Discovery of hypoxia-mediated alternative splicing isoforms with impact in cellular metabolism and cancer

Abstract

Oxygen is essential for eukaryotic life. Over millions of years, it was necessary for organisms to evolve and integrate elaborate oxygen sensing and adaptation systems into cellular pathways to ensure cellular survival during periods of oxygen deprivation (hypoxia). Cellular hypoxia occurs both physiologically, e.g. during embryonic development and tissue growth, as well as in various disease contexts ranging from stroke or heart failure to cancer biology. Especially solid tumors are often poorly vascularized and thus harbor cancer cells experiencing intra-tumor hypoxia. These hypoxic cancer cells are of high interest in oncology research with respect to their roles in tumor survival, immune system evasion, metastasis and resistance to therapy. The most important and well-studied proteins involved in oxygen sensing and signaling include hypoxia-inducible factors (HIFs) and their biochemical regulators. HIF proteins are a class of transcriptional activators which, under hypoxic conditions, accumulate in the nucleus and bind to hypoxia-responsive elements in DNA to regulate gene expression of thousands of genes involved in varied cellular pathways, including angiogenesis, proliferation, metabolism, apoptosis and DNA repair. Although many transcriptional targets of hypoxia-inducible factors (HIFs) have been quite well characterized, hypoxia is also reported to impact on multiple post-transcriptional processes, including alternative splicing of RNA. Recently discovered hypoxia-dependent alternative splicing events are thought to introduce an additional layer of complexity to hypoxia biology but their overall contribution to cancer development, survival and resistance to therapy remain poorly understood. In this study, we set out to identify HIF dependent alternative splicing events after whole transcriptome sequencing in pancreatic cancer cells exposed to hypoxia with and without downregulation of the aryl hydrocarbon receptor -nuclear-translocator (ARNT), a protein required for HIFs to form a transcriptionally active dimer. We discover and validate a set of hypoxia-dependent alternative splicing events in detail in human pancreatic cancer cells as well as patient-derived human pancreatic cancer organoids. We investigate potential biological functions in these novel, hypoxia-dependent RNA transcripts. Finally, we report the discovery of a hypoxia-dependent long isoform of the UDP-N-acetylglucosamine transporter SLC35A3 with potential impact on cellular metabolism.