Hypoxia-induced CTCF promotes EMT in breast cancer.

Cancer cells experiencing hypoxic stress employ epithelial-mesenchymal transition (EMT) to undergo metastasis through rewiring of the chromatin landscape, epigenetics, and importantly, gene expression. Here, we showed that hypoxia modulates the epigenetic landscape on CTCF promoter and upregulates its expression. Hypoxia-driven epigenetic regulation, specifically DNA demethylation mediated by TET2, is a prerequisite for CTCF induction. Mechanistically, in hypoxic conditions, Hypoxia-inducible factor 1-alpha (HIF1α) binds to the unmethylated CTCF promoter, causing transcriptional upregulation. Further, we uncover the pivotal role of CTCF in promoting EMT as loss of CTCF abrogated invasiveness of hypoxic breast cancer cells. These findings highlight the functional contribution of HIF1α-CTCF axis in promoting EMT in hypoxic breast cancer cells. Lastly, CTCF expression is alleviated and the potential for EMT is diminished when the HIF1α binding is particularly disrupted through the dCas9-DNMT3A system-mediated maintenance of DNA methylation on the CTCF promoter. This axis may offer a unique therapeutic target in breast cancer.

PKM2 dictates the poised chromatin state of PFKFB3 promoter to enhance breast cancer progression.

The hypoxic milieu is a critical modulator of aerobic glycolysis, yet the regulatory mechanisms between the key glycolytic enzymes in hypoxic cancer cells are largely unchartered. In particular, the M2 isoform of pyruvate kinase (PKM2), the rate-limiting enzyme of glycolysis, is known to confer adaptive advantages under hypoxia. Herein, we report that non-canonical PKM2 mediates HIF-1α and p300 enrichment at PFKFB3 hypoxia-responsive elements (HREs), causing its upregulation. Consequently, the absence of PKM2 activates an opportunistic occupancy of HIF-2α, along with acquisition of a poised state by PFKFB3 HREs-associated chromatin. This poised nature restricts HIF-2α from inducing PFKFB3 while permitting the maintenance of its basal-level expression by harboring multiple histone modifications. In addition, the clinical relevance of the study has been investigated by demonstrating that Shikonin blocks the nuclear translocation of PKM2 to suppress PFKFB3 expression. Furthermore, TNBC patient-derived organoids and MCF7 cells-derived xenograft tumors in mice exhibited substantial growth inhibition upon shikonin treatment, highlighting the vitality of targeting PKM2. Conclusively, this work provides novel insights into the contributions of PKM2 in modulating hypoxic transcriptome and a previously unreported poised epigenetic strategy exhibited by the hypoxic breast cancer cells for ensuring the maintenance of PFKFB3 expression.

Hypoxia-induced loss of SRSF2-dependent DNA methylation promotes CTCF-mediated alternative splicing of VEGFA in breast cancer.

Alternative splicing of vascular endothelial growth factor A (VEGFA) generates numerous isoforms with unique roles in tumor angiogenesis, and investigating the underlying mechanism during hypoxia necessitates diligent pursuance. Our research systematically demonstrated that the splicing factor SRSF2 causes the inclusion of exon-8b, leading to the formation of the anti-angiogenic VEGFA-165b isoform under normoxic conditions. Additionally, SRSF2 interacts with DNMT3A and maintains methylation on exon-8a, inhibiting CCCTC-binding factor (CTCF) recruitment and RNA polymerase II (pol II) occupancy, causing exon-8a exclusion and decreased expression of pro-angiogenic VEGFA-165a. Conversely, SRSF2 is downregulated by HIF1α-induced miR-222-3p under hypoxic conditions, which prevents exon-8b inclusion and reduces VEGFA-165b expression. Furthermore, reduced SRSF2 under hypoxia promotes hydroxymethylation on exon-8a, increasing CTCF recruitment, pol II occupancy, exon-8a inclusion, and VEGFA-165a expression. Overall, our findings unveil a specialized dual mechanism of VEGFA-165 alternative splicing, instrumented by the cross-talk between SRSF2 and CTCF, which promotes angiogenesis under hypoxic conditions.

Can a signature molecular-profile define disparate survival in BRAF-positive Gliosarcoma and identify novel targets for therapeutic intervention?

Purpose: Gliosarcoma (GS) has a low incidence but is aggressively invasive, with poor-survival. Even though GS is recognized as a different subgroup from glioblastoma (GB), there is no molecular panel available to define its clinical outcome. The objective was to identify the molecular imprint of GS in terms of expression of human telomerase reverse transcriptase (hTERT), high mobility group A1 (HMGA-1), kinesin superfamily protein-14 (KIF-14), epidermal growth factor receptor (EGFR) markers with reference to disparate prognosis and identify plausible targets for intervention. Materials and Methods: We retrieved 9-GS samples from a cohort of 57-GB patients during a 36 months study period and compared them with 10 molecularly typed GB-samples and 15 controls. Conventional-immunohistochemistry (IHC) was used for histopathology of GS and immunofluorescence-IHC was performed for quantification of identified marker-panel. Statistical tools for non-parametric data were used for inferring results. Results: GS was confirmed by reticulin-staining and positivity for glial fibrillary acidic protein, Vimentin, smooth muscle actin. Immune-reactivity for BRAF-V600Ewas present in both glial and sarcomatous cells and negative expression of isocitrate dehydrogenase, ATRX, TP53.Comparison between GS, GB, and control tissues showed that the expression of markers reached significance (P < 0.0001), without the influence of confounders. Significant correlation of EGFR was found with hTERT (r = 0.77), HMGA-1 (r = 0.72), KIF-14 (r = 0.82) suggesting that their combined analysis can define prognosis. To establish the diagnostic accuracy (threshold ≥80% specificity), AUC for EGFR was 0.78 (>3.95), KIF-14 0.97 (>7.45), hTERT 0.63 (>23.86), and HMGA-1 0.53 (>15.45). Conclusion: This is the first evidence-based investigation presenting differential expression of proliferation and stemness markers hTERT, HMGA-1, KIF-14 in-correlation with EGFR, indicating a plausible-association between survival and disease-progression in individual GS-cases. It can serve as a model for further studies in this glioma-subgroup and the designing of a target panel for personalized treatment.

E2F1 and epigenetic modifiers orchestrate breast cancer progression by regulating oxygen-dependent ESRP1 expression.

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.

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

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-ß 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-ß signaling. Notably, we found that the exosomal delivery of TGF-ß contributes to the elevation of TGF-ß signaling under hypoxia. Moreover, our results show that in addition to hMENA, hypoxia-induced TGF-ß 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.

The HNRNPA2B1-MST1R-Akt axis contributes to epithelial-to-mesenchymal transition in head and neck cancer.

The deregulation of splicing factors and alternative splicing are increasingly viewed as major contributory factors in tumorigenesis. In this study, we report overexpression of a key splicing factor, heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1), and thereby misregulation of alternative splicing, which is associated with the poor prognosis of head and neck cancer (HNC). The role of HNRNPA2B1 in HNC tumorigenesis via deregulation of alternative splicing is not well understood. Here, we found that the CRISPR/Cas9-mediated knockout of HNRNPA2B1 results in inhibition of HNC cells growth via the misregulation of alternative splicing of MST1R, WWOX, and CFLAR. We investigated the mechanism of HNRNPA2B1-mediated HNC cells growth and found that HNRNPA2B1 plays an important role in the alternative splicing of a proto-oncogene, macrophage stimulating 1 receptor (MST1R), which encodes for the recepteur d'origine nantais (RON), a receptor tyrosine kinase. Our results indicate that HNRNPA2B1 mediates the exclusion of cassette exon 11 from MST1R, resulting in the generation of RON∆165 isoform, which was found to be associated with the activation of Akt/PKB signaling in HNC cells. Using the MST1R-minigene model, we validated the role of HNRNPA2B1 in the generation of RON∆165 isoform. The depletion of HNRNPA2B1 results in the inclusion of exon 11, thereby reduction of RON∆165 isoform. The decrease of RON∆165 isoform causes inhibition of Akt/PKB signaling, which results in the upregulation of E-cadherin and downregulation of vimentin leading to the reduced epithelial-to-mesenchymal transition. The overexpression of HNRNPA2B1 in HNRNPA2B1 knockout cells rescues the expression of the RON∆165 isoform and leads to activation of Akt/PKB signaling and induces epithelial-to-mesenchymal transition in HNC cells. In summary, our study identifies HNRNPA2B1 as a putative oncogene in HNC that promotes Akt/PKB signaling via upregulation of RON∆165 isoform and promotes epithelial to mesenchymal transition in head and neck cancer cells.