Marija Ostojić, Ana Đurić, Kristina Živić, Jelena Grahovac
Abstract
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.
Introduction
Nischarin was first identified in the year 2000 as a novel protein interacting with the α5 integrin subunit involved in the control of cell migration [1]. Soon it was recognized that it was the same protein as the imidazoline receptor antisera-selected protein (IRAS), at the time studied as a novel target in drug discovery [2]. Implications of involvement in regulation of cell movement and its potential as a druggable receptor made it an interesting target in cancer research. Over the past 20 years nischarin (NISCH) role has been studied mostly in the breast, ovarian [3] and lung cancer [4, 5]. Seminal work on elucidating NISCH role in breast cancer initiation and progression has been done by the Alahari group [6]. They have shown that NISCH was involved in breast epithelial cell migration and invasion through regulation of the Rac driven signaling and interaction with multiple proteins involved in formation of focal adhesions and invadopodia [7–11]. It was suggested that NISCH functions as a scaffolding protein integrating extracellular to intracellular signaling. Alahari group also developed a NISCH mutant mouse and showed that NISCH can interact with and activate AMPK thus having a role in the regulation of cell metabolism [12]. When crossed with the MMTV-PyMT mice (a mouse strain in which the oncogenic polyoma virus middle T antigen is driven by the mouse mammary tumor virus promoter), NISCH mutant mice had increased breast tumor growth and metastasis [13]. They further developed a NISCH exon 5 and 6 knock-out mouse and showed that NISCH KO mouse embryonic fibroblasts had increased migration, but lower oxygen production rates and lower ATP production [14]. This confirmed that NISCH was involved in several biological processes important for cancer progression. Of importance, a difference in metabolic phenotype of male and female NISCH KO mice was observed, in body fat distribution, insulin resistance and glucose tolerance [15].
Materials and methods
Nischarin expression in healthy and tumor tissues
Nischarin endogenous expression was assessed using an interactive web resource The Human Protein Atlas (HPA) [20, 21]. The HPA RNA-seq tissue data is reported as nTPM (normalized protein-coding transcripts per million), corresponding to mean values of the different individual samples from each tissue. For HPA protein expression overview, NISCH protein levels were detected in 45 tissues using the HPA023189 antibody. Protein levels are reported as not detected, low, medium, or high, based on the combination of the staining intensity and fraction of stained cells.
Results
Nischarin expression in healthy and cancer tissues
Nischarin mRNA and protein were expressed in all the examined HPA healthy human tissues (S1 Fig). While the majority of analyzed tissues showed medium NISCH expression, protein staining had a high score in cerebellum, adrenal gland, bronchus, rectum, gall bladder, heart muscle, and the skin.
Discussion
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 could suppress breast cancer cell survival and motility in vitro and growth in vivo [7, 18, 69, 70]. NISCH was found to be downregulated 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 considering sex-related differences.
Acknowledgments
We thank Dr Miljana Tanić for the meaningful suggestions for the improvement of the study design. The results shown here were in part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga.
Citation: Ostojić M, Đurić A, Živić K, Grahovac J (2024) 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. PLoS ONE 19(5): e0299685. https://doi.org/10.1371/journal.pone.0299685
Editor: Chen Li, Free University of Berlin, GERMANY
Received: February 14, 2024; Accepted: May 8, 2024; Published: May 23, 2024
Copyright: © 2024 Ostojić et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The datasets presented and analyzed in the current study are publicly available in online repositories: • The Human Protein Atlas: https://www.proteinatlas.org/ENSG00000010322-NISCH/tissue https://www.proteinatlas.org/ENSG00000010322-NISCH/pathology • Gene Expression Profiling Interactive Analysis, version2 (GEPIA2): http://gepia2.cancer-pku.cn • UALCAN portal: http://ualcan.path.uab.edu/ • UniProt: https://www.uniprot.org/ https://www.uniprot.org/uniprotkb/Q9Y2I1 • Ensembl: https://www.ensembl.org/ • cBioPortal platform: http://www.cbioportal.org/ • Mexpress: https://mexpress.be • Broad Institute website: https://gdac.broadinstitute.org/ • TCGA Research Network: https://www.cancer.gov/tcga.
Funding: This research was supported by the Science Fund of the Republic of Serbia, PROMIS Grant No. 6056979, REPANCAN to JG; by the Ministry of Education, Science and Technological Development of the Republic of Serbia Agreement No. 451-03-68/2022-14/200043 to all authors; and the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 891135 to JG.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0299685#abstract0
