Hypoxia-induced TGF-β–RBFOX2–ESRP1 axis regulates human MENA alternative splicing and promotes EMT in breast cancer

Abstract

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.

Regulation of Alternative Splicing during Epithelial-Mesenchymal Transition

“…SRSF1 promotes the production of mesenchymal ∆Ron, while hnRNP A1 inhibits its pro- duction [Bonomi et al, 2013]. The ratio of ESRP1 and RBFOX2 expression dictates the outcome of hMENA exon 11a splicing in breast cancer [Ahuja et al, 2020]. In CD44 alternative splicing, inclusion of variable exons is promoted by ESRP1, hnRNPF, and AKAP8 in the epithelial state, while skipping of variable exons is enhanced by hnRNPM (Fig.…”

“…In recent years, RNA-seq has been extensively involved in discovering global alternative splicing changes in processes including EMT. Numerous splicing events have been identified, and their encoding genes are commonly involved in actin cytoskeleton regulation, cell-cell junctions, migration, and wound healing [Shapiro et al, 2011;Vanharanta et al, 2014;Li et al, 2018;Pillman et al, 2018;Ahuja et al, 2020]. While at this stage only a handful of these splicing changes have been connected to functional properties, it will be important to investigate these many other alternative splicing events on their biological roles in EMT in the future.…”

Alternative RNA splicing in tumour heterogeneity, plasticity and therapy

“…This gives rise to an isoform with impaired Wnt transactivation activity, directly linking ESRP1 to reduced Wnt signalling, a hallmark of the epithelial cell phenotype (Weise et al, 2010). RBFOX2, the expression of which is induced upon EMT, is an important regulator of mesenchymal splicing events (Ahuja et al, 2020;Braeutigam et al, 2014;Venables et al, 2013). Interestingly, while being required for inducing a mesenchymal cell-like splicing programme, RBFOX2 is dispensable for the TGFβ-induced phenotypic changes associated with EMT (Braeutigam et al, 2014).…”

Actin Cytoskeleton and Regulation of TGFβ Signaling: Exploring Their Links

“…hMENA spliced variants have been linked to TGFβ-induced EMT process and TGFβ is involved in the regulation of splicing of ENAH gene. TGFβ inhibits the expression of the epithelial splicing factor epithelial splicing regulatory proteins 1/2 (ESRP1/2) and primes the switching of hMENA isoforms by excluding the exon 11a in mammary epithelial and breast cancer cells [ 33 , 34 , 188 , 189 , 190 ]. In addition, TGFβ induces the expression of polypyrimidine tract-binding protein 1 (PTBP1), involved in exon 11a skipping, enhancing migration and invasion of lung cancer cells as well as EMT features in A549 cells [ 191 ].…”