Hypoxic microenvironment heralds epithelial–mesenchymal transition (EMT), invasion and metastasis in solid tumors. Deregulation of alternative splicing (AS) of several cancer-associated genes has been instrumental in hypoxia-induced EMT. Our study in breast cancer unveils a previously unreported mechanism underlying hypoxia-mediated AS of hMENA, a crucial cytoskeleton remodeler during EMT. We report that the hypoxia-driven depletion of splicing regulator ESRP1 leads to skipping of hMENA exon 11a producing a pro-metastatic isoform, hMENAΔ11a. The transcriptional repression of ESRP1 is mediated by SLUG, which gets stimulated via hypoxia-driven TGF-β signaling. Interestingly, RBFOX2, an otherwise RNA-binding protein, is also found to transcriptionally repress ESRP1 while interacting with SLUG. Similar to SLUG, RBFOX2 gets upregulated under hypoxia via TGF-β signaling. Notably, we found that the exosomal delivery of TGF-β contributes to the elevation of TGF-β signaling under hypoxia. Moreover, our results show that in addition to hMENA, hypoxia-induced TGF-β signaling contributes to global changes in AS of genes associated with EMT. Overall, our findings reveal a new paradigm of hypoxia-driven AS regulation of hMENA and insinuate important implications in therapeutics targeting EMT.

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Non-coding RNAs (ncRNAs) are functional RNA molecules that are transcribed from mammalian genome but lack protein coding capacity. Nearly 80% of the human genome constitutes non-coding elements such as small non-coding RNAs, sncRNAs (miRNA, piRNA, SiRNA, SnRNA) and long non-coding RNAs, lncRNAs (linc RNA, NAT, eRNA, circ RNA, ceRNAs, PROMPTS). These ncRNAs have been extensively studied and are known to mediate the regulation of gene expression. In recent decades, lncRNAs have emerged as pivotal molecules that participate in the post-transcriptional regulation by acting as a signal, guide, scaffold and decoy molecules in addition to their role(s) in transcription. ncRNAs are known to play critical roles in defining DNA methylation patterns, imprinting as well as chromatin remodeling, thus having a substantial effect in epigenetic signaling. The expression of lncRNAs is regulated in a tissue specific and developmental stage specific manner and their mis-regulation is often associated with tumorigenesis. Henceforth, this chapter focuses mainly on the role(s) of ncRNAs in transcriptional and post-transcriptional regulation and their relevance in cancers.

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Epithelial splicing regulatory protein 1 (ESRP1) is an RNA binding protein that governs the alternative splicing events related to epithelial phenotypes. ESRP1 contributes significantly at different stages of cancer progression. ESRP1 expression is substantially elevated in carcinoma in situ compared to the normal epithelium, whereas it is drastically ablated in cancer cells within hypoxic niches, which promotes epithelial to mesenchymal transition (EMT). Although a considerable body of research sought to understand the EMT-associated ESRP1 downregulation, the regulatory mechanisms underlying ESRP1 upregulation in primary tumors remained largely uncharted. This study seeks to unveil the regulatory mechanisms that spatiotemporally fine-tune the ESRP1 expression during breast carcinogenesis. Our results reveal that an elevated expression of transcription factor E2F1 and increased CpG hydroxymethylation of the E2F1 binding motif conjointly induce ESRP1 expression in breast carcinoma. However, E2F1 fails to upregulate ESRP1 despite its abundance in oxygen-deprived breast cancer cells. Mechanistically, impelled by the hypoxia-driven reduction of tet methylcytosine dioxygenase 3 (TET3) activity, CpG sites across the E2F1 binding motif lose the hydroxymethylation marks while gaining the de novo methyltransferase-elicited methylation marks. These two oxygen-sensitive epigenetic events work in concert to repel E2F1 from the ESRP1 promoter, thereby diminishing ESRP1 expression under hypoxia. Furthermore, E2F1 skews the cancer spliceome by upregulating splicing factor SRSF7 in hypoxic breast cancer cells. Our findings provide previously unreported mechanistic insights into the plastic nature of ESRP1 expression and insinuate important implications in therapeutics targeting breast cancer progression.

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