Hypoxia is a Key Driver of Alternative Splicing in Human Breast Cancer Cells

Jian Han,Lorenz Poellinger,Hiroyuki Kato,Sudhakar Jha,Grace Sushin Chia,Jia Li,Henry Yang,Jolene Caifeng Ho,Kian Leong Lee

doi:10.1038/s41598-017-04333-0

Abstract

Adaptation to hypoxia, a hallmark feature of many tumors, is an important driver of cancer cell survival, proliferation and the development of resistance to chemotherapy. Hypoxia-induced stabilization of hypoxia-inducible factors (HIFs) leads to transcriptional activation of a network of hypoxia target genes involved in angiogenesis, cell growth, glycolysis, DNA damage repair and apoptosis. Although the transcriptional targets of hypoxia have been characterized, the alternative splicing of transcripts that occurs during hypoxia and the roles they play in oncogenesis are much less understood. To identify and quantify hypoxia-induced alternative splicing events in human cancer cells, we performed whole transcriptome RNA-Seq in breast cancer cells that are known to provide robust transcriptional response to hypoxia. We found 2005 and 1684 alternative splicing events including intron retention, exon skipping and alternative first exon usage that were regulated by acute and chronic hypoxia where intron retention was the most dominant type of hypoxia-induced alternative splicing. Many of these genes are involved in cellular metabolism, transcriptional regulation, actin cytoskeleton organisation, cancer cell proliferation, migration and invasion, suggesting they may modulate or be involved in additional features of tumorigenic development that extend beyond the known functions of canonical full-length transcripts.

Highlights

In addition to the transcriptional regulation of a vast network of thousands of hypoxia target genes[13], hypoxia regulates post-transcriptional modifications, including alternative splicing of pre-mRNA which remains poorly understood
RNA-Seq was carried out on total RNA extracted from MCF7 human breast cancer (ER+, PR+, HER2−) cells cultured in normoxia (21% O2, 24 h), acute (1% O2, 4 h) and chronic hypoxia (1% O2, 24 h) for n = 1 replicate
Previous studies on hypoxia-regulated alternative splicing in breast cancer cells have been limited to single gene examples such as for Cysteine rich 61 (CYR61) and CD4419, 20

Summary

Introduction

In addition to the transcriptional regulation of a vast network of thousands of hypoxia target genes[13], hypoxia regulates post-transcriptional modifications, including alternative splicing of pre-mRNA which remains poorly understood. Some of the splicing targets, including LDHA, TNFSF13 and ARHGAP4 for intron retention, MARCH7, PCBP2 and LRCH3 for exon skipping and VGLL4, AHNAK and NFE2L1 that are subjected to alternative first exon usage may potentially contribute to cancer cell hypoxic adaptation by altering cellular metabolism, transcriptional regulation, actin cytoskeleton organization and promoting cancer cell proliferation, migration and invasion. The identification of these splicing targets provides novel insights into the oncogenic processes driving breast cancer cells and potentially new markers and therapeutic targets in the management of the disease

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Results

Conclusion