Hypoxia-induced changes in intragenic DNA methylation correlate with alternative splicing in breast cancer

ABSTRACT

The tumor microenvironment is marked by gradients in the level of oxygen and nutrients, with oxygen levelsreaching a minimum at the core of the tumor, a condition known as tumor hypoxia. Mediated by members of theHIF family of transcription factors, hypoxia leads to a more aggressive tumor phenotype by transactivation ofseveral genes as well as reprogramming of pre-mRNA splicing. Intragenic DNA methylation, which is known toaffect alternative splicing in cancer, could be one of several reasons behind the changes in splicing patterns underhypoxia. Here, we have tried to establish a correlation between intragenic DNA methylation and alternative usageof exons in tumor hypoxia. First, we have generated a custom hypoxia signature consisting of 34 genes that are upregulated under hypoxia and are direct targets of HIF-1a. Using this gene expression signature, we have successfully stratified publicly available breast cancer patient samples into hypoxia positive and hypoxia negativegroups followed by mining of differentially spliced isoforms between these groups. The Hypoxia Hallmarksignature from MSigDB was also used independently to stratify the same tumor samples into hypoxic andnormoxic. We found that 821 genes were showing differential splicing between samples stratified using a customsignature, whereas, 911 genes were showing differential splicing between samples stratified using the MSigDBsignature. Finally, we performed multiple correlation tests between the methylation levels (b) of microarrayprobes located within 1 kilo base pairs of isoform-specific exons using those exons’ expression levels in the samepatient samples in which the methylation level was recorded. We found that the expression level of one of theexons of DHX32 and BICD2 significantly correlated with the methylation levels, and we were also able to predictpatient survival (p-value 0.02 for DHX32 and 0.0024 for BICD2). Our findings provide new insights into thepotential functional role of intragenic DNA methylation in modulating alternative splicing during hypoxia.