Ananya Palo

Serine protease inhibitor b5 (SERPINB5) is a tumor suppressor gene that plays a critical role in various cellular processes. In gallbladder cancer (GBC), SERPINB5's aberrant expression is reported but its role in genetic predisposition is not known. We enrolled 270 cases and 296 controls and genotyped them for single nucleotide polymorphisms (SNPs) using direct DNA sequencing, followed by genotype–phenotype analysis in GBC and other cancer cell lines. Luciferase assay was done to determine the role of rs2289521 SNP on expression regulation. We found that two SERPINB5 variants rs2289519 and rs2289521 are significantly associated with GBC and contribute to genetic predisposition. The TT genotype of variant rs2289519 was found to be significantly associated ( p = 0.008) with GBC in a recessive model. C allele of rs2289521 increased the risk for GBC significantly at genotypic (CT, p = 0.026) and allelic ( p = 0.04) levels. In silico analysis and luciferase assay uncovered the probable regulatory role of the rs2289521 variant on expression. Genotype–phenotype correlation in GBC and breast cancer cell lines showed reduced expression of SERPINB5 in the presence of C allele that was consistent with the result of luciferase assay. Overall, our study reveals the genetic association of two SERPINB5 variants with GBC and rs2289521's possible role in the regulation of expression.

Multiple molecular subtypes and distinct clinical outcomes in breast cancer, necessitate specific therapy. Moreover, despite the improvements in breast cancer therapy, it remains the fifth cause of cancer-related deaths, indicating the involvement of unknown genes. To identify novel contributors and molecular subtype independent therapeutic options, we report reduced expression of FRG1 in breast cancer patients, which regulates GM-CSF expression via direct binding to its promoter. Reduction in FRG1 expression enhanced EMT and increased cell proliferation, migration, and invasion, in breast cancer cell lines. Loss of FRG1 increased GM-CSF levels which activated MEK/ERK axis and prevented apoptosis by inhibiting p53 in an ERK-dependent manner. FRG1 depletion in the mouse model increased tumor volume, phospho-ERK, and EMT marker levels. The therapeutic potential of anti-GM-CSF therapy was evident by reduced tumor size, when tumors with decreased FRG1 were treated with anti-GM-CSF mAb. We found an inverse expression pattern of FRG1 and phospho-ERK levels in breast cancer patient tissues, corroborating the in vitro and mouse model-based findings. Our findings first time elucidate the role of FRG1 as a metastatic suppressor of breast cancer by regulating the GM-CSF/MEK-ERK axis.

FRG1 is the primary candidate gene for Fascioscapulohumeral Muscular Dystrophy. So far, its role has been reported in muscle development, vasculogenesis, angiogenesis, and tumorigenesis. Mechanistically studies suggest FRG1's role in RNA biogenesis which may have implications in multiple physiological processes and diseases, including tumorigenesis. Its probable role as hnRNP and association with NMD-related genes prompted us to look into FRG1's effect on NMD gene expression and the mechanism. Using microarray profiling in cell lines, we found that FRG1 altered the mRNA surveillance pathway and associated pathways, such as RNA transport and spliceosome machinery molecules. Multiple sequence alignment of core factors, namely, UPF1, UPF3B, and SMG1, showed conserved stretches of nucleotide sequence 'CTGGG'. Structural modeling followed by EMSA, ChIP-qPCR, and luciferase reporter assays showed 'CTGGG' as a FRG1 binding site. Analysis of the publicly available datasets showed that the expression of FRG1 correlates with NMD genes in different tissue types. We validated the effect of FRG1 on NMD gene transcription by qRT-PCR. Overall, FRG1 might be a transcriptional regulator of NMD genes.

FRG1 has a role in tumorigenesis and angiogenesis. Our preliminary analysis showed that FRG1 mRNA expression is associated with overall survival (OS) in certain cancers, but the effect varies. In cervix and gastric cancers, we found a clear difference in the OS between the low and high FRG1 mRNA expression groups, but the difference was not prominent in breast, lung, and liver cancers. We hypothesized that FRG1 expression level could affect the functionality of the correlated genes or vice versa, which might mask the effect of a single gene on the OS analysis in cancer patients. We used the multivariate Cox regression, risk score, and Kaplan Meier analyses to determine OS in a multigene model. STRING, Cytoscape, HIPPIE, Gene Ontology, and DAVID (KEGG) were used to deduce FRG1 associated pathways. In breast, lung, and liver cancers, we found a distinct difference in the OS between the low and high FRG1 mRNA expression groups in the multigene model, suggesting an independent role of FRG1 in survival. Risk scores were calculated based upon regression coefficients in the multigene model. Low and high-risk score groups showed a significant difference in the FRG1 mRNA expression level and OS. HPF1, RPL34, and EXOSC9 were the most common genes present in FRG1 associated pathways across the cancer types. Validation of the effect of FRG1 mRNA expression level on these genes by qRT-PCR supports that FRG1 might be an upstream regulator of their expression. These genes may have multiple regulators, which also affect their expression, leading to the masking effect in the survival analysis. In conclusion, our study highlights the role of FRG1 in the survivability of cancer patients in tissue-specific manner and the use of multigene models in prognosis.