mazvita maziveyi and suresh k. alahari · mazvita maziveyi and suresh k. alahari abstract tumor...

Review

Breast Cancer Tumor Suppressors: A SpecialEmphasis on Novel Protein NischarinMazvita Maziveyi and Suresh K. Alahari

Abstract

Tumor suppressor genes regulate cell growth and preventspontaneous proliferation that could lead to aberrant tissuefunction. Deletions and mutations of these genes typically leadto progression through the cell-cycle checkpoints, as well asincreased cell migration. Studies of these proteins are importantas theymay provide potential treatments for breast cancers. In thisreview, we discuss a comprehensive overview on Nischarin, anovel protein discovered by our laboratory. Nischarin, or imida-zoline receptor antisera-selected protein, is a protein involved in a

vast number of cellular processes, including neuronal protectionand hypotension. TheNISCH promoter experiences hypermethy-lation in several cancers, whereas some highly aggressive breastcancer cells exhibit genomic loss of the NISCH locus. Further-more, we discuss data illustrating a novel role of Nischarin asa tumor suppressor in breast cancer. Analysis of this new para-digm may shed light on various clinical questions. Finally, thetherapeutic potential of Nischarin is discussed. Cancer Res; 75(20);4252–9. �2015 AACR.

IntroductionBreast cancer initiation and progression involve several genetic

events that can activate oncogenes and/or abrogate the function oftumor suppressor genes. Tumor suppressor genes are commonlylost or deleted in cancers, facilitating the initiation and progres-sion of cancer through several biological events, including cellproliferation, cell death, cellmigration, and cell invasion.Usually,cancer mortality occurs due to complications of metastasis ratherthan the mass effect of the primary tumor, and several tumorsuppressors regulate metastasis. Genetic modifications throughallelic loss are one of the important factors for deregulation oftumor suppressor genes. Importantly, promoter hypermethyla-tion of several tumor suppressors has been shown to be associatedwith tumor progression. In addition, several signaling mechan-isms are dysregulated in breast cancer as a result of mutations inthese genes. Among the tumor suppressors, BRCA1/2, p53, PTEN,ATM, Rb, LKB, Nm23, and p16 have been studied in great detailand discussed inmany review articles (1–3). This article primarilyemphasizes the novel tumor suppressor Nischarin (Fig. 1) andhow it regulates cell migration, cell invasion, tumor growth, andmetastasis through various signaling pathways and interactionswith other proteins. Caretaker genes, such as BRCA1, are geneswhose loss does not directly inhibit tumor growth (1, 4, 5).Nischarin imposes its tumor-suppressive functions through itsinteractions with other proteins; thus, it is a caretaker tumorsuppressor gene. For example, it interacts with p21 activatedkinase 1 (PAK1) and integrin a5 to prevent cell migration

(6, 7). It also interacts with LIM kinase (LIMK) in order to preventcytoskeletal reorganization (8). Typically, scaffold proteins suchas Nischarin are characterized as caretaker genes because theireffects on tumor growth are indirect.

Discovery of NischarinThe first function attributed to Nischarin was its role as an

integrina5b1 binding protein (9). In this way,Nischarin is able toregulate cell motility, specifically by anatomization of cell signal-ing proteins that contribute to tumor cell migration and invasion(10). The structural and functional domainsofNischarinpromoteits interaction with 17 known proteins to influence cell adhesion,cell migration, vesicle trafficking, apoptosis, glucose metabolism,and cell signaling. Thus far, diseases associated with the NISCHgene include hypertension, xerostomia, morphine dependence,depression, anxiety, ventricular hypertrophy, congestive heartfailure, rosacea, several cancers (11). Its location at 3p21.1 putsit in a category of tumor suppressor genes that are associated withthe development of many cancers (12). Highly aggressive breastcancer cells frequently exhibit genomic loss of the NISCH locus(12), whereas the NISCH promoter is hypermethylated in lungcancers (13). Nischarin mRNA and protein expression is high instage 0 human breast specimens but reduced in stage I–IV breastcancer specimens (12).

NISCH RegulationThe structural and functional domains of Nischarin

Nischarin was first characterized 15 years ago by SK Alahariand colleagues (10). The 37,955 bases of the full-length NISCHgene are regulated by the transcription factors max1, sp1,COUP, olf-1, COUP-TF, pax-4a, ATF, and c-Myc (11). Soonafter, the human homolog of Nischarin was discovered asimidazoline receptor antisera-selected protein (IRAS; ref. 14).Human IRAS has 80% homology with rodent Nischarin butinterestingly, the integrin a5-binding sites of Nischarin and

Department of Biochemistry and Molecular Biology, Louisiana StateUniversity Health Sciences Center, New Orleans, Louisiana.

Corresponding Author: Suresh K. Alahari, Louisiana State University HealthSciences Center, 1901 Perdido Street, New Orleans, LA 70112. Phone: 504-568-4734; Fax: 504-568-2093; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-1395

�2015 American Association for Cancer Research.

CancerResearch

Cancer Res; 75(20) October 15, 20154252

on October 17, 2020. © 2015 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 21, 2015; DOI: 10.1158/0008-5472.CAN-15-1395

http://cancerres.aacrjournals.org/

IRAS are 100% identical (Fig. 2; ref. 14). In humans, Nischarin/IRAS was first discovered as an I1-imidazoline receptor, whichare expressed in both neurons and astrocytes (14, 15). Humanand mouse Nischarin differ in the alanine/proline rich region,which is removed in human Nischarin (Fig. 2).

Nischarin is a cytosolic protein that anchors itself to the innerlayer of the plasma membrane and has been found to interactwith both cytosolic and intermembrane proteins (16). HumanNischarin has four isoforms that are achieved by alternativesplicing (9). Isoform 1 encodes the full-length protein and ishighly expressed in neural and endocrine tissue (Fig. 2; ref. 9).Isoform 2 has amino acids 1–511 spliced and is expressed inthe brain (9). Isoform 3, also known as IRAS-L, is highlyexpressed in the brain, missing amino acids 584–1504, andhas a modified sequence in amino acids 511–583 (9). Isoform4, also known as IRAS-S, is also highly expressed in the brain,has amino acids 516–1504 spliced out, and has a change inamino acids between 512 and 515 (9). Isoform 1 is 166,629Da, isoform 2 is 110,194 Da, isoform 3 is 63,997 Da, andisoform 4 is 56,867 Da (9).

Mouse Nischarin has seven isoforms that are achieved byalternative splicing (Fig. 2; ref. 17). Isoform 1 is full-lengthNischarin, which is 1,593 amino acids (17). Isoform 2 ismissing amino acids 348–500 and isoform 3 is missing aminoacids 1–245 (17). Amino acids 437–472 of isoform 4 differfrom the canonical sequence and it is also missing amino acids473–1593 (17). Amino acids 332–334 of isoform 5 have asequence difference and amino acids 335–1593 are missing(17). Isoform 6 also has a sequence difference between 513 and

516, as well as amino acids 517–1593 missing (17). Isoform 7has a long amino acid sequence difference between 122–153and amino acids 143–1593 missing (17).

Furthermore, the N-terminus of Nischarin contains a phox(PX) domain from amino acids 11–121 (Fig. 1; ref. 18). ThisPX domain is necessary for plasma membrane and vesiculartargeting of Nischarin (19). Both Nischarin's PX domain, andits coiled-coil domain (634–695) are essential for endosomaltargeting and interaction with phosphatidylinositol 3-phosphatePI3P in endosomes enriched in this phospholipid (Fig. 1; ref. 19).Though the interaction of PI3P and Nischarin alone is notsufficient for endosomal targeting, this interaction occurs aroundregion 2–133 of Nischarin. Although regions 120–695 are nec-essary for Nischarin to be targeted to the endosomes, mutation ofamino acids 49 and 50 inhibit endosomal targeting (18).

Interestingly, other regions of Nischarin have been found tointeract with other signaling molecules as well. For example,amino acids 1–624 of Nischarin have been found to stronglyinteract with p21 activated kinase 1 (PAK1) to prevent cellmigration (Fig. 1; ref. 6). Positions 416–624 of Nischarin aresufficient to interact with LIMK in order to prevent cytoskeletalreorganization (Fig. 1; ref. 8). LKB1 interacts with positions 416–624 of Nischarin to prevent cancer progression (Fig. 1; ref. 20). Inaddition, Rab14 and insulin receptor substrates 1–4 interact withthe c-terminal domain of Nischarin (Fig. 1; refs. 21, 22). Amongthe known binding partners of Nischarin, its interaction withintegrin a5 is the best characterized.

Integrins are cell adhesion proteins with a and b transmem-brane heterodimers that play a major role in transmitting

© 2015 American Association for Cancer Research

Rac1PAK1

Serine phosphorylation

Tyrosine phosphorylation

Threonine phosphorylation

Ubiquitination

Acetylation

LIMKLKB1

Rac1

Rab 14IRS 1–4

PXNH2

1 300

Leucine-Rich repeats IntegrinBinding

Alanine/proline

rich COOHGlutaminerich

Coiled-Coil

600 900 1504

PI3PPI3P

Figure 1.The predicted post-translational modifications of Nischarin. Ubiquitination of Nischarin is predicted to occur at K1009, K1015, K1290, K1299, and K1303. Acetylationis predicted to occur at K1015. The predicted human Nischarin phosphorylation sites are S246, S250, T252, S477, S541, S546, S883, S1022, S1038, T1282,S1284, Y1293, Y1294, and Y1307. Both Nischarin's PX domain, and its coiled-coil domain are essential for endosomal targeting and interaction withphosphatidylinositol 3-phosphate (PI3P) in PI3P-enriched endosomes. Amino acids 1–624 of Nischarin strongly interact with p21-activated kinase 1 (PAK1).LKB1 interacts with positions 416–624 of Nischarin. Insulin receptor substrates 1–4 interact with the C-terminal domain of Nischarin. Positions 416–624 ofNischarin are sufficient to interact with LIMK. Residues 464 to 562 of Nischarin interact with the integrin a5 cytoplasmic tail. Rab14 interacts with Nischarin'sC-terminus. Both the N- and C-terminus of Nischarin interact with Rac1.

Tumor Suppressor Nischarin

www.aacrjournals.org Cancer Res; 75(20) October 15, 2015 4253




signals from outside of the cell membrane to the inside of thecell and vice versa. The cytosolic portion of the transmembraneheterodimer interacts with a number of cytoskeletal proteinsand signaling molecules, including Ras and MAPK (21). Extra-cellular signals trigger signaling cascades that modulate cellbehaviors such as cytoskeletal remodeling. The membraneproximal region of the integrin a5 subunit has been shownto interact with Nischarin to inhibit cell migration (Fig. 3;refs. 7, 10). Residues 464 to 562 of Nischarin interact withresidues 1017 to 1030 of the a5 cytoplasmic tail, also known asthe membrane proximal region (12). More specifically, Tyr1018 and Lys1022 are crucial points for this a5–Nischarin

interaction (7). When Nischarin is overexpressed, a5 integrinpromoter activity decreases (12). The exact mechanism ofNischarin downregulation of a5-integrin is unknown (12). Ithas been hypothesized that the leucine zipper domain ofNischarin interacts with other leucine zipper-containing tran-scription factors to influence the gene expression of integrin a5(12). Increased expression of integrin a5b1 has been linked toreduced tumor growth rates, regulation of muscle cell growth,and reduced apoptosis (23). Because Nischarin has been foundto regulate the expression of other proteins, it is important tocharacterize its domains and interacting partners in order tobetter understand global tissue expression patterns. On the


NH21

COOH1504

COOH1593

COOH1441

PX Leucine-rich

NH21

NH21

PX Leucine-rich

Coiled-Coil

Glutaminerich

Integrinbinding

NH21

NH21

PX Leucine-rich

COOH153

NH21

PX

COOH472

NH21

PX Leucine-rich

Coiled-Coil

Glutaminerich

Integrinbinding

COOH992

COOH583

NH21

PX Leucine-rich

COOH515

Coiled-Coil

Glutaminerich

Integrinbinding

Alanine/proline

rich

NH21

COOH516

NH21

PX Leucine-rich

COOH1259

Leucine-rich

Coiled-Coil

Glutaminerich

Integrinbinding

Alanine/proline

rich

PX LRR

COOH334

NH21

PX LRR

Coiled-Coil

Glutaminerich

Integrinbinding

Alanine/proline

rich

1

2

3

4

5

6

7

Human Mouse

Figure 2.The structural domains of human and mouse Nischarin. The human homolog of Nischarin was discovered as IRAS. Human Nischarin has 80% homology withrodent Nischarin. Human Nischarin has four isoforms and mouse Nischarin has seven isoforms that are all achieved by alternative splicing.

Maziveyi and Alahari

Cancer Res; 75(20) October 15, 2015 Cancer Research4254




basis of Nischarin interaction with several proteins, we believeit functions as a scaffolding protein.

Nischarin tissue expressionIn cell lines, Nischarin mRNA levels are highest in multiple

rodent neuronal, epithelial, and fibroblast cell lines (10). Further-more, its expression levels have been noted in humans, rodents,chicken, lizards, zebrafish, cows, dogs, opossums, chimpanzees,platypus, sea squirts, fruit flies, mosquitos, worms, and the Africanclawed frog (11). Specifically,Nischarin expressionhas been foundin the embryo, diencephalon, hindbrain, midbrain, future spinalcord, hypothalamus, neocortex, ganglionic eminence, hippocam-pus, cerebellum, spinal cord, meninges, choroid plexus, basalganglia, amygdale, cerebral cortex, brainstem, olfactory bulb, ret-ina, capmesenchyme, renal interstitiumgroup,ovaries,primarysexcord, female and male associated reproductive structures, heart,liver, lung, metanephros, and skeletal muscle (Table 1; ref. 24). Inthe breast, Nischarin expression is normal in stage 0 breast speci-mensbut reduced in stage I–IVbreast cancer specimens (12).Giventhat thederegulationofNischarin is a frequent eventunderlying thedevelopment ofmultiple diseases, understanding themechanismscontributing to its regulation is imperative.

The predicted post-translational modifications of NischarinProtein ubiquitination is important for many biologic process-

es, including immunity and cell differentiation (25). It has beenlinked to many disease progressions, including metabolic syn-

dromes, muscle dystrophy, cancer, neurodegeneration, autoim-munity, and inflammatory diseases (25). Ubiquitination ofNischarin is predicted to occur at K1009, K1015, K1290,K1299, and K1303 (Fig. 1; ref. 26). Acetylation is predicted tooccur at K1015 (Fig. 1; ref. 26). Lysine conjugation has been foundto target large macromolecular complexes involved in processessuch as nuclear transport, actin nucleation, chromatin remodel-ing, cell cycle, and splicing (27). Protein phosphorylation isimportant and necessary for protein signal transduction. Thepredicted human Nischarin phosphorylation sites are S246,S250, T252, S477, S541, S546, S883, S1022, S1038, T1282,S1284, Y1293, Y1294, and Y1307 (Fig. 1; ref. 26).

Nischarin Participates in Insulin SignalingThe carboxy terminus of Nischarin has been found to bind to

the carboxy terminus of insulin receptor substrate 4 (IRS-4; Fig. 1;ref. 22). The IRS family is a family of adaptor proteins that arerecruited and phosphorylated after insulin binds to and activatesthe insulin tyrosine receptor kinase (IR). Phosphorylated IRS isactivated and participates in a number of signaling cascades thathave growth andmitogenic effects (22). Overexpression of humanNischarin in human embryonic kidney 293 cells induces a 4-foldincrease of insulin-stimulated activation of ERK (22). The tyrosinephosphorylationof the IRS-4 andSrchomology2domain-contain-ing (shc) initiates this ERK activation in response to insulin (22).IRS and shc then bind to growth factor receptor-bound protein


Focal adhesions

EGF

EGF

EGFR

RAS

Cofilin

P

P P

MLCK

LIMK

PAK1

ERK

LKB1

Nischarin

ERK

Rac1

NF-κBMEK

FAK

Cell attachment

CytoskeletalremodelingTumor growth

cell survival

Nucleus

Migration

α5B1Integrins

Figure 3.The tumor-suppressive function ofNischarin. Nischarin decreases FAKphosphorylation levels due todecreased a5-integrin expression.Reduced FAK phosphorylation thusprevents ERK activation via Ras andMEK, resulting in decreased cellsurvival. Nischarin andLKB1 interact toreduce tumor growth, regulate cellmigration,metastasis, and anchorage-independent growth. Nischarindirectly interactswith PAK1 to preventits kinase activity and decreasecytoskeletal remodeling. PAK targetscell migration signaling pathwaysthrough MLCK and LIMK.Phosphorylation of LIMK through PAKinhibits cofilin, an actin severingprotein, thus leading to actin filamentassembly. Nischarin binds to thekinase domain of active LIMK todeactivate it and to prevent actinfilament assembly during cytoskeletalreorganization.






2 (Grb2) for activation (22). Grb2 then associates with Son ofsevenless homolog (Sos), the guanine nucleotide exchange proteinfor Ras (22). Sos elevates the GTP-bound form of Ras, whichresults in increased ERK activation and then cell growth (22).ERK inactivation prevents cell survival, growth, and differenti-ation of the cancerous cells (12). A weaker interaction is alsoseen between Nischarin and IRS-1, IRS-2, and IRS-3 (22).

Alternatively, the tyrosine phosporylation of IRS4promotes theactivation of the PI3K signaling cascade. PI3K is a key kinase thatplays a role in themitogenic andmetabolic effects of insulin (28).After PI3K activation, the substrates PI(3,4,5)P3, PI(3,4)P2, and PI(3)P recruit PI3K-dependent serine/threonine kinases (PDK1)and Akt to the plasma membrane for activation (28). ActivatedAkt regulates a number of cellular processes, including the acti-vation of the Glut 4 transporter that participates in glucose uptake(28). Akt also activates p70s6k, a serine/threonine kinase thatregulates protein synthesis (29). Akt activation also induces

synthesis of SREBP 1 and 2 to promote fatty acid synthesis(30). Taken together, this shows that Nischarin participates ininsulin signaling to increase cell survival.

Nischarin in the BrainNischarin is highly expressed on the leading edge of neurons

(31). Silencing of Nischarin in both rat and mouse neurons hasbeen found to increase neuronal migration (31). Nischarin isexpressed by mature neurons to prevent them from furthermigration (31).Neuronalmigration is necessary in the embryonicperiod for normal brain formation (31). Because Nischarin hashigher expression in layers IV–V of the cortex, it is expressed bymature neurons that no longer need to migrate (31).

Nischarin is a neuroprotective proteinNischarin has been found to induce neuronal apoptosis

through the PI3K and protein kinase B (PKB) pathways. Lipo-polysaccharide (LPS) is a proinflammatory component found onthe outer membrane of Gram negative bacteria (32). Uponinjection of LPS, proinflammation factors are produced andapoptosis occurs downstream of the release (32). One studyshowed that upon intracerebroventricular injection of LPS intomale Sprague–Dawley rats, Nischarin levels progressivelyincreased for one day then gradually decreased from 3 to 7 days(32). Colocalization was also seen between Nischarin, Bcl-2–associated death promoter (BAD), and pAKT, which indicatesan upregulation in the PI3K/AKT pathway (32). These resultsdemonstrate that Nischarin is a neuroprotective protein (32).

Nischarin levels are increased in the amygdala in response toanxiety

During an anxiety response, the genes participating in thesynthesis of neurotransmitters are typically up regulated (33).There are also a number of genes involved in signal transductionthat are upregulated (33). Abetter understanding of themolecularmechanisms of anxiety disorders can lead to the discovery ofmoreeffective drugs. The amygdala has long been found to regulateemotional behavior and vigilance (33). It processes the input ofemotional stimuli and influences the output of the behavioralresponse (33). Cat odor exposure to rats induces a behavioralresponse consistent with anxiety (33). Rho GTPase-activatingprotein and Rho-specific guanine nucleotide exchange factor arepresent in the amygdala of a normal rat because of their involve-ment in the guidance of growth cones (33). After cat odorexposure to the rats, the proteins involved in that pathway wereno longer detected (33). Also, a 1.2-fold increase was seen in theexpression of Nischarin during the anxiety response (33). It isproposed that during an anxiety response, Nischarin inhibits theactivity of the Rho GTPase pathway (33). Nischarin is alsoassociated with morphine dependence, depression, and braindisease (11), suggesting that it is an important regulator in thebrain.

Nischarin Plays a Role in HypotensionThe generation of pERK1/2 in the rostral ventrolateral

medulla (RVLM) has implications in I1R-activated hypotension(34). In PC12 cells, this I1R activation depends on nischarinto generate pERK1/2 levels (34). Nischarin knockdown in theRVLM abolishes I1 receptor activation in the RVLM and

Table 1. Nischarin RNA tissue expression


Major tissueSystem

Internal organs Small intestineColonAdipocyteKidneyLiverLung

Bone marrowWhole bloodWhite blood cellsLymph nodeThymus

BrainCortexCerebellumRetinaSpinal cord

OvaryUterusPlacentaProstateTestis

PancreasThyroidSalivary glandAdrenal glandBreastSkin

Immune system

Nervous

Reproductive system

Secretory system

NOTE: Nischarin RNA is expressed in the small intestine, colon, adipocytes,kidneys, liver, lungs, bone marrow, whole blood, white blood cells, lymphnodes, thymus, brain, cortex, cerebellum, retina, spinal cord, ovaries, uterus,placenta, prostate, testis, pancreas, thyroid, salivary glands, adrenalglands, breast, and skin.






produces a hypotensive response (34). Rilmendine, a drug usedto treat hypertension, elicits its hypotensive effects by increas-ing the production of pERK1/2 in the RVLM (34). Centraladministration of rilmenidine was attenuated by rats that hadreduced Nischarin expression, compared with the control(34). Rats with Nischarin antisense ODNs abrogated I1R acti-vation by abolishing pERK1/2 levels and hypotensive respon-ses (34). These findings indicate that Nischarin participates inpERK1/2-mediated hypotension.

Nischarin in CancerNischarin is a tumor suppressor of ovarian cancer

Nischarin expression is downregulated inovarian cancer tissues(35). This decreased expression of Nischarin is associated withinvasiveness, tumor stage, lymph node metastasis, and histologictumor grade (35). Interestingly, ovarian cancer patients withNischarin expression have a better overall survival than non-expressing patients (35). TheNISH promoter is hypermethylatedin 36.7% of ovarian cancers (35). Overexpression of this tumorsuppressor in an ovarian cancer cell line imposes a G1 phase arrestand cyclin D1 downregulation, which leads to decelerated cellproliferation (35). It has been shown that Nischarin exerts itstumor-suppressive effects through FAK inovarian cancer (35). Therole of Nischarin in ovarian cancer has recently been discoveredand further studies are needed to understand the tumor-suppres-sive effect of Nischarin in ovarian cancer.

Nischarin is a tumor suppressor of breast cancerNischarin is known to control cell migration by antagonizing

the actions of cell signaling proteins that contribute to tumor cellmigration and invasion (10). This protein maps at 3p21.1 and ithas been shown that regions of chromosome 3p are associatedwith the development of many cancers (12). Highly aggressivebreast cancer cells exhibit genomic loss of the NISCH locus andNischarin promoter methylation is seen in 30% of breast cancers(12). A study of 962 human breast cancer patients from TCGArevealed that 0.3% of breast invasive carcinomas exhibit a dele-tion of Nischarin, and 0.7% of breast invasive carcinomas have amutated Nischarin (36).

Loss of Nischarin plays a significant role in breast cancer cellprogression. Correspondingly, Nischarin mRNA is highlyexpressed in normal breast tissue but poorly expressed in humanbreast cancer specimens (12). Highly invasive breast cancer celllines such as MDA-MB-231 exhibit low Nischarin expressionlevels, moderately invasive breast cancer cell lines such asMCF-7 exhibit higher Nischarin expression levels, and nontu-morigenic cells such as MCF-10A have the highest amount ofNischarin expression (12). Restoring Nischarin expression inaggressive breast cancer cell lines decreases focal adhesion kinase(FAK) phosphorylation levels due to decreased a5-integrinexpression (12). Reduced FAK phosphorylation thus preventsERK activation, resulting in decreased cell survival (12).

Analysis of human breast cancer patient tumors revealed thattissues with lymph node metastasis have significantly decreasedlevels of Nischarin than patients without lymph node metastasis(37). In addition, LOH studies performed using microsatellitemarkers in DNA samples from 18 human breast cancers and theirnormal tissue counterparts, revealed that LOH is seen in 50% ofhuman breast cancer patients, which results in decreasedNischarin expression (12). Therefore, promoter methylation and

LOH are the leading causes of reduced Nischarin expression inbreast cancers (12).

Nischarin inhibits cell migration and invasionNischarinhas been found to interactwith anumber of signaling

proteins such as integrin a5, PAK1, LIMK1, Rab14, LKB1, andRac1. Liver kinase B1 (LKB1) is a tumor suppressor that has a rolein cell polarity and the regulation of metabolism through themTOR pathway (38). Like Nischarin, LKB1 inhibits PAK phos-phorylation to prevent actin filament assembly during cytoskel-etal reorganization (20). LKB1 interacts with amino acids 416–624 ofNischarin to increase its kinase activity (20). Nischarin andLKB1 both reduce tumor growth, regulate cell migration, metas-tasis, and anchorage-dependent growth (Fig. 3; ref. 20).

Rac1 is a Rho GTPase family member that regulates a numberof signaling pathways, including the organization and assemblyof actin in response to the extracellular environment. Severaldownstream effectors exert the biologic effects of Rac1. Rac1activates NF-kB transcription factors, leading to inflammatoryresponses, cell growth, and apoptotic suppression (39). Both theN- and C-terminus of Nischarin interacts with Rac1 in its activestate to disrupt the NF-kB pathway and thus repress cyclin D1, thepromoter associated with malignancy (Fig. 3; ref. 40). Also,reduced activation of Rac1 by Nischarin results in reduced tumorgrowth (12).

Nischarin has been found to inhibit the Rac1 effector, p21activated kinase (PAK), to prevent Rac1 driven cell migration(41). Nischarin directly interacts with PAK1 and prevents itskinase activity (Fig. 3; ref. 40). PAK targets cell migrationsignaling pathways through myosin light chain kinase (MLCK)and LIMK (40). The activation of MLCK through PAK phos-phorylates myosin light chain, which regulates actin cyto-skeletal dynamics (40). LIMK is highly expressed in cancercells and has been highly regarded as an oncogene (8). Phos-phorylation of LIMK through PAK inhibits cofilin, an actinsevering protein, thus leading to actin filament assembly (41).Nischarin binds to the kinase domain of active LIMK to deac-tivate it and prevents actin filament assembly during cyto-skeletal reorganization (Fig. 3; ref. 8).

Nischarin also interacts with a number of signaling pro-teins to inhibit apoptosis (12). In PC12 and Cos7 cell lines,Nischarin has been found to inhibit the activation of caspase-3,a critical apoptosis mediator (42). Upon staurosporine andthapsigargin treatment, Nischarin-transfected cells showdecrease in apoptotic activity (42). This inhibition of apoptosisleads to increased cell survival. It is not clear how Nischarin caninhibit tumor growth as well as apoptosis, which needs furtherinvestigation.

Therapeutic Importance of NischarinNischarin's strong tumor-suppressive effect can be used to

reverse the invasive capacity of cancer cells,making it an appealingoption in developing future cancer treatments. It has been welldemonstrated that the loss of Nischarin leads to increased focaladhesions, cytoskeletal organization, cell migration, tumorgrowth, and cell survival. Increasing expression of Nischarin intumor cells would reduce the invasive and migratory capacity ofcancer cells. Also, because LOHwas seen in 50% of human breastcancer patients, Nischarin could be used as a clinical biomarkerfor patients.






To compensate for lost or reduced Nischarin function intumors, it is possible to create peptide-based drugs that mimicNischarin's natural interactions. Peptide-based drugs are advan-tageous for high potency, high selectivity, and low toxicity treat-ment (43). Designing a small peptide-based drug that mimics thedifferent domains of Nischarin could decrease the migratoryeffects of the cancer cells. For example, designing a peptide drugwith the amino acids of Nischarin that are necessary to bindintegrina5would decrease integrina-5–mediated cell migration.

In conclusion, Nischarin has been shown to be an importantprotein in the maintenance of normal cell function, and itsdysfunction is widely implicated in human disease. Its expressionis most evident within cells of the immune, nervous, secretory,muscular, and reproductive systems (11). A role for Nischarin inmany cell processes has now been described, but its most well-characterized functions involve its regulation of cell migration,

which it achieves by interacting with a number of proteins. Over30 research papers have been published on Nischarin and furtherstudies of this proteinwill increase current knowledge in the fieldsof cancer biology, cellmigration, apoptosis, vesicle trafficking, celladhesion, signal transduction, and hypertension. This dynamicscaffolding protein is ideal for studying cell signaling pathways ina vast number of diseases. A deeper understanding of Nischarin-mediated pathways will help predict disease progression andprovide better therapeutic targets for breast cancer patients.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Received May 21, 2015; revised July 2, 2015; accepted July 2, 2015;published OnlineFirst September 21, 2015.

References1. Oliveira AM, Ross JS, Fletcher JA. Tumor suppressor genes in breast

cancer: the gatekeepers and the caretakers. Am J Clin Pathol 2005;124:S16–28.

2. Varma AK, Brown RS, Birrane G, Ladias JA. Structural basis for cell cyclecheckpoint control by the BRCA1-CtIP complex. Biochemistry 2005;44:10941–6.

3. Zilfou JT, Lowe SW. Tumor suppressive functions of p53. Cold SpringHarbPerspect Biol 2009;1:a001883.

4. Yu V. Caretaker Brca1: keeping the genome in the straight and narrow.Breast Cancer Res 2000;2:82–5.

5. Deng CX, Scott F. Role of the tumor suppressor gene Brca1 in geneticstability and mammary gland tumor formation. Oncogene 2000;19:1059–64.

6. Alahari SK, Reddig PJ, Juliano RL. The integrin-binding protein Nischarinregulates cell migration by inhibiting PAK. EMBO J 2004;23:2777–88.

7. Alahari SK, Nasrallah H. A membrane proximal region of the integrinalpha5 subunit is important for its interaction with nischarin. Biochem J2004;377:449–57.

8. Ding Y, Milosavljevic T, Alahari SK. Nischarin inhibits LIM kinase toregulate cofilin phosphorylation and cell invasion. Mol Cell Biol 2008;28:3742–56.

9. Piletz JE, Deleersnijder W, Roth BL, Ernsberger P, Zhu H, Ziegler D. IRASsplice variants. Ann N Y Acad Sci 2003;1009:419–26.

10. Alahari SK, Lee JW, JulianoRL.Nischarin, a novel protein that interactswiththe integrin alpha5 subunit and inhibits cell migration. J Cell Biol2000;151:1141–54.

11. Gene Cards. Nisch Gene 2015 [cited 2015 Jul 16]. Available from: http://www.genecards.org/cgi-bin/carddisp.pl?gene¼NISCH.

12. Baranwal S, Wang Y, Rathinam R, Lee J, Jin L, McGoey R, et al. Molecularcharacterization of the tumor-suppressive function of nischarin in breastcancer. J Natl Cancer Inst 2011;103:1513–28.

13. Ostrow KL, Hoque MO, Loyo M, Brait M, Greenberg A, Siegfried JM,et al. Molecular analysis of plasma DNA for the early detection of lungcancer by quantitative methylation-specific PCR. Clin Cancer Res 2010;16:3463–72.

14. Dontenwill M, Pascal G, Piletz JE, ChenM, Baldwin J, Ronde P, et al. IRAS,the human homologue ofNischarin, prolongs survival of transfected PC12cells. Cell Death Differ 2003;10:933–5.

15. Keller B, Garcia-Sevilla JA. Immunodetection and subcellular distributionof imidazoline receptor proteins with three antibodies in mouse andhuman brains: effects of treatments with I1- and I2-imidazoline drugs.J Psychopharmacol 2015:1–17

16. NCBI. NISCH 2015 [cited 2015 Jul 16]. Available from: http://www.ncbi.nlm.nih.gov/gene/11188.

17. UniProt. Q80TM9 (Nisch_Mouse) 2015 [cited 2015 Jul 16]. Availa-ble from: http://www.uniprot.org/uniprot/Q80TM9.

18. UniProt. Q9Y2I1 2015 [cited 2015 Jul 16]. Available from: http://www.uniprot.org/uniprot/Q9Y2I1.

19. Lim KP, Hong W. Human Nischarin/imidazoline receptor antisera-selected protein is targeted to the endosomes by a combined actionof a PX domain and a coiled-coil region. J Biol Chem 2004;279:54770–82.

20. Jain P, Baranwal S, Dong S, Struckhoff AP, Worthylake RA, Alahari SK.Integrin-binding protein nischarin interacts with tumor suppressor liverkinase B1 (LKB1) to regulate cell migration of breast epithelial cells. J BiolChem 2013;288:15495–509.

21. Kuijl C, Pilli M, Alahari SK, JanssenH, Khoo PS, Ervin KE, et al. Rac and RabGTPases dual effector Nischarin regulates vesicle maturation to facilitatesurvival of intracellular bacteria. EMBO J 2013;32:713–27.

22. Sano H, Liu SC, Lane WS, Piletz JE, Lienhard GE. Insulin receptorsubstrate 4 associates with the protein IRAS. J Biol Chem 2002;277:19439–47.

23. Giancotti FG, Ruoslahti E. Integrin signaling. Science 1999;285:1028–32.24. Smith CM, Finger JH, Hayamizu TF, McCright IJ, Xu J, Berghout J, et al. The

mouse Gene Expression Database (GXD): 2014 update. Nucleic Acids Res2014;42:D818–24.

25. Popovic D, Vucic D, Dikic I. Ubiquitination in disease pathogenesis andtreatment. Nat Med 2014;20:1242–53.

26. Phosphositeplus. NISCH (human) 2015 [cited 2015 Jul 16]. Availa-ble from: http://www.phosphosite.org/proteinAction.do?id¼15675&showAllSites¼true.

27. Choudhary C, Kumar C, Gnad F, NielsenML, RehmanM,Walther TC, et al.Lysine acetylation targets protein complexes and co-regulates major cel-lular functions. Science 2009;325:834–40.

28. Czech MP, Corvera S. Signaling mechanisms that regulate glucose trans-port. J Biol Chem 1999;274:1865–8.

29. Kawasome H, Papst P, Webb S, Keller GM, Johnson GL, Gelfand EW, et al.Targeted disruption of p70(s6k) defines its role in protein synthesis andrapamycin sensitivity. Proc Natl Acad Sci U S A 1998;95:5033–8.

30. Porstmann T, Griffiths B, Chung YL, Delpuech O, Griffiths JR, Down-ward J, et al. PKB/Akt induces transcription of enzymes involved incholesterol and fatty acid biosynthesis via activation of SREBP. Onco-gene 2005;24:6465–81.

31. Ding Y, ZhangR, ZhangK, LvX,ChenY, Li A, et al.Nischarin is differentiallyexpressed in rat brain and regulates neuronalmigration. PLoSONE2013;8:e54563.

32. Wu X, Xu W, Cui G, Yan Y, Wu X, Li L, et al. The expression pattern ofNischarin after lipopolysaccharides (LPS)-induced neuroinflammation inrats brain cortex. Inflamm Res 2013;62:929–40.

33. Koks S, LuukH, Nelovkov A, Areda T, Vasar E. A screen for genes induced inthe amygdaloid area during cat odor exposure. Genes Brain Behav2004;3:80–9.

34. Zhang J, Abdel-Rahman AA. Inhibition of nischarin expression attenuatesrilmenidine-evoked hypotension and phosphorylated extracellular signal-regulated kinase 1/2 production in the rostral ventrolateralmedulla of rats.J Pharmacol Exp Ther 2008;324:72–8.






35. Li J, He X, Dong R, Wang Y, Yu J, Qiu H. Frequent loss of NISCH promotestumor proliferation and invasion in ovarian cancer via inhibiting the FAKsignal pathway. Mol Cancer Ther 2015;14:1202–12.

36. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al.Integrative analysis of complex cancer genomics and clinical profiles usingthe cBioPortal. Sci Signal 2013;6:pl1.

37. Chen J, FengWL, MoWJ, Ding XW, Xie SN. Expression of integrin-bindingprotein Nischarin in metastatic breast cancer. Mol Med Rep 2015;12:77–82.

38. Shackelford DB, Shaw RJ. The LKB1-AMPK pathway: metabolism andgrowth control in tumour suppression. Nat Rev Cancer 2009;9:563–75.

39. Gastonguay A, Berg T, Hauser AD, Schuld N, Lorimer E, Williams CL. Therole of Rac1 in the regulation of NF-kappaB activity, cell proliferation, and

cell migration in non-small cell lung carcinoma. Cancer Biol Ther2012;13:647–56.

40. Reddig PJ, Xu D, Juliano RL. Regulation of p21-activated kinase-inde-pendent Rac1 signal transduction by nischarin. J Biol Chem 20053;280:30994–1002.

41. Alahari SK. Nischarin inhibits Rac induced migration and invasion ofepithelial cells by affecting signaling cascades involving PAK. Exp Cell Res2003;288:415–24.

42. Dontenwill M, Piletz JE, Chen M, Baldwin J, Pascal G, Ronde P, et al.IRAS is an anti-apoptotic protein. Ann N Y Acad Sci 2003;1009:400–12.

43. Craik DJ, Fairlie DP, Liras S, Price D. The future of peptide-based drugs.Chem Biol Drug Des 2013;81:136–47.






2015;75:4252-4259. Published OnlineFirst September 21, 2015.Cancer Res Mazvita Maziveyi and Suresh K. Alahari Protein NischarinBreast Cancer Tumor Suppressors: A Special Emphasis on Novel

Updated version

10.1158/0008-5472.CAN-15-1395doi:

Access the most recent version of this article at:

Cited articles

http://cancerres.aacrjournals.org/content/75/20/4252.full#ref-list-1

This article cites 37 articles, 18 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/75/20/4252.full#related-urls

This article has been cited by 4 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/75/20/4252To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-15-1395

http://cancerres.aacrjournals.org/content/75/20/4252.full#ref-list-1

http://cancerres.aacrjournals.org/content/75/20/4252.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/75/20/4252


mazvita maziveyi and suresh k. alahari · mazvita maziveyi and suresh k. alahari abstract tumor...

Documents