Breast Cancer Tumor Suppressors: a Special Emphasis on Novel Protein Nischarin Mazvita Maziveyi and Suresh K

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

Cancer Review Research

Breast Cancer Tumor Suppressors: A Special Emphasis on Novel Protein Nischarin Mazvita Maziveyi and Suresh K. Alahari

Abstract

Tumor suppressor genes regulate cell growth and prevent vast number of cellular processes, including neuronal protection spontaneous proliferation that could lead to aberrant tissue and hypotension. The NISCH promoter experiences hypermethy- function. Deletions and mutations of these genes typically lead lation in several cancers, whereas some highly aggressive breast to progression through the cell-cycle checkpoints, as well as cancer cells exhibit genomic loss of the NISCH locus. Further- increased cell migration. Studies of these proteins are important more, we discuss data illustrating a novel role of Nischarin as as they may provide potential treatments for breast cancers. In this a tumor suppressor in breast cancer. Analysis of this new para- review, we discuss a comprehensive overview on Nischarin, a digm may shed light on various clinical questions. Finally, the novel protein discovered by our laboratory. Nischarin, or imida- therapeutic potential of Nischarin is discussed. Cancer Res; 75(20); zoline receptor antisera-selected protein, is a protein involved in a 4252–9. 2015 AACR.

Introduction (6, 7). It also interacts with LIM kinase (LIMK) in order to prevent cytoskeletal reorganization (8). Typically, scaffold proteins such Breast cancer initiation and progression involve several genetic as Nischarin are characterized as caretaker genes because their events that can activate oncogenes and/or abrogate the function of effects on tumor growth are indirect. tumor suppressor genes. Tumor suppressor genes are commonly lost or deleted in cancers, facilitating the initiation and progression of cancer through several biological events, including cell Discovery of Nischarin proliferation, cell death, cell migration, and cell invasion. Usually, The first function attributed to Nischarin was its role as an cancer mortality occurs due to complications of metastasis rather integrin a5b1 binding protein (9). In this way, Nischarin is able to than the mass effect of the primary tumor, and several tumor regulate cell motility, specifically by anatomization of cell signal- suppressors regulate metastasis. Genetic modifications through ing proteins that contribute to tumor cell migration and invasion allelic loss are one of the important factors for deregulation of (10). The structural and functional domains of Nischarin promote tumor suppressor genes. Importantly, promoter hypermethyla- its interaction with 17 known proteins to influence cell adhesion, tion of several tumor suppressors has been shown to be associated cell migration, vesicle trafficking, apoptosis, glucose metabolism, with tumor progression. In addition, several signaling mechan- and cell signaling. Thus far, diseases associated with the NISCH isms are dysregulated in breast cancer as a result of mutations in gene include hypertension, xerostomia, morphine dependence, these genes. Among the tumor suppressors, BRCA1/2, p53, PTEN, depression, anxiety, ventricular hypertrophy, congestive heart ATM, Rb, LKB, Nm23, and p16 have been studied in great detail failure, rosacea, several cancers (11). Its location at 3p21.1 puts and discussed in many review articles (1–3). This article primarily it in a category of tumor suppressor genes that are associated with emphasizes the novel tumor suppressor Nischarin (Fig. 1) and the development of many cancers (12). Highly aggressive breast how it regulates cell migration, cell invasion, tumor growth, and cancer cells frequently exhibit genomic loss of the NISCH locus metastasis through various signaling pathways and interactions (12), whereas the NISCH promoter is hypermethylated in lung with other proteins. Caretaker genes, such as BRCA1, are genes cancers (13). Nischarin mRNA and protein expression is high in whose loss does not directly inhibit tumor growth (1, 4, 5). stage 0 human breast specimens but reduced in stage I–IV breast Nischarin imposes its tumor-suppressive functions through its cancer specimens (12). interactions with other proteins; thus, it is a caretaker tumor suppressor gene. For example, it interacts with p21 activated kinase 1 (PAK1) and integrin a5 to prevent cell migration NISCH Regulation The structural and functional domains of Nischarin Nischarin was first characterized 15 years ago by SK Alahari Department of Biochemistry and Molecular Biology, Louisiana State and colleagues (10). The 37,955 bases of the full-length NISCH University Health Sciences Center, New Orleans, Louisiana. gene are regulated by the transcription factors max1, sp1, Corresponding Author: Suresh K. Alahari, Louisiana State University Health COUP, olf-1, COUP-TF, pax-4a, ATF, and c-Myc (11). Soon Sciences Center, 1901 Perdido Street, New Orleans, LA 70112. Phone: 504-568- after, the human homolog of Nischarin was discovered as 4734; Fax: 504-568-2093; E-mail: [email protected] imidazoline receptor antisera-selected protein (IRAS; ref. 14). doi: 10.1158/0008-5472.CAN-15-1395 Human IRAS has 80% homology with rodent Nischarin but 2015 American Association for Cancer Research. interestingly, the integrin a5-binding sites of Nischarin and

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Rac1 Rac1 PAK1 LIMK Rab 14 LKB1 IRS 1–4 PI3P PI3P Coiled- Coil Alanine/ PX Leucine-Rich repeats Integrin Binding proline NH2 Glutamine COOH rich rich 1 300 600 900 1504

Serine phosphorylation Ubiquitination

Tyrosine phosphorylation Acetylation

Threonine phosphorylation

Figure 1. The predicted post-translational modiﬁcations of Nischarin. Ubiquitination of Nischarin is predicted to occur at K1009, K1015, K1290, K1299, and K1303. Acetylation is 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 with phosphatidylinositol 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 of Nischarin are sufﬁcient to interact with LIMK. Residues 464 to 562 of Nischarin interact with the integrin a5 cytoplasmic tail. Rab14 interacts with Nischarin's C-terminus. Both the N- and C-terminus of Nischarin interact with Rac1.

IRASare100%identical(Fig.2;ref.14).Inhumans,Nischarin/ 516, as well as amino acids 517–1593 missing (17). Isoform 7 IRAS was first discovered as an I1-imidazoline receptor, which has a long amino acid sequence difference between 122–153 are expressed in both neurons and astrocytes (14, 15). Human and amino acids 143–1593 missing (17). and mouse Nischarin differ in the alanine/proline rich region, Furthermore, the N-terminus of Nischarin contains a phox which is removed in human Nischarin (Fig. 2). (PX) domain from amino acids 11–121 (Fig. 1; ref. 18). This Nischarin is a cytosolic protein that anchors itself to the inner PX domain is necessary for plasma membrane and vesicular layer of the plasma membrane and has been found to interact targeting of Nischarin (19). Both Nischarin's PX domain, and with both cytosolic and intermembrane proteins (16). Human its coiled-coil domain (634–695) are essential for endosomal Nischarin has four isoforms that are achieved by alternative targeting and interaction with phosphatidylinositol 3-phosphate splicing (9). Isoform 1 encodes the full-length protein and is PI3P in endosomes enriched in this phospholipid (Fig. 1; ref. 19). highly expressed in neural and endocrine tissue (Fig. 2; ref. 9). Though the interaction of PI3P and Nischarin alone is not Isoform 2 has amino acids 1–511 spliced and is expressed in sufficient for endosomal targeting, this interaction occurs around the brain (9). Isoform 3, also known as IRAS-L, is highly region 2–133 of Nischarin. Although regions 120–695 are nec- expressed in the brain, missing amino acids 584–1504, and essary for Nischarin to be targeted to the endosomes, mutation of has a modified sequence in amino acids 511–583 (9). Isoform amino acids 49 and 50 inhibit endosomal targeting (18). 4, also known as IRAS-S, is also highly expressed in the brain, Interestingly, other regions of Nischarin have been found to has amino acids 516–1504 spliced out, and has a change in interact with other signaling molecules as well. For example, amino acids between 512 and 515 (9). Isoform 1 is 166,629 amino acids 1–624 of Nischarin have been found to strongly Da, isoform 2 is 110,194 Da, isoform 3 is 63,997 Da, and interact with p21 activated kinase 1 (PAK1) to prevent cell isoform 4 is 56,867 Da (9). migration (Fig. 1; ref. 6). Positions 416–624 of Nischarin are Mouse Nischarin has seven isoforms that are achieved by sufficient to interact with LIMK in order to prevent cytoskeletal alternative splicing (Fig. 2; ref. 17). Isoform 1 is full-length reorganization (Fig. 1; ref. 8). LKB1 interacts with positions 416– Nischarin, which is 1,593 amino acids (17). Isoform 2 is 624 of Nischarin to prevent cancer progression (Fig. 1; ref. 20). In missing amino acids 348–500 and isoform 3 is missing amino addition, Rab14 and insulin receptor substrates 1–4 interact with acids 1–245 (17). Amino acids 437–472 of isoform 4 differ the c-terminal domain of Nischarin (Fig. 1; refs. 21, 22). Among from the canonical sequence and it is also missing amino acids the known binding partners of Nischarin, its interaction with 473–1593 (17). Amino acids 332–334 of isoform 5 have a integrin a5 is the best characterized. sequence difference and amino acids 335–1593 are missing Integrins are cell adhesion proteins with a and b transmem- (17). Isoform 6 also has a sequence difference between 513 and brane heterodimers that play a major role in transmitting

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Human Mouse

Coiled- Coiled- Coil Coil Alanine/ 1 PX Leucine-rich Integrin PX Leucine-rich Integrin Glutamine binding Glutamine binding proline NH2 rich COOH NH2 rich rich COOH 1 1504 1 1593

Coiled- Coiled- Coil Coil Alanine/ 2 Integrin PX LRR Integrin Glutamine binding Glutamine binding proline NH2 rich COOH NH2 rich rich COOH 1 992 1 1441

Coiled- Coil Alanine/ 3 PX Leucine-rich Leucine-rich Integrin Glutamine binding proline NH2 COOH NH2 rich rich COOH 1 583 1 1259

4 PX Leucine-rich PX Leucine-rich NH2 COOH NH2 COOH 1 515 1 472

5 PX LRR

NH2 COOH 1 334

6 PX Leucine-rich

NH2 COOH 1 516

7 PX

NH2 COOH 1 153

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

signals from outside of the cell membrane to the inside of the interaction (7). When Nischarin is overexpressed, a5integrin cell and vice versa. The cytosolic portion of the transmembrane promoter activity decreases (12). The exact mechanism of heterodimer interacts with a number of cytoskeletal proteins Nischarin downregulation of a5-integrin is unknown (12). It and signaling molecules, including Ras and MAPK (21). Extra- has been hypothesized that the leucine zipper domain of cellular signals trigger signaling cascades that modulate cell Nischarin interacts with other leucine zipper-containing tran- behaviors such as cytoskeletal remodeling. The membrane scription factors to inﬂuence the gene expression of integrin a5 proximal region of the integrin a5 subunit has been shown (12). Increased expression of integrin a5b1 has been linked to to interact with Nischarin to inhibit cell migration (Fig. 3; reduced tumor growth rates, regulation of muscle cell growth, refs. 7, 10). Residues 464 to 562 of Nischarin interact with and reduced apoptosis (23). Because Nischarin has been found residues 1017 to 1030 of the a5 cytoplasmic tail, also known as to regulate the expression of other proteins, it is important to the membrane proximal region (12). More speciﬁcally, Tyr characterize its domains and interacting partners in order to 1018 and Lys1022 are crucial points for this a5–Nischarin better understand global tissue expression patterns. On the

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Focal adhesions Integrins Cell attachment B1 α5 F EG F EG Figure 3. The tumor-suppressive function of P P Nischarin. Nischarin decreases FAK phosphorylation levels due to EGFR ERK decreased a5-integrin expression. FAK Reduced FAK phosphorylation thus Nischarin prevents ERK activation via Ras and MEK, resulting in decreased cell PAK1 MLCK survival. Nischarin and LKB1 interact to RAS Rac1 reduce tumor growth, regulate cell migration, metastasis, and anchorage- independent growth. Nischarin directly interacts with PAK1 to prevent MEK ERK NF-κB LKB1 LIMK Migration its kinase activity and decrease cytoskeletal remodeling. PAK targets cell migration signaling pathways through MLCK and LIMK. P Phosphorylation of LIMK through PAK Cytoskeletal inhibits cofilin, an actin severing Cofilin Tumor growth remodeling protein, thus leading to actin filament assembly. Nischarin binds to the cell survival kinase domain of active LIMK to deactivate it and to prevent actin Nucleus filament assembly during cytoskeletal reorganization.

basis of Nischarin interaction with several proteins, we believe dromes, muscle dystrophy, cancer, neurodegeneration, autoim- it functions as a scaffolding protein. munity, and inflammatory diseases (25). Ubiquitination of Nischarin is predicted to occur at K1009, K1015, K1290, Nischarin tissue expression K1299, and K1303 (Fig. 1; ref. 26). Acetylation is predicted to In cell lines, Nischarin mRNA levels are highest in multiple occur at K1015 (Fig. 1; ref. 26). Lysine conjugation has been found rodent neuronal, epithelial, and fibroblast cell lines (10). Further- to target large macromolecular complexes involved in processes more, its expression levels have been noted in humans, rodents, such as nuclear transport, actin nucleation, chromatin remodel- chicken, lizards, zebrafish, cows, dogs, opossums, chimpanzees, ing, cell cycle, and splicing (27). Protein phosphorylation is platypus, sea squirts, fruit flies, mosquitos, worms, and the African important and necessary for protein signal transduction. The clawed frog (11). Specifically, Nischarin expression has been found predicted human Nischarin phosphorylation sites are S246, in the embryo, diencephalon, hindbrain, midbrain, future spinal S250, T252, S477, S541, S546, S883, S1022, S1038, T1282, cord, hypothalamus, neocortex, ganglionic eminence, hippocam- S1284, Y1293, Y1294, and Y1307 (Fig. 1; ref. 26). pus, cerebellum, spinal cord, meninges, choroid plexus, basal ganglia, amygdale, cerebral cortex, brainstem, olfactory bulb, ret- Nischarin Participates in Insulin Signaling ina,capmesenchyme, renal interstitium group, ovaries,primarysex cord, female and male associated reproductive structures, heart, The carboxy terminus of Nischarin has been found to bind to liver, lung, metanephros, and skeletal muscle (Table 1; ref. 24). In the carboxy terminus of insulin receptor substrate 4 (IRS-4; Fig. 1; the breast, Nischarin expression is normal in stage 0 breast speci- ref. 22). The IRS family is a family of adaptor proteins that are mens but reduced in stage I–IV breast cancer specimens (12). Given recruited and phosphorylated after insulin binds to and activates that the deregulation of Nischarin is a frequent event underlying the the insulin tyrosine receptor kinase (IR). Phosphorylated IRS is development of multiple diseases, understanding the mechanisms activated and participates in a number of signaling cascades that contributing to its regulation is imperative. have growth and mitogenic effects (22). Overexpression of human Nischarin in human embryonic kidney 293 cells induces a 4-fold The predicted post-translational modifications of Nischarin increase of insulin-stimulated activation of ERK (22). The tyrosine Protein ubiquitination is important for many biologic process- phosphorylation of the IRS-4 and Src homology 2 domain-contain- es, including immunity and cell differentiation (25). It has been ing (shc) initiates this ERK activation in response to insulin (22). linked to many disease progressions, including metabolic syn- IRS and shc then bind to growth factor receptor-bound protein

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Table 1. Nischarin RNA tissue expression synthesis of SREBP 1 and 2 to promote fatty acid synthesis (30). Taken together, this shows that Nischarin participates in System Major tissue insulin signaling to increase cell survival. Internal organs Small intestine Nischarin in the Brain Colon Nischarin is highly expressed on the leading edge of neurons Adipocyte (31). Silencing of Nischarin in both rat and mouse neurons has Kidney been found to increase neuronal migration (31). Nischarin is Liver expressed by mature neurons to prevent them from further Lung migration (31). Neuronal migration is necessary in the embryonic Immune system Bone marrow period for normal brain formation (31). Because Nischarin has Whole blood higher expression in layers IV–V of the cortex, it is expressed by White blood cells mature neurons that no longer need to migrate (31). Lymph node Thymus Nischarin is a neuroprotective protein Nischarin has been found to induce neuronal apoptosis Nervous Brain through the PI3K and protein kinase B (PKB) pathways. Lipo- Cortex polysaccharide (LPS) is a proinflammatory component found on Cerebellum the outer membrane of Gram negative bacteria (32). Upon Retina injection of LPS, proinflammation factors are produced and Spinal cord apoptosis occurs downstream of the release (32). One study Reproductive system Ovary showed that upon intracerebroventricular injection of LPS into – Uterus male Sprague Dawley rats, Nischarin levels progressively Placenta increased for one day then gradually decreased from 3 to 7 days (32). Colocalization was also seen between Nischarin, Bcl-2– Prostate associated death promoter (BAD), and pAKT, which indicates Testis an upregulation in the PI3K/AKT pathway (32). These results Secretory system Pancreas demonstrate that Nischarin is a neuroprotective protein (32). Thyroid Salivary gland Nischarin levels are increased in the amygdala in response to Adrenal gland anxiety Breast During an anxiety response, the genes participating in the Skin synthesis of neurotransmitters are typically up regulated (33). There are also a number of genes involved in signal transduction © 2015 American Association for Cancer Research that are upregulated (33). A better understanding of the molecular mechanisms of anxiety disorders can lead to the discovery of more effective drugs. The amygdala has long been found to regulate NOTE: Nischarin RNA is expressed in the small intestine, colon, adipocytes, kidneys, liver, lungs, bone marrow, whole blood, white blood cells, lymph emotional behavior and vigilance (33). It processes the input of fl nodes, thymus, brain, cortex, cerebellum, retina, spinal cord, ovaries, uterus, emotional stimuli and in uences the output of the behavioral placenta, prostate, testis, pancreas, thyroid, salivary glands, adrenal response (33). Cat odor exposure to rats induces a behavioral glands, breast, and skin. response consistent with anxiety (33). Rho GTPase-activating protein and Rho-specific guanine nucleotide exchange factor are present in the amygdala of a normal rat because of their involve- 2 (Grb2) for activation (22). Grb2 then associates with Son of ment in the guidance of growth cones (33). After cat odor sevenless homolog (Sos), the guanine nucleotide exchange protein exposure to the rats, the proteins involved in that pathway were for Ras (22). Sos elevates the GTP-bound form of Ras, which no longer detected (33). Also, a 1.2-fold increase was seen in the results in increased ERK activation and then cell growth (22). expression of Nischarin during the anxiety response (33). It is ERK inactivation prevents cell survival, growth, and differenti- proposed that during an anxiety response, Nischarin inhibits the ation of the cancerous cells (12). A weaker interaction is also activity of the Rho GTPase pathway (33). Nischarin is also seen between Nischarin and IRS-1, IRS-2, and IRS-3 (22). associated with morphine dependence, depression, and brain Alternatively, the tyrosine phosporylation of IRS4 promotes the disease (11), suggesting that it is an important regulator in the activation of the PI3K signaling cascade. PI3K is a key kinase that brain. plays a role in the mitogenic and metabolic 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) Nischarin Plays a Role in Hypotension and Akt to the plasma membrane for activation (28). Activated The generation of pERK1/2 in the rostral ventrolateral Akt regulates a number of cellular processes, including the acti- medulla (RVLM) has implications in I1R-activated hypotension vation of the Glut 4 transporter that participates in glucose uptake (34). In PC12 cells, this I1R activation depends on nischarin (28). Akt also activates p70s6k, a serine/threonine kinase that to generate pERK1/2 levels (34). Nischarin knockdown in the regulates protein synthesis (29). Akt activation also induces RVLM abolishes I1 receptor activation in the RVLM and

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produces a hypotensive response (34). Rilmendine, a drug used LOH are the leading causes of reduced Nischarin expression in to treat hypertension, elicits its hypotensive effects by increas- breast cancers (12). ing the production of pERK1/2 in the RVLM (34). Central administration of rilmenidine was attenuated by rats that had Nischarin inhibits cell migration and invasion reduced Nischarin expression, compared with the control Nischarin has been found to interact with a number of signaling (34). Rats with Nischarin antisense ODNs abrogated I1R acti- proteins such as integrin a5, PAK1, LIMK1, Rab14, LKB1, and vation by abolishing pERK1/2 levels and hypotensive respon- Rac1. Liver kinase B1 (LKB1) is a tumor suppressor that has a role ses (34). These findings indicate that Nischarin participates in in cell polarity and the regulation of metabolism through the pERK1/2-mediated hypotension. mTOR pathway (38). Like Nischarin, LKB1 inhibits PAK phosphorylation to prevent actin filament assembly during cytoskeletal reorganization (20). LKB1 interacts with amino acids 416– Nischarin in Cancer 624 of Nischarin to increase its kinase activity (20). Nischarin and Nischarin is a tumor suppressor of ovarian cancer LKB1 both reduce tumor growth, regulate cell migration, metas- Nischarin expression is downregulated in ovarian cancer tissues tasis, and anchorage-dependent growth (Fig. 3; ref. 20). (35). This decreased expression of Nischarin is associated with Rac1 is a Rho GTPase family member that regulates a number invasiveness, tumor stage, lymph node metastasis, and histologic of signaling pathways, including the organization and assembly tumor grade (35). Interestingly, ovarian cancer patients with of actin in response to the extracellular environment. Several Nischarin expression have a better overall survival than non- downstream effectors exert the biologic effects of Rac1. Rac1 expressing patients (35). The NISH promoter is hypermethylated activates NF-kB transcription factors, leading to inflammatory in 36.7% of ovarian cancers (35). Overexpression of this tumor responses, cell growth, and apoptotic suppression (39). Both the suppressor in an ovarian cancer cell line imposes a G1 phase arrest N- and C-terminus of Nischarin interacts with Rac1 in its active and cyclin D1 downregulation, which leads to decelerated cell state to disrupt the NF-kB pathway and thus repress cyclin D1, the proliferation (35). It has been shown that Nischarin exerts its promoter associated with malignancy (Fig. 3; ref. 40). Also, tumor-suppressive effects through FAK in ovarian cancer (35). The reduced activation of Rac1 by Nischarin results in reduced tumor role of Nischarin in ovarian cancer has recently been discovered growth (12). and further studies are needed to understand the tumor-suppres- Nischarin has been found to inhibit the Rac1 effector, p21 sive effect of Nischarin in ovarian cancer. activated kinase (PAK), to prevent Rac1 driven cell migration (41). Nischarin directly interacts with PAK1 and prevents its Nischarin is a tumor suppressor of breast cancer kinaseactivity(Fig.3;ref.40).PAKtargetscellmigration Nischarin is known to control cell migration by antagonizing signaling pathways through myosin light chain kinase (MLCK) the actions of cell signaling proteins that contribute to tumor cell and LIMK (40). The activation of MLCK through PAK phos- migration and invasion (10). This protein maps at 3p21.1 and it phorylates myosin light chain, which regulates actin cyto- has been shown that regions of chromosome 3p are associated skeletal dynamics (40). LIMK is highly expressed in cancer with the development of many cancers (12). Highly aggressive cells and has been highly regarded as an oncogene (8). Phos- breast cancer cells exhibit genomic loss of the NISCH locus and phorylation of LIMK through PAK inhibits cofilin, an actin Nischarin promoter methylation is seen in 30% of breast cancers severing protein, thus leading to actin filament assembly (41). (12). A study of 962 human breast cancer patients from TCGA NischarinbindstothekinasedomainofactiveLIMKtodeac- revealed that 0.3% of breast invasive carcinomas exhibit a dele- tivate it and prevents actin filament assembly during cyto- tion of Nischarin, and 0.7% of breast invasive carcinomas have a skeletal reorganization (Fig. 3; ref. 8). mutated Nischarin (36). Nischarin also interacts with a number of signaling pro- Loss of Nischarin plays a significant role in breast cancer cell teins to inhibit apoptosis (12). In PC12 and Cos7 cell lines, progression. Correspondingly, Nischarin mRNA is highly Nischarin has been found to inhibit the activation of caspase-3, expressed in normal breast tissue but poorly expressed in human a critical apoptosis mediator (42). Upon staurosporine and breast cancer specimens (12). Highly invasive breast cancer cell thapsigargin treatment, Nischarin-transfected cells show lines such as MDA-MB-231 exhibit low Nischarin expression decrease in apoptotic activity (42). This inhibition of apoptosis levels, moderately invasive breast cancer cell lines such as leads to increased cell survival. It is not clear how Nischarin can MCF-7 exhibit higher Nischarin expression levels, and nontu- inhibit tumor growth as well as apoptosis, which needs further morigenic cells such as MCF-10A have the highest amount of investigation. Nischarin expression (12). Restoring Nischarin expression in aggressive breast cancer cell lines decreases focal adhesion kinase (FAK) phosphorylation levels due to decreased a5-integrin Therapeutic Importance of Nischarin expression (12). Reduced FAK phosphorylation thus prevents Nischarin's strong tumor-suppressive effect can be used to ERK activation, resulting in decreased cell survival (12). reverse the invasive capacity of cancer cells, making it an appealing Analysis of human breast cancer patient tumors revealed that option in developing future cancer treatments. It has been well tissues with lymph node metastasis have significantly decreased demonstrated that the loss of Nischarin leads to increased focal levels of Nischarin than patients without lymph node metastasis adhesions, cytoskeletal organization, cell migration, tumor (37). In addition, LOH studies performed using microsatellite growth, and cell survival. Increasing expression of Nischarin in markers in DNA samples from 18 human breast cancers and their tumor cells would reduce the invasive and migratory capacity of normal tissue counterparts, revealed that LOH is seen in 50% of cancer cells. Also, because LOH was seen in 50% of human breast human breast cancer patients, which results in decreased cancer patients, Nischarin could be used as a clinical biomarker Nischarin expression (12). Therefore, promoter methylation and for patients.

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To compensate for lost or reduced Nischarin function in which it achieves by interacting with a number of proteins. Over tumors, it is possible to create peptide-based drugs that mimic 30 research papers have been published on Nischarin and further Nischarin's natural interactions. Peptide-based drugs are advan- studies of this protein will increase current knowledge in the fields tageous for high potency, high selectivity, and low toxicity treat- of cancer biology, cell migration, apoptosis, vesicle trafficking, cell ment (43). Designing a small peptide-based drug that mimics the adhesion, signal transduction, and hypertension. This dynamic different domains of Nischarin could decrease the migratory scaffolding protein is ideal for studying cell signaling pathways in effects of the cancer cells. For example, designing a peptide drug a vast number of diseases. A deeper understanding of Nischarin- with the amino acids of Nischarin that are necessary to bind mediated pathways will help predict disease progression and integrin a5 would decrease integrin a-5–mediated cell migration. provide better therapeutic targets for breast cancer patients. In conclusion, Nischarin has been shown to be an important protein in the maintenance of normal cell function, and its dysfunction is widely implicated in human disease. Its expression Disclosure of Potential Conflicts of Interest is most evident within cells of the immune, nervous, secretory, No potential conflicts of interest were disclosed. muscular, and reproductive systems (11). A role for Nischarin in many cell processes has now been described, but its most well- Received May 21, 2015; revised July 2, 2015; accepted July 2, 2015; characterized functions involve its regulation of cell migration, published OnlineFirst September 21, 2015.

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www.aacrjournals.org Cancer Res; 75(20) October 15, 2015 4259

Breast Cancer Tumor Suppressors: A Special Emphasis on Novel Protein Nischarin

Mazvita Maziveyi and Suresh K. Alahari

Cancer Res 2015;75:4252-4259. Published OnlineFirst September 21, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-1395

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