and increasing NK cell apoptosis. J Biol Chem. 2008 Mar 28;283(13):8202-10

Nishikawa H, Kato T, Hirayama M, Orito Y, Sato E, Harada N, Gnjatic S, Old LJ, Shiku H. Regulatory T cell-resistant CD8+ T cells induced by glucocorticoid-induced tumor necrosis factor receptor signaling. Cancer Res. 2008 Jul 15;68(14):5948-54

Baessler T, Krusch M, Schmiedel BJ, Kloss M, Baltz KM, Wacker A, Schmetzer HM, Salih HR. Glucocorticoid-induced tumor necrosis factor receptor-related protein ligand subverts immunosurveillance of acute myeloid leukemia in humans. Cancer Res. 2009 Feb 1;69(3):1037-45

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cell dependent anti-tumor immunity. Cancer Immunol Immunother. 2009 Jul;58(7):1057-69

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Piao J, Kamimura Y, Iwai H, Cao Y, Kikuchi K, Hashiguchi M, Masunaga T, Jiang H, Tamura K, Sakaguchi S, Azuma M. Enhancement of T-cell-mediated anti-tumour immunity via the ectopically expressed glucocorticoid-induced tumour necrosis factor receptor-related receptor ligand (GITRL) on tumours. Immunology. 2009 Aug;127(4):489-99

Vecchiarelli A, Pericolini E, Gabrielli E, Agostini M, Bistoni F, Nocentini G, Cenci E, Riccardi C. The GITRL-GITR system alters TLR-4 expression on DC during fungal infection. Cell Immunol. 2009;257(1-2):13-22

You S, Poulton L, Cobbold S, Liu CP, Rosenzweig M, Ringler D, Lee WH, Segovia B, Bach JF, Waldmann H, Chatenoud L. Key role of the GITR/GITRLigand pathway in the development of murine autoimmune diabetes: a potential therapeutic target. PLoS One. 2009 Nov 20;4(11):e7848

Gonçalves-Sousa N, Ribot JC, deBarros A, Correia DV, Caramalho I, Silva-Santos B. Inhibition of murine gammadelta lymphocyte expansion and effector function by regulatory alphabeta T cells is cell-contact-dependent and sensitive to GITR modulation. Eur J Immunol. 2010 Jan;40(1):61-70


Placke T, Kopp HG, Schmiedel BJ, Salih HR. TNFSF18 (tumor necrosis factor (ligand) superfamily, member 18). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):67-71.

Gene Section Review




USF1 (upstream transcription factor 1) Adrie JM Verhoeven

Cardiovascular Research School (COEUR), Department of Biochemistry, Erasmus MC, Rotterdam,

Netherlands (AJMV)


Online updated version : http://AtlasGeneticsOncology.org/Genes/USF1ID45856ch1q23.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI USF1ID45856ch1q23.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Other names: bHLHb11, FCHL, FCHL1,

HYPLIP1, MLTF, MLTF1, UEF

HGNC (Hugo): USF1

Location: 1q23.3

Local order:

From centromere to telomere:

F11R (F11 receptor) (on reverse strand), TSTD1

(thiosulfate sulfurtransferase (rhodanese)-like

domain containing 1) (on reverse strand), USF1

(upstream transcription factor 1) (on reverse

strand), ARHGAP30 (Rho GTPase activating

protein 30) (on reverse strand), PVRL4 (poliovirus

receptor-related 4) (on reverse strand), KLHDC9

(kelch domain containing 9) (on plus strand),

PFDN2 (prefoldin subunit 2) (on reverse strand).

Note

USF1 is a bHLH-ZIP transcription factor which

forms homo-dimers or heterodimers with USF2, a

highly homologous bHLH-ZIP transcription factor.

USF1 and USF2 homo- and heterodimers are

similarly active in affecting transcription of most

target genes. USF2 homodimers may have

additional effects.

DNA/RNA

Description

The human USF1 gene on chromosome 1q23 spans

6.73 kb and 11 exons.

Transcription

The mRNA is about 1870 nt. Translation is from a

start codon in exon 2 and ends at a stop codon in

exon 11, and results in a 310 amino acid protein

product. In a splice variant, an alternative donor

splice site within exon 4 is used; translation from

this variant mRNA is from an in-frame start codon

in exon 5, and results in a 251 amino acid protein

product (Saito et al., 2003).

Human USF1 gene diagram. Exons 1 through 11 are depicted by boxes, the open reading frames of the USF1 protein and the

splice variant are shown by dark and light green colour code, respectively. The approximate positions of two functional SNPs are also indicated.

USF1 (upstream transcription factor 1) Verhoeven AJM


Functional domains of the USF1 protein. The A1 domain is important for E-box dependent transactivation, the USR (USF-

specific region) and A2 domains are important for E-box and initiator element (Inr)-dependent transactivation (Roy et al., 1997). Post-translational modifications that affect USF1 function are indicated. The protein product of the splice variant lacks the first 59

amino acids, dimerizes with full-length USF1 protein, which results in its inactivation (Saito et al., 2003).

Protein

Description

USF1 belongs to the bHLH-Zip class of

transcription factors. The bHLH-ZIP domains are

important for DNA binding and dimerization. USF

homo- and heterodimers activate transcription of

target genes through binding either at distal E-box

elements or at pyrimidine-rich Inr elements in the

core promoter (Roy et al., 1997). Whole genome

ChIP-chip analysis in human hepatoma HepG2

cells showed that USF1 and USF2 bind

predominantly to CACGTGAC elements (Rada-

Iglesias et al., 2008). In addition, USF2 but not

USF1 binds to pyrimidine rich elements, suggesting

that transactivation through Inr elements is mainly

through USF2. Transactivation activity critically

depends on post-translational modification of

USF1. DNA binding to the E-box element is

increased by phosphorylation of USF1 by the cdk1,

p38 stress-activated kinase, protein kinase A and

protein kinase C pathway (Corre and Galibert,

2005), whereas phosphorylation through the

PI3Kinase pathway leads to loss of DNA binding

activity to the ApoAV promoter (Nowak et al.,

2005). Cellular stress stimuli such as DNA damage,

oxidative stress and heavy metal exposure, induce

p38-mediated phosphorylation at T153

and increased

USF1 transactivation activity. Upon increased

and/or prolonged stress exposure, USF1

phosphorylated at T153

becomes acetylated at K199

with concomitant loss of transactivation activity

(Corre et al., 2009). In fasting-refeeding cycles,

insulin increases the transactivation activity of

USF1 via DNA-PK mediated phosphorylation of

residue S262

and subsequent acetylation at K237

(Wong et al., 2009).

Expression

The USF1 gene is ubiquitously expressed (Sirito et

al., 1994).

Localisation

The USF1 protein is located in the nucleus.

Function

USF1 has been shown to play an important role in

transcriptional regulation of a huge number of

seemingly unrelated genes (Corre and Galibert,

2005; Rada-Iglesias et al., 2008), consistent with

the abundant distribution of E-box like elements in

the genome. Whole-genome ChIP analysis in

HepG2 cells identified 2518 USF1 binding sites in

chromatin context, of which 41 % were located

within 1 kb of a transcription start site (Rade-

Iglesias et al., 2008). USF1 binding signals strongly

correlate with target gene expression levels,

suggesting that USF1 plays an important role in

transcription activation. USF1 physically interacts

with histone modifying enzymes, transcription

preinitiation complex factors, coactivator and

corepressor proteins (Corre and Galibert, 2005;

Huang et al., 2007; Corre et al., 2009; Wong et al.,

2009). In addition, USF1 interacts with other

transcription factors to achieve cooperative

transcriptional activation of individual genes (Corre

and Galibert, 2005). USF1 also plays a crucial role

in chromatin barrier insulator function, in which

euchromatin regions are protected from

heterochromatin-induced gene silencing (Huang et

al., 2007). USFs recruit histone modifying enzymes

to the insulator element, which modify the adjacent

nucleosomes thereby maintaining chromatin in an

open state and preventing heterochromatin spread.

Similarly, USFs main function at enhancer

elements may be to render the adjacent region

accessible for binding of other, bona fide

transcription factors, by the recruitment of histone

modifying enzymes (Huang et al., 2007).

Tumor suppression: Several lines of evidence

support the hypothesis that USF1 may act as a

tumor suppressor. First, USF1 is involved in the

transcriptional activation of several tumor

suppressor genes (e.g. p53, APC, BRCA2, PTEN,

SSeCKS) (Corre and Galibert, 2005; Pezzolesi et

al., 2007; Bu and Gelman, 2007), and represses

expression of human telomerase reverse

transcriptase TERT (McMurray and McCance,

2003; Chang et al., 2005). Second, USF1 is

involved in cell cycle control (Cogswell et al.,

1995) and overexpression of USF1 slows G2/M

transition in thyrocytes and thyroid carcinoma cells

(Jung et al., 2007). Third, USF1 overexpression

leads to a strong reduction in cell proliferation in

Ha-Ras/c-Myc transformed fibroblasts (Luo and

Sawadogo, 1996). Fourth, USF1 transactivation

activity is completely lost in three out of six

transformed breast cell lines (Ismail et al., 1999).

Fifth, USF1 antagonizes some activities of the

oncoprotein c-Myc, possibly by competing for the

same DNA binding sites (Luo and Sawadogo, 1996;

McMurray and McCance, 2003). Definitive proof



that USF1 is a tumor suppressor protein, e.g.

showing that USF1 knockdown increases cell

proliferation and tumor formation, however, is still

missing. This proof may be hard to gain, as USF2

may compensate for USF1 loss, and USF2 appears

to have a broader antiproliferative function than

USF1 (Luo and Sadawogo, 1996; Sirito et al., 1998;

Vallet et al., 1998).

Homology

The USF1 gene is widely conserved with orthologs

identified in Ciona intestinalis and Drosophila

melanogaster.

Mutations

Note

Of the 121 SNPs in the USF1 gene collected in the

dbSNP database, only the rs4126997 T>C

polymorphism causes a non-synchronous mutation

(V15

A missense), but data on allele frequency or

functional effects are not available. The two SNPs

that are shown to be functional, rs2073658 A>G in

intron 7 (heterozygosity 0.296) and rs3737787 C>T

in the 3'-UTR (heterozygosity 0.309), are in almost

complete linkage disequilibrium. The minor allele

is accompanied by normal USF1 expression in

human muscle and fat tissue but loss of insulin-

induced upregulation of USF1 mRNA and known

USF1 target genes (Naukkarinen et al., 2005;

Naukkarinen et al., 2009), as well as reduced

insulin-mediated anti-lipolytic activity (Kantartzis

et al., 2007).

Implicated in

Carcinogenesis

Note

Given the suggestive evidence for a role of USF1 in

tumor suppression, one may anticipate that

carcinogenesis will evolve from loss of USF1

transactivation activity, either as a result of

mutations in the USF1 gene or of posttranslational

modification of USF1 protein. This has not been

reported yet. Alternatively, tumor suppressor genes

may lose responsivity to USF1 by mutations in the

DNA binding element or by changes in local DNA

methylation. This is exemplified by the observation

of a classic Cowden syndrome patient with early

onset breast cancer and reduced PTEN activity,

which appears to be due to a specific germline

mutation of an E-box element in the PTEN gene

and loss of USF1 binding (Pezzolesi et al., 2007).

Familial combined hyperlipidemia (FCHL)

Disease

FCHL is the most common genetic form of

hyperlipidemia and is associated with increased risk

of premature cardiovascular disease. Affected

persons characteristically show elevation of both

cholesterol and triglycerides in the blood, which is

due to increased VLDL and LDL levels. This is

often accompanied by elevated apoB100 and low

HDL levels, and a preponderance of small dense

LDL particles (Naukkarinen et al., 2006). FCHL is

genetically heterogeneous. One of the loci that is

linked to FCHL is 1q21-q23. Pajukanta et al. (2004)

showed that the dyslipidemia observed in FCHL is

linked to the USF1 gene. The disease is associated

with a common haplotype of non-coding SNPs

within the USF1 gene. Carriers of the risk allele

show lack of insulin-induced increase of USF1

expression in skeletal muscle and fat tissue

(Naukkarinen et al., 2009). As USF1 is involved in

regulation of numerous genes of glucose and lipid

metabolism (Corre and Galibert, 2005), non-

responsive USF1 expression may lead to increased

production and reduced metabolism of plasma

lipids and lipoproteins.

References Sirito M, Lin Q, Maity T, Sawadogo M. Ubiquitous expression of the 43- and 44-kDa forms of transcription factor USF in mammalian cells. Nucleic Acids Res. 1994 Feb 11;22(3):427-33

Cogswell JP, Godlevski MM, Bonham M, Bisi J, Babiss L. Upstream stimulatory factor regulates expression of the cell cycle-dependent cyclin B1 gene promoter. Mol Cell Biol. 1995 May;15(5):2782-90

Luo X, Sawadogo M. Antiproliferative properties of the USF family of helix-loop-helix transcription factors. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1308-13

Roy AL, Du H, Gregor PD, Novina CD, Martinez E, Roeder RG. Cloning of an inr- and E-box-binding protein, TFII-I, that interacts physically and functionally with USF1. EMBO J. 1997 Dec 1;16(23):7091-104

Sirito M, Lin Q, Deng JM, Behringer RR, Sawadogo M. Overlapping roles and asymmetrical cross-regulation of the USF proteins in mice. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3758-63

Vallet VS, Casado M, Henrion AA, Bucchini D, Raymondjean M, Kahn A, Vaulont S. Differential roles of upstream stimulatory factors 1 and 2 in the transcriptional response of liver genes to glucose. J Biol Chem. 1998 Aug 7;273(32):20175-9

Ismail PM, Lu T, Sawadogo M. Loss of USF transcriptional activity in breast cancer cell lines. Oncogene. 1999 Sep 30;18(40):5582-91

McMurray HR, McCance DJ. Human papillomavirus type 16 E6 activates TERT gene transcription through induction of c-Myc and release of USF-mediated repression. J Virol. 2003 Sep;77(18):9852-61

Saito T, Oishi T, Yanai K, Shimamoto Y, Fukamizu A. Cloning and characterization of a novel splicing isoform of USF1. Int J Mol Med. 2003 Aug;12(2):161-7

Pajukanta P, Lilja HE, Sinsheimer JS, Cantor RM, Lusis AJ, Gentile M, Duan XJ, Soro-Paavonen A, Naukkarinen J, Saarela J, Laakso M, Ehnholm C, Taskinen MR, Peltonen L. Familial combined hyperlipidemia is associated with upstream transcription factor 1 (USF1). Nat Genet. 2004 Apr;36(4):371-6

Chang JT, Yang HT, Wang TC, Cheng AJ. Upstream stimulatory factor (USF) as a transcriptional suppressor of



human telomerase reverse transcriptase (hTERT) in oral cancer cells. Mol Carcinog. 2005 Nov;44(3):183-92

Corre S, Galibert MD. Upstream stimulating factors: highly versatile stress-responsive transcription factors. Pigment Cell Res. 2005 Oct;18(5):337-48

Naukkarinen J, Gentile M, Soro-Paavonen A, Saarela J, Koistinen HA, Pajukanta P, Taskinen MR, Peltonen L. USF1 and dyslipidemias: converging evidence for a functional intronic variant. Hum Mol Genet. 2005 Sep 1;14(17):2595-605

Nowak M, Helleboid-Chapman A, Jakel H, Martin G, Duran-Sandoval D, Staels B, Rubin EM, Pennacchio LA, Taskinen MR, Fruchart-Najib J, Fruchart JC. Insulin-mediated down-regulation of apolipoprotein A5 gene expression through the phosphatidylinositol 3-kinase pathway: role of upstream stimulatory factor. Mol Cell Biol. 2005 Feb;25(4):1537-48

Naukkarinen J, Ehnholm C, Peltonen L. Genetics of familial combined hyperlipidemia. Curr Opin Lipidol. 2006 Jun;17(3):285-90

Bu Y, Gelman IH. v-Src-mediated down-regulation of SSeCKS metastasis suppressor gene promoter by the recruitment of HDAC1 into a USF1-Sp1-Sp3 complex. J Biol Chem. 2007 Sep 14;282(37):26725-39

Huang S, Li X, Yusufzai TM, Qiu Y, Felsenfeld G. USF1 recruits histone modification complexes and is critical for maintenance of a chromatin barrier. Mol Cell Biol. 2007 Nov;27(22):7991-8002

Jung HS, Kim KS, Chung YJ, Chung HK, Min YK, Lee MS, Lee MK, Kim KW, Chung JH. USF inhibits cell proliferation through delay in G2/M phase in FRTL-5 cells. Endocr J. 2007 Apr;54(2):275-85

Kantartzis K, Fritsche A, Machicao F, Stumvoll M, Machann J, Schick F, Häring HU, Stefan N. Upstream transcription factor 1 gene polymorphisms are associated with high antilipolytic insulin sensitivity and show gene-gene interactions. J Mol Med. 2007 Jan;85(1):55-61

Pezzolesi MG, Zbuk KM, Waite KA, Eng C. Comparative genomic and functional analyses reveal a novel cis-acting PTEN regulatory element as a highly conserved functional E-box motif deleted in Cowden syndrome. Hum Mol Genet. 2007 May 1;16(9):1058-71

Rada-Iglesias A, Ameur A, Kapranov P, Enroth S, Komorowski J, Gingeras TR, Wadelius C. Whole-genome maps of USF1 and USF2 binding and histone H3 acetylation reveal new aspects of promoter structure and candidate genes for common human disorders. Genome Res. 2008 Mar;18(3):380-92

Corre S, Primot A, Baron Y, Le Seyec J, Goding C, Galibert MD. Target gene specificity of USF-1 is directed via p38-mediated phosphorylation-dependent acetylation. J Biol Chem. 2009 Jul 10;284(28):18851-62

Naukkarinen J, Nilsson E, Koistinen HA, Söderlund S, Lyssenko V, Vaag A, Poulsen P, Groop L, Taskinen MR, Peltonen L. Functional variant disrupts insulin induction of USF1: mechanism for USF1-associated dyslipidemias. Circ Cardiovasc Genet. 2009 Oct;2(5):522-9

Wong RH, Chang I, Hudak CS, Hyun S, Kwan HY, Sul HS. A role of DNA-PK for the metabolic gene regulation in response to insulin. Cell. 2009 Mar 20;136(6):1056-72


Verhoeven AJM. USF1 (upstream transcription factor 1). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):72-75.





WNK2 (WNK lysine deficient protein kinase 2) Peter Jordan

Departamento de Genetica, Instituto Nacional de Saude Dr Ricardo Jorge, Avenida Padre Cruz, 1649-

016 Lisboa, Portugal (PJ)


Online updated version : http://AtlasGeneticsOncology.org/Genes/WNK2ID41867ch9q22.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI WNK2ID41867ch9q22.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Other names: KAIA302, KIAA1760, NY-CO-43,

P/OKcl.13, PRKWNK2, SDCCAG43

HGNC (Hugo): WNK2

Location: 9q22.31

Local order: The WNK2 gene is covered by BAC

clones RP11-370F5, RP11-480F4 and RP11-165J3

and is flanked by the NINJ1 (telomeric) and

C9orf129 (centromeric) genes.

DNA/RNA

Description

The human WNK2 gene is composed of 31 exons

spanning 136 Kbp on chromosome 9q22.31. The

promoter region contains a 700 bp CpG island

between 1103 bp and 396 bp upstream of the ATG

translation start codon. A second CpG island spans

exon 1 from 135 bp upstream of the ATG

translation start codon until 638 bp downstream of

the ATG and close to the end of exon 1.

Transcription

Two major alternative transcripts exist depending

on the terminal exon chosen.

One variant uses exons 1-30, has a coding sequence

of 6894 bp and yields WNK2(1-2297) (related to

clone KIAA1760, Acc. Nb. AB051547). The other

variant skips exon 30 and includes exon 31, has a

coding sequence of 6765 bp and yields WNK2(1-

2254) (related to clone Kaia302; Acc. Nb.

AK000694). Both terminal exons 30 and 31 carry

their own 3'-untranslated regions and

polyadenylation signals. In addition, there is

evidence for alternative splicing in other exons and

in a tissue-specific manner.

Pseudogene

None known.

Protein

Description

Amino acids: 2297 or 2254. Molecular Weight:

243000 Daltons. The WNK2 protein encodes a

cytoplasmic serine-threonine kinase that lacks a

lysine in subdomain II required for ATP-binding in

most protein kinases and instead uses an alternative

lysine in subdomain I. WNK kinase form a separate

family branch, most closely related to kinases

MEKK, Raf and PAK.

Human WNK2 gene structure. The gene spans 136 Kbp, contains 31 exons and localizes to chromosome 9q22.31. Exons (vertical boxes) and separating introns are shown in proportion to their sizes; however, intron scale differs from exon scale.

WNK2 (WNK lysine deficient protein kinase 2) Jordan P


Diagram of the WNK2 protein in scale. The sequence contains a catalytic domain near the N-terminus and a coiled coil

domain near the C-terminus. Except for three short homology regions shared with the three other human WNK kinases, no other functional domains are known. The two splicing variants WNK2(1-2297) and WNK2(1-2254) differ in the C-terminal protein

sequence.

Expression

WNK2 is preferentially expressed in heart, skeletal

muscle and brain but also in small intestine, colon

and liver. Loss of expression was reported in a large

percentage of human gliomas (Hong et al., 2007)

and grade II and III meningiomas (Jun et al., 2009)

due to extensive methylation in the CpG island at

the 5' end of the WNK2 gene. In contrast, promoter

methylation was rare in other tumor types. This

finding makes WNK2 a candidate tumor suppressor

gene in brain tumors.

Localisation

The subcellular localization of GFP-tagged WNK2

in HeLa cells was predominantly cytoplasmic. Part

of the endogenous WNK2 pool in HT29 colorectal

cells localized to the plasma membrane and

overexpression of a WNK2(1922-2156) that

contains the coiled-coil domain was targeted to the

plasma membrane.

Function

Human WNK2 modulates the activation level of

ERK1 and ERK2. Experimental depletion of

WNK2 or overexpression of a kinase-dead

WNK2K207M mutant led to increased phospho-

ERK1/2 levels when a basal ERK stimulation was

present but not, for example, in serum-free culture

conditions (Moniz et al., 2007). This increase in

ERK1/2 activation promoted cell cycle progression

through G1/S and sensitized cells to respond to

lower concentrations of EGF. From these data one

might predict that loss of WNK2 expression will

promote cell cycle progression in tumor cells.

Interestingly, WNK2 expression is silenced in a

significant percentage of human gliomas (Hong et

al., 2007) suggesting that this pathway may be used

in some tumor types to promote cell proliferation.

The molecular mechanism through which a

reduction in WNK2 expression can increase

ERK1/2 activation involves phosphorylation of

MEK1 at serine 298, a modification that increases

MEK1 affinity towards ERK1/2. Apparently,

WNK2 affects PAK1 activation via Rac1 and

PAK1 is the kinase responsible for MEK1 S298

phosphorylation (Moniz et al., 2008).

Homology

The catalytic domain of WNK2 is 90% identical to

WNK1, 91% identical to WNK3 and 81% identical

to WNK4. The remaining sequence of WNK2 has

little homology to other WNK members except for

three small WNK homology regions (Holden et al.,

2004; Moniz et al., 2007). These include an acidic

motif (residues 586-597) to which hereditary

mutations in WNK4 cluster (Wilson et al., 2001),

residues 1186-1261 without any recognizable motif,

and residues 1918-1988 including a coiled-coil

domain.

Mutations

Note

At present it is unclear whether the observed

somatic mutations have a functional impact on the

WNK2 protein or confer any selective advantage to

tumors cells.

WNK2 (WNK lysine deficient protein kinase 2) Jordan P


Tissue Histology/Type cDNA Protein Mutation Ref

Colorectal adenocarcinoma c.1964delC p.P655fs*2 Frameshift

deletion Greenman et al., 2007

Brain glioblastoma c.3799G>A p.A1267T Missense Parsons et al., 2008

Stomach adenocarcinoma c.4269delC p.S1424fs*5 Frameshift


Lung neuroendocrine

carcinoma c.5009G>A p.G1670E Missense

Greenman et al., 2007; Davies

et al., 2005

Lung adenocarcinoma c.6089G>T p.S2030I Missense Greenman et al., 2007; Davies

et al., 2005

Ovary serous carcinoma c.1528G>T p.V510L Missense Greenman et al., 2007

Ovary mucinous carcinoma c.6798delC p.T2267fs*31 Frameshift


Germinal

No germinal mutations described.

Somatic

Somatic mutations in WNK2 have been found in

the course of large scale tumor genome sequencing

efforts (see Table above).

Heterozygous somatic mutations in the WNK2 gene

identified by large-scale tumor sequencing.

Implicated in

Brain tumors

Note

Promoter methylation leads to loss of expression.

Disease

Glioma and meningioma.

Prognosis

Unknown.

Colon cancer

Note

WNK2 clone was isolated as a serologically defined

colon cancer antigen 43; WNK2 is expressed in

colon.

To be noted Possible role in invasion due to the effect of WNK2

on Rho-GTPases. WNK2 controls (through a yet

unknown mechanism) the activation of RhoA,

which in turn determines the activation of Rac1 in a

reciprocal manner. Experimental depletion of

WNK2 leads to reduced RhoA and increased Rac1

activation.

References Scanlan MJ, Chen YT, Williamson B, Gure AO, Stockert E, Gordan JD, Türeci O, Sahin U, Pfreundschuh M, Old LJ. Characterization of human colon cancer antigens recognized by autologous antibodies. Int J Cancer. 1998 May 29;76(5):652-8

Ito M, Shichijo S, Tsuda N, Ochi M, Harashima N, Saito N, Itoh K. Molecular basis of T cell-mediated recognition of pancreatic cancer cells. Cancer Res. 2001 Mar 1;61(5):2038-46

Wilson FH, Disse-Nicodème S, Choate KA, Ishikawa K et al. Human hypertension caused by mutations in WNK kinases. Science. 2001 Aug 10;293(5532):1107-12

Holden S, Cox J, Raymond FL. Cloning, genomic organization, alternative splicing and expression analysis of the human gene WNK3 (PRKWNK3). Gene. 2004 Jun 23;335:109-19

Davies H, Hunter C, Smith R, Stephens P, et al. Somatic mutations of the protein kinase gene family in human lung cancer. Cancer Res. 2005 Sep 1;65(17):7591-5

Greenman C, Stephens P, Smith R, Dalgliesh GL, Hunter C, et al. Patterns of somatic mutation in human cancer genomes. Nature. 2007 Mar 8;446(7132):153-8

Hong C, Moorefield KS, Jun P, Aldape KD, Kharbanda S, Phillips HS, Costello JF. Epigenome scans and cancer genome sequencing converge on WNK2, a kinase-independent suppressor of cell growth. Proc Natl Acad Sci U S A. 2007 Jun 26;104(26):10974-9

Moniz S, Veríssimo F, Matos P, Brazão R, Silva E, Kotelevets L, Chastre E, Gespach C, Jordan P. Protein kinase WNK2 inhibits cell proliferation by negatively modulating the activation of MEK1/ERK1/2. Oncogene. 2007 Sep 6;26(41):6071-81

Moniz S, Matos P, Jordan P. WNK2 modulates MEK1 activity through the Rho GTPase pathway. Cell Signal. 2008 Oct;20(10):1762-8

Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, et al. An integrated genomic analysis of human glioblastoma multiforme. Science. 2008 Sep 26;321(5897):1807-12

Jun P, Hong C, Lal A, Wong JM, McDermott MW, Bollen AW, Plass C, Held WA, Smiraglia DJ, Costello JF. Epigenetic silencing of the kinase tumor suppressor WNK2 is tumor-type and tumor-grade specific. Neuro Oncol. 2009 Aug;11(4):414-22

Moniz S, Jordan P. Emerging roles for WNK kinases in cancer. Cell Mol Life Sci. 2010 Apr;67(8):1265-76


Jordan P. WNK2 (WNK lysine deficient protein kinase 2). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):76-78.

Leukaemia Section Mini Review




t(1;2)(p36;p21) Jean-Loup Huret

Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers,

France (JLH)


Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0102p36p21ID1542.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI t0102p36p21ID1542.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Clinics and pathology

Disease

Myelodysplastic syndrome (MDS) in most cases,

acute lymphoblastic leukemia (ALL) in one case.

Phenotype/cell stem origin

At least 3 of the 6 available cases were treatment

related myelodysplastic syndromes (t-MDS)

(Roulston et al., 1998; Mauritzson et al., 2002;

Masuya et al., 2002), and 2 other cases were MDS

(Horiike et al., 1988; Storlazzi et al., 2008).

Clinics

A 38-year-old male patient presented with a

treatment related myelodysplastic syndrome (t-

MDS) evolving towards an acute myeloid leukemia

(t-AML). Previous treatment included

topoisomerase inhibitors for a Hodgkin disease 36

months before diagnosis of the t-MDS (Roulston et

al., 1998). A t-MDS was diagnosed in a 76-year-old

female patient previously treated with radiotherapy

for uterine cancer 29 years ago. She died 26 months

after diagnosis of the t-MDS (Mauritzson et al.,

2002). A 49-year-old female patient was diagnosed

with t-MDS (FAB refractory anemia (RA)); she had

been treated with etoposide 2 years previously for

M1-AML; the patient died 6.5 years after onset of

the t(1;2). Other chromosome anomalies appeared

during course of the disease, as well as an unrelated

clone (Masuya et al., 2002). A 67-year-old female

patient had a chronic myelomonocytic leukemia

(CMML) with a normal karyotype; she received

hydroxyurea. Three years later, a refractory anemia

with excess of blasts-2 (RAEB-2) and a t(1;2) was

diagnosed. The patient died one month later

(Storlazzi et al., 2008).

Refractory anemia with excess of blasts (RAEB)

was diagnosed in a 69-year-old male patient. The

patient was still alive 15 months after diagnosis

(Horiike et al., 1988). A T-cell acute lymphoblastic

leukemia (T-ALL) was found in a 1-year-old child

(Mathew et al., 2001).

Cytogenetics

Cytogenetics morphological

In two cases, the t(1;2) was the sole anomaly

(Horiike et al., 1988; Storlazzi et al., 2008). In

contrast, complex karyotype were present in the 4

other cases. inv(14)(q11q32) was present in the T-

ALL case (Mathew et al., 2001); del(5q) was found

in two cases (Roulston et al., 1998; Mauritzson et

al., 2002) and del(7q) in one case (Masuya et al.,

2002). Other remarkable anomalies were:

t(14;21)(q22;q22) with RUNX1 involvement

(Roulston et al., 1998), +8, +12, +13 appearing

during course of the disease (Masuya et al., 2002);

there was also, in the latter case, an unrelated clone

with t(11;12)(p15;q13).

Genes involved and proteins

Note

In only one case were the genes involved in the

translocation studied (Storlazzi et al., 2008).

PRDM16

Location

1p36

Protein

Transcription activator; PRDM16 forms a

t(1;2)(p36;p21) Huret JL


transcriptional complex with CEBPB. PRDM16

plays a downstream regulatory role in mediating

TGFB signaling (Bjork et al., 2010). PRDM16

induces brown fat determination and differentiation

(Kajimura et al., 2010).

FLJ42875

Location

1p36

DNA/RNA

2 transcript variants; non-coding RNA of unknown

function.

Result of the chromosomal anomaly

Fusion protein

Description

PRDM16 (both long and short isoforms) and

FLJ42875 are overexpressed. The sequence on

chromosome 2 upregulating these 2 genes is

unknown.

References Horiike S, Taniwaki M, Misawa S, Abe T. Chromosome abnormalities and karyotypic evolution in 83 patients with myelodysplastic syndrome and predictive value for prognosis. Cancer. 1988 Sep 15;62(6):1129-38

Roulston D, Espinosa R 3rd, Nucifora G, Larson RA, Le Beau MM, Rowley JD. CBFA2(AML1) translocations with novel partner chromosomes in myeloid leukemias: association with prior therapy. Blood. 1998 Oct 15;92(8):2879-85

Mathew S, Rao PH, Dalton J, Downing JR, Raimondi SC. Multicolor spectral karyotyping identifies novel translocations in childhood acute lymphoblastic leukemia. Leukemia. 2001 Mar;15(3):468-72

Masuya M, Katayama N, Inagaki K, Miwa H, Hoshino N, Miyashita H, Suzuki H, Araki H, Mitani H, Nishii K, Kageyama S, Minami N, Shiku H. Two independent clones in myelodysplastic syndrome following treatment of acute myeloid leukemia. Int J Hematol. 2002 Feb;75(2):182-6

Mauritzson N, Albin M, Rylander L, Billström R, Ahlgren T, Mikoczy Z, Björk J, Strömberg U, Nilsson PG, Mitelman F, Hagmar L, Johansson B. Pooled analysis of clinical and cytogenetic features in treatment-related and de novo adult acute myeloid leukemia and myelodysplastic syndromes based on a consecutive series of 761 patients analyzed 1976-1993 and on 5098 unselected cases reported in the literature 1974-2001. Leukemia. 2002 Dec;16(12):2366-78

Storlazzi CT, Albano F, Guastadisegni MC, Impera L, Mühlematter D, Meyer-Monard S, Wuillemin W, Rocchi M, Jotterand M. Upregulation of MEL1 and FLJ42875 genes by position effect resulting from a t(1;2)(p36;p21) occurring during evolution of chronic myelomonocytic leukemia. Blood Cells Mol Dis. 2008 May-Jun;40(3):452-5

Kajimura S, Seale P, Kubota K, Lunsford E, Frangioni JV, Gygi SP, Spiegelman BM. Initiation of myoblast to brown fat switch by a PRDM16-C/EBP-beta transcriptional complex. Nature. 2009 Aug 27;460(7259):1154-8

Bjork BC, Turbe-Doan A, Prysak M, Herron BJ, Beier DR. Prdm16 is required for normal palatogenesis in mice. Hum Mol Genet. 2010 Mar 1;19(5):774-89


Huret JL. t(1;2)(p36;p21). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):79-80.

Leukaemia Section Short Communication




t(2;18)(q11;q21) Jean-Loup Huret


France (JLH)


Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0218q11q21ID2158.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI t0218q11q21ID2158.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology


Disease

Non Hodgkin lymphoma.


One case to date, a 65-year-old female patient with

a follicular lymphoma stage II-a (Impera et al.,

2008).

Evolution

Complete remission was obtained.

Cytogenetics

Additional anomalies

A complex karyotype was found, with +11, and

other anomalies.


AFF3

Location

2q11.2

Protein

AFF3 belongs to a family of putative transcription

factors also comprising AFF1 (AF4, FEL, MLLT2)

in 4q21, AFF2 (FMR2, FRAXE) in Xq28 and

AFF4 (AF5Q31) in 5q31. AFF3 has been found a

susceptibility gene in autoimmune diseases, namely

rheumatoid arthritis, psoriatic arthritis, and juvenile

idiopathic arthritis (Barton et al., 2009; Castelino

and Barton, 2010; Hinks et al., 2010). AFF3 is

deleted in Nievergelt syndrome, an autosomal

dominant mesomelic dysplasia (Steichen-Gersdorf

et al., 2008). AFF3 was also found expressed in

20% of mammary tumor cells but not in normal

acini in a study (To et al., 2005).

BCL2

Location

18q21.33

Protein

Antiapoptotic protein.


Hybrid gene

Description

Fusion of AFF3 exon 1 to BCL2 exon 2.

Fusion protein

Oncogenesis

Leads to the overexpression of BCL2.

References To MD, Faseruk SA, Gokgoz N, Pinnaduwage D, Done SJ, Andrulis IL. LAF-4 is aberrantly expressed in human breast cancer. Int J Cancer. 2005 Jul 1;115(4):568-74

Impera L, Albano F, Lo Cunsolo C, Funes S, Iuzzolino P, Laveder F, Panagopoulos I, Rocchi M, Storlazzi CT. A novel fusion 5'AFF3/3'BCL2 originated from a t(2;18)(q11.2;q21.33) translocation in follicular lymphoma. Oncogene. 2008 Oct 16;27(47):6187-90

Steichen-Gersdorf E, Gassner I, Superti-Furga A, Ullmann R, Stricker S, Klopocki E, Mundlos S. Triangular tibia with fibular aplasia associated with a microdeletion on 2q11.2 encompassing LAF4. Clin Genet. 2008 Dec;74(6):560-5

Barton A, Eyre S, Ke X, Hinks A, Bowes J, Flynn E, Martin P, Wilson AG, Morgan AW, Emery P, Steer S, Hocking LJ, Reid DM, Harrison P, Wordsworth P, Thomson W, Worthington J. Identification of AF4/FMR2 family, member

t(2;18)(q11;q21) Huret JL


3 (AFF3) as a novel rheumatoid arthritis susceptibility locus and confirmation of two further pan-autoimmune susceptibility genes. Hum Mol Genet. 2009 Jul 1;18(13):2518-22

Castelino M, Barton A. Genetic susceptibility factors for psoriatic arthritis. Curr Opin Rheumatol. 2010 Mar;22(2):152-6

Hinks A, Eyre S, Ke X, Barton A, Martin P, Flynn E, Packham J, Worthington J, Thomson W. Association of the AFF3 gene and IL2/IL21 gene region with juvenile idiopathic arthritis. Genes Immun. 2010 Mar;11(2):194-8


Huret JL. t(2;18)(q11;q21). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):81-82.

Leukaemia Section Short Communication




t(2;21)(q11;q22) Jean-Loup Huret


France (JLH)


Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0221q11q22ID1551.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI t0221q11q22ID1551.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology


Disease

T-cell acute lymphoblastic leukemia.


One case to date, a 6-year-old boy (Chinen et al.,

2008).

Evolution

Complete remission was obtained. An allogenic

bone marrow transplantation was performed, and

the patient had remained in complete remission for

17 months at the time of the report.

Cytogenetics

Additional anomalies

A complex karyotype was found.


AFF3

Location

2q11.2

Protein

AFF3 belongs to a family of putative transcription

factors also comprising AFF1 (AF4, FEL, MLLT2)

in 4q21, AFF2 (FMR2, FRAXE) in Xq28 and

AFF4 (AF5Q31) in 5q31. AFF3 has been found a

susceptibility gene in autoimmune diseases, namely

rheumatoid arthritis, psoriatic arthritis, and juvenile

idiopathic arthritis (Barton et al., 2009; Castelino

and Barton, 2010; Hinks et al., 2010). AFF3 is

deleted in Nievergelt syndrome, an autosomal

dominant mesomelic dysplasia (Steichen-Gersdorf

et al., 2008). AFF3 was also found expressed in

20% of mammary tumor cells but not in normal

acini in a study (To et al., 2005).

RUNX1

Location

21q22.3

Protein

Transcription factor (activator) for various

hematopoietic-specific genes.


Hybrid gene

Description

Fusion of RUNX1 exon 7 to AFF3 exon 8.

References To MD, Faseruk SA, Gokgoz N, Pinnaduwage D, Done SJ, Andrulis IL. LAF-4 is aberrantly expressed in human breast cancer. Int J Cancer. 2005 Jul 1;115(4):568-74

Chinen Y, Taki T, Nishida K, Shimizu D, Okuda T, Yoshida N, Kobayashi C, Koike K, Tsuchida M, Hayashi Y, Taniwaki M. Identification of the novel AML1 fusion partner gene, LAF4, a fusion partner of MLL, in childhood T-cell acute lymphoblastic leukemia with t(2;21)(q11;q22) by bubble PCR method for cDNA. Oncogene. 2008 Apr 3;27(15):2249-56

Steichen-Gersdorf E, Gassner I, Superti-Furga A, Ullmann R, Stricker S, Klopocki E, Mundlos S. Triangular tibia with fibular aplasia associated with a microdeletion on 2q11.2 encompassing LAF4. Clin Genet. 2008 Dec;74(6):560-5

Barton A, Eyre S, Ke X, Hinks A, Bowes J, Flynn E, Martin P, Wilson AG, Morgan AW, Emery P, Steer S, Hocking LJ, Reid DM, Harrison P, Wordsworth P, Thomson W, Worthington J. Identification of AF4/FMR2 family, member

t(2;21)(q11;q22) Huret JL


3 (AFF3) as a novel rheumatoid arthritis susceptibility locus and confirmation of two further pan-autoimmune susceptibility genes. Hum Mol Genet. 2009 Jul 1;18(13):2518-22

Castelino M, Barton A. Genetic susceptibility factors for psoriatic arthritis. Curr Opin Rheumatol. 2010 Mar;22(2):152-6

Hinks A, Eyre S, Ke X, Barton A, Martin P, Flynn E, Packham J, Worthington J, Thomson W. Association of the AFF3 gene and IL2/IL21 gene region with juvenile idiopathic arthritis. Genes Immun. 2010 Mar;11(2):194-8


Huret JL. t(2;21)(q11;q22). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):83-84.

Solid Tumour Section Mini Review




Liver: Nested stromal epithelial tumor Y Albert Yeh

North Shore University Hospital, Long Island Jewish Medical Center, Hofstra University School of

Medicine, New York, USA (YAY)


Online updated version : http://AtlasGeneticsOncology.org/Tumors/NestStromEpithLiverID6243.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI NestStromEpithLiverID6243.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity

Alias

Ossifying malignant mixed epithelial and stromal

tumor; Ossifying stromal-epithelial tumor;

Desmoplastic nested spindle cell tumor; Calcifying

nested stromal-epithelial tumors of the liver

Note

Nested stromal epithelial tumor (NSET) of the liver

is an extremely rare non-hepatocytic tumor of the

liver and is characterized by nests of spindle and

epithelioid cells with occasional calcification and

ossification.

Synonyms include ossifying malignant mixed

epithelial and stromal tumor, ossifying stromal-

epithelial tumor, desmoplastic nested spindle cell

tumor, and calcifying nested stromal-epithelial

tumors of the liver.


Epidemiology

Twenty four cases of NSET of the liver have been

reported. NSET occurs in patients with age range

from 2 to 33 years old. The tumor affects mainly

children, and females are more frequently affected

than males.

Gross and histopathological characteristics of NSET. Grossly, the tumor has a yellow-tan and bulging lobulated appearance. A satellite tumor nodule is present in the periphery (A). Microscopically, nested tumor cells have oval nuclei, stippled chromatin,

and inconspicuous nucleoli. The interface between epithelioid and spindle cells is shown (arrows). Courtesy of Sergey V Brodsky.

Liver: Nested stromal epithelial tumor Yeh YA


Clinics

Patients are presented with palpable abdominal

tumors. Cushing syndrome has been reported in a

few patients.

Pathology

Macroscopically, NSETs predominantly occur in

the right lobe of the liver and are unencapsulated,

well circumscribed tumors range in size from 2.8 to

30 cm in greatest dimension. Satellite tumor

nodules also have been reported. Tumors have a

yellow-tan and lobulated appearance.

Microscopically, NSETs are characterized by

organoid arrangement of cellular nests composed of

spindle and epithelioid cells embedded in a

desmoplastic or fibrocytic/myofibroblastic stroma,

within which proliferation of bile ductules is noted.

Areas of myxoid and cystic degeneration or

necrosis are sometimes encountered within or

adjacent to the cellular nests. Focal psammomatous

calcification or osteoid formation is present in some

tumors. Lymphovascular invasion is occasionally

seen.

The spindle cells within the cellular nests are

arranged in short fascicles with a somewhat

whorled pattern.

The nested cells are characterized by plump nuclei,

stippled chromatin, and inconspicuous nucleoli.

Scattered mitotic figures with abnormal forms are

identified.

Immunohistochemically, the tumor cells are stained

positive for cytokeratin AE1/AE3, keratin CK19

(focal), EMA, CD117 (c-kit), CD56, CD99, ACTH,

chromogranin, synaptophysin, neuron-specific

enolase, and S100 (focally weak in epithelioid

cells). Vimentin stain is positive in the nested

spindled cell and stroma. Muscle specific actin and

smooth muscle actin immunostains highlight

stromal myofibroblastic cells. Alpha-fetoprotein

and p53 are negative.

Cytogenetics

Chromosomal analysis of one tumor reveals an

abnormal karyotype of 60-63,XXX,-1,-4,-5,-

others,+2mar.

Prognosis

Most patients are doing well with no tumor

recurrence in 6 months to 14 years. Tumor

recurrence has been observed in two of twenty four

patients. One case with aggressive clinical behavior

and extrahepatic lymph node metastasis has been

reported.

Immunohistochemical stains of NSET. Tumor cells are stained positive for immunostains including ACTH (A) (Courtesy of

Milton J Finegold), b-catenin (B), cyclin D1 (C), Ki-67 (30% of nuclear staining) (D), p21ras (E), topoisomerase II (F).

Liver: Nested stromal epithelial tumor Yeh YA


References Ishak KG, Goodman ZD, Stocker JT.. Miscellaneous malignant tumors. In: Rosai J, Sobin LH, eds. Tumors of the liver and intrahepatic bile ducts. Armed forces Institute of Pathology, Washington DC, 2001:271-2.

Heywood G, Burgart LJ, Nagorney DM. Ossifying malignant mixed epithelial and stromal tumor of the liver: a case report of a previously undescribed tumor. Cancer. 2002 Feb 15;94(4):1018-22

Heerema-McKenney A, Leuschner I, Smith N, Sennesh J, Finegold MJ. Nested stromal epithelial tumor of the liver: six cases of a distinctive pediatric neoplasm with frequent calcifications and association with cushing syndrome. Am J Surg Pathol. 2005 Jan;29(1):10-20

Hill DA, Swanson PE, Anderson K, Covinsky MH, Finn LS, Ruchelli ED, Nascimento AG, Langer JC, Minkes RK, McAlister W, Dehner LP. Desmoplastic nested spindle cell tumor of liver: report of four cases of a proposed new entity. Am J Surg Pathol. 2005 Jan;29(1):1-9

Brodsky SV, Sandoval C, Sharma N, Yusuf Y, Facciuto ME, Humphrey M, Yeh YA, Braun A, Melamed M, Finegold MJ. Recurrent nested stromal epithelial tumor of the liver

with extrahepatic metastasis: case report and review of literature. Pediatr Dev Pathol. 2008 Nov-Dec;11(6):469-73

Makhlouf HR, Abdul-Al HM, Wang G, Goodman ZD. Calcifying nested stromal-epithelial tumors of the liver: a clinicopathologic, immunohistochemical, and molecular genetic study of 9 cases with a long-term follow-up. Am J Surg Pathol. 2009 Jul;33(7):976-83

Meir K, Maly A, Doviner V, Gross E, Weintraub M, Rabin L, Pappo O. Nested (ossifying) stromal epithelial tumor of the liver: case report. Pediatr Dev Pathol. 2009 May-Jun;12(3):233-6

Grazi GL, Vetrone G, d'Errico A, Caprara G, Ercolani G, Cescon M, Ravaioli M, Del Gaudio M, Vivarelli M, Zanello M, Pinna AD. Nested stromal-epithelial tumor (NSET) of the liver: a case report of an extremely rare tumor. Pathol Res Pract. 2010 Apr 15;206(4):282-6

Oviedo Ramírez MI, Bas Bernal A, Ortiz Ruiz E, Bermejo J, De Alava E, Hernández T. Desmoplastic nested spindle cell tumor of the liver in an adult. Ann Diagn Pathol. 2010 Feb;14(1):44-9


Yeh YA. Liver: Nested stromal epithelial tumor. Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):85-87.





Inhibitors of the heat shock protein 90: from cancer clinical trials to neurodegenerative diseases Jean François Peyrat, Samir Messaoudi, Jean Daniel Brion, Mouad Alami

Université Paris-Sud, CNRS, BioCIS-UMR 8076, Laboratoire de Chimie Thérapeutique, Faculté de

Pharmacie, 5 rue J.-B. Clément, Châtenay-Malabry, F-92296, France (JFP, SM, JDB, MA)


Online updated version : http://AtlasGeneticsOncology.org/Deep/HSP90inCancerTreatmentID20086.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI HSP90inCancerTreatmentID20086.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

I) Introduction

The 90-kDa heat shock protein 90, Hsp90, belongs

to the family of molecular chaperone responsible

for the conformational maturation or reparation of

other proteins, referred to as "clients", into

biologically active structures (Pearl and Prodromou,

2006). Hsp90 exerts its essential ATP dependant

chaperone function to more than three hundred

client proteins involved in cell growth,

differentiation and survival (Workman, 2004;

Chiosis et al., 2004; Sreedhar et al., 2004b; Zhang

and Burrows, 2004; Neckers, 2002). Many of them,

more than forty, include overexpressed or mutant

oncogenic proteins ErbB2/HER2 (Miller et al.,

1994; An et al., 1997), Braf (Grbovic et al., 2006),

Akt/PKB (Sato et al., 2000), muted p53

(Blagosklonny et al., 1996), transcription factors:

hormone steroid receptors GR (Grad and Picard,

2007) ER and AR, angiogenic factors HIF-1α

(Picard, 2006; Kuduk et al., 2000; Kuduk et al.,

1999; Johnson and Toft, 1995), telomerase

(Forsythe et al., 2001; Akalin et al., 2001) which

are associated with the six hallmarks of cancer

(Figure 1).

Under non-stress conditions the quaternary

structure of Hsp90 is now well established to be a

dimeric complex, and its abundance is

approximately 1% of the total protein contents.

Each monomer consists in three domains: the N-

terminal domain (NTD), a middle domain (MD)

implicated in client protein binding, and a C-

terminal dimerization domain (CTD) (Figure 2)

(Harris et al., 2004; Shiau et al., 2006).

Figure 1: Hsp90 protein partners and clients destabilized by Hsp90 inhibition (Jackson et al., 2004).

http://atlasgeneticsoncology.org/Genes/ERBB2ID162ch17q11.html

http://atlasgeneticsoncology.org/Genes/BRAFID828.html

http://atlasgeneticsoncology.org/Genes/AKT1ID355ch14q32.html

http://atlasgeneticsoncology.org/Genes/P53ID88.html

http://atlasgeneticsoncology.org/Genes/ARID685chXq12.html

Inhibitors of the heat shock protein 90: from cancer clinical trials to neurodegenerative diseases

Peyrat JF, et al.


Figure 2: Structure of the full length yeast Hsp90, in complex with non hydrolysable ATP analogue (Hsp90 ATP) and structure of

the full length E. coli Hsp90 without nucleotide (Hsp90 apo) (Shiau et al., 2006).

In the human proteome, several isoforms of Hsp90

have been isolated, Hsp90α (inducible form) and

Hsp90β (constitutive form) localized in cytoplasm

(Sreedhar et al., 2004a), while Grp94 (glucose-

regulated protein) and TRAP-1 (HSP75/tumor

necrosis factor receptor associated protein 1) are

localized in the endoplasmic reticulum and in

mitochondria respectively (Csermely et al., 1998;

Maloney and Workman, 2002). To acquire its full

active molecular chaperone activity, Hsp90

operates with molecular co-chaperones and partner

proteins to form a series of multimeric protein

complexes (Figure 3) including Hsp70, peptidyl-

prolyl isomerases, immunophilins (FKBP51 and

FKBP52) and the cyclophilin CYP40. Others co-

chaperones such as p23, recently identified as a

prostaglandine E2-Syntase, plays an important role

in the activity of a number of transcription factors

of the steroid/thyroid receptor family (Chan et al.,

2008; Grad et al., 2006).

It is now well established, that Hsp90 needs to bind

ATP in a pocket located in the N-terminal domain

to exert its function. Thus, the Hsp90 protein

function may be inhibited by molecules competing

with ATP binding (such as geladanamycin: GA,

Figure 3), thereby freezing the chaperone cycle,

which in turn decreases the affinity of Hsp90 for

client proteins and leads to 26S proteasome-

mediated oncogenic client protein degradation

(Sepp-Lorenzino et al., 1995). N-terminal domain

Hsp90 inhibitors block cancer cell proliferation in

vitro and cancer growth in vivo (Sharp and

Workman, 2006).

To date, the full crystal structure of Hsp90 in

complex with a non hydrolysable ATP analogue

(Ali et al., 2006), and of the full length E.Coli

Hsp90 without nucleotide (apo-Hsp90) (Shiau et

al., 2006), have yet been reported (Figure 2).

Furthermore, an interesting recent study

investigated Hsp90 conformational changes in

solution, shows a long range effects between Hsp90

domains, as the binding of co-chaperones (or

inhibitors) at NTD induce conformational changes

in the MD and CTD (Phillips et al., 2007). The C-

terminal domain has been implicated biochemically

as the site of a possible second, cryptic ATP-

binding site on Hsp90. Its contribution to the

overall regulation of chaperone function is not

clear, but the antibiotic novobiocin (Nvb) (c.f.

structure in Figure 15) has been reported to bind

this site and alter the conformation of the chaperone

(Yun et al., 2004).

Since pharmacological inhibition of Hsp90 by

several families of small molecules leading to the

degradation of oncogenic proteins, Hsp90 has

become a target of interest against cancer and

allowed the development of numerous small

inhibitors (Biamonte et al., 2010).

II) Hsp90 health and cancer (Powers and

Workman, 2007)

Hsp90 has probably been most widely

acknowledged as a therapeutic target for the

treatment of cancer (Mitsiades et al., 2007).

Although there is no evidence of Hsp90 mutations

in malignancy, there is increasing support for the

view that this molecular chaperone plays an

important role in the development, maintenance and

progression of cancers.

One of the principal debates concerning the

inhibition of the highly abundant Hsp90 is the

selectivity of inhibitors for the chaperone protein in

malignant cells (Kamal et al., 2003). Some works

suggest that Hsp90 inhibitors could provide an

exploitable therapeutic index (Banerji, 2005).

Firstly, it has been reported that inhibitors were

significantly more sensitive to Hsp90 in cancer

cells (Neckers and Neckers, 2005; Powers and

Workman, 2006; Chiosis, 2006; Whitesell et al.,

1994; Neckers, 2006). In support to this surprising

observation, Kamal et al. showed that the activity

state of the Hsp90 chaperone machine was different

in tumor cells.


Peyrat JF, et al.


Figure 3: Hsp90 cycle: GA: geldanamycin analogue; 40=Hsp40; 70=Hsp70; IP: immunophillin; HIP: Hsp70-interacting protein; HOP=Hsp70/Hsp90 organizing protein (Biamonte et al., 2010).

Indeed, the Hsp90 is entirely bound in an active

complex with co-chaperones, whereas most Hsp90

in normal tissues resides in a free, uncomplexed

state (Workman, 2004; Kamal et al., 2003).

Furthermore, Hsp90 is constitutively expressed at

higher levels (2-10 fold) in tumor cells compared

with their normal counterparts. This higher Hsp90

activity is probably due to the

overexpression/amplification of mutated Hsp90

clients, and this is in correlation with the higher

level of cochaperones of Hsp90 observed in

cancerous cells.

Finally, the selective sensitivity of transformed

cells for Hsp90 inhibitors may be partly due to the

selective accumulation of these drugs in cancer

cells since the in vivo observation of Hsp90

inhibitors in murine model system showed higher

concentration in tumor tissue (Chiosis and Neckers,

2006).

Consequently, the Hsp90 has emerged as an

exciting target for the development of cancer

chemotherapeutics. However, despite the numerous

molecules which have prompted a phase I clinical

trial, it remains to be verified if Hsp90 inhibitors

will provide adequate treatment in clinic.

III) Hsp90 inhibitors (Messaoudi, 2008)

The Hsp90 protein function may be inhibited with

molecules that bind the ATP pocket, or its

chaperone activity may be disturbed by small

molecules binders interfering with domains in the

C-terminus or median region. Although Hsp90

function provides an attractive target for the

treatment of cancer, the feasibility and efficacy of

the inhibitors approach has just begun to be

explored in clinic.

Direct inhibitors of Hsp90 have been divided into

two groups:

A) N-terminal domain binders

1) Ansamycin macrolactames

1.a) Quinone derivatives

Geldanamycin (Figure 4), was isolated from the

broth of Streptomyces hygroscopicus in 1970s (De

Boer et al., 1970). Further studies have shown that

GA revert the phenotype of v-src oncogene

transformed cells. However, this ability was not due

to a direct action of the Src kinase activity, but to an

inhibition of Hsp90. Subsequent

immunoprecipitation and X-ray cristallographic

studies have shown that GA competes with ATP

and binds to the N-terminal domain site of Hsp90,

leading the Hsp90 multichaperone complexes to the

ubiquitin-mediated proteasome degradation (Roe et

al., 1999; Stebbins et al., 1997). Since this

observation, GA was used to identify additional

Hsp90 substrates and to understand the role of

Hsp90 in promoting malignant transformation.

Although GA provided very promising antitumor

effects, it showed several pharmacologic limitations

as poor solubility, limited in vivo stability and high

hepatotoxicity in some of the human tumor models

(Neckers, 2006; Supko et al., 1995). Thus, the 17-

position of GA has been an attractive focal point for

the synthesis of GA analogues. Structure-activity

relationship (SAR) studies have shown that

structurally and sterically diverse 17-substituents

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can be introduced without destroying antitumor

activity. Then, further derivatives of GA, with

similar biological behaviour but a better toxicity

profile, were synthesized (Schulte and Neckers,

1998). Therefore, new C-17 substituted derivatives

17-AAG (17-allyl-17-desmethoxygeldanamycin,

also designed KOS953, CNF 1010, tanespimycin,

Figure 4) and 17-DMAG (17-(2-

dimethylaminoethylamino)-17-

desmethoxygeldanamycin, KOS1022,

alvespimycin, Figure 4) (Snader et al., 2002; Solit

et al., 2007) were brought to the fore by displaying

a significant enhancement of the

chemical/metabolic stability.

17-AAG can be used in single agent or in

combination with other cancer therapeutics

(KOS953/bortezomib (Anderson, 2007; Richardson

et al., 2007), KOS953/trastuzumab (Modi et al.,

2007), 17-AAG/Paclitaxel (Sain et al., 2006), 17-

AAG/cisplatin (McCollum et al., 2008)).

To enhance the pharmacokinetics and dynamics of

17-AAG, Kosan Biosciences Incorporated has

developed a DMSO-free formulation (KOS953)

contained cremophor, which is actually in Phase I

clinical testing.

Although 17-AAG and its numerous formulations

have shown some encouraging clinical responses,

they present important drawbacks (e.g.; liver

toxicity and cumbersome formulation) that may

limit their clinical applications whereas 17-DMAG

exhibits a better water solubility and oral

bioavailability (Ronnen et al., 2006). However,

although clinical trials in myeloid leukemia seemed

to be promising, the 17-DMAG was discontinued in

2008 (ClinicalTrials.gov).

1.b) Hydroquinone derivatives

In a different approach, Infinity Pharmaceuticals

has developed IPI504 (retaspimycin or 17-AAG

hydroquinone, Figure 4) (Adams et al., 2005; Sydor

et al., 2006), a new GA analogue, in which the

quinone moiety was replaced by a dihydroquinone

one. Indeed, the preclinical data suggested that the

hepatotoxicity of 17-AAG was attributable to the

ansamycin benzoquinone moiety, prone to

nucleophilic attack. Furthermore, it was recently

reported that the hydroquinone form binds Hsp90

with more efficiency than the corresponding

quinone form (Maroney et al., 2006). In biological

conditions, the hydroquinone form can interconvert

with GA, depending on redox equilibrium existing

in cell. It has been recently proposed, that NQ01

(NAD(P)H: quinone oxidoreductase) can produce

the active hydroquinone from the quinone form of

IPI504 (Chiosis, 2006). However, Infinity

Pharmaceuticals showed that if the overexpression

of NQ01 increased the level of hydroquinone and

cell sensitivity to IPI504, it has no significant effect

on its growth inhibitory activity. These results

suggest that NQ01 is not a determinant of IPI504

activity in vivo (Douglas et al., 2009).

1.c) Clinical trials

In 2007, results of the phase I clinical trial of

tanespimicyn (KOS953) with bortezomib in

patients with relapsed refractory multiple myeloma

were reported (Solit and Chiosis, 2008; Taldone et

al., 2008). Dose escalations in the trial ranged from

100 to 340 mg/m2 for tanespimycin, and from 0.7 to

1.3 mg/m2 for bortezomib. Results showed that two

patients, on the 41 enrolled, exhibited stable disease

after two cycles, and 18 of them demonstrated a

response to combination (Richardson et al., 2007).

Moreover, the tanespimicyn was co-administrated

with trastuzumab on 25 patients treated with up to

450 mg/m2 of drug on a weekly schedule. This

combination induced a regression of 21, 22 and

25% in three patients, which had failed trastuzumab

therapy, with HER2-amplified breast cancer (Modi

et al., 2007).

Figure 4: GA, 17-AAG, 17-DMAG and IPI504.

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In 2008, Infinity Pharmaceuticals reported the

results of a dose escalation Phase I/II clinical trial

of retaspimycin hydrochloride in patients with

metastatic and/or unresectable gastrointestinal

stromal tumors (GIST) on a twice weekly schedule

(400 mg/m2). 4 of the 18 patients enrolled, achieved

a partial response and 11/18 achieved stable

disease. These results had initiated the phase III

clinical trial of the study in 2008. However, Infinity

Pharmaceuticals reported on April 2009, the

decision to end its phase III study (RING trial) of

IPI504 hydrochloride in patients with refractory

gastrointestinal stromal tumors (Infinity Press

Release). The trial was based on 46 patients whose

tumors persist despite treatment with Gleevec

(imatinib) and Sutent (sunitinib). Resulting data

showed a higher than anticipated mortality rate. In

this heavily pretreated population, IPI504 was not

tolerated (400 mg/m2 or placebo in 21 days cycles

as a 30 min intravenous infusion twice weekly for 2

weeks followed by a 1 week rest) and the study was

terminated early.

Nevertheless, the IPI504 is still evaluating in phase

II trials in patients with non-small cell lung cancer,

and in combination with herceptin (trastuzumab) in

patients with HER-2 positive metastatic breast

cancer.

In the same month, KOSAN, acquired by BMS in

2008, reported that the phase III clinical trial

concerning the KOS953 or tanespimycin, in

combination with Bortezomib in patients with

multiple myeloma in first relapse has been

suspended. This was probably a precaution as the

metabolization of tanespimycin leads to IPI504.

Conforma Therapeutics/Biogen idec developed a

hydroquinone form of the 17-AAG (CNF1010),

trapped as HCl salt, which was in clinical phase I

against chronic lymphocytic leukemia. However, to

date, this program is terminated. Moreover, the

CNF1010 had started a phase III trial against GIST

in 2008. This study was also suspended due to the

anticipated mortality rate of patients enrolled

(ClinicalTrials.gov). Parallel efforts to improve the

solubility and bioavailability of 17-AAG have led

the NCI and Kosan to develop 17-DMAG

(KOS1022) as a second generation alternative

which has entered Phase I clinical testing (Santi et

al., 2007). Promising results were obtained in

patients with chemotherapy refractory acute

myelogenous leukemia, as 3 of 17 patients had a

complete response to therapy (Lancet et al., 2006).

However, researches were given up in 2008, as the

17-DMAG presents an unusable toxicity profile.

1.d) Other analogues

Diverse derivatives of 17-AAG bearing non-redox-

active phenol group designed by Kosan Biosciences

were reported (Tian et al., 2007). Amongst them,

KOSN1559 was claimed as the most potent Hsp90

inhibitor (e.g.; SKBr3 Cell Line IC50=860 nM,

Kd=16 nM) (Figure 5). To date, no clinical trial had

been reported with this compound.

Figure 5: Structure of KOSN 1559.

2) Purines

2.a) Purines analogues

Limitations in the clinical use of 17-AAG and 17-

DMAG have prompted the discovery of novel

Hsp90 ATPase inhibitors with improved "drug-

like" structural characteristics and better

pharmacological profiles. To this end, structure-

based design and high-throughput screening

approaches performed at the Memorial Sloane

Kettering Institute, have been taken to identify new

chemotypes that inhibit Hsp90 ATPase activity. A

significant breakthrough in the preparation of

synthetic Hsp90 inhibitor was the PU3 (Figure 6).

On the basis of X-ray analysis and molecular

modelling, Chiosis's group, showed that PU3 was

designed to place the purine moiety into the same

spatial orientation as adenine ring of ATP in the

nucleotide pocket of Hsp90 (Chiosis et al., 2001).

PU3 presented molecular signature of Hsp90

inhibition, including the degradation of HER2, even

if its affinity for Hsp90 is moderate.

Chiosis's group and Conforma therapeutics/Biogen

Idec optimized this class of compounds leading to

new analogues bearing a thioether bridge to connect

the purine nucleus to substituted phenyl rings.

Among them, the PUH58 (Figure 7) (Llauger et al.,

2005), an 8-arylsulfanyl analogue of PU3, has been

identified as the most potent and selective purine.

Further efforts in optimization of this lead

compound led to the development of PU24F-Cl,

(Figure 7) which presents a higher affinity (30

times more than PU3) for the N-terminus of the

Hsp90, and low micromolar activity in a cell

proliferation assay (Chiosis et al., 2002; Vilenchik

et al., 2004). In an in vivo experiment in MCF-7

tumor bearing mice, PU24FCl led to 70% inhibition

when administered at a dose of 200 mg/kg every

second day for 30 days (Vilenchik et al., 2004).

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Figure 6: Co-crystal structures of ADP (left, in green) and PU3 (right, in magenta) with Hsp90.

Figure 7: Structures of PU24FCl and analogues.

Additional investigations concerning the

pharmacophore of this family were done. It was

demonstrated that the presence of an amino group

on the C2 position of the purine nucleus respects

the global size of the molecule in regard to the

parent one (PU3). In addition, it offers multiple

possibilities for hydrogen bonding, and thus

allowed the connection of the benzyl group on the

N-9 rather than the C-8 of the purine. Thus,

Conforma-Biogen idec have identifed the BIIB021

(originally named CNF 2024, Figure 7) (Kasibhatla

et al., 2007) which displayed a binding affinity of

1.7 nM (4.6 nM for 17-AAG) and induces

degradation of HER2 with an IC50 of 38 nM in

MCF-7 cells (Lundgren et al., 2009). BIIB021

compound was entered in clinical trials in 2005.

2.b) Clinical trial

Currently, BIIB021 is the only purine analogue

evaluated in phase I/II clinical trials in combination

with trastuzumab (herceptin) against breast cancer,

with an aromatase inhibitor (exemestane) in

metastatic HER2-advanced breast cancer, or alone

in subjects with gastrointestinal stromal tumors.

Results from the phase I trials showed that BIIB021

was well tolerated (800 mg twice weekly) and

induced a significant inhibition of the HER2 (Elfiky

et al., 2008).

3) Pyrazole and isoxazole derivatives

3.a) Pyrazole analogues

In 2004, CCT018159 (Figure 8), the first Hsp90

inhibitor in the pyrazole series, was identified by

Workman et al. from a library of 60000

compounds, using a high throughput screening

(HTS), in the Cancer Research UK Centre for

Cancer Therapeutics (Rowlands, 2004). This

compound inhibits the N-terminal ATPase activity

of yeast and human Hsp90 with an IC50 of 7.1 and

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Peyrat JF, et al.


al., 2007a). Further HTS studies undertaken by

Genomics institute of the Novartis Research

Foundation (GNF) and based on time-resolved

fluorescence resonance energy transfer (TR-FRET)

had allowed identifying two leads, G3129 and

G3130 (Figure 8), amongst the one million

molecules screened (Kreusch et al., 2005).

However, both compounds exhibited relatively poor

binding affinity to N-terminal domain of the Hsp90

(Kd=680 and 280 nM respectively). In SkBr3 breast

cancer cells, G3130 caused the degradation of

HER2 (IC50=30 µM) while G3129 was ineffective.

In addition, the co-crystal structures of G3129 and

G3130, bound to the N-terminus of human Hsp90α,

were reported (Kreusch et al., 2005). From this

study, it was showed that the resorcinol ring bound

Hsp90 in a similar way than that of radicicol, a

resorcylic lactone that inhibits Hsp90.

Figure 8: Structure of analogues G3129 and G3130.

Figure 9: Co-crystal structure of G3130 bound to the N-terminus of human Hsp90α.

Figure 10: VER49009 and VER50589.


Peyrat JF, et al.


Furthermore, the 5-ethyl appendage projected into

the aromatic pocket that accommodates the benzyl

group of the purine analogues described before. The

pyrazole provides hydrogen bound acceptor, and

the imidazole (G3130) occupies the same pocket as

the quinone of GA (Figure 9).

Based on these data, medicinal chemistry efforts led

to the identification of the more potent analogue of

CCT018159, the VER49009 (IC50 ATPase

activity=0.14 µM, Figure 10), where the amide

group was a key in forming a new interaction with

the residue Gly97 of the protein (Dymock et al.,

2005).

Synta Pharmaceuticals Corp. had reported another

class of triazoles analogues as modulators of Hsp90

(Figure 11) (Ying et al., 2009). It has been shown

that STA-9090, an unspecified new resorcinol-

containing triazole compound (Lin et al., 2008),

inhibits the activity of Hsp90 protein from 10 to

100 µM and thereby leading to degradation of

Hsp90 client proteins such as HER2 gene product

(Ying et al., 2009). More recently, it had been

shown that the STA-1474, a highly soluble

phosphate prodrug of STA-9090, exhibits very

interesting biologic activity against osteosarcoma

cell lines (McCleese et al., 2009).

Figure 11: Pyrazoles reported by Synta Pharmaceuticals.

3.b) Isoxazole analogues

Further optimization of potency, pharmakinetic and

pharmacodynamic properties of VER49009, were

undertaken by Vernalis Ltd. (in collaboration with

Novartis) to offer a series of isoxazole resorcinol

inhibitors. One of these was the VER50589 (Figure

10) which exhibited a higher affinity (Kd=5 nM)

than VER49009 (Kd=78 nM) (Sharp et al., 2007b).

Thus, the pyrazole to isoxazole switch does not

affect the critical hydrogen bound of the pyrazole

resorcinol unit that anchors this class of inhibitors

to the Hsp90 NH2-terminal ATP site (Brough et al.,

2008). Moreover, VER50589 also showed

improved cellular uptake over VER49009.

Brough et al. from Vernalis, reported recently the

identification of new diarylisoxazole compound

VER52296 (Figure 12) (Brough et al., 2008; Eccles

et al., 2008). The areas of interest for SAR studies

were the 5' positon on the resorcinol ring, and the

para substitution of the phenyl group on the

isoxazole ring. It has been shown with the X-ray

cristal structure, that the replacement of the

chlorine, in regard to VER50589, by an isopropyl

group, results in an additional hydrophobic

interaction with Leu107 in the flexible lipophilic

pocket of the N-terminal site of Hsp90. Additional

hydrophobic interactions were also observed with

Thr109 and Gly135 from the morpholine moiety

present in VER52296/NVP-AUY922 (Figure 12).

This latter, subsequently developed by Novartis,

was found to be very potent in the Hsp90

Fluorescence Polarization binding assay (IC50=21

nM) and displays an average GI50 of 2-40 nM in

antiproliferation assays against different human

tumor cell lines (Brough et al., 2008). In addition,

as evaluated by cassette dosing to mice bearing

subcutaneous HCT116 human colon cancer,

VER52296/NVP-AUY922 was retained in HCT116

xenograft tumors when administered i.p., at

concentrations well above the GI50. Further in vivo

characterization in a human colon cancer xenograft

model, VER52296/NVP-AUY922, also inhibits

tumor growth by ~50% when dosed at 50 mg/kg i.p.

daily. Moreover VER52296/NVP-AUY922 induces

the degradation of HER2 with an IC50 of 7 nM. In

addition, VER52296/NVP-AUY922 was tested in

several xenografts (colon, glioblastoma, breast,

ovarian, prostate) and a therapeutic response was

observed in each case (Cheung et al., 2005; Jensen

et al., 2008; Eccles et al., 2008).

Figure 12: Structure of VER52296/NVP-AUY922.

3.c) Clinical trials

The VER52296/NVP-AUY922 is the sole isoxazole

analogue currently in phase I/II clinical trials as a

single agent or in combination with bortezomib or

dexamethasone, in patients with relapsed or

refractory multiple myeloma. AUY922 is also used

as a single agent, in advanced solid malignancies

and efficacy in HER2+ or ER

+ locally advanced or

metastatic breast cancer patients. In this latter phase

I/II trial, VER52296/NVP-AUY922 was

intravenously administrated once a week schedule.

The maximum dose reported is 54 mg/m2. At 40

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mg/m2, VER52296 induces an up regulation (4-19

fold) of Hsp70 and a 20% reduction in soluble

HER2 was achieved by 74% of patients.

Noteworthy, the STA9090 (Figure 11) is currently

enrolling patients in several phase I/II clinical trials

against solid tumor, myeloid leukemia, non-small

cell lung cancer and gastrointestinal stromal tumor.

4) Dihydroindazolone derivatives

4.a) SNX2112 and SNX5422

In 2007, Serenex Inc./Pfizer had started a phase I

clinical trial program for the SNX5422 (Figure 13).

Using a new screening platform, the compounds

retained on the ATP-affinity column were analyzed

by mass spectrometry leading to the identification

of a poorly soluble analogue, SNX2112, which will

become the glycine prodrug SNX5422. The

SNX2112 showed higher activity to bind Hsp90

(Ki=1 nM) than that of 17-DMAG and induced the

degradation of HER2 with an IC50 of 20 nM. Based

on the observation that breast cancer cell lines with

HER2 amplification are more sensitive to 17-AAG,

the SNX2112 was tested using a panel of breast,

lung, and ovarian cancer cell lines. In all cell lines

studied, SNX2112 inhibited cell proliferation with

IC50 ranging from 10 to 50 nmol/L. In contrast to

17-AAG, the sensitivity of cancer cell lines to

SNX2112 in vitro did not correlate with the level of

HER2 expression (Chandarlapaty et al., 2008;

Huang et al., 2006). This compound uniformly

targets both the pro-proliferation pathways driven

by HER2 and ERK as well as the anti-apoptotic Akt

pathway (Okawa et al., 2009). Indeed, it exhibits

potent in vivo antitumor activity that extends

significantly the effects observed with GA

analogues.

4.b) Clinical trial

SNX-5422 is currently in a phase I clinical trial in

treating patients with solid tumor or lymphoma that

has not responded to treatment. SNX5422 is equally

tested to treat solid tumor cancer and lymphomas,

and, in subjects with refractory hematological and

solid tumor malignancies. In 2008, results of a

phase I dose-escalation study of SNX5422 reported

that this compound was well tolerated at 21 mg/m2.

5) Other inhibitors

Another class of ATP Hsp90 inhibitors bearing a

resorcinol moiety is radicicol (Rd) (Figure 14), a

natural resorcylic lactone, isolated from the fungus

Monocillium nordinii and Monosporium bonorden.

Rd, also known as monorden, has been described to

reverse the Src-transformed morphology of

fibroblast (Whitesell et al., 1994). This effect was

first attributed to the inhibition of the oncogenic Src

(v-Src), and later proved to act as an inhibitor of

Hsp90 despite its difference in structure to GA.

Moreover, Rd was found to compete with GA for

binding to the NTD of the chaperone, suggesting

that Rd shares the geldanamycin binding site. This

compound is a potent and specific inhibitor of the

ATPase activity of Hsp90 with nanomolar affinity

(Kd=19 nM). This causes destabilization of Hsp90

client proteins (v-Src, Raf-1, ErbB2 and Ras), many

of which are essential for tumor cell growth.

Although, the in vitro antitumoral activity of Rd is

very promising however, its in vivo activity is very

weak probably because of its chemical instability in

serum and its rapid conversion into inactive

metabolites due to the electrophilic nature of the

dienone moiety.

Therefore, synthetic efforts have been directed to

identify radicicol derivatives with improved in vivo

activity (Proisy et al., 2006). To date, Kyowa Hakko

described novel oxime-derivatives of Rd, including

KF55823 and KF25706 (Soga et al., 2003) (Figure

14). Although these compounds exhibit potent

antitumor activities in preclinical models and do not

seem to cause hepatotoxicity, their clinical

evaluations of these compounds has not been

pursued.

Figure 13: Structure of SNX5422 and SNX2112.

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Figure 14: Resorcylic inhibitors.

Figure 15: Structures of novobiocin, chlorobiocin and coumermycin A1.

B) C-terminal domain binders

In 1991, Csermely, Kahn and co-workers reported

the presence of a C-terminal ATP binding site on

Hsp90 which becomes accessible when the N-

terminal Bergerat pocket is occupied (Sõti et al.,

2002). A decade later, it has been shown that Nvb

interacts with an ATP-binding domain in the C-

terminus of Hsp90 (Marcu et al., 2000a).

Biochemical studies on the CTD of Hsp90 have

identified an allosteric regulation process with the

N-terminus site, where the occupancy of one site

blocks the interaction of the ligand with the other

site (Garnier et al., 2002). The structure of the full

length and middle and C-terminal construction of

Hsp90 with different nucleotide states (apo, ATP)

have shown that there is a hinge region between the

middle and C-terminal region of Hsp90. The

conformation of this region is dictated by the status

of the nucleotide at the N-terminal site. This

observation is in accordance with the allosteric

regulation of ATP binding. It suggests that the

putative secondary ATP site could be located at the

immediate proximity of the hinge.

IV) Coumarin inhibitors

Coumarin group antibiotics, such as novobiocin

(Nvb), coumermycin A1 (Kd=10 nM) and

clorobiocin (Figure 15), are potent inhibitors of the

bacterial ATP binding gyrase B, a type II DNA

topoisomerase (Gormley et al., 1996). Their affinity

for gyrase is considerably higher than that of

modern fluoroquinolones. These antibiotics have

been isolated from various Streptomyces species

(Lanoot et al., 2002) and all possess a 3-amino-4-

hydroxycoumarin moiety as a key structural feature.

Nvb is licensed as an antibiotic for clinical use

(Albamycin; Pharmacia-Upjohn) and for the

treatment of infections with multi-resistant gram-

positive bacteria such as Staphylococcus aureus and

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Rappa et al., 2000). It had been demonstrated that

the interaction of Nvb with Hsp90 induces

alteration in the affinity of the chaperone for GA

and Rd and causes in vitro and in vivo depletion of

key regulatory Hsp90-dependant kinases including

v-Src, Raf-1 and ErbB2 (e.g., ErbB2 in SkBr3

breast cancer cells ~700 µM). In addition, Nvb was

found to bind the C-terminal nucleotide binding

region of Hsp90, albeit with a lower affinity than

with gyrase B. Moreover, Nvb disrupts the

interaction of both the cochaperones p23 and

HSC70 with the Hsp90 complex.

In 2005, the first attempt to improve the inhibitory

activity of Nvb against Hsp90 was reported (Yu et

al., 2005; Blagg et al., 2006). These authors have

highlighted the crucial role of the noviose moiety at

the 7-position of the coumarin ring for the

biological activity. Compound A4 lacking the 4-

hydroxyl of the coumarin moiety and containing an

N-acetyl side chain in lieu of the benzamide was the

most active compound. This compound was

identified as Hsp90 inhibitor that induced

degradation of Hsp90-dependent client proteins at

70-fold lower concentration than Nvb. Recently, in

continuation of their structural modification studies,

the same authors reported that 3'-descarbamoyl-4-

deshydroxynovobiocin DHN2 (Figure 16) and

compound KU135 (Shelton et al., 2009) proved to

be a more effective and selective Hsp90 inhibitor

(degradation of ErbB2 and p53 between 0.1 and 1.0

µM) (Burlison et al., 2006).

Our group reported a novel series of 3-

aminocoumarin analogues (Le Bras et al., 2007a;

Le Bras et al., 2007b) lacking the noviose moiety as

a class of highly potent Hsp90 inhibitors. A

representative example of this new class of

inhibitors is 4TCNA (Figure 17) (Le Bras et al.,

2007b).

In these analogues, the introduction of a tosyl

substituent on C-4 position of coumarin nucleus

(4TCNA) contributed to a significant extent for

maximal activity despite weaker water solubility.

Moreover, this lead has a particular implication in

apoptotic process. Thus, 4TCNA promotes

apoptosis through activation of caspases 7 and 8 in

ER-positive MCF-7 human breast cancer cells,

whereas in Ishikawa endometrial adenocarcinoma

cells, it induced apoptosis that was associated with

caspase activation and cleavage of PARP.

Furthermore, characterization of its mode of action

revealed that 4TCNA induced-cleavage of the p23,

recently identified as a prostaglandine E2-Syntase,

which plays an important role in activity of a

number of transcription factors of steroids/thyroid

receptors family. These results demonstrate that this

new denoviose compound presents originality in

regard to Nvb osidic derivatives already known.

In another study based on a simplified 3-

aminocoumarin scaffold, we also demonstrated that

4-tosyl-7-deshydroxycyclonovobiocic acid

(4TDHCNA) (Figure 17) (Radanyi et al., 2008),

exhibit increased inhibitory activity against the

Hsp90 protein folding process (MCF7 IC50=50

µM).

This result shows that removal of C7/C8

substituents is not detrimental for Hsp90 inhibitory

activity and strongly enhances the capacity of

4DHTCNA to inhibit Hsp90. This compound was

identified to be the most potent representative of the

new family of simplified coumarins. Results from

this study suggest that 4TDHCNA and 4TCNA,

which exerted similar biological profile may be

considered interesting compounds for the

development of more potent novobiocin analogues.

More recently, results from our group allowed the

identification of a new family of novobiocin

analogues in which the coumarin unit has been

replaced by a 2-quinoleinone moiety (unpublished

results). The quinolone-scaffold represents a

platform for the creation of easily synthesizable

soluble molecules. Compound 4-tosyl-3[(chroman-

6-yl) carboxylamino]-2-quinolon (4TCCQ, Figure

17) (IC50=5-8 µM) is 100-fold more potent than the

parent natural compound (novobiocin) and 6-fold

more active than the synthetic analogue 4TCNA.

Additionally, 4TCCQ induces the degradation of

ERα and strongly induces the cell death in MCF-7

breast cancer cell line.

Overall, these data provides compelling evidence

for the continued development of novobiocin-based

C-terminal domain Hsp90 inhibitors as promising

alternative to N-terminal domain inhibitors.

Figure 16: Structures of A4, DHN2 and KU135.


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Figure 17: Structures of 4TCNA, 4TDHCNA and 4TTCQ.

Figure 18: Tau-Hsp90 in AD.

V) HSP 90 in Alzheimer's disease

Hsp90, a molecular chaperone, has come into its

own as a tantalizing target for cancer therapies.

However, its important functions of stabilization,

rematuration, disaggregation of many client

proteins could be exploitable in others diseases.

Indeed, neurodegenerative diseases are

characterized by the accumulation of misfolded

proteins that results in plaque formation. These

proteins rely upon HSP's for their refolding and

viability. Recently, it was suggested that Hsp90

may play a crucial role in maintaining pathogenic

changes that lead to neurodegenerative diseases

(Luo et al., 2008). Furthermore, the inhibition of

Hsp90 by 17-AAG derivatives and geldanamycin,

induces the HSP induction via HSF-1 activation,

resulting in neuroprotective activities. In the

Alzheimer's disease, the most common tauopathy,

in addition to β-amyloid deposition, there is an

accumulation of abnormal species of

hyperphosphorylated protein tau which leads to the

formation of toxic neurofibrillary tangles (Luo et

al., 2007; Dickey et al., 2007). This

hyperphosphorylation is caused by abnormal

kinases (CdK4, GSK-3β) activities resulting in

dissociation of transformed tau from microtubules,

aggregation and formation of neurofibrillary tangles

which can block the synaptic transmission (Figure

18).

Thus, the decrease of hyperphosphorylated tau

levels through refolding or degradation may

provide a possible therapeutic strategy against AD.

In this purpose, Dickey and Luo have presented

evidence that the stability of p35, (neuronal

activator of CdK4) and P301L mutant (most

common mutation in Alzheimer disease) are

maintaining by Hsp90.

Dou et al. 2007 reported that Hsp90 associates with

GSK-3β, regulating its stability and function,

preventing its degradation by the proteasome and so

allowing the increase of tau hyperphosphorylation.

Thus, the use of Hsp90 inhibitors leads to a

destabilization of GSK-3β and to a decrease of

hyperphosphorylated tau protein.

Dickey et al. 2007 demonstrated that CHIP (a tau

ubiquitin ligase) is intimately linked to tau

degradation following Hsp90 inhibition and that

this process is specific for promoting degradation of

only aberrant phosphorylated tau due to the fact that

the Hsp90 complex, in AD brain, presents higher

affinity for inhibitors than in unaffected brain

tissue.

http://atlasgeneticsoncology.org/Genes/CDK4ID238ch12q14.html

http://atlasgeneticsoncology.org/Genes/GSK3BID40761ch3q13.html


Peyrat JF, et al.


Recently, compound A4 (Figure 16) was found to

exhibit significant protection against the Aβ-

induced toxicity at low concentrations (Lu, 2009).

These results suggest that novobiocin analogues

may represent an effective class of novel

compounds for treatment of AD.

VI) Conclusion

Since the first discovery of natural analogues, GA

and RD, the search for inhibitors of Hsp90 has

generated considerable interest as evidenced by the

number of compounds in clinical evaluations (Table

1).

However, if the first clinical results were very

encouraging, it seems that currently the

development of Hsp90 inhibitors experiencing

some difficulties, especially due to their toxicity.

Stopping clinical trials of IPI504, which

represented the most advanced HSP90 inhibitors, is

the unfortunate illustration of that. Thus, many

efforts are still needed in the understanding of the

administration of these agents but also in the

synthesis of new molecules. Moreover, the

involvement of Hsp90 in other non-oncological

diseases such as Alzheimer's disease shows the

importance of acquiring new and more potent

inhibitors with suitable pharmacological and

pharmacokinetic profiles.

Table 1: Current clinical trials.

Condition: R: recruiting, ANR: active, not recruiting, C: completed, NYR: not yet recruiting, S: suspended, T: terminated.

Therapy: ADL: Advanced Dedifferentiated Liposarcoma, AGC: Advanced Gastric Cancer, AM: Advanced Malignancies, AML: Acute Myeloid Leukemia, AST: Advanced Solid Tumors, BC: Breast Cancer, CLL: B-Cell Chronic Lymphotic Leukemia, CLL:

Chronic Lymphocytic Leukemia, CML: Chronic Myelogenous Leukemia, ALL: Acute Lymphoid Leukemia, MM: Multiple Myeloma, MetM: Metastatic Myeloma, NSCLC: Non Small Cell Lung Cancer, PC: Prostate Cancer, GIST: Gastrointestinal Stroma Tumor,

RRMM: Relapsed or Refractory Multiple Myeloma, ST: Solid Tumors, UST: Unresectable Solid Tumors.


Peyrat JF, et al.


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LDI-PCR in Cancer Translocation Mapping Björn Schneider, Hans G Drexler, Roderick AF MacLeod

DSMZ, German Collection of Microorganisms and Cell Cultures, Department of Human and Animal

Cell Cultures, Inhoffenstr. 7b, 38124 Braunschweig, Germany (BS, HGD, RAFM)


Online updated version : http://AtlasGeneticsOncology.org/Deep/LDI-PCRinCancerID20087.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI LDI-PCRinCancerID20087.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract Identification of genes in oncogenic chromosome translocations by Fluorescence In Situ Hybridization (FISH)

screening using genomic tilepath clones, is often laborious, notably if the region of interest is gene-dense. Other

molecular methods for partner identification also suffer limitations; for instance, genomic PCR screening

requires prior knowledge of both sets of breakpoints, while Rapid Amplification of cDNA Ends (RACE) is

limited to translocations causing mRNA fusion and delivers no breakpoint data. With Long Distance Inverse

(LDI)-PCR, however, it is possible to identify unknown translocation partners and to map breakpoints at the

base-pair level. Applying LDI-PCR merely requires approximate sequence information on one partner, rendering

it ideal for use in combination with FISH to extend and refine cytogenetic breakpoint data.

Introduction Recurrent chromosomal rearrangements

characterize many different types of cancer.

Specific cytogenetic translocations are key events,

widely considered to be diagnostically and

prognostically significant in leukemia and

lymphoma, and increasingly so in solid tumors

(Mitelman et al., 2007). Hitherto, most cancer

genes have been identified following analysis of

recurrent chromosome translocations (Futreal et al.,

2004). The pathological significance and usefulness

of such rearrangements depend on two key features:

a) whether rearrangements display distinct patterns

of recurrence within specific tumors, e.g.

t(8;14)(q24;q32) which is restricted to B-cell

neoplasia; and b) how clustered are the

chromosomal breakpoints therein. The significance

accorded to breakpoint data depends on their

ascertainment precision, from megabase- and

kilobase-, down to single base-pair levels, when

ascertained by classical cytogenetics, fluorescence

in situ hybridization (FISH), and sequence-based

methods, respectively.

Chromosome translocations fall into three broad

categories. The first causes the physical fusion of

the two mRNAs expressed by the participating

genes, thus creating novel fusion proteins translated

from exons emanating from both genes, e.g. BCR

(at [chromosome]-22-[band]-q11) with ABL1 (at

9q34) fused by t(9;22)(q34;q11) in chronic myeloid

leukemia (CML) and in some cases of acute

lymphoblastic leukemias (ALL) (Turhan, 2008a,b).

The second category also fuses mRNA from genes

at separate loci but, in this case, serving to

deregulate a developmentally silenced partner by

exchanging promoters with more active partners,

e.g. BCL6 (at 3q27) which is activated by

translocations with any one of many partners,

chiefly in diffuse large B-cell lymphoma (DLBCL)

(Knezevich, 2007).

The third class of chromosome translocation again

results in the activation of the normally silent

partner, this time by juxtaposition with another

constitutively active partner without mRNA fusion,

e.g. the neighboring homeobox genes, TLX3 (at

5q35.1) and NKX2-5 (at 5q35.2). According to the

proximity of the breakpoint involved, either (but

not both) genes may be activated in T-cell ALL by

the recurrent t(5;14)(q35;q32.2) by which these are

juxtaposed with regulatory regions from BCL11B

http://atlasgeneticsoncology.org/Anomalies/t0814ID1050.html

http://atlasgeneticsoncology.org/Genes/BCR.html

http://atlasgeneticsoncology.org/Genes/ABL.html

http://atlasgeneticsoncology.org/Genes/BCL6ID20.html

http://atlasgeneticsoncology.org/Anomalies/DLCLID2076.html

http://atlasgeneticsoncology.org/Genes/TLX3ID398.html

http://atlasgeneticsoncology.org/Genes/NKX25ID42958ch5q35.html

http://atlasgeneticsoncology.org/Anomalies/t0514q35q32ID1386.html

http://atlasgeneticsoncology.org/Genes/BCL11BID392.html

LDI-PCR in Cancer Translocation Mapping Schneider B, et al.


(at 14q32.2) to stimulate transcription (Bernard et

al., 2001; MacLeod et al., 2003; Nagel et al., 2007).

Some "promiscuous" genes engage with multiple

partners: notably MLL with 64 known partners

(Meyer et al., 2009b), BCL6 with 28 (Knezevich,

2007), RUNX1 with 39 (Huret and Senon, 2003),

NUP98 with 29 (Kearney, 2002), and the IgH-locus

with 40 (Lefranc, 2003).

Although, promiscuity may reflect the dependence

of tumors on the inappropriate oncogene expression

without overly caring how deregulation is

accomplished, the role of the partner genes has

come under renewed scrutiny. Choice of partner

gene may not only reveal in which types of

precancerous cells primary oncogenic

rearrangements occur, but also by looking for

conserved DNA or protein motifs, yield clues to the

mechanisms underlying their formation or their

functional contribution to neoplasia. In addition, the

roles of biologically important genes, e.g. BCL11B,

a key regulator of both differentiation and survival

during thymocyte development, are often first

rendered visible by their participation in cancer

rearrangements (MacLeod et al., 2003).

Both the identities of each partner gene and their

precise breakpoints at the DNA base-pair level,

may be useful not only to characterize potential

fusion genes/products but also to ascertain whether

additional non protein-coding genomic entities,

such as chromosomal fragile sites (Schneider et al.,

2008), microRNA loci, putative genes, unspliced

"expressed sequence tags", or regulatory non-

coding regions may be involved.

Clues to the biological mechanisms generating

chromosome rearrangements are given by

breakpoint sequences, including T- and B-cell

receptor gene (VDJ) rearrangement, Alu-mediated

recombination, non-homologous end joining, etc.

VDJ genes are flanked by recombination signal

sequences composed of heptamers followed, in

turn, by a spacer containing either 12 or 23

unconserved nucleotides and a conserved nonamer.

Spacers of 12 nucleotides undergo physiological

recombination with those containing 23 in order to

obey the so-called "12/23 rule". The presence of

these or related sequences has been reported in

connection with cancer translocations to reveal how

physiologic processes may be abused to cause

genomic rearrangements (Gu et al., 1992).

Genomic fusion sequences may also be used to aid

cell line authentication - an omnipresent problem

confronting cell culturists, given that an

unexpectedly (and unacceptably) high percentage of

new cell lines has been misidentified, or cross-

contaminated by older cell lines (MacLeod et al.,

1999). While mRNA fusion sequences are

constrained by splicing, their genomic equivalents

allow sufficient variation to provide "fingerprints"

unique to individual cell lines to serve as potential

identifiers. For analyzing patient tumors,

knowledge of the exact fusion sequence allows

design of patient-specific quantitative (q)PCR used

for monitoring minimal residual disease with high

sensitivity (Burmeister et al., 2006), to follow up

therapeutic responses thus enabling early detection

of relapse.

Figure 1: Amplifying Genomic Fusions of Unknown Sequence. The schema summarizes how the genomic DNA is first restricted, then re-ligated to the circular template, and how the resulting amplicon should appear. Note unknown region (red) flanked by

known sequences (black). R: restriction site, BP: breakpoint; arrows: forward (FW) and reverse (REV) primers.

http://atlasgeneticsoncology.org/Genes/MLL.html

http://atlasgeneticsoncology.org/Genes/AML1.html

http://atlasgeneticsoncology.org/Genes/NUP98.html

http://atlasgeneticsoncology.org/Genes/IgHID40.html



Cancer gene promiscuity enables oncogenic

chromosome rearrangements, "smoking guns" of

cancer genes, to be distinguished from random

changes. FISH is initially used to confirm

rearrangement of a contextually appropriate

oncogene residing at the locus in question. Hence, a

breakpoint at 9q34 might throw suspicion onto

NOTCH1 in a T-cell-, ABL1 in myeloid- neoplasia,

and NUP214 in either entity. While such an

approach is less helpful among solid tumors where

oncogene rearrangements are less informative, at

this locus TSC1 might be deemed a candidate in

tuberous sclerosis cells. Even when the index

breakpoint is precisely known, determination of its

partner by FISH requires time-consuming and

laborious procedures for those not afforded

immediate blanket tilepath-clone coverage with

which to quarter the region of interest.

PCR screening with hit-lists of known and potential

partner genes is quite as laborious as FISH, and is

liable to miss unknown translocation partners, or

those with breakpoints lying outside their respective

cluster regions. When there are grounds to suspect

transcriptional fusion (as among partners of genes

prone to this type of gene rearrangement, e.g.

ABL1, ETV6, NUP98, etc.) a mRNA-based

method, rapid amplification of cDNA ends

(RACE), may be used to detect novel fusion

partners (Frohman et al., 1988). A drawback of

RACE is its inability to supply genomic breakpoint

data, and the risk of overlooking some splice

variants. Hence, the technique of choice for

identifying unknown partner genes and their

breakpoints should not require prior knowledge of

the partner gene, yet provide breakpoint data at the

DNA base pair level.

Long Distance Inverse (LDI)-PCR satisfies these

needs. LDI-PCR was developed from the earlier

inverse-PCR (Ochman et al., 1988) to allow the

amplification of large DNA fragments comprised of

known and unknown sequences (Willis et al., 1997)

using re-ligated circular restriction fragments as

templates. Primers are set in opposition within the

known sequence. The unknown sequence is flanked

on both sides by known sequences following re-

ligation in the resultant amplicon (Fig. 1). When a

restriction fragment length polymorphism (RFLP)

distinguishing the wild type and derivative alleles is

generated by the genomic alteration, the two

resulting amplicons should be separable by gel

electrophoresis, enabling their respective sequences

to be compared (Fig. 2). Sequencing with one of the

PCR-primers directed towards the restriction site

allows immediate identification of the partner gene.

Sequencing in the other direction allows precise

mapping of the breakpoint.

Figure 2: How to Interpret LDI-PCR Gels. Left figure shows the wild type configuration where twin circular templates identical in size would yield a single band by agarose gel electrophoresis. Translocation bearing cells (right figure) yield both wild type and

derivative templates, differing in size and detectible as two bands on the gel. The derivative band is indicated by an arrow. Known regions are outlined in black, unknown in red. R: Restriction site, REV: reverse primer, FW: forward primer.

http://atlasgeneticsoncology.org/Genes/NOTCH1ID30ch9q34.html

http://atlasgeneticsoncology.org/Genes/CAN.html

http://atlasgeneticsoncology.org/Genes/TSC1ID183.html

http://atlasgeneticsoncology.org/Genes/ETV6ID38.html



While easier to perform for genes with well

defined, short breakpoint cluster regions, LDI-PCR

may be applied to any gene or region involved in a

translocation and has, therefore, been applied to a

wide variety of translocations involving both

frequently rearranged promiscuous oncogenes, but

also as single events. Table 1 gives an overview of

genes analyzed by LDI-PCR according to literature

databases and the respective references.

Limitations are set by the performance of the DNA

polymerase since lengthier fragments may resist

amplification, and by the placement of the RFLP, as

fragments similar in size cannot be readily

distinguished by gel electrophoresis. If primary

patient tumor material is analyzed, it should be

noted that the samples used for analysis not only

contain tumor material, but also normal bystander

cells devoid of tumor rearrangement. Detection

attempted at lower tumor infiltration rates risk false

negative results.

In contrast to other PCR methods suitable for

detection of unknown fusion sequences, such as

panhandle PCR (Megonigal et al., 2000) or

analogous techniques requiring adaptor ligations

(reviewed in Tonooka and Fujishima, 2009), LDI-

PCR is independent of any additional adaptors or

anchors which have to be ligated to the restricted

fragments, reducing the number of steps required,

while remaining sufficiently flexible to allow a

wide choice of restriction enzymes.

In the future, translocation analysis by next

generation sequencing should overcome these

limitations and suitable algorithms have been

developed to recognize novel derivative breakpoint-

flanking sequences and thereby identify novel

cancer translocations and other synonymous

rearrangements, including a subset of fusogenic

microdeletions (Campbell et al., 2008).

Methology In principle, LDI-PCR utilizes digested and re-

ligated circular templates, which are of different

sizes, due to RFLP caused by genomic

rearrangements. This size difference renders the

amplicons separable by gel electrophoresis (Fig. 2).

For a successful analysis, the LDI-PCR has to be

designed carefully. The sequence covering the

genomic region of interest should be selected from

a genome browser (ENSEMBL, UCSC, NCBI) and

then pasted into the query box of a restriction map

generator (BioEdit, multiple online tools: SMS,

RestrictionMapper). Restriction enzymes should be

chosen to yield fragments in a size range of 2-5 kb.

If using a double-digest strategy with enzymes

producing sticky ends, these ends must be

compatible. Ensure that both enzymes perform well

in the same buffer and at the same temperature.

Primer pairs have to be designed in such a way that

one primer is directed towards the restriction site,

the other one in the opposite direction (see Figures).

The sequence lying between the primer tails is not

subject to amplification, so the gap should not be

excessive, ideally 30-50 bp. A breakpoint lying

therein cannot be detected unless another primer

pair, e.g. at the other end of the restriction fragment

is used. For longer fragments (greater than 5 kb,

say) use of a primer set consisting of one forward

and multiple reverse primers (or vice versa) can be

helpful. The oligonucleotides should be ~30 bp

with a Tm ~65°C and a GC-content of 40-60%.

For LDI-PCR template preparation high quality

genomic DNA should be used, meaning high purity

(260/280 1.8-2.0 and 260/230 > 2) and high

integrity without degradation. One microgram of

DNA is then digested with 30-50 U of each

restriction enzyme in the presence of the

appropriate digestion buffer in a total volume of

100 μl for 3-4 h at the temperature suitable for the

chosen enzymes (mostly 37°C), followed by heat

inactivation (where applicable) and purification,

preferably with a column based purification kit.

Phenol / chloroform purification followed by

precipitation may also be performed, but residual

phenol can disturb downstream processes. To form

the circular templates, the restriction fragments are

then religated with 5 U T4 ligase overnight at 4-8°C

in a total volume of 80 μL, terminated by heat

inactivation. These conditions favor the desired

self-ligation.

The PCR is performed best using a PCR kit suitable

for amplification of long templates and using 5 μL

(62.5 ng) of the digested and re-ligated DNA. The

PCR products are analyzed by gel electrophoresis.

Discrepant bands not corresponding to the

calculated amplicon size may represent amplicons

of translocated fragments. These are excised from

the gel, purified and subjected to sequence analysis

and, unless artefacts, may reveal the translocation

partner and the exact breakpoint of the

rearrangement subject to analysis.

http://www.ensembl.org/

http://genome.cse.ucsc.edu/

http://www.ncbi.nlm.nih.gov/gene/

http://www.mbio.ncsu.edu/BioEdit/bioedit.html

http://www.bioinformatics.org/sms2/rest_map.html

http://www.restrictionmapper.org/



Gene No. of Partners References describing LDI-PCR analysis

ALK 14 (Allouche, 2010) Ma et al., 2000

ANTXR1 1 (Oberthuer et al., 2005) Oberthuer et al., 2005

API2 1 (Mathijs and Marynen, 2001) Dierlamm et al., 1999

BCL6 28 (Knezevich, 2007)

Akasaka H et al., 2000; Akasaka T et al., 2000; Kurata et

al., 2002; Akasaka et al., 2003; Chen et al., 2003;

Montesinos-Rongen et al., 2003; Chen et al., 2006;

Schneider et al., 2008

BRD4 1 (Collin, 2007) Haruki et al., 2005

CALL 1 (Frints et al., 2003) Frints et al., 2003

E2A 5 (Huret, 1997) Wiemels et al., 2002a

ETV6 28 (Knezevich, 2005) Wiemels et al., 1999a; Wiemels et al., 1999b; Wiemels

and Greaves, 1999; Wiemels et al., 2008

IGH 40 (Lefranc, 2003)

Willis et al., 1997; Willis et al., 1998; Nardini et al., 2000;

Satterwhite et al., 2001; Sonoki et al., 2001; Bichi et al.,

2002; Sanchez-Izquierdo et al., 2003; Sonoki et al., 2004;

Akasaka et al., 2007; Lenz et al., 2007; Souabni et al.,

2007; d'Amore et al., 2008; Ishizaki et al., 2008; Russell et

al., 2008; Vieira et al., 2008; Vinatzer et al., 2008; Nagel

et al., 2009; Russell et al., 2009; Yin et al., 2009; Hu et al.,

2010

let-7a-2, miR-100 1 (Bousquet, 2008) Bousquet et al., 2008

MLH1 1 (Meyer et al., 2009b) Meyer et al., 2009a

MLL 64 (Meyer et al., 2009b)

Blanco et al., 2001; Meyer et al., 2005; Teuffel et al.,

2005; Attarbaschi et al., 2006; Burmeister et al., 2006;

Matsuda et al., 2006; Meyer et al., 2006a; Meyer et al.,

2006b; Strehl et al., 2006; Burmeister et al., 2008;

Balgobind et al., 2009; Bueno et al., 2009; Burmeister et

al., 2009; Matsuda et al., 2009; Meyer and Marschalek,

2009; Cóser et al., 2010; De Braekeleer et al., 2010; Lee et

al., 2010

NOTCH1 2 (Suzuki et al., 2009) Suzuki et al., 2009

PAX5 6 (Strehl, 2005) An et al., 2008; An et al., 2009

PDGFRA 6 (Dessen, 2009) Cools et al., 2003

PDGFRB 20 (Vizmanos, 2005) Walz et al., 2007; Walz et al., 2009

RUNX1 / AML1 39 (Huret and Senon, 2003) Xiao et al., 2001; Wiemels et al., 2002b

Table 1: Genes involved in translocations analyzed by LDI-PCR, numbers of yet known translocation partner genes and

references wherein the analysis of the particular gene is described.

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Case Report Section Paper co-edited with the European LeukemiaNet




Unbalanced rearrangement der(9;18)(p10;q10) in a patient with polycythemia vera Xinjie Xu, Xueyan Chen, Elizabeth A Rauch, Eric B Johnson, Kate J Thompson, Jennifer

JS Laffin, Gordana Raca, Daniel F Kurtycz

University of Wisconsin-Madison, School of Medicine and Public Health, Department of Pediatrics,

University of Wisconsin Cytogenetic Services, Wisconsin State Laboratory of Hygiene, Madison, WI,

USA (XX, JJSL); University of Wisconsin-Madison, Department of Pathology and Laboratory

Medicine, Madison, WI, USA (XC); University of Wisconsin Cytogenetic Services, Wisconsin State

Laboratory of Hygiene, Madison, WI, USA (EAR, EBJ, KJT); University of Wisconsin-Madison,

School of Medicine and Public Health, Department of Pathology and Laboratory Medicine, University

of Wisconsin Cytogenetic Services, Wisconsin State Laboratory of Hygiene, Madison, WI, USA

(GR); University of Wisconsin-Madison, School of Medicine and Public Health, Department of

Pathology and Laboratory Medicine, Wisconsin State Laboratory of Hygiene, Madison, WI, USA

(DFK)


Online updated version : http://AtlasGeneticsOncology.org/Reports/der0918XuID100044.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI der0918XuID100044.txt This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Clinics

Age and sex

69 years old female patient.

Previous history

No preleukemia ; no previous malignancy ; no

inborn condition of note.

Organomegaly

no hepatomegaly , splenomegaly , no enlarged

lymph nodes , no central nervous system

involvement (there was no apparent central nervous

system involvement at diagnosis).

Blood WBC : 15.2X 10

9/l

HB : 11.7g/dl

Platelets : 894X 109/l

Blasts : 0% peripheral

Bone marrow : 2% blasts

Cyto-Pathology Classification

Cytology

NA

Immunophenotype

NA

Rearranged Ig Tcr

NA

Diagnosis

Polycythemia vera

Survival

Date of diagnosis: 03-2005

Treatment: Phlebotomy

Complete remission : NA

Treatment related death : no

Relapse : NA

Status: Alive. Last follow up: 03-2010

Survival: 62 months

Karyotype

Sample: Bone marrow biopsy Sep 17th 2009.

Culture time: analysis was performed on overnight

colcemid and 24-hour cultures.

Banding: 400 band level.

Unbalanced rearrangement der(9;18)(p10;q10) in a patient with polycythemia vera

Xu X, et al.


Results:

46,XX,+9,der(9;18)(p10;q10)[11]/46,XX[9]

Karyotype of a metaphase from the follow up specimen

from September 2009 (four years after the initial diagnosis), 24-hour culture.

FISH confirmation for an extra copy of 9p.

Comments

We describe a new case of der(9;18)(p10;q10)

detected in a patient with polycythemia vera (PV).

This rare rearrangement has been reported in five

cases of PV and one case of therapy associated

acute myeloid leukemia (t-AML) after essential

thrombocythemia (ET). Two of the five cases of PV

showed progression from PV to post-polycythemic

fibrosis, suggesting an association between this

cytogenetic abnormality and disease progression.

The patient presented in this report was diagnosed

with PV in 2005. Fluorescence In Situ

Hybridization (FISH) testing for the BCR/ABL

translocation was performed at diagnosis and was

negative. Subsequent molecular analysis detected

the presence of the JAK2 V617F mutation. The

patient had a bone marrow biopsy in September of

2009, due to worsening anemia which was at that

time attributed to excessive phlebotomy.

Cytogenetic analysis showed the presence of the

der(9;18)(p10;q10) in eleven out of twenty

analyzed cells. At the later follow-up visit in

February 2010, progression to the spent phase of

PV was suspected, based on the worsening of the

patient's clinical presentation. However, the next

bone marrow biopsy from March 2010 only

revealed mildly increased reticulin fibrosis. In

summary, although the patient currently does not

have pathohistological signs of progression,

clinically she is exhibiting worsening of the disease.

Gain of function of the JAK2 gene at 9p24 has a

crucial role in the pathogenesis of

myeloproliferative neoplasms. It has been proposed

that the der(9;18)(p10;q10) contributes to the

pathogenesis of PV through the gain of 9p, leading

to an extra copy of the JAK2 gene. For the patient

presented in this report the clinical significance of

the der(9;18)(p10;q10) cannot be fully interpreted

due to the absence of the cytogenetic results at

diagnosis. However, concurrence between the

detection of this cytogenetic abnormality and

worsening of the patient's symptoms suggests that

the der(9;18)(p10;q10) may have been an early

marker of the disease evolution.

Gain of 9p resulting from +i(9)(p10) has been

reported in two cases of PV, further indicating this

gain as a recurrent finding in PV. Our patient is

known to carry the activating JAK2 V617F

mutation. One can hypothesize that in combination

with this mutation, gain of an extra copy of either

the mutated or the normal JAK2 allele through

formation of the der(9;18)(p10;q10) contributed to

the progression of the patient's disease. Our report

therefore further suggests the association between

the unbalanced rearrangement der(9;18)(p10;q10)

and an advanced stage of polycythemia vera.

References Chen Z, Notohamiprodjo M, Guan XY, Paietta E, Blackwell S, Stout K, Turner A, Richkind K, Trent JM, Lamb A, Sandberg AA. Gain of 9p in the pathogenesis of polycythemia vera. Genes Chromosomes Cancer. 1998 Aug;22(4):321-4

Bacher U, Haferlach T, Schoch C. Gain of 9p due to an unbalanced rearrangement der(9;18): a recurrent clonal abnormality in chronic myeloproliferative disorders. Cancer Genet Cytogenet. 2005 Jul 15;160(2):179-83

Ohyashiki K, Kodama A, Ohyashiki JH. Recurrent der(9;18) in essential thrombocythemia with JAK2 V617F is highly linked to myelofibrosis development. Cancer Genet Cytogenet. 2008 Oct;186(1):6-11


Xu X, Chen X, Rauch EA, Johnson EB, Thompson KJ, Laffin JJS, Raca G, Kurtycz DF. Unbalanced rearrangement der(9;18)(p10;q10) in a patient with polycythemia vera. Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1):114-115.



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