Contribution of Nischarin/IRAS in CNS development, injury and diseases.

BACKGROUND: Murine Nischarin and its human homolog IRAS are scaffold proteins highly expressed in the central nervous system (CNS). Nischarin was initially discovered as a tumor suppressor protein, and recent studies have also explored its potential value in the CNS. Research on IRAS has largely focused on its effect on opioid dependence. Although the role of Nischarin/IRAS in the physiological function and pathological process of the CNS has gradually attracted attention and the related research results are expected to be applied in clinical practice, there is no systematic review of the role and mechanisms of Nischarin/IRAS in the CNS so far. AIM OF REVIEW: This review will systematically analyze the role and mechanism of Nischarin/IRAS in the CNS, and provide necessary references and possible targets for the treatment of neurological diseases, thereby broadening the direction of Nischarin/IRAS research and facilitating clinical translation. KEY SCIENTIFIC CONCEPTS OF REVIEW: The pathophysiological processes affected by dysregulation of Nischarin/IRAS expression in the CNS are mainly introduced, including spinal cord injury (SCI), opioid dependence, anxiety, depression, and autism. The molecular mechanisms such as factors regulating Nischarin/IRAS expression and signal transduction pathways regulated by Nischarin/IRAS are systematically summarized. Finally, the clinical application of Nischarin/IRAS has been prospected.

Moxonidine ameliorates cardiac injury in rats with metabolic syndrome by regulating autophagy.

AIMS: Reduced cardiac autophagy, ischemic injury, sympathetic overactivity, and apoptosis all contribute to metabolic syndrome (MetS)-associated cardiovascular risks. NR4A2, an orphan nuclear receptor NR4A family member, induces autophagy while suppressing apoptosis in myocardial infarction. Moxonidine, a sympathoinhibitor imidazoline1 receptor (I1R) agonist, has beneficial metabolic and hemodynamic effects; however, whether autophagy and/or NR4A2 signaling are involved in moxonidine's cardiovascular effects via I1R activation, is unknown, and is the aim of this study. MATERIALS AND METHODS: To induce MetS, rats were fed 3 % salt in their diet and 10 % fructose in their drinking water for 12 weeks. MetS-rats were given either moxonidine (6 mg/kg/day, gavage), efaroxan (I1R antagonist, 0.6 mg/kg/day, i.p), both treatments, or vehicles for the last two weeks. Blood pressure, lipid profile, and glycemic control were evaluated. Histopathological examination, circulating cardiac troponin I (c-TnI), proinflammatory interleukin-6 (IL-6), apoptosis (active caspase-3 and Fas-immunostaining), interstitial fibrosis [transforming growth factor-ß1 (TGF-ß1), Mallory's trichrome staining], and extracellular matrix remodeling [matrix metalloproteinase-9 (MMP-9)], were used to assess cardiac pathology. Cardiac NR4A2 and its downstream factor, p53, as well as autophagic flux markers, SQSTM1/p62, LC3, and Beclin-1 were also determined. KEY FINDINGS: Moxonidine significantly ameliorated MetS-induced metabolic and hemodynamic derangements and the associated cardiac pathology. Moxonidine restored NR4A2 and p53 myocardial levels and enhanced autophagic flux via modulating SQSTM1/p62, LC3, and Beclin-1. Efaroxan reversed the majority of the moxonidine-induced improvements. SIGNIFICANCE: The current study suggests that autophagy modulation via I1R activation is involved in moxonidine-mediated cardiac beneficial effects in MetS.

Nischarin Deletion Reduces Oxidative Metabolism and Overall ATP: A Study Using a Novel NISCHΔ5-6 Knockout Mouse Model.

Nischarin (Nisch) is a cytosolic scaffolding protein that harbors tumor-suppressor-like characteristics. Previous studies have shown that Nisch functions as a scaffolding protein and regulates multiple biological activities. In the current study, we prepared a complete Nisch knockout model, for the first time, by deletion of exons 5 and 6. This knockout model was confirmed by Qrt-PCR and Western blotting with products from mouse embryonic fibroblast (MEF) cells. Embryos and adult mice of knockouts are significantly smaller than their wild-type counterparts. Deletion of Nisch enhanced cell migration, as demonstrated by wound type and transwell migration assays. Since the animals were small in size, we investigated Nisch's effect on metabolism by conducting several assays using the Seahorse analyzer system. These data indicate that Nisch null cells have lower oxygen consumption rates, lower ATP production, and lower levels of proton leak. We examined the expression of 15 genes involved in lipid and fat metabolism, as well as cell growth, and noted a significant increase in expression for many genes in Nischarin null animals. In summary, our results show that Nischarin plays an important physiological role in metabolic homeostasis.

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.

Benzofuranyl-2-imidazoles as imidazoline I2 receptor ligands for Alzheimer's disease.

Recent findings unveil the pharmacological modulation of imidazoline I2 receptors (I2-IR) as a novel strategy to face unmet medical neurodegenerative diseases. In this work, we report the chemical characterization, three-dimensional quantitative structure-activity relationship (3D-QSAR) and ADMET in silico of a family of benzofuranyl-2-imidazoles that exhibit affinity against human brain I2-IR and most of them have been predicted to be brain permeable. Acute treatment in mice with 2-(2-benzofuranyl)-2-imidazole, known as LSL60101 (garsevil), showed non-warning properties in the ADMET studies and an optimal pharmacokinetic profile. Moreover, LSL60101 induced hypothermia in mice while decreased pro-apoptotic FADD protein in the hippocampus. In vivo studies in the familial Alzheimer's disease 5xFAD murine model with the representative compound, revealed significant decreases in the protein expression levels of antioxidant enzymes superoxide dismutase and glutathione peroxidase in hippocampus. Overall, LSL60101 plays a neuroprotective role by reducing apoptosis and modulating oxidative stress.

Isoginkgetin derivative IP2 enhances the adaptive immune response against tumor antigens.

The success of cancer immunotherapy relies on the induction of an immunoprotective response targeting tumor antigens (TAs) presented on MHC-I molecules. We demonstrated that the splicing inhibitor isoginkgetin and its water-soluble and non-toxic derivative IP2 act at the production stage of the pioneer translation products (PTPs). We showed that IP2 increases PTP-derived antigen presentation in cancer cells in vitro and impairs tumor growth in vivo. IP2 action is long-lasting and dependent on the CD8+ T cell response against TAs. We observed that the antigen repertoire displayed on MHC-I molecules at the surface of MCA205 fibrosarcoma is modified upon treatment with IP2. In particular, IP2 enhances the presentation of an exon-derived epitope from the tumor suppressor nischarin. The combination of IP2 with a peptide vaccine targeting the nischarin-derived epitope showed a synergistic antitumor effect in vivo. These findings identify the spliceosome as a druggable target for the development of epitope-based immunotherapies.

Small Molecule Targets TMED9 and Promotes Lysosomal Degradation to Reverse Proteinopathy.

Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.

The role of IRAS/Nischarin involved in the development of morphine tolerance and physical dependence.

Morphine is a potent opioid analgesic used to alleviate moderate or severe pain, but the development of drug tolerance and dependence limits its use in pain management. Our previous studies showed that the candidate protein for I1 imidazoline receptor, imidazoline receptor antisera-selected (IRAS)/Nischarin, interacts with µ opioid receptor (MOR) and modulates its trafficking. However, there is no report of the effect of IRAS on morphine tolerance and physical dependence. In the present study, we found that IRAS knockout (KO) mice showed exacerbated analgesic tolerance and physical dependence compared to wild-type (WT) mice by chronic morphine treatment. Chronic morphine treatment down-regulated the expression of MOR in spinal cord of IRAS KO mice, while had no significant effect on MOR expression in WT mice. We observed the compensatory increase of cAMP accumulation in spinal cord after morphine tolerance, and this change was more significant in KO mice than WT mice. Furthermore, KO mice showed more elevation in the phosphorylation of AMPA receptor GluR1-S845 than WT mice, while the total expression of GluR1 remained unchanged after morphine dependence. Altogether, these data suggest that IRAS may play an important role in the development of morphine tolerance and physical dependence in vivo through modulating MOR expression, as well as AMPA GluR1-S845 phosphorylation, which might be one of the mechanisms underlying the development of opiate addiction.

Effects of imidazoline I2 receptor agonists on reserpine-induced hyperalgesia and depressive-like behavior in rats.

Pharmacotherapies for fibromyalgia treatment are lacking. This study examined the antinociceptive and antidepressant-like effects of imidazoline I2 receptor (I2R) agonists in a reserpine-induced model of fibromyalgia in rats. Rats were treated for 3 days with vehicle or reserpine. The von Frey filament test was used to assess the antinociceptive effects of I2 receptor agonists, and the forced swim test was used to assess the antidepressant-like effects of these drugs. 2-BFI (3.2-10 mg/kg, intraperitoneally), phenyzoline (17.8-56 mg/kg, intraperitoneally), and CR4056 (3.2-10 mg/kg, intraperitoneally) all dose-dependently produced significant antinociceptive effects, which were attenuated by the I2R antagonist idazoxan. Only CR4056 significantly reduced the immobility time in the forced swim test in both vehicle-treated and reserpine-treated rats. These data suggest that I2R agonists may be useful to treat fibromyalgia-related pain and comorbid depression.

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.

Ameliorative effects of clonidine on ethanol induced kidney injury in rats: Potential role for imidazoline-1 receptor.

Chronic alcoholism is a risk factor for kidney injury. Clonidine is an α2-adrenergic receptor/imidazoline-1 receptor agonist that can reduce blood pressure and maintain renal functions. This study aims to investigate the possible ameliorative effects of clonidine on ethanol induced kidney injury and its mechanism of action. Kidney injury was induced in rats by adding ethanol to drinking water for eight weeks. Clonidine effects on kidney functions and histopathology were measured. Moreover, phentolamine (α-adrenergic receptor antagonist), efaroxan (imidazoline-1 receptor antagonist) and rilmenidine (imidazoline-1 receptor agonist) were used to clarify the role of imidazoline-1 receptor in mediating renal ameliorative effects. Also, the effect of clonidine on liver functions and metabolic changes, in addition to renal oxidative stress, inflammatory and apoptotic pathways were measured. Results showed that, clonidine improved renal functions and reduced ethanol induced renal inflammation and fibrosis. On the other hand, efaroxan, only, blocked clonidine effects on kidney functions. Rilmenidine decreased kidney injury like clonidine. Both clonidine and rilmenidine increased renal nischarin gene expression. Furthermore, clonidine improved liver functions, increased serum insulin and decreased serum advanced glycation end products (metabolic markers). Also, clonidine reduced renal oxidative stress as reflected by decreased myeloperoxidase, malondialdehyde, inducible nitric oxide synthase and total nitric oxide levels and increased superoxide dismutase level. Moreover, clonidine reduced renal tumor necrosis factor-α (inflammatory marker) and caspase-3 (apoptotic marker) levels, while increased renal prostaglandine E2 and interleukin-10 levels (anti-inflammatory markers). In conclusion, clonidine can reduce ethanol induced kidney injury, at least in part, by stimulating imidazoline-1 receptor signaling.

Nischarin regulates focal adhesion and Invadopodia formation in breast cancer cells.

BACKGROUND: During metastasis, tumor cells move through the tracks of extracellular matrix (ECM). Focal adhesions (FAs) are the protein complexes that link the cell cytoskeleton to the ECM and their presence is necessary for cell attachment. The tumor suppressor Nischarin interacts with a number of signaling proteins such as Integrin α5, PAK1, LIMK1, LKB1, and Rac1 to prevent cancer cell migration. Although previous findings have shown that Nischarin exerts this migratory inhibition by interacting with other proteins, the effects of these interactions on the entire FA machinery are unknown. METHODS: RT-PCR, Western Blotting, invadopodia assays, and immunofluorescence were used to examine FA gene expression and determine whether Nischarin affects cell attachment, as well as the proteins that regulate it. RESULTS: Our data show that Nischarin prevents cell migration and invasion by altering the expression of key focal adhesion proteins. Furthermore, we have found that Nischarin-expressing cells have reduced ability to attach the ECM, which in turn leads to a decrease in invadopodia-mediated matrix degradation. CONCLUSIONS: These experiments demonstrate an important role of Nischarin in regulating cell attachment, which adds to our understanding of the early events of the metastatic process in breast cancer.

A mouse-to-man candidate gene study identifies association of chronic otitis media with the loci TGIF1 and FBXO11.

Chronic otitis media with effusion (COME) is the most common cause of hearing loss in children, and known to have high heritability. Mutant mouse models have identified Fbxo11, Evi1, Tgif1, and Nisch as potential risk loci. We recruited children aged 10 and under undergoing surgical treatment for COME from 35 hospitals in the UK, and their nuclear family. We performed association testing with the loci FBXO11, EVI1, TGIF1 and NISCH and sought to replicate significant results in a case-control cohort from Finland. We tested 1296 families (3828 individuals), and found strength of association with the T allele at rs881835 (p = 0.006, OR 1.39) and the G allele at rs1962914 (p = 0.007, OR 1.58) at TGIF1, and the A allele at rs10490302 (p = 0.016, OR 1.17) and the G allele at rs2537742 (p = 0.038, OR 1.16) at FBXO11. Results were not replicated. This study supports smaller studies that have also suggested association of otitis media with polymorphism at FBX011, but this is the first study to report association with the locus TGIF1. Both FBX011 and TGIF1 are involved in TGF-ß signalling, suggesting this pathway may be important in the transition from acute to chronic middle ear inflammation, and a potential molecular target.

Dexmedetomidine relieves formaldehyde-induced pain in rats through both α2 adrenoceptor and imidazoline receptor.

Dexmedetomidine (DEX) is a highly selective α2 adrenergic receptor agonist. In this study, we investigated the analgesic effect and the underlying mechanisms of DEX on inflammatory visceral pain in rats. Twenty-five male Sprague Dawley (SD) rats were randomly divided into 5 groups, including control, sham, low dose DEX, medium dose DEX and high dose DEX group. Pain was induced with 10% formalin and scored every 15min till 2 h-post the induction. Hematoxylin-eosin (HE) staining was used to evaluate the toxicity of DEX on spinal cord neurons. Acetycholine (Ach) and noradrenaline (NA) levels were determined by using ELISA method. The expressions of natural nitric oxide synthase (nNOS), protein kinase γ (PKCγ) and protease-activated receptor 2 (PAR2) were determined by using western blot. DEX treatment relieved formaldehyde-induced pain in rats in a dose-dependent manner. Furthermore, DEX showed little neuro-toxicity on the spinal cord neurons, even at the highest dosage used in our study. Ach level was significantly increased in Sham group compared with control group. DEX treatment decreased NA levels and increased Ach levels in the incubation medium of spinal cord sections. Western blot analysis showed that the expression of nNOS, PKCγ and PAR2 was significantly decreased in DEX group compared with Sham group, whereas these effects of DEX on nNOS, PKCγ and PAR2 were blocked by both yohimbine and idazoxan, indicating that the analgesic effect of DEX is mediated by both α2 adrenergic receptor and imidazoline receptor. Yohimbine and idazoxan treatment significantly enhanced pain scores compared to DEX group, and which antagonizes the effects DEX. In conclusion, our study demonstrated that DEX could inhibit formaldehyde-induced pain by inhibiting nNOS, PKCγ and PAR2 expression through α2 adrenergic receptor and imidazoline receptor.

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.

New 2-Aminothiazoline derivatives lower blood pressure of spontaneously hypertensive rats (SHR) via I1-imidazoline and alpha-2 adrenergic receptors activation.

2-Aminothiazolines share an isosteric relationship with imidazolines and oxazolines with antihypertensive activity mainly mediated by the imidazoline I1-receptor. In the present work, we have prepared five aminothiazolines, following a previously described synthetic pathway. Aminothiazolines derived from dicyclopropylmethylamine (ATZ1) and cyclohexylamine (3) are unprecedented in the literature. Competitive radioligand assay was carried out with all synthetic compounds, and the I1 receptor affinity in comparison to rilmenidine in PC12 cells was determined. Surprisingly, the rilmenidine isoster (ATZ1) showed no I1-receptor interaction. Diethyl (ATZ4) and 2-ethyl-hexylamine (ATZ5) derivatives bind to the receptor with 11.98 and 10.94nmol/l, respectively. These compounds were selected for in vivo experiments. Both compounds reduced the blood pressure of spontaneously hypertensive rats (SHR). The hypotensive effect of these compounds was abrogated in the presence of α2 adrenergic (yohimbine) and I1 (efaroxan) receptor antagonists suggesting that both aminothiazolines bind to the adrenergic and imidazoline receptors. Lipinski's descriptors of the synthesized aminothiazolines were calculated and are similar to the known imidazoline I1 receptor ligands. 3D-Similarity between ATZ5 and agmatine, the natural imidazoline receptor ligand, was also observed.

Rilmenidine suppresses proliferation and promotes apoptosis via the mitochondrial pathway in human leukemic K562 cells.

Imidazoline I1 receptor signaling is associated with pathways that regulate cell viability leading to varied cell-type specific phenotypes. We demonstrated that the antihypertensive drug rilmenidine, a selective imidazoline I1 receptor agonist, modulates proliferation and stimulates the proapoptotic protein Bax thus inducing the perturbation of the mitochondrial pathway and apoptosis in human leukemic K562 cells. Rilmenidine acts through a mechanism which involves deactivation of Ras/MAP kinases ERK, p38 and JNK. Moreover, rilmenidine renders K562 cells, which are particularly resistant to chemotherapeutic agents, susceptible to the DNA damaging drug doxorubicin. The rilmenidine co-treatment with doxorubicin reverses G2/M arrest and triggers apoptotic response to DNA damage. Our data offer new insights into the pathways associated with imidazoline I1 receptor activation in K562 cells suggesting rilmenidine as a valuable tool to deepen our understanding of imidazoline I1 receptor signaling in hematologic malignancies and to search for medicinally active agents.

Bidirectional effects of dexmedetomidine on human platelet functions in vitro.

Platelets express the imidazoline (I)-receptor, I1 and I2, as well as the α2-adrenoceptor. Although dexmedetomidine, a selective α2-adrenoceptor agonist with some affinity for the I-receptor is expected to affect platelet function, the effects of dexmedetomidine on platelet functions remain unclear. In the present study, we investigated the effects of dexmedetomidine on human platelet functions in vitro. The effects of dexmedetomidine on platelet aggregation were examined using aggregometers. The formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in platelets was measured by an enzyme immunoassay. In addition, P-selectin expression in platelets was estimated by flow cytometry. We showed that dexmedetomidine enhances platelet aggregation. But in the presence of yohimbine, an α2-antagonist, dexmedetomidine suppressed platelet aggregation. Efaroxan, an I1-antagonist, and methylene blue, a soluble guanylate cyclase inhibitor, abolished the suppressive effect of dexmedetomidine, whereas idazoxan, an I2-antagonist, showed no effect. Dexmedetomidine suppressed cAMP formation and enhanced P-selectin expression in platelets, and these effects were inhibited by yohimbine. Dexmedetomidine increased cGMP formation in platelets in the presence of yohimbine, and this increase was suppressed by efaroxan. These results demonstrated that dexmedetomidine has both enhancing and suppressive effects on human platelet functions through its action on the α2-adrenoceptor and on the I1-imidazoline receptor, respectively.

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

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.

Frequent Loss of NISCH Promotes Tumor Proliferation and Invasion in Ovarian Cancer via Inhibiting the FAK Signal Pathway.

NISCH encodes the imidazoline receptor Nischarin and is a known tumor suppressor in many human malignancies; however, its roles in ovarian cancer are still largely unknown. Here, we aim to investigate the biologic functions of NISCH in ovarian cancer. We found that NISCH was significantly downregulated, which correlated considerably with advanced tumor stage, poor differentiation, lymph node metastasis, and the serous/mucinous subtypes in a panel of ovarian cancer tissues. Moreover, NISCH gene silencing was mainly the product of promoter hypermethylation, which could be reversed by treatment with 5-aza-dC. In vitro, NISCH overexpression suppressed cell proliferation and colony formation by hindering cell-cycle progression, whereas the opposite was observed in NISCH knockdown counterparts. In vivo, abundant NISCH expression hindered the growth of HO8910 xenografts, whereas NISCH knockdown accelerated the growth of SKOV3 xenografts. In addition, NISCH significantly attenuated cell invasion by inhibiting the phosphorylation of FAK and ERK, which could be neutralized by PF-562271 (a FAK/Pyk2 inhibitor). Accordingly, NISCH knockdown xenografts exhibited increased peritoneal/pelvic metastases that were not present in counterparts treated with PF-562271. Furthermore, NISCH expression in primary ovarian cancer cells predicted a cellular resistance to PF-562271. In conclusion, we showed that NISCH was frequently silenced by promoter hypermethylation in human ovarian cancer. NISCH manipulated cellular proliferation and invasion by arresting cell cycle and inhibiting the FAK signal. Our findings revealed the biologic functions of NISCH in ovarian cancer, and might be useful for treating patients with aberrant expression of NISCH.