03.29.12 - slu phd admissions seminar
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Cadm1, an Inherited Modifier of Metastasis that Suppresses Metastasis by Interacting with the Cell Mediated Immunity
Farhoud FarajiKent Hunter
March 29, 2012
Agenda
1. Introduction 2. Genetic analysis reveals a locus on mouse Chr9
that is associated with metastasis3. Validating Cadm1 as a metastasis-associated
gene4. Elucidating the mechanism of Cadm1-mediated
metastasis suppression
Breast Cancer
• Most common malignancy in women
• In 2011: ~230,000 cases~40,000 deaths (#2 killer after lung)
• >90% of deaths are related to metastatic disease
Cancer.orgHunter & Crawford. Cancer Res. 2006
Metastasis Biology
Valastyan & Weinberg. Cell. 2011
Metastasis Biology
Valastyan & Weinberg. Cell. 2011
Highly Complex Process•Numerous Gene Expression Programs•Tumor Phenotypic Transitions•Niche Remodeling•Intercellular Communication
Endpoint: Reaching and adapting to foreign environment
Where is potential for metastatic susceptibility encoded?
The Genetic Background
Definition:– Complement of genetic variation that
distinguishes each of us as an individual
More specifically:– Germline polymorphisms
• SNP, Indel, CNV
Why do some breast cancer patients develop metastatic disease, whereas
other patients, with seemingly similar tumors, don’t?
Our Question
Does the genetic background play a role in metastasis?
MMTV-PyMT Transgenic mouse•Mammary tumors
100% penetrance (9wks)•Pulmonary metastasis
>90% penetrance (100days)•FVB inbred background•Luminal-like, ER+
Credit: Robert Cardiff, UC Davis Transgenic Mouse Webpage http://www-mp.ucdavis.edu/tgmice/firststop.html
The Experiment: Does the genetic background play a role in metastasis?
DadMom
Constants: Oncogenic driver (PyMT)Paternal genotype (FVB)
Variable: Maternal genotype
The Observation:
Altering the Maternal Genotype Results in a Non-binary Phenotype
Metastasis has a heritable component independent of the oncogenic driver
Phenotypic continuum implicates the interactions of two or more genes in the metastatic process
Therefore:• Metastasis can be considered a complex trait
• Genetic tools can be used to identify genomic elements involved in metastatic susceptibility
Our System
Two approaches to investigate metastatic breast cancer
1. Identify candidate genes – associated with metastasis • Genetic screen
2. Validate – are candidate genes causative?
• In vivo metastasis assay
1) Outcross between inbred strains of varying metastatic propensity
2) Cross to FVB-PyMT3) Determine phenotype and genotype4) Which segments of genome segregate with metastasis?
Identification by Forward Genetic Screen
Mvt-1 and 6DT1 – murine mammary tumor cell lines•Driver: Myc•Pulmonary metastasis
>90% penetrance (30 days)•Luminal-like, ER+
In vivo metastasis assay•Orthotopic graft of isogenic murine mammary tumor cells
•Immune-competent
Validation
Agenda
1. Introduction 2. Genetic analysis reveals a locus on mouse Chr9
that is associated with metastasis3. Validating Cadm1 as a metastasis-associated
gene4. Elucidating the mechanism of Cadm1-mediated
metastasis suppression
FVB: Metastasis-Prone
C58 and NZB: Metastasis-Resistant
Genetic Screen
NZB Metastasis QTL C58 Metastasis QTL
x
FVB/N-PyMTNZB or C58
P
F1
x
N2
Measure: Primary Tumor Burden Pulmonary Metastases
Genotype: Determine segments of genome segregating with metastasis
NZB/C58 + FVB Backcross Reveals QTL peak on Chromosome 9
Subcongenic Analysis Resolves Chr. 9 Susceptibility Locus to 49-67Mb
* p < 0.05
Subcongenic Tumor Burden Subcongenic Metastasis
Hundreds of genes on Chr 9 49-67Mb
Filters• Haplotype structure: [C58=NZB] ≠ FVB• Differentially expressed between NZB and FVB
Candidate list• Cadm1, Pias1, Zbtb16
Agenda
1. Introduction 2. Genetic analysis reveals a locus on mouse Chr9 that
is associated with metastasis3. Validating Cadm1 as a metastasis-associated gene4. Elucidating the mechanism of Cadm1-mediated
metastasis suppression
Exon Sequencing Reveals Synonymous SNP in Exon 2
524 586530 540 550 560 570« NZB(524)
« FVB(520)
Consensus(524)
rs327213609
Cadm1 is Differentially Expressed in FVB and NZB Mice
Mammary Tumor Normal Whole Lung
p = 0.076 *
* p < 0.05(NZB Chr9)
Level of Cadm1 Expression Predicts Survival in Patient Data Sets of Breast Cancer
GOBO: ER-positive Tumors
anti-V5
anti-Cadm1
anti-β-actin
6DT1 Vector
6DT1 Cadm1-V5
Mvt-1 Vecto
r
Mvt-1 Cadm1-V5
Ectopic Expression of Cadm1 in Mvt1 and 6DT1
Overexpression is within physiological range.
** p < 0.01
Cadm1 Expression Reduces Pulmonary Metastasis in vivo
6DT1 results demonstrate a metastasis-specific role for Cadm1
Cadm1 Expression Reduces Pulmonary Metastasis in vivo
Metastases from Cadm1 Expressing Primary Tumors Do Not Express the Cadm1 Transgene
Down-regulation of Cadm1 may be prerequisite for metastasis formation.
Stable Knockdown of Cadm1 by shRNA
Cadm1
β-actin
6DT1 shSc
rambled
6DT1 shCadm1 14
6DT1 shCadm1 15
Primary Tumor Burden
Tum
or M
ass
(g)
0.0
0.5
1.0
1.5
*
Tumor-Normalized Metastases
Met
asta
ses
Per
Gra
m T
umor
6DT1 Scr
6DT1 shRNA 14
6DT1 shRNA 150
50
100
150
* * *
Pulmonary Metastases
Sur
face
Met
asta
sis
Cou
nt
0
20
40
60
80
100
* * p = 0.07
Cadm1 Knockdown Promotes Pulmonary Metastasis
* p < 0.05
** p < 0.01
Taken together, results confirm a causative role for Cadm1 in metastasis.
Agenda
1. Introduction 2. Genetic analysis reveals a locus on mouse Chr9
that is associated with metastasis3. Validating Cadm1 as a metastasis-associated
gene4. Elucidating the mechanism of Cadm1-mediated
metastasis suppression
Cadm1 Initially Identified as the Gene Underlying Locus Frequently Deleted in Cancer
• Observations:– 11q23 is deleted in NSCLC– Introduction 11q23.2 “completely suppresses
tumor formation”• Cadm1 was identified as the responsible gene
Murakami et al. PNAS. 1998Kuramochi et al. Nat Genet. 2001
Loss of Cadm1 Associated with Poor Outcome in Numerous Solid Cancers
Melanoma:– Loss/down-regulation (by promoter methylation)
• Increased tumor stage• Significantly shorter disease-free survival
Similar results in:-Neuroblastoma, Meningioma-Nasopharyngeal, Esophageal, Gastric, HCC-Pancreatic, Prostate, NSCLC-Ovarian, Cervical
You et al. Melanoma Res. 2010Murakami. Cancer Sci. 2005
Cadm1 is an adhesion molecule
• Single-pass integral membrane protein• Homotypic cell-cell adhesion
– Epithelial structure – adherens jn, hemidesmosome
• Heterotypic cell-cell adhesion– Immunological synapse– Cell Differentiation
• Synaptogenesis• Spermatogenesis• T-Lymphocyte Maturation Hagiyama et al. J Immunol. 2011
Wakayama et al. Anat Sci Int. 2009Ito et al. Hepatology. 2007
Sakurai-Yageta et al. Biochem Biophys Res Commun. 2009
Could Cadm1 Suppress Metastasis by Regulating Motility or Invasion Properties?
• 4.1 Binding Motif– Dal-1 – Tumor suppressor– Ezrin – Metastasis associated
• PDZ Binding motif– Tiam1 – Metastasis associated
• Rac GEF – involved in motility
Busam et al. JBC. 2011Wong et al. PNAS. 2007
Fujita et al. Am J Pathol. 2007
Cadm1 Expression: • No significant impact on tumor cell in vitro
properties:– In vitro Growth– In vitro Motility– In vitro Invasion– 2D and 3D Morphology
Summary of Cadm1 Effect onIn Vitro Properties
Metastasis Biology
Valastyan & Weinberg. Cell. 2011
•Effect of Cadm1 expression on metastasis might be distinct from early steps of metastatic cascade
21 days
Does Cadm1 Expression Have an Effect on Late Events of the Metastatic Cascade?
Cadm1 Reduces Pulmonary Metastasis of Tail Vein Injected Tumor Cells
Pulmonary Metastases
Sur
face
Met
asta
sis
Cou
nt
Mvt-1 Vector
Mvt-1 Cadm1
6DT1 Vector
6DT1 Cadm1-20
0
20
40
60
* p < 0.05
* *
•Metastasis inhibitory effect of Cadm1 is not restricted to the early stages of metastasis.
Extracellular ligand:– CRTAM - expressed on activated CTLs
• increased secretion of IFNγ and IL-2 by activated CD8+ T-cells
• Enhanced NK-cell cytotoxicity Galibert et al. J Biol Chem. 2005Boles et al. Blood. 2005
Cadm1 and Immunity
NK cellsNKT cells
CD8+ T-cellsTumor Cell
Could Effects of Cadm1 on Metastasis be Mediated by the Immune System?
Nude mouse– FoxN1 null– Athymic– Immunophenotype
• Mature T-cells – Absent• B-cells – Present • NK-cells, APCs – Present and Functional
Cadm1 Expression Has No Effect on Metastasis or Tumorigenesis in Athymic Mice
ImmuneCompetentHost:
Primary Tumor Burden
Tu
mo
r M
ass
(g
)
Mvt
-1 V
ecto
r
Mvt
-1 C
adm
1
6DT1
Vecto
r
6DT1
Cadm
10.0
0.5
1.0
1.5
Pulmonary Surface Metastases
Met
asta
sis
Co
un
t
Mvt
-1 V
ecto
r
Mvt
-1 C
adm
1
6DT1
Vecto
r
6DT1
Cadm
10
20
40
60
80
AthymicHost:
Hypothesis
1) CD8+ CTL-mediated tumor killing2) NK-cell recruitment, activation, and tumor killing3) IFN-γ-mediated tumor cytostaticity and/or killing
Tumor Cell CD8+ T-cell
CRTAMCadm1
IFN-γIL-2
MHCITCR
CD8
Kuipers et al. Blood. 2011Giangreco et al. J Immunol. 2012
Two Primary Questions:
1. Are CD8+ T-Cells Involved in Cadm1-Mediated Metastasis Suppression?
2. Are either IL-2 or IFN-γ involved?
CD8+ Cell Depletion in Immune Competent Mice • Study in Progress• Design:
_____IgG_____ ___anti-CD8___ Mvt1 Mvt1 Mvt1 Mvt1
Vector Cadm1 Vector Cadm1
1) Are CD8+ T-Cells Involved inCadm1-Mediated Metastasis Suppression?
30 days
Fat Pad Injection
Harvest: •Lung•Lymph Nodes
Make Single Cell Suspension of Living Cells
2) Do mice bearing Cadm1+ tumors show increased IFN-γ secreting lymphocytes?
2) Do mice bearing Cadm1+ tumors show increased IFN-γ secreting lymphocytes?
anti-IFN-γ
anti-CD3
IFN-γ ELISPOT – Draining Lymph Nodes
6DT1 Vector
6DT1 Cadm1
Mvt1 Vector
Mvt1 Cadm1
Tumor cells expressing Cadm1 may induce lymphocytes in draining lymph nodes to secrete IFN-γ secretion
Future Plans
• If CD8+ T-cell depletion rescues metastatic phenotype:– Do CD8+ T-cells directly induce tumor cytotoxicity?
• In vitro co-culture cytotoxicity assay by Cr51 release+/- Crtam blocking antibody+/- IFN-γ blocking antibodyELISA on supernatant for IL-2, IFN-γ
• Is Crtam involved?– Cadm1 orthotopic transplant in Crtam KO mouse
Future Plans
• If CD8+ T-cell depletion does not rescue metastatic phenotype: – CD4+ T-cell, NKT-cell depletion– Investigate role of stromal FoxN1 in Cadm1-
mediated metastasis suppression• Sort tumor stromal cells and check FoxN1 expression
– BMDC, CAF, Endothelial cells, etc.
SummaryCadm1:1. Is an inherited modifier of metastatic risk in mice2.Is a metastasis suppressor in mice3.Metastasis suppression may involve host cell-
mediated immunity4.Expression levels in human tumor samples
predicts prognosis
ConclusionTumor-autonomous expression of Cadm1 impacts
tumor metastatic capability through non-tumor-autonomous mechanisms.
Cadm1 may sensitize tumor cells to immune surveillance and result in lymphocyte-mediated cytotoxicity.
Implications
The preponderance of data in the literature linking promoter hypermethylation of Cadm1 to advanced stage may indicate a critical role for loss of Cadm1 expression in cancer immunoediting.
AcknowledgementsDr. Glenn Merlino• Dr. Chi-Ping Day• Dr. Raza Zaidi
Dr. Lalage Wakefield• Dr. Yuan Yang
Dr. Li Yang• Dr. Yanli Pang
Dr. Kent Hunter• Dr. Jude Alsarraj• Dr. Ling Bai• Dr. Natalie Goldberger• Dr. Luanne Lukes• Renard Walker• Dr. Scott Winter• Dr. Thos Geiger
Thanks for your attention
Questions?
Thanks for your attention
Questions?
Cadm1 localizes to cell-cell junctionsMvt1 Vector
Mvt1 Cadm1
6DT1 Vector
6DT1 Cadm1
No Significantly Impact on Cell Morphology or Actin Stress Fibers
Mvt1 Cadm1
Mvt1 Vector
6DT1 Cadm1
6DT1 Vector
No Significant Change in 3D Culture Growth Properties in
Mvt-1 Vector Mvt-1 Cadm1
6DT1 Vector 6DT1 Cadm1
No Significant Change in Motility in vitro
•Mvt1 Vector•Mvt1 Cadm1
•6DT1 Vector•6DT1 Cadm1
Tumor Cells
Matrigel-Coatedmembrane
Chemoattractant Rich Media
Chemoattractant Depleted Media
Transwell Migration and Invasion Assay
TranswellInsert
No Significant Change in in vitro Migration or Invasion by Transwell Assay
Cadm1 Expression Does Not Impact Tumor Cell Proliferation in vitro
•Mvt1 Vector•Mvt1 Cadm1
•6DT1 Vector•6DT1 Cadm1
IFN-γ ELISPOT – Lung
6DT1 Vector
6DT1 Cadm1
Mvt1 Vector
Mvt1 Cadm1
p=0.027 p=0.003
Level of Cadm1 Expression Predicts Survival in Patient Data Sets of Breast Cancer
524 586530 540 550 560 570« NZB(524)
« FVB(520)
Consensus(524)
rs327213609
Exon Sequencing Reveals Synonymous SNP in Exon 2
IiExonIntron Intron
IiExon
PCR AmplifyTOPO Clone
Sanger Sequencing
IF CD8+ T-Cells play a major role, expect:
Mvt1 Mvt1 Mvt1 Mvt1 Vector Cadm1 Vector Cadm1 IgG anti-CD8
1) Are CD8+ T-Cells Involved inCadm1-Mediated Metastasis Suppression?
Met
asta
ses
Implications
Schreiber et al. Science. 2011
Cadm1’s Divergent Role in Hematogenous Malignancies
Myeloid & Lymphoblastic Leukemia:– High expression - better survival
T-cell Leukemia and Lymphoma (ATLL, HTLV)– High expression – poor prognosis
• Increased tumor infiltration• More aggressive tumors
Kuipers et al. Blood. 2011Paulson et al. Br J Haematol. 2009
Nakahata et al. Leukemia. 2012
Chromosome 9 Metastasis QTL
NZB
C58