Extensive global alternative splicing induced by hypoxia across four major cancer types

Abstract

Background: Hypoxia, lack of oxygen, is a well-known cancer phenotype associated with poor prognosis and therapeutic resistance. Understanding the tumour molecular response to hypoxia is key to developing effective therapies and generating robust clas-sifiers. We and others have previously demonstrated large transcriptional changes, up to 10-15% of the coding genome, in response to hypoxia. Whilst several studies described the gene expression changes in response to hypoxia, there is no comprehensive study addressing the extent different isoforms of the same gene are differentially regulated. Methods: Here, we investigated which transcripts are regulated in response to hypoxia, and whether there was significant alternative splicing across different tumour types. Twenty-one cell lines were used covering the main subtypes of four solid cancer types (prostate, breast, pancreas, colon), and cultured under normoxia and hypoxia (1% oxygen, 24 hours) in triplicate. Messenger RNA was sequenced in the 126 samples (polyA selection), and estimations made of differential gene expression, transcript-level differential expression and alternative splicing. Results: Of the 198,503 transcripts considered, 11826, 11604, 8686 and 5719 transcripts were upregulated and 11179, 12308, 9390 and 6220 were downregulated in the prostate, breast, pancreas, and colon cancer cell lines respectively. Of these, 861 were significantly up-regulated, and 1,484 down-regulated across all 21 cell lines and four cancer types indicating a significant common transcriptional response to hypoxia. Notably, this transcript-level analysis identified nearly all genes which could be detected in a gene-level analysis (96%-99% of genes depending on cancer types), but also many more (43%-58% of new genes) which were not discoverable in a gene-level analysis. Interestingly, there was a high number of genes (728, enrichment p < 0.00001) which showed regulation across all cancer types, although the specific isoforms regulated in each cancer were different. We observed that up to about 40% of the genes regulated by hypoxia, showed two or more transcripts differentially expressed. In agreement with this, alternative splicing analysis indicated large global changes in transcript architecture. These included transcripts in important hypoxia-regulated genes, such as VEGFA-204, transcript of the VEGFA gene, where three alternative splicing events could be detected, but also events and genes not previously linked to hypoxia. A meta-analysis merging the results from gene-, isoform-and exon-level analyses identified 10 top alternative splicing events which were highly significant across all cancers and 21 cell lines. Importantly, a pathway analysis revealed that the pathways affected were similar across cancer types and regulation of transcription was the top one, followed by signal transduction, vesicle-mediated transport, glycolysis and cell cycle. Conclusions: This study shows for the first time the extent of hypoxia alternative splicing and differential isoform regulation in multiple cancer types, highlighting differences and commonalities, and opening an important new avenue to understand how cancers adapt to hypoxia.