Tumor suppressor genes regulate cell growth and prevent spontaneous proliferation that could lead to aberrant tissue function. Deletions and mutations of these genes typically lead to progression through the cell-cycle checkpoints, as well as increased cell migration. Studies of these proteins are important as they may provide potential treatments for breast cancers. In this review, we discuss a comprehensive overview on Nischarin, a novel protein discovered by our laboratory. Nischarin, or imidazoline receptor antisera-selected protein, is a protein involved in a vast number of cellular processes, including neuronal protection and hypotension. The NISCH promoter experiences hypermethylation in several cancers, whereas some highly aggressive breast cancer cells exhibit genomic loss of the NISCH locus. Furthermore, we discuss data illustrating a novel role of Nischarin as a tumor suppressor in breast cancer. Analysis of this new paradigm may shed light on various clinical questions. Finally, the therapeutic potential of Nischarin is discussed. Cancer Res; 75(20); 4252–9. ©2015 AACR.
Breast cancer initiation and progression involve several genetic events that can activate oncogenes and/or abrogate the function of 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 proliferation, cell death, cell migration, and cell invasion. Usually, cancer mortality occurs due to complications of metastasis rather than the mass effect of the primary tumor, and several tumor suppressors regulate metastasis. Genetic modifications through allelic loss are one of the important factors for deregulation of tumor suppressor genes. Importantly, promoter hypermethylation of several tumor suppressors has been shown to be associated with tumor progression. In addition, several signaling mechanisms are dysregulated in breast cancer as a result of mutations in these genes. Among the tumor suppressors, BRCA1/2, p53, PTEN, ATM, Rb, LKB, Nm23, and p16 have been studied in great detail and discussed in many review articles (1–3). This article primarily emphasizes the novel tumor suppressor Nischarin (Fig. 1) and how it regulates cell migration, cell invasion, tumor growth, and metastasis through various signaling pathways and interactions with other proteins. Caretaker genes, such as BRCA1, are genes whose loss does not directly inhibit tumor growth (1, 4, 5). Nischarin imposes its tumor-suppressive functions through its interactions with other proteins; thus, it is a caretaker tumor suppressor gene. For example, it interacts with p21 activated kinase 1 (PAK1) and integrin α5 to prevent cell migration (6, 7). It also interacts with LIM kinase (LIMK) in order to prevent cytoskeletal reorganization (8). Typically, scaffold proteins such as Nischarin are characterized as caretaker genes because their effects on tumor growth are indirect.
Discovery of Nischarin
The first function attributed to Nischarin was its role as an integrin α5β1 binding protein (9). In this way, Nischarin is able to regulate cell motility, specifically by anatomization of cell signaling proteins that contribute to tumor cell migration and invasion (10). The structural and functional domains of Nischarin promote its 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 NISCH gene include hypertension, xerostomia, morphine dependence, depression, anxiety, ventricular hypertrophy, congestive heart failure, rosacea, several cancers (11). Its location at 3p21.1 puts it in a category of tumor suppressor genes that are associated with the development of many cancers (12). Highly aggressive breast cancer cells frequently exhibit genomic loss of the NISCH locus (12), whereas the NISCH promoter is hypermethylated in lung cancers (13). Nischarin mRNA and protein expression is high in stage 0 human breast specimens but reduced in stage I–IV breast cancer specimens (12).
The structural and functional domains of Nischarin
Nischarin was first characterized 15 years ago by SK Alahari and colleagues (10). The 37,955 bases of the full-length NISCH gene are regulated by the transcription factors max1, sp1, COUP, olf-1, COUP-TF, pax-4a, ATF, and c-Myc (11). Soon after, the human homolog of Nischarin was discovered as imidazoline receptor antisera-selected protein (IRAS; ref. 14). Human IRAS has 80% homology with rodent Nischarin but interestingly, the integrin α5-binding sites of Nischarin and IRAS are 100% identical (Fig. 2; ref. 14). In humans, Nischarin/IRAS was first discovered as an I1-imidazoline receptor, which are expressed in both neurons and astrocytes (14, 15). Human and 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 inner layer of the plasma membrane and has been found to interact with both cytosolic and intermembrane proteins (16). Human Nischarin has four isoforms that are achieved by alternative splicing (9). Isoform 1 encodes the full-length protein and is highly expressed in neural and endocrine tissue (Fig. 2; ref. 9). Isoform 2 has amino acids 1–511 spliced and is expressed in the brain (9). Isoform 3, also known as IRAS-L, is highly expressed in the brain, missing amino acids 584–1504, and has a modified sequence in amino acids 511–583 (9). Isoform 4, also known as IRAS-S, is also highly expressed in the brain, has amino acids 516–1504 spliced out, and has a change in amino acids between 512 and 515 (9). Isoform 1 is 166,629 Da, isoform 2 is 110,194 Da, isoform 3 is 63,997 Da, and isoform 4 is 56,867 Da (9).
Mouse Nischarin has seven isoforms that are achieved by alternative splicing (Fig. 2; ref. 17). Isoform 1 is full-length Nischarin, which is 1,593 amino acids (17). Isoform 2 is missing amino acids 348–500 and isoform 3 is missing amino acids 1–245 (17). Amino acids 437–472 of isoform 4 differ from the canonical sequence and it is also missing amino acids 473–1593 (17). Amino acids 332–334 of isoform 5 have a sequence 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 7 has a long amino acid sequence difference between 122–153 and 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). This PX domain is necessary for plasma membrane and vesicular targeting of Nischarin (19). Both Nischarin's PX domain, and its coiled-coil domain (634–695) are essential for endosomal targeting and interaction with phosphatidylinositol 3-phosphate PI3P in endosomes enriched in this phospholipid (Fig. 1; ref. 19). Though the interaction of PI3P and Nischarin alone is not sufficient for endosomal targeting, this interaction occurs around region 2–133 of Nischarin. Although regions 120–695 are necessary for Nischarin to be targeted to the endosomes, mutation of amino acids 49 and 50 inhibit endosomal targeting (18).
Interestingly, other regions of Nischarin have been found to interact with other signaling molecules as well. For example, amino acids 1–624 of Nischarin have been found to strongly interact with p21 activated kinase 1 (PAK1) to prevent cell migration (Fig. 1; ref. 6). Positions 416–624 of Nischarin are sufficient to interact with LIMK in order to prevent cytoskeletal reorganization (Fig. 1; ref. 8). LKB1 interacts with positions 416–624 of Nischarin to prevent cancer progression (Fig. 1; ref. 20). In addition, Rab14 and insulin receptor substrates 1–4 interact with the c-terminal domain of Nischarin (Fig. 1; refs. 21, 22). Among the known binding partners of Nischarin, its interaction with integrin α5 is the best characterized.
Integrins are cell adhesion proteins with α and β transmembrane heterodimers that play a major role in transmitting signals from outside of the cell membrane to the inside of the cell and vice versa. The cytosolic portion of the transmembrane heterodimer interacts with a number of cytoskeletal proteins and signaling molecules, including Ras and MAPK (21). Extracellular signals trigger signaling cascades that modulate cell behaviors such as cytoskeletal remodeling. The membrane proximal region of the integrin α5 subunit has been shown to interact with Nischarin to inhibit cell migration (Fig. 3; refs. 7, 10). Residues 464 to 562 of Nischarin interact with residues 1017 to 1030 of the α5 cytoplasmic tail, also known as the membrane proximal region (12). More specifically, Tyr 1018 and Lys1022 are crucial points for this α5–Nischarin interaction (7). When Nischarin is overexpressed, α5 integrin promoter activity decreases (12). The exact mechanism of Nischarin downregulation of α5-integrin is unknown (12). It has been hypothesized that the leucine zipper domain of Nischarin interacts with other leucine zipper-containing transcription factors to influence the gene expression of integrin α5 (12). Increased expression of integrin α5β1 has been linked to reduced tumor growth rates, regulation of muscle cell growth, and reduced apoptosis (23). Because Nischarin has been found to regulate the expression of other proteins, it is important to characterize its domains and interacting partners in order to better understand global tissue expression patterns. On the basis of Nischarin interaction with several proteins, we believe it functions as a scaffolding protein.
Nischarin tissue expression
In cell lines, Nischarin mRNA levels are highest in multiple rodent neuronal, epithelial, and fibroblast cell lines (10). Furthermore, 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 African clawed frog (11). Specifically, Nischarin expression has been found in the embryo, diencephalon, hindbrain, midbrain, future spinal cord, hypothalamus, neocortex, ganglionic eminence, hippocampus, cerebellum, spinal cord, meninges, choroid plexus, basal ganglia, amygdale, cerebral cortex, brainstem, olfactory bulb, retina, cap mesenchyme, renal interstitium group, ovaries, primary sex cord, female and male associated reproductive structures, heart, liver, lung, metanephros, and skeletal muscle (Table 1; ref. 24). In the breast, Nischarin expression is normal in stage 0 breast specimens but reduced in stage I–IV breast cancer specimens (12). Given that the deregulation of Nischarin is a frequent event underlying the development of multiple diseases, understanding the mechanisms contributing to its regulation is imperative.
The predicted post-translational modifications of Nischarin
Protein ubiquitination is important for many biologic processes, including immunity and cell differentiation (25). It has been linked to many disease progressions, including metabolic syndromes, muscle dystrophy, cancer, neurodegeneration, autoimmunity, and inflammatory diseases (25). Ubiquitination of Nischarin is predicted to occur at K1009, K1015, K1290, K1299, and K1303 (Fig. 1; ref. 26). Acetylation is predicted to occur at K1015 (Fig. 1; ref. 26). Lysine conjugation has been found to target large macromolecular complexes involved in processes such as nuclear transport, actin nucleation, chromatin remodeling, cell cycle, and splicing (27). Protein phosphorylation is important and necessary for protein signal transduction. The predicted 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 Signaling
The 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 are recruited and phosphorylated after insulin binds to and activates the insulin tyrosine receptor kinase (IR). Phosphorylated IRS is activated and participates in a number of signaling cascades that have growth and mitogenic effects (22). Overexpression of human Nischarin in human embryonic kidney 293 cells induces a 4-fold increase of insulin-stimulated activation of ERK (22). The tyrosine phosphorylation of the IRS-4 and Src homology 2 domain-containing (shc) initiates this ERK activation in response to insulin (22). IRS and shc then bind to growth factor receptor-bound protein 2 (Grb2) for activation (22). Grb2 then associates with Son of sevenless homolog (Sos), the guanine nucleotide exchange protein for Ras (22). Sos elevates the GTP-bound form of Ras, which results in increased ERK activation and then cell growth (22). ERK inactivation prevents cell survival, growth, and differentiation of the cancerous cells (12). A weaker interaction is also seen between Nischarin and IRS-1, IRS-2, and IRS-3 (22).
Alternatively, the tyrosine phosporylation of IRS4 promotes the activation of the PI3K signaling cascade. PI3K is a key kinase that 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) and Akt to the plasma membrane for activation (28). Activated Akt regulates a number of cellular processes, including the activation of the Glut 4 transporter that participates in glucose uptake (28). Akt also activates p70s6k, a serine/threonine kinase that regulates 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 in insulin signaling to increase cell survival.
Nischarin in the Brain
Nischarin is highly expressed on the leading edge of neurons (31). Silencing of Nischarin in both rat and mouse neurons has been found to increase neuronal migration (31). Nischarin is expressed by mature neurons to prevent them from further migration (31). Neuronal migration is necessary in the embryonic period for normal brain formation (31). Because Nischarin has higher expression in layers IV–V of the cortex, it is expressed by mature neurons that no longer need to migrate (31).
Nischarin is a neuroprotective protein
Nischarin has been found to induce neuronal apoptosis through the PI3K and protein kinase B (PKB) pathways. Lipopolysaccharide (LPS) is a proinflammatory component found on the outer membrane of Gram negative bacteria (32). Upon injection of LPS, proinflammation factors are produced and apoptosis occurs downstream of the release (32). One study showed that upon intracerebroventricular injection of LPS into male Sprague–Dawley rats, Nischarin levels progressively increased 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 indicates an upregulation in the PI3K/AKT pathway (32). These results demonstrate that Nischarin is a neuroprotective protein (32).
Nischarin levels are increased in the amygdala in response to anxiety
During an anxiety response, the genes participating in the synthesis of neurotransmitters are typically up regulated (33). There are also a number of genes involved in signal transduction 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 emotional behavior and vigilance (33). It processes the input of emotional stimuli and influences the output of the behavioral response (33). Cat odor exposure to rats induces a behavioral 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 involvement in the guidance of growth cones (33). After cat odor exposure to the rats, the proteins involved in that pathway were no longer detected (33). Also, a 1.2-fold increase was seen in the expression of Nischarin during the anxiety response (33). It is proposed that during an anxiety response, Nischarin inhibits the activity of the Rho GTPase pathway (33). Nischarin is also associated with morphine dependence, depression, and brain disease (11), suggesting that it is an important regulator in the brain.
Nischarin Plays a Role in Hypotension
The 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 nischarin to generate pERK1/2 levels (34). Nischarin knockdown in the RVLM abolishes I1 receptor activation in the RVLM and produces a hypotensive response (34). Rilmendine, a drug used to treat hypertension, elicits its hypotensive effects by increasing the production of pERK1/2 in the RVLM (34). Central administration of rilmenidine was attenuated by rats that had reduced Nischarin expression, compared with the control (34). Rats with Nischarin antisense ODNs abrogated I1R activation by abolishing pERK1/2 levels and hypotensive responses (34). These findings indicate that Nischarin participates in pERK1/2-mediated hypotension.
Nischarin in Cancer
Nischarin is a tumor suppressor of ovarian cancer
Nischarin expression is downregulated in ovarian cancer tissues (35). This decreased expression of Nischarin is associated with invasiveness, tumor stage, lymph node metastasis, and histologic tumor grade (35). Interestingly, ovarian cancer patients with Nischarin expression have a better overall survival than non-expressing patients (35). The NISH promoter is hypermethylated in 36.7% of ovarian cancers (35). Overexpression of this tumor suppressor in an ovarian cancer cell line imposes a G1 phase arrest and cyclin D1 downregulation, which leads to decelerated cell proliferation (35). It has been shown that Nischarin exerts its tumor-suppressive effects through FAK in ovarian cancer (35). The role of Nischarin in ovarian cancer has recently been discovered and further studies are needed to understand the tumor-suppressive effect of Nischarin in ovarian cancer.
Nischarin is a tumor suppressor of breast cancer
Nischarin is known to control cell migration by antagonizing the actions of cell signaling proteins that contribute to tumor cell migration and invasion (10). This protein maps at 3p21.1 and it has been shown that regions of chromosome 3p are associated with the development of many cancers (12). Highly aggressive breast cancer cells exhibit genomic loss of the NISCH locus and Nischarin promoter methylation is seen in 30% of breast cancers (12). A study of 962 human breast cancer patients from TCGA revealed that 0.3% of breast invasive carcinomas exhibit a deletion of Nischarin, and 0.7% of breast invasive carcinomas have a mutated Nischarin (36).
Loss of Nischarin plays a significant role in breast cancer cell progression. Correspondingly, Nischarin mRNA is highly expressed in normal breast tissue but poorly expressed in human breast cancer specimens (12). Highly invasive breast cancer cell lines such as MDA-MB-231 exhibit low Nischarin expression levels, moderately invasive breast cancer cell lines such as MCF-7 exhibit higher Nischarin expression levels, and nontumorigenic cells such as MCF-10A have the highest amount of Nischarin expression (12). Restoring Nischarin expression in aggressive breast cancer cell lines decreases focal adhesion kinase (FAK) phosphorylation levels due to decreased α5-integrin expression (12). Reduced FAK phosphorylation thus prevents ERK activation, resulting in decreased cell survival (12).
Analysis of human breast cancer patient tumors revealed that tissues with lymph node metastasis have significantly decreased levels of Nischarin than patients without lymph node metastasis (37). In addition, LOH studies performed using microsatellite markers in DNA samples from 18 human breast cancers and their normal tissue counterparts, revealed that LOH is seen in 50% of human breast cancer patients, which results in decreased Nischarin expression (12). Therefore, promoter methylation and LOH are the leading causes of reduced Nischarin expression in breast cancers (12).
Nischarin inhibits cell migration and invasion
Nischarin has been found to interact with a number of signaling proteins such as integrin α5, PAK1, LIMK1, Rab14, LKB1, and Rac1. Liver kinase B1 (LKB1) is a tumor suppressor that has a role in cell polarity and the regulation of metabolism through the mTOR pathway (38). Like Nischarin, LKB1 inhibits PAK phosphorylation to prevent actin filament assembly during cytoskeletal reorganization (20). LKB1 interacts with amino acids 416–624 of Nischarin to increase its kinase activity (20). Nischarin and LKB1 both reduce tumor growth, regulate cell migration, metastasis, and anchorage-dependent growth (Fig. 3; ref. 20).
Rac1 is a Rho GTPase family member that regulates a number of signaling pathways, including the organization and assembly of actin in response to the extracellular environment. Several downstream effectors exert the biologic effects of Rac1. Rac1 activates NF-κB transcription factors, leading to inflammatory responses, cell growth, and apoptotic suppression (39). Both the N- and C-terminus of Nischarin interacts with Rac1 in its active state to disrupt the NF-κB pathway and thus repress cyclin D1, the promoter associated with malignancy (Fig. 3; ref. 40). Also, reduced activation of Rac1 by Nischarin results in reduced tumor growth (12).
Nischarin has been found to inhibit the Rac1 effector, p21 activated kinase (PAK), to prevent Rac1 driven cell migration (41). Nischarin directly interacts with PAK1 and prevents its kinase activity (Fig. 3; ref. 40). PAK targets cell migration signaling pathways through myosin light chain kinase (MLCK) and LIMK (40). The activation of MLCK through PAK phosphorylates myosin light chain, which regulates actin cytoskeletal dynamics (40). LIMK is highly expressed in cancer cells and has been highly regarded as an oncogene (8). Phosphorylation of LIMK through PAK inhibits cofilin, an actin severing protein, thus leading to actin filament assembly (41). Nischarin binds to the kinase domain of active LIMK to deactivate it and prevents actin filament assembly during cytoskeletal reorganization (Fig. 3; ref. 8).
Nischarin also interacts with a number of signaling proteins 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 and thapsigargin treatment, Nischarin-transfected cells show decrease in apoptotic activity (42). This inhibition of apoptosis leads to increased cell survival. It is not clear how Nischarin can inhibit tumor growth as well as apoptosis, which needs further investigation.
Therapeutic Importance of Nischarin
Nischarin's strong tumor-suppressive effect can be used to reverse the invasive capacity of cancer cells, making it an appealing option in developing future cancer treatments. It has been well demonstrated that the loss of Nischarin leads to increased focal adhesions, cytoskeletal organization, cell migration, tumor growth, and cell survival. Increasing expression of Nischarin in tumor cells would reduce the invasive and migratory capacity of cancer cells. Also, because LOH was seen in 50% of human breast cancer patients, Nischarin could be used as a clinical biomarker for patients.
To compensate for lost or reduced Nischarin function in tumors, it is possible to create peptide-based drugs that mimic Nischarin's natural interactions. Peptide-based drugs are advantageous for high potency, high selectivity, and low toxicity treatment (43). Designing a small peptide-based drug that mimics the different domains of Nischarin could decrease the migratory effects of the cancer cells. For example, designing a peptide drug with the amino acids of Nischarin that are necessary to bind integrin α5 would decrease integrin α-5–mediated cell migration.
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 is most evident within cells of the immune, nervous, secretory, muscular, and reproductive systems (11). A role for Nischarin in many 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. Over 30 research papers have been published on Nischarin and further studies of this protein will increase current knowledge in the fields of cancer biology, cell migration, apoptosis, vesicle trafficking, cell adhesion, signal transduction, and hypertension. This dynamic scaffolding protein is ideal for studying cell signaling pathways in a vast number of diseases. A deeper understanding of Nischarin-mediated pathways will help predict disease progression and provide better therapeutic targets for breast cancer patients.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.