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.

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 RONΔ165 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 RONΔ165 isoform. The depletion of HNRNPA2B1 results in the inclusion of exon 11, thereby reduction of RONΔ165 isoform. The decrease of RONΔ165 isoform causes inhibition of Akt/PKB signaling, which results in the upregulation of Ecadherin 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 RONΔ165 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 RONΔ165 isoform and promotes epithelial to mesenchymal transition in head and neck cancer cells.

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.

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.

BACKGROUND AND AIMS: The study was done to know the epidemiology, clinicopathological aspects of carcinoma breast patients in our population. MATERIALS AND METHODS: The epidemiological and clinicopathological data pertaining to demography and risk factors for carcinoma breast were analyzed in patients attending tertiary care hospital of North India from January 2012 to June 2013. Thorough Clinical and physical examination was done, FNAC was done for diagnosis of cancer Breast. RESULTS: In our study mean age of our female breast cancer patients was found to be lower compared to the western world, with an average difference of one decade. Majority of the patients were from urban background. Lump in the breast was a dominant symptom. Familial breast cancer was uncommon. Left sided breast cancer was slightly preponderant. CONCLUSION: Most common symptom was lump in breast among postmenopausal women from urban area. Lack of education was responsible for their delayed presentation. Left upper and outer quadrant being the commonest site, Infiltrating duct carcinoma was the most common type. Modified radical mastectomy was found to be a safe operative procedure. Adjuvant chemotherapy and hormonal therapy was found very effective in early stages. Overall survival for stage IV was 60% for 1 year follow up. INTRODUCTION: The World Cancer Report issued by the International Agency for Research on Cancer (IARC), tells us that cancer rates are set to increase at an alarming rate globally. Cancer rates could increase by 50% to 15 million new cases in the year 2020. 1 Worldwide breast cancer is the most frequent cancer in women and represents the second leading cause of cancer death among women (after lung cancer). 2, 3 Presently, 75, 000 new cases occur in Indian women every year. 4 This figure must be viewed against the backdrop that the National Cancer Registry and the Hospital-based Tumor Registries hardly sample 3% of the total population. Locally advanced breast cancer (LABC) constitutes more than 50 to 70% of the patients presenting for treatment. 4 The information on the epidemiology of breast cancer in India is very limited, except for a few reports on limited samples. India is rapidly stepping towards industrialization vis-à-vis urbanization resulting in change of lifestyle factors. These factors possibly contributed to a gradual increase in the incidence of breast cancer in the country. The burden of breast cancer will continue to grow not only in terms of the absolute number of cases but also in terms of incidence. Late diagnosis is a major factor for increased mortality as the majority of the patients present in advanced or metastatic stage. This is primarily attributed to lack of access to medical facilities, virtually non-existent breast cancer screening programs, lack of awareness and social-cultural attitudes. Accordingly, five-year survival rates have been poorer, reported as 42% and 48% in two population-based studies. 5, 6 Early breast cancer (EBC)