Nischarin Expression Research Articles

Preliminary investigation on the mechanism of baicalein regulating the effects of Nischarin on invasion and apoptosis of human breast cancer cells MCF-7 through Wnt3α/β-catenin pathway

BackgroundBreast cancer (BC) remains the leading cause of cancer-related mortality in women. Here, we investigate the anti-tumor effects of baicalein on human BC cells (MCF-7 cells) and explore if it regulates the Nischarin protein via Wnt3α/β-catenin signaling pathway. MethodsWe employed Wnt3α and DKK-1 to activate and inhibit the Wnt/β-catenin signaling pathway, respectively. We used CCK-8 cell viability, flow cytometry apoptosis, wound-healing and transwell migration/invasion assays. Further, using western blotting and real-time quantitative PCR (q-PCR) we analyzed expression levels of Nischarin, MMP-9, Wnt/β-catenin pathway (β-catenin, Axin 1), and apoptotic pathway (Bax, Bcl-2) proteins and their mRNAs. ResultsWe found that baicalein inhibits MCF-7 cell viability and promotes apoptosis (evidenced by increased Bax and decreased Bcl-2 expressions) in a concentration-dependent manner. It also inhibits TPA-induced migration and invasion, and downregulates MMP-9 expression. Baicalein reverses the increase in cell viability caused by Wnt3α-induced Wnt/β-catenin pathway activation. Conversely, baicalein counteracts the increase in apoptosis caused by DKK-1 mediated inhibition of the Wnt/β-catenin pathway. Additionally, baicalein upregulates Nischarin expression via modulating the Wnt/β-catenin pathway as indicated by the antagonistic effects of Wnt3α and DKK-1 on this effect of baicalein. ConclusionBaicalein exerts anti-tumor effects on MCF-7 cells through the Wnt3α/β-catenin signaling pathway, and promotes apoptosis and inhibits migration and invasion. The upregulation of Nischarin by baicalein further suggests a potential therapeutic target for BC treatment.

Analysis of the nischarin expression across human tumor types reveals its context-dependent role and a potential as a target for drug repurposing in oncology.

Nischarin was reported to be a tumor suppressor that plays a critical role in breast cancer initiation and progression, and a positive prognostic marker in breast, ovarian and lung cancers. Our group has found that nischarin had positive prognostic value in female melanoma patients, but negative in males. This opened up a question whether nischarin has tumor type-specific and sex-dependent roles in cancer progression. In this study, we systematically examined in the public databases the prognostic value of nischarin in solid tumors, regulation of its expression and associated signaling pathways. We also tested the effects of a nischarin agonist rilmenidine on cancer cell viability in vitro. Nischarin expression was decreased in tumors compared to the respective healthy tissues, most commonly due to the deletions of the nischarin gene and promoter methylation. Unlike in healthy tissues where it was located in the cytoplasm and at the membrane, in tumor tissues nischarin could also be observed in the nuclei, implying that nuclear translocation may also account for its cancer-specific role. Surprisingly, in several cancer types high nischarin expression was a negative prognostic marker. Gene set enrichment analysis showed that in tumors in which high nischarin expression was a negative prognostic marker, signaling pathways that regulate stemness were enriched. In concordance with the findings that nischarin expression was negatively associated with pathways that control cancer growth and progression, nischarin agonist rilmenidine decreased the viability of cancer cells in vitro. Taken together, our study lays a ground for functional studies of nischarin in a context-dependent manner and, given that nischarin has several clinically approved agonists, provides rationale for their repurposing, at least in tumors in which nischarin is predicted to be a positive prognostic marker.

Nischarin expression may have differing roles in male and female melanoma patients.

Due to the development of resistance to previously effective therapies, there is a constant need for novel treatment modalities for metastatic melanoma. Nischarin (NISCH) is a druggable scaffolding protein reported as a tumor suppressor and a positive prognostic marker in breast and ovarian cancers through regulation of cancer cell survival, motility and invasion. The aim of this study was to examine the expression and potential role of nischarin in melanoma. We found that nischarin expression was decreased in melanoma tissues compared to the uninvolved skin, and this was attributed to the presence of microdeletions and hyper-methylation of the NISCH promoter in the tumor tissue. In addition to the previously reported cytoplasmic and membranous localization, we observed nischarin in the nuclei in melanoma patients' tissues. NISCH expression in primary melanoma had favorable prognostic value for female patients, but, unexpectedly, high NISCH expression predicted worse prognosis for males. Gene set enrichment analysis suggested significant sex-related disparities in predicted association of NISCH with several signaling pathways, as well as with different tumor immune infiltrate composition in male and female patients. Taken together, our results imply that nischarin may have a role in melanoma progression, but that fine-tuning of the pathways it regulates is sex-dependent. KEY MESSAGES: Nischarin is a tumor suppressor whose role has not been investigated in melanoma. Nischarin expression was downregulated in melanoma tissue compared to the normal skin. Nischarin had the opposite prognostic value in male and female melanoma patients. Nischarin association with signaling pathways differed in females and males. Our findings challenge the current view of nischarin as a universal tumor suppressor.

Effects of Nischarin on the Migration Ability of Hepatocellular Carcinoma Cells

To clarify the effect of Nischarin on the biological behavior of hepatocellular carcinoma cells, two human hepatocellular carcinoma cell lines QGY-7701 and HepG2 were selected. The migration ability of the cells was determined with human normal hepatocyte LO2 as control. Western blotting was used to detect the differential expression of Nischarin and its downstream signals. The expression of Nischarin was knocked down by plasmid transfection to study the changes of cell migration ability, so as to evaluate the effect of Nischarin on hepatocellular carcinoma cells. The results of cell scratch experiment showed that compared with LO2 cells, the migration ability of HepG2 was significantly enhanced (<I>P </I>< 0.05), but there was no significant difference in the migration ability between QGY-7701 and LO2 cells. The results of Western blotting showed that the expression of Nischarin in HepG2 was significantly lower than that in LO2 cells (<I>P</I> < 0.05). Quantitative RT-PCR was used to detect the mRNA level of Nischarin in cells and found that the Nischarin mRNA level of HepG2 was significantly lower than that of LO2 (<I>P</I> < 0.01). When HepG2 cells were transfected with Nis-shRNA to knock down the expression of Nischarin, the cell migration ability was significantly increased (<I>P</I> < 0.05). While Nischarin overexpression significantly inhibited the phosphorylation of LIMK1 and cofilin, the important molecules of Rho-GTPase signaling pathway. These results strongly suggest that Nischarin is an endogenous protein with the ability to inhibit the migration of hepatocellular carcinoma cells. In conclusion, we found a protein that can inhibit the migration of hepatocellular carcinoma cells, which may have potential value in the diagnosis and treatment of hepatocellular carcinoma, but its molecular mechanism needs to be further studied.

Abstract PO-040: Nischarin is expressed in pancreatic ductal adenocarcinoma and is a potential target for drug repurposing

Abstract The objective of this study was to examine the expression of Nischarin (NISCH) in pancreatic ductal adenocarcinoma (PDAC), and its potential as a target for drug repurposing. NISCH has been described as a tumor suppressor in breast and ovarian cancers, and there are several clinically approved agonists for this receptor. NISCH has also been reported as a scaffolding protein with a role in cell adhesion, invasion and metabolism – aspects of the PDAC biology that are of interest for therapeutical intervention. We examined mRNA and protein expression of NISCH in publicly available datasets, patient tumor samples, representative PDAC cell lines and patient-derived stellate cells. We found that NISCH is a prognostic marker in PDAC patients and that it is expressed in both cancer cells and cancer-associated stroma. Gene set enrichment analysis showed that NISCH expression was associated with the regulation of cell motility, cell cycle and energy metabolism. Indeed, NISCH knockdown in cancer cells induced reorganization of the actin cytoskeleton. Next, we tested the effects of three clinically approved NISCH agonists (rilmenidine, clonidine and moxonidine) on cancer cells and cancer-associated fibroblasts (CAFs). We found that rilmenidine, agonist with the highest affinity for the NISCH receptor, had the most potent effect. Rilmenidine impaired cancer cell adhesion, limited migration and decreased production of pro-metastatic cytokines and extracellular matrix deposition in cancer cell-CAF co-cultures. Our study lays a ground for more extensive examination of the role of NISCH in PDAC and implies that NISCH agonists may be good candidates for drug repurposing in this type of cancer. Citation Format: Jelena Grahovac, Marijana Pavlovic, Marija Ostojic, Kristina Zivic, Daniel Galun, Tatjana Srdic-Rajic. Nischarin is expressed in pancreatic ductal adenocarcinoma and is a potential target for drug repurposing [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-040.

Prenatal exposure to di (2-ethylhexyl) phthalate causes autism-like behavior through inducing Nischarin expression in the mouse offspring

Epidemiologic evidence has suggested a relationship between di (2-ethylhexyl) phthalate (DEHP) prenatal exposure and autism spectrum disorders (ASD), but the underlying mechanisms are still at large unknown. In this study, pregnant mice were intragastrically administered with DEHP once a day from GD 3 to GD 17 and the neurobehavioral changes of offspring were evaluated. In addition to the repetitive stereotyped behaviors, DEHP at the concentration of 50 mg/kg/day and above significantly impaired the sociability of the offspring (P < 0.05) and decreased the density of dendritic spines of pyramidal neurons in the prefrontal cortex (P < 0.05). At the same time, the expression of Nischarin protein in prefrontal lobe increased (P < 0.05). Similarly, after 12-h incubation of DEHP at the concentration of 100 nM, the total spine density, especially the mushroom and stubby spine populations, significantly decreased in the primary cultured prefrontal cortical neurons (P < 0.05). However, the inhibitory effect of DEHP were reversed by knockdown of Nischarin expression. Collectively, these results suggest that prenatal DEHP exposure induces Nischarin expression, causes dendritic spine loss, and finally leads to autism-like behavior in mouse offspring.

Expression of nischarin, an imidazoline 1 receptor candidate protein, in the ventrolateral medulla of newborn rats

The activation of imidazoline 1 (I1) receptors is suggested to stimulate the respiratory drive in newborn rats. Here, we immunohistochemically examined whether nischarin, an I1 receptor candidate protein, is expressed in the ventrolateral medulla, where cardiorespiratory centers are located. Newborn rats (age, 3–5 days) were deeply anesthetized with isoflurane; the brainstem was dissected, sectioned sagittally, and labeled with nischarin. Nischarin-associated signals were observed broadly throughout the newborn rat brainstem, including at motor nuclei (motor trigeminal nucleus and facial nucleus), sensory nuclei (lateral superior olive, medial and spinal vestibular nuclei, cuneate nucleus, spinal trigeminal nucleus, and solitary nucleus), and the rostral and caudal ventrolateral medullar regions. In particular, the rostral ventrolateral medulla included a layer of aggregated nischarin expression along the ventral surface, and the layer was in close contact with GFAP-positive processes. In addition, some Phox2b-positive neurons were positive for nischarin in the region. Our results reveal nischarin expression in the newborn rat brainstem and suggest that I1 receptor activation at the level of the ventrolateral medulla contributes to central chemoreception and respiratory control in newborn rats.

Nischarin downregulation attenuates cell injury induced by oxidative stress via Wnt signaling.

Nischarin (NISCH) is a key protein functioning as a molecular scaffold and thereby hosting interactions with several protein partners. Here, we aimed to investigate whether NISCH downregulation could protect rat pheochromocytoma (PC12) cells against oxidative stress-induced injury using a model of cell injury induced by hydrogen peroxide (H2O2). Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis rate was evaluated using flow cytometry. The expressions of apoptosis-related proteins Bax, Bcl-2, caspase-3 and NISCH were examined via Western blot analysis and immunofluorescence staining analyses. The expressions of NISCH, glycogen synthase kinase-3β (GSK-3β) and T-cell factor-1 (TCF-1) were examined using Western blot analysis. The results showed that incubation of H2O2 for 48 h significantly decreased the cell viability, increased the cell apoptosis rate and the NISCH expression in PC12 cells, whereas NISCH downregulation blocked the effects of H2O2 on cells. In addition, the expression of Bcl-2 was significantly reduced, and the expression of Bax and caspase-3 were significantly increased by H2O2 treatment. However, these effects were partially inhibited by the downregulation of NISCH. Furthermore, H2O2 significantly weakened the transduction of Wnt signaling, including the increases of GSK-3β and TCF-1 expressions and the decrease of β-catenin expression, while NISCH downregulation attenuated the effect of H2O2 on Wnt signaling. Moreover, inhibition of the Wnt pathway further decreased the cell viability and promoted the cell apoptosis induced by H2O2 in PC12 cells. Our results suggest that NISCH downregulation may protect cells against oxidative stress-induced injury through regulating the transduction of Wnt signaling.

Effect of imidazoline-1 receptor agonists on renal dysfunction in rats associated with chronic, sequential fructose and ethanol administration.

Insulin resistance and chronic alcoholism are risk factors for renal dysfunction. This study investigated the therapeutic effects of two imidazoline-1 receptor (I1R) agonists on renal dysfunction in rats after chronic, sequential fructose and ethanol administration. Daily drinking water was supplemented with fructose (10%, w/v) for 12weeks and then with ethanol (20%, v/v) for another 8weeks. Rats were treated with rilmenidine and clonidine in the last two weeks of the study. Blood glucose and serum insulin (sIns) levels, lipid profiles, kidney function and renal histopathology were evaluated at the end of the experiment. Additionally, renal gene expression of nischarin, phosphatidylcholine-specific phospholipase C (PC-PLC) and prostaglandin E2 (PGE2) were measured. Renal levels of superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS) and total NO (tNO) were detected, and we determined the relative renal gene expression levels of alpha smooth muscle actin (α-SMA), hydroxyproline, interleukin 10 (IL-10), tumour necrosis factor alpha (TNF-α) and caspase-3. The results showed significant deterioration of blood glucose, sIns, lipid profiles, kidney function and renal histopathology in fructose/ethanol-fed rats. Additionally, markers of inflammation, fibrosis, apoptosis and oxidative stress were upregulated. The administration of rilmenidine or clonidine significantly improved blood glucose and sIns levels and reduced renal dysfunction. Our work showed that chronic, sequential fructose and ethanol administration induced fasting hyperglycaemia and renal impairment, and these effects were ameliorated by I1R agonists.

Tizanidine (Hydrochloride) Inhibits A549 Lung Cancer Cell Proliferation and Motility Through Regulating Nischarin.

PurposeTizanidine hydrochloride (TZN) is a centrally acting α2-adrenergic agonist. In this study, we aimed to explore the role of TZN on human lung cancer and to elucidate its underlying mechanisms.MethodsThe effect of TZN treatment in A549 cell proliferation, migration, invasion and apoptosis was evaluated by CCK8, transwell and flow cytometer assays. The expression of apoptosis-related proteins and the activation of AKT and Wnt3a/β-catenin pathways were detected by Western blot. From the data of DrugBank, TZN could act as an agonist to target Nischarin in humans. We next investigated the function of Nischarin receptor in lung cancer and its role in the anti-tumor activity of TZN.ResultsThe treatment of TZN inhibited the proliferation, migration and invasion of A549 cells, and induced apoptosis. These results were further confirmed by that TZN treatment increased the Bax/Bcl-2 ratio in A549 cells. We also observed that TZN treatment changed the expression and phosphorylation of proteins of AKTand Wnt3a/β-catenin signaling pathway members. By bioinformatics analysis, we found that Nischarin was down-regulated in human lung cancer tissues and patients with high Nischarin expression had a better survival. Moreover, Nischarin functioned as a tumor suppressor in the survival and metastasis of A549 cells through the regulation of AKT and Wnt3a/β-catenin pathways. Knockdown of Nischarin promoted the proliferation, invasion, migration of A549 cells and inhibited the apoptosis, which were reversed by the TZN treatment.ConclusionSummary, our data revealed that treatment of TZN inhibited the growth of lung cancer cell line A549 and may be used as a novel strategy for lung cancer therapy.

Nischarin Regulates Secretion of Exosomes and Cancer Progression.

The intercellular exchange of exosomes may play a regulatory function in tumor progression and metastasis. Maziveyi and colleagues demonstrated that Nischarin regulated the secretion of exosomes from breast cancer cells. Loss of Nischarin expression increased exosome production and promoted tumor cell growth and migration, supporting that Nischarin can influence the behavior of surrounding cancer cells. This study identified a novel function of the tumor suppressor Nischarin in exosome biology and cancer progression.See related article by Maziveyi et al., p. 2152.

Development of insulin resistance in Nischarin mutant female mice.

NISCH-STAB1 is a newly identified locus correlated to human waist-hip ratio (WHR), which is a risk indicator of developing obesity-associated diabetes. Our previous studies have shown that Nisch mutant male mice increased glucose tolerance in chow-fed conditions. Thus we hypothesized that Nisch mutant mice will have changes in insulin resistance, adipocytes, hepatic steatosis when mice are fed with high-fat diet (HFD). Insulin resistance was assessed in Nisch mutant mice and WT mice fed with high-fat diet (60% by kCal) or chow diet. Whole-body energy metabolism was examined using an indirect calorimeter. Adipose depots including inguinal white adipose tissue (WAT), perigonadal WAT, retroperitoneal WAT, and mesenteric WAT were extracted. Area and eqdiameter of each adipocyte were determined, and insulin signaling was examined as well. Paired samples of subcutaneous and omental visceral adipose tissue were obtained from 400 individuals (267 women, 133 men), and examined the expression of Nischarin. We found that insulin signaling was impaired in major insulin-sensitive tissues of Nisch mutant female mice. When mice were fed with HFD for 15 weeks, the Nisch mutant female mice not only developed severe insulin resistance and decreased glucose tolerance compared with wild-type control mice, but also accumulated more white fat, had larger adipocytes and developed severe hepatic steatosis than wild-type control mice. To link our animal studies to human diseases, we further analyzed Nischarin expression in the paired human samples of visceral and subcutaneous adipose tissue from Caucasians. In humans, we found that Nischarin expression is attenuated in adipose tissue with obesity. More importantly, we found that Nischarin mRNA inversely correlated with parameters of obesity, fat distribution, lipid and glucose metabolism. Taken together, our data revealed sexual dimorphism of Nischarin in body fat distribution, insulin resistance, and glucose tolerance in mice.

Nischarin attenuates apoptosis induced by oxidative stress in PC12 cells.

Nischarin (NISCH) is a cytoplasmic protein known to serve an inhibitory role in breast cancer cell apoptosis, migration and invasion. Recently, NISCH has been reported to be involved in the regulation of spinal cord injury (SCI). However, the molecular mechanism is still unclear. Oxidative stress contributes to tissue injury and cell apoptosis during the development of various diseases, including SCI. The aim of the present study was to investigate the role of NISCH in the regulation of apoptosis induced by oxidative stress in PC12 cells. H2O2 was used to establish an oxidative stress model in PC12 cells. Apoptosis levels were examined using flow cytometry analysis, and the expression of NISCH, Bcl-2, Bcl-2-associated X (Bax) and caspase-3 were examined using western blot and immunofluorescence staining analyses. The results demonstrated that treatment with 100 µM H2O2 significantly increased the apoptotic rate and expression of NISCH in PC12 cells. At 48 h following incubation with 100 µM H2O2, NISCH downregulation partially inhibited apoptosis of PC12 cells. In addition, the expression of Bcl-2 was significantly reduced and the expression of Bax and caspase-3 were significantly increased by H2O2 treatment. These effects were also partially inhibited by the downregulation of NISCH. The authors of the present study therefore hypothesize that NISCH may function as a pro-apoptotic protein that participates in the regulation of oxidative stress, and NISCH downregulation may protect cells from oxidative stress-induced apoptosis.

Expression of long noncoding RNA MALAT1 correlates with increased levels of Nischarin and inhibits oncogenic cell functions in breast cancer.

Malat1 is a long noncoding RNA with a wide array of functions, including roles in regulating cancer cell migration and metastasis. However, the nature of its involvement in control of these oncogenic processes is incompletely understood. In the present study, we investigate the role of Malat1 and the effects of Malat1 KO in a breast cancer cell model. Our selection of Malat1 as the subject of inquiry followed initial screening experiments seeking to identify lncRNAs which are altered in the presence or absence of Nischarin, a gene of interest previously discovered by our lab. Nischarin is a well characterized tumor suppressor protein and actively represses cell proliferation, migration, and invasion in breast cancer. Our microarray screen for lncRNAs revealed multiple lncRNAs to be significantly elevated in cells ectopically expressing Nischarin compared to control cancer cells, which have only marginal Nisch expression. Using these cells, we assess how the link between Nischarin and Malat1 affects cancer cell function, finding that Malat1 confers an inhibitory effect on cell growth and migration which is lost following Malat1 KO, but in a Nisch-dependent context. Specifically, Malat1 KO in the background of low Nischarin expression had a limited effect on cell functions, while Malat1 KO in cells with high levels of Nischarin led to significant increases in cell proliferation and migration. In summary, this project provides further clarity concerning the function of Malat1, specifically in breast cancer, while also indicating that the Nischarin expression context is an important factor in the determining how Malat1 activity is governed in breast cancer.

Expression of Nischarin negatively correlates with estrogen receptor and alters apoptosis, migration and invasion in human breast cancer

Nischarin, a novel integrin binding protein, has been demonstrated its negative effects on cell migration and invasion. However, the biological role of Nischarin in breast cancer has not been fully elucidated yet. Our study aimed to analyze the association between Nischarin expression and clinical features of breast cancer patients, and further investigate the role of Nischarin in breast cancer cells apoptosis, migration and invasion. Results showed that Nischarin expression was significantly lower in breast cancer tissues (37.8%, 23/67) than in normal tissues (61.8%, 21/34; P < 0.05), and the expression of Nischarin significantly negatively correlated with estrogen receptor status. Similarly, Nischarin expression was highest in normal breast cell line HBL-100 while triple-negative breast cancer cell line MDA-MB-231 had the lowest expression of Nischarin. Further experiments demonstrated that overexpression of Nischarin may induce apoptosis, and inhibit cell migration and invasion. The present data confirmed that Nishcharin might be a novel tumor suppressor and plays an important role in breast cancer cell apoptosis and metastasis, which can be used as a potential therapeutic target for breast cancer treatment.

Nischarin-siRNA delivered by polyethylenimine-alginate nanoparticles accelerates motor function recovery after spinal cord injury.

A previous study by our group found that inhibition of nischarin promotes neurite outgrowth and neuronal regeneration in Neuro-2a cells and primary cortical neurons. In recent years, more and more studies have shown that nanomaterials have good prospects in treatment of spinal cord injury. We proposed that small interfering RNA targeting nischarin (Nis-siRNA) delivered by polyethyleneimine-alginate (PEI-ALG) nanoparticles promoted motor function recovery in rats with spinal cord injury. Direct microinjection of 5 μL PEI-ALG/Nis-siRNA into the spinal cord lesion area of spinal cord injury rats was performed. From day 7 after surgery, Basso, Beattie and Bresnahan score was significantly higher in rats from the PEI-ALG/Nis-siRNA group compared with the spinal cord injury group and PEI-ALG/Control-siRNA group. On day 21 after injection, hematoxylin-eosin staining showed that the necrotic area was reduced in the PEI-ALG/Nis-siRNA group. Immunohistochemistry and western blot assay results confirmed successful inhibition of nischarin expression and increased protein expression of growth-associated protein-43 in the PEI-ALG/Nis-siRNA group. These findings suggest that a complex of PEI-ALG nanoparticles and Nis-siRNA effectively suppresses nischarin expression, induces expression of growth-associated protein-43, and accelerates motor function recovery after spinal cord injury.

Inhibition of Nischarin Expression Promotes Neurite Outgrowth through Regulation of PAK Activity.

Nischarin is a cytoplasmic protein expressed in various organs that plays an inhibitory role in cell migration and invasion and the carcinogenesis of breast cancer cells. We previously reported that Nischarin is highly expressed in neuronal cell lines and is differentially expressed in the brain tissue of adult rats. However, the physiological function of Nischarin in neural cells remains unknown. Here, we show that Nischarin is expressed in rat primary cortical neurons but not in astrocytes. Nischarin is localized around the nucleus and dendrites. Using shRNA to knockdown the expression of endogenous Nischarin significantly increases the percentage of neurite-bearing cells, remarkably increases neurite length, and accelerates neurite extension in neuronal cells. Silencing Nischarin expression also promotes dendrite elongation in rat cortical neurons where Nischarin interacts with p21-activated kinase 1/2 (PAK1/2) and negatively regulates phosphorylation of both PAK1 and PAK2. The stimulation of neurite growth observed in cells with decreased levels of Nischarin is partially abolished by IPA3-mediated inhibition of PAK1 activity. Our findings indicate that endogenous Nischarin inhibits neurite outgrowth by blocking PAK1 activation in neurons.

Expression of integrin-binding protein Nischarin in metastatic breast cancer.

The present study aimed to investigate the expression of Nischarin protein in primary breast cancer (PBC), and to evaluate its role in tumor metastasis. Paired specimens of breast cancer tissues and adjacent normal tissues were surgically obtained from 60 patients with PBC at the Zhejiang Cancer Hospital (Hangzhou, China). Nischarin protein concentrations were determined by an ELISA assay. Breast cancer tissues exhibited a significantly lower concentration of Nischarin (5.86±3.19 ng/ml) compared with that of the adjacent noncancerous tissues (9.25±3.65 ng/ml; P<0.001). Furthermore, cancer tissue from patients with lymph node metastasis had significantly lower levels of Nischarin protein (4.69±2.40 ng/ml) than those of patients without lymph node metastasis (7.04±3.47 ng/ml; P=0.004). There was no significant difference in Nischarin protein expression levels between patients with grade I, II or III PBC (grade I, 5.44±3.57 ng/ml; grade II, 6.42±3.85 ng/ml and grade III, 5.10±1.18 ng/ml; P=0.765). The significant differences in the expression of Nischarin between: i) Cancer tissue and noncancerous tissue and ii) patients with and without lymph node metastasis, suggested that Nischarin may have a significant role in tumor occurrence and metastasis of breast cancer. Nischarin expression may therefore be used as a marker to predict the invasiveness and metastasis of PBC.

Prooncogenic Factors miR-23b and miR-27b Are Regulated by Her2/Neu, EGF, and TNF-α in Breast Cancer

miRNAs (miR) are a critical class of small (21-25 nucleotides) noncoding endogenous RNAs implicated in gene expression regulation. We identified miR-23b and miR-27b as miRNAs that are highly upregulated in human breast cancer. We found that engineered knockdown of miR-23b and miR-27b substantially repressed breast cancer growth. Nischarin (NISCH) expression was augmented by knockdown of miR-23b as well as miR-27b. Notably, these miRNAs and Nischarin were inversely expressed in human breast cancers, underscoring their biologic relevance. We showed the clinical relevance of the expression of these miRNAs and showed that high expression of miR-23b and miR-27b correlates with poor outcome in breast cancer. Moreover, intraperitoneally delivered anti-miR-27b restored Nischarin expression and decreased tumor burden in a mouse xenograft model of human mammary tumor. Also, we report for the first time that HER2/neu (ERBB2), EGF, and TNF-α promote miR-23b/27b expression through the AKT/NF-κB signaling cascade. Nischarin was found to regulate miR-27b/23b expression through a feedback loop mechanism by suppressing NF-κB phosphorylation. Because anti-miR-27b compounds that suppress miR-27b inhibit tumor growth, the anti-miR-27b seems to be a good candidate for the development of new antitumor therapies.

Nischarin Is Differentially Expressed in Rat Brain and Regulates Neuronal Migration

Nischarin is a protein known to inhibit breast cancer cell motility by regulating the signaling of the Rho GTPase family. However, little is known about its location and function in the nervous system. The aim of the present study was to investigate the regional and cellular expression and functions of Nischarin in the adult rodent brain. As assessed by real-time PCR, Western blot analysis and immunostaining, we found that Nischarin was widely distributed throughout the brain, with a higher expression in the cerebral cortex and hippocampus. Double-labeling showed that Nischarin was expressed in neurons and was mainly located in the perinuclear region and F-actin-rich protrusions. The expression pattern of Nischarin in the brain was thought to be closely associated with its function. This was verified by our findings from cell migration assays that Nischarin regulated neuronal migration. These results provide a preliminary survey of the distribution of Nischarin in different regions and cell types in the rat brain. This might help to elucidate its physiological roles, and to evaluate its potential clinical implications.