Nischarin was so far known as a novel tumor suppressor gene whose downregulation promotes tumorigenesis (13), tumor progression (3, 6, 18), and poor survival in breast, ovarian and lung cancer patients (3, 5, 7, 18). It was reported that exogenous expression of NISCH can suppress breast cancer cell survival and motility in vitro and growth in vivo (7, 18, 45, 46). NISCH was found to be down-regulated in breast and ovarian cancer tissues compared to the healthy counterparts and was found to be a marker of better prognosis (3, 6, 18). Surprisingly, although NISCH expression was downregulated in melanoma tissue compared to the uninvolved skin, our group found that it was a favorable prognostic marker only in female melanoma patients, but not in males (19). Bearing in mind that most findings about NISCH role in cancer stemmed from the breast cancer research and that most of the examined patient data were from females, we aimed to perform a multidimensional pan-cancer analysis of nischarin in both sexes. We examined NISCH mRNA and protein expression, prognostic value, transcriptional regulation, as well as its potential role in cancer progression, by examining publicly available datasets, and taking into account sex-related differences.
NISCH was expressed at both the mRNA and protein level in all the examined healthy human tissues, and its expression was significantly decreased in most of the analyzed solid tumor types, except thymoma where it was increased. Thymoma is a rare type of tumor characterized by unique features in comparison to other tumors of the epithelial origin. It rarely spreads beyond the thymus, has great histological heterogeneity and is often associated with immune diseases (47), which makes it an outlier in pan-cancer analysis.
In addition to the dominantly cytoplasmic and membranous localization that was reported in healthy and breast cancer cells, our group reported that NISCH was also present in the cell nucleus in melanoma tissues (19). We examined the subcellular distribution of NISCH across tumors in the Human Protein Atlas and found that only in breast and endometrial cancer NISCH localization was restricted to the cytoplasmic and membranous. In the rest of the tumor types NISCH could be also be found in the nucleus. This is a very interesting discovery because nuclear translocation of tumor suppressors is quite common in cancer to promote tumor development and inference of their localization is often implemented for diagnostic purposes (48). The novel localization could also account for cancer-specific nischarin role and should be investigated in functional studies. We analyzed the expression of NISCH by tumor type, and it is possible that analysis by molecular subtypes would reveal further intricacies of the NISCH role in cancer progression.
We next analyzed the distribution of the 4 transcripts coding NISCH isoforms, and confirmed that isoform 1 coding the full-length protein was the dominant in both solid tumors and matching healthy tissues. The expression of transcript coding isoform 1 decreased in all cancer types except thymoma; and all other investigated transcripts had the similar pattern of decrease without the isoform switch between the healthy and the tumor tissue.
Even though NISCH expression was decreased across most cancer types compared to the healthy tissues, higher expression in tumors was not a universally positive prognostic marker. Surprisingly, even within adenocarcinomas NISCH prognostic value was inconsistent: it was positive prognostic marker in LUAD and PAAD, negative in COAD and PRAD, and had no prognostic value in READ and STAD. The difference between COAD and READ can be of exceptional importance since it was previously determined that the carcinogenic risk of the rectal mucosa to develop cancer is significantly higher than that of the colon mucosa (25). NISCH was previously reported to be a positive prognostic marker in breast cancer (6), but in our examination of the TCGA dataset, it was not statistically significant. This may be due to the heterogeneity of breast cancer samples in the set. Okpechi et al (6) described that NISCH mRNA expression was lower in basal than in the luminal breast cancer, in ER negative compared to the ER positive and in PR negative compared to the PR positive tumor samples, but higher in HER negative tumors compared to the HER positive. They also reported that NISCH expression decreases with breast cancer stage and grade. This implies that NISCH prognostic role should further be examined in each tumor type by stage, grade and molecular subtype. Surprisingly, our analysis implied that the higher NISCH mRNA expression was unfavorable prognostic marker in ovarian cancer patients, which was in contrast to the report by Li et al (3). In that study, based on the immunohistochemical staining of NISCH in a tissue array consisting of 89 samples, it was found that increased NISCH protein levels were a positive prognostic marker.
Most of the cancers had lower NISCH levels compared to the healthy tissues, but only in some of them lower nischarin expression impacted the overall survival. To investigate the differences between tumors with the opposite prognostic value of NISCH, we examined the mutational status of NISCH gene and expression regulation mechanisms in cancer types in which the prognostic value, either negative or positive, was significant. Mutations in the NISCH gene were present across the length of the gene in most of the examined cancers in this study, but had no significant impact on the NISCH mRNA expression level. A missense change L972P in the NISCH gene was previously shown to be an important factor in the development of otitis media and consequential conductive hearing loss in the mouse model (49). Nevertheless, even though there was a possibility that mutations could affect some of the protein-protein interactions between NISCH and other signaling molecules, and disrupt its reported antitumor effects, they were present at a very low frequency and with no specific clustering to be considered as a cause for negative prognostic value of NISCH in a subset of tumors. In terms of the down-regulation of NISCH expression, methylation of the NISCH promoter was an important factor in BLCA, LIHC, LUAD, KIRC, KIRP, PRAD, and TGCT, and shallow deletions were a common important mechanism for all the examined tumor types. This was not surprising, as studies have shown that allelic loss from several distinct regions on chromosome 3p, including 3p21–22 where NISCH gene is located, are the earliest and most frequent genomic abnormalities involved in a wide spectrum of epithelial cancers including lung, breast, kidney, head and neck, ovary, cervix, colon, pancreas, esophagus and bladder (50). Copy-number alteration frequency was the highest in KIRC and COAD, where NISCH had negative prognostic value, but similarly high CNA levels were noted in HNSC, in which NISCH was a favorable prognostic marker. Paradoxically, high levels of chromosomal instability can be both tumor suppressive, owing to the frequent generation of unviable karyotype, and tumor propagating, leading to high intratumoral heterogeneity, therapeutic resistance and poor prognosis (51). The complexity of this problem is the subject of many ongoing studies that attempt to better exploit the dynamic process of chromosomal instability in order to create new therapeutic opportunities in cancer.
It is worth mentioning that expression of NISCH may also be regulated by microRNAs. In HNSC, it was reported that miR-2355-5p decreased NISCH expression, leading to higher tumor cell proliferation (52), and knockdown of miR-23b and miR-27b was reported to upregulate NISCH and repress breast cancer growth (53). However, OncomiR (an online resource that explores the miRNomes across TCGA cancers) (54) did not show significant correlations of NISCH mRNA with miRNAs in any of the cancers we examined.
It is possible that even without the mutations present, protein can have a tumor suppressive role that is context-dependent (55, 56). We performed GSEA to look for the differences in associated gene networks in tumor types where NISCH had negative versus positive prognostic value. In line with the NISCH so far described biological roles (7–12), and regardless of the tumor type, decreased NISCH expression was associated with activation of metabolic pathways that allow increased tumor growth. In addition, in “NISCH low” phenotype, regardless of the prognostic role, pathways that characterize survival in hypoxic and nutrient deprived conditions like reactive oxygen species pathway, unfolded protein response (57), and DNA repair (58) were enriched. The pathways that were repeatedly associated with high NISCH expression in cancer types in which it had negative prognostic value were Wnt, Hedgehog and Notch signaling. It has been reported that NISCH may regulate some aspects of Wnt signaling (59) but the association of NISCH with Notch and Hedgehog signaling is a novel finding. Common characteristic for these three pathways is that they are cell-fate determining and their crosstalk is important in the maintenance of the cancer stem cell phenotype (43). Whether there is a functional connection between nischarin and Wnt-Notch-Hedgehog is worthy of a functional study. The GSEA that we performed generated a plethora of cues that are worth investigating in functional assays in each tumor type separately, as NISCH role seems to be context dependent.
Prompted by our previous study on melanoma, we set out to examine possible sex-related differences in NISCH prognostic role, but have only found differences in survival by sex in two other cancer types: glioblastoma and thyroid cancer. We have also not found a NISCH-associated common gene network in GSEA analysis by sex. Male sex is associated with increased cancer risk and worse survival in many cancer types (60–63), and it could be that the differences in sample representation (in terms of grade, type etc.) in groups by sex were confounding our results. Nevertheless, this issue is worth further investigation, as a difference in metabolic phenotype of male and female NISCH KO mice was recently reported (15).
In contrast to the findings that exogenous expression of NISCH in breast cancer cells suppresses cell survival, in vitro studies from the early 2000s on the function of IRAS (then considered a human homologue of mouse nischarin) support the opposite claim. Overexpression of NISCH delayed apoptosis induced by a variety of stimuli (64, 65), partially through activation of the PI3 kinase pathway. Nischarin was also shown to bind insulin receptor substrate protein, activate ERK and promote survival (66). Again, it is possible that NISCH signaling in terms of the cell survival is context-dependent, as is the case with some other genes which can act as both tumor-suppressors and proto-oncogenes (e.g. TGF-β, BRCA1, p16, p14, p53, etc.) (67). Of importance, there are several FDA-approved antihypertensives with imidazoline ring that are nischarin agonists: clonidine, moxonidine and rilmenidine (68); as well as several endogenous ligands that are present in the brain tissue: agmatine, harmane, harmalan and imidazoleacetic acid-ribotide (69). Rilmenidine and rilmenidine-derived compounds were shown to induce apoptosis in breast (70) and leukemic cells in vitro (71). Agmatine was found to have anti-proliferative (72) and anti-metastatic effects in vitro (73), and to suppress tumor growth of sarcomas and melanomas in mouse models (72). Tizanidine hydrochloride reversed the proliferation, invasion, and migration of A549 cells caused by nischarin knockdown (5). Therefore, NISCH agonists present a great opportunity for testing as anti-cancer agents, at least in tumors in which NISCH is predicted to be a positive prognostic marker. Taken together, our study highlights several novel findings with regards to the nischarin biology that are prompting further investigation: the nuclear localization in cancer, negative prognostic value in several cancer types that questions the tumor suppressor role, and association with signaling pathways that regulate stemness in these cancer types.