CTCF and BORIS-mediated autophagy regulation via alternative splicing of BNIP3L in breast cancer.

Autophagy is a pivotal regulatory and catabolic process, induced under various stressful conditions, including hypoxia. However, little is known about alternative splicing of autophagy genes in the hypoxic landscape in breast cancer. Our research unravels the hitherto unreported alternative splicing of BNIP3L, a crucial hypoxia-induced autophagic gene. We showed that BNIP3L, under hypoxic condition, forms two isoforms, a full-length isoform (BNIP3L-F) and a shorter isoform lacking exon 1 (BNIP3L-Δ1). The hypoxia-induced BNIP3L-F promotes autophagy, while under normoxia, the BNIP3L-Δ1 inhibits autophagy. We discovered a novel dimension of hypoxia-mediated epigenetic modification that regulates the alternative splicing of BNIP3L. Here, we showed differential DNA methylation of BNIP3L intron 1, causing reciprocal binding of epigenetic factor CCCTC-binding factor (CTCF) and its paralog BORIS. Additionally, we highlighted the role of CTCF and BORIS impacting autophagy in breast cancer. The differential binding of CTCF and BORIS results in alternative splicing of BNIP3L forming BNIP3L-F and BNIP3L-Δ1, respectively. The binding of CTCF on unmethylated BNIP3L intron 1 under hypoxia results in RNA Pol-II pause and inclusion of exon 1, promoting BNIP3L-F and autophagy. Interestingly, the binding of BORIS on methylated BNIP3L intron 1 under normoxia also results in RNA Pol-II pause but leads to the exclusion of exon 1 from BNIP3L mRNA. Finally, we reported the critical role of BORIS-mediated RNA Pol-II pause, which subsequently recruits SRSF6, redirecting the proximal splice-site selection, promoting BNIP3L-Δ1, and inhibiting autophagy. Our study provides novel insights into the potential avenues for breast cancer therapy by targeting autophagy regulation, specifically under hypoxic condition.

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.

Unfolding the role of autophagy in the cancer metabolism.

Autophagy is considered an indispensable process that scavenges toxins, recycles complex macromolecules, and sustains the essential cellular functions. In addition to its housekeeping role, autophagy plays a substantial role in many pathophysiological processes such as cancer. Certainly, it adapts cancer cells to thrive in the stress conditions such as hypoxia and starvation. Cancer cells indeed have also evolved by exploiting the autophagy process to fulfill energy requirements through the production of metabolic fuel sources and fundamentally altered metabolic pathways. Occasionally autophagy as a foe impedes tumorigenesis and promotes cell death. The complex role of autophagy in cancer makes it a potent therapeutic target and has been actively tested in clinical trials. Moreover, the versatility of autophagy has opened new avenues of effective combinatorial therapeutic strategies. Thereby, it is imperative to comprehend the specificity of autophagy in cancer-metabolism. This review summarizes the recent research and conceptual framework on the regulation of autophagy by various metabolic pathways, enzymes, and their cross-talk in the cancer milieu, including the implementation of altered metabolism and autophagy in clinically approved and experimental therapeutics.