Hypoxia-driven splicing into noncoding isoforms regulates the DNA damage response.

Danish Memon,Crispin J Miller,Christopher SF Smowton,Keren Dawson,Wei Xing,Caroline Dive

doi:10.1038/npjgenmed.2016.20

Abstract

Tumour hypoxia is associated with poor patient outcome and resistance to therapy. It is accompanied by widespread changes in gene expression mediated largely through the transcription factors HIF1/2/3α. Hypoxia impacts on multiple pathways throughout the cell and has widespread effects on phenotype. Here we use sample-specific annotation approaches to determine the changes in transcript architecture that arise as result of alternative splicing in hypoxic cells. Using in vivo data generated from a time course in reduced oxygenation we identified genome-wide switching between coding and noncoding isoforms, including a significant number of components of the DNA damage response pathway. Notably, HDAC6, a master regulator of the cytotoxic response, and TP53BP1, which sits at the nexus of the double-strand break repair pathway, both underwent a marked transition towards an intron-retention pattern with a concomitant decline in protein levels. These transitions from coding to noncoding isoforms were recapitulated in a large and independent cohort of 499 colorectal samples taken from The Cancer Genome Atlas (TCGA). The set of altered genes was enriched for multiple components of the Fanconi Anaemia, nucleotide excision and double-strand break repair pathways, and together correlating with tumour status at last contact. Altogether, these data demonstrate a new role for hypoxia-driven alternative splicing in regulating DNA damage response, and highlight the importance of considering alternative splicing as a critical factor in our understanding of human disease.

Highlights

Hypoxia occurs within the majority of solid tumours and is associated with poor patient outcome and chemo- and radioresistance.[1,2]
Hypoxia has multiple impacts on tumour biology including selection of altered cell signalling, angiogenesis, vasculogenesis, changes in central metabolism, suppression of immune reactivity, enhanced receptor tyrosine kinase signalling and down regulation of DNA repair pathways, promotion of prosurvival phenotypes and increased proclivity for invasion and metastasis;[3,4] extensively reviewed in references 5,6. Many of these hypoxia responses are characterised by widespread alterations in transcription profiles driven largely by stabilisation of the transcription factor subunit hypoxia inducible factor 1α (HIF1α).[7,8]
Hypoxia mediated transcriptional regulation is controlled by other factors including HIF2α9 and HIF3α.10. Signalling through both the growth factor receptor pathways and energy depletion pathways (5-AMP-activated protein kinase; AMPK) converge on the tuberous sclerosis complex (TSC1/2) leading to complex patterns of spatial and temporal regulation in response to stress. These signals feed into mechanistic target of rapamycin, which, in the context of hypoxia, leads to the rapid suppression of protein synthesis,[11] presumably in order to conserve energy.[12]

Summary

INTRODUCTION

Hypoxia occurs within the majority of solid tumours and is associated with poor patient outcome and chemo- and radioresistance.[1,2] Hypoxia arises both because disorganisation within tumour microvasculature lengthens intracapillary distances beyond the diffusion range of oxygen and because transient disruptions to blood flow provoke periods of acute oxygen starvation. Signalling through both the growth factor receptor pathways (phosphatidylinositol 3-kinase; PI3K, ERK) and energy depletion pathways (5-AMP-activated protein kinase; AMPK) converge on the tuberous sclerosis complex (TSC1/2) leading to complex patterns of spatial and temporal regulation in response to stress These signals feed into mechanistic target of rapamycin (mTOR), which, in the context of hypoxia, leads to the rapid suppression of protein synthesis,[11] presumably in order to conserve energy.[12] Levels of hypoxia vary between and within tumours, correlate with patient outcomes, and can lead to differences in response to therapy.[13] A better understanding of heterogeneity in hypoxia-driven changes in gene expression will inform strategies for precision medicine,[14] raising the need for reliable biomarkers of tumour hypoxia. Published in partnership with the Center of Excellence in Genomic Medicine Research

RESULTS

Findings

DISCUSSION