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 levels reaching a minimum at the core of the tumor, a condition known as tumor hypoxia. Mediated by members of the HIF family of transcription factors, hypoxia leads to a more aggressive tumor phenotype by transactivation of several genes as well as reprogramming of pre-mRNA splicing. Intragenic DNA methylation, which is known to affect alternative splicing in cancer, could be one of several reasons behind the changes in splicing patterns under hypoxia.Here, we have tried to establish a correlation between intragenic DNA methylation and alternative usage of exons in tumor hypoxia. First, we have generated a custom hypoxia signature consisting of 34 genes that are up-regulated under hypoxia and are direct targets of HIF-1α. Using this gene expression signature, we have successfully stratified publicly available breast cancer patient samples into hypoxia positive and hypoxia negative groups followed by mining of differentially spliced isoforms between these groups. The Hypoxia Hallmark signature from MSigDB was also used independently to stratify the same tumor samples into hypoxic and normoxic. We found that 821 genes were showing differential splicing between samples stratified using a custom signature, whereas, 911 genes were showing differential splicing between samples stratified using the MSigDB signature. Finally, we performed multiple correlation tests between the methylation levels (α) of microarray probes located within 1 kilo base pairs of isoform-specific exons using those exons' expression levels in the same patient samples in which the methylation level was recorded. We found that the expression level of one of the exons of DHX32 and BICD2 significantly correlated with the methylation levels, and we were also able to predict patient survival (p-value: 0.02 for DHX32 and 0.0024 for BICD2). Our findings provide new insights into the potential functional role of intragenic DNA methylation in modulating alternative splicing during hypoxia.

Hypoxia-induced TGF-β–RBFOX2–ESRP1 axis regulates human MENA alternative splicing and promotes EMT in breast cancer

“…The HTA 2.0 array data from MCF7 and HCC1806 cell lines show a significant exclusion (P = 4.22 × 10 −5 and 0.0026, respectively) of exon 11a from hMENA pre-mRNA under hypoxia ( Supplementary Figure S1A ). To validate the cell line data, we stratified 165 tumor samples from a breast cancer patient microarray profile (GSE76250) ( 23 ) as hypoxic or normoxic based on the HYPOXIA_HALLMARK ( 25 ) gene signature using a method described previously ( 26 ). It appeared that the exon 11a is significantly excluded (P = 1.19 × 10 −5 ) in samples stratified as hypoxic as compared to those stratified as normoxic ( Figure 1A ).…”

“…It emerged that the hypoxia-associated genes were significantly enriched in the tumor set ( Supplementary Figure S2A ). Next, we stratified the tumor samples into two groups (Hypoxic_high and Hypoxic_low) based on the degree of hypoxia activation using a method described previously ( 26 ) ( Supplementary Figure S2B ), followed by GSEA to check for the enrichment of various signaling pathways implicated in EMT ( 37 ) ( Supplementary Table S2 ). Notably, expression of the TGF-β signaling pathway genes was found to be significantly enriched in tumor samples with a higher degree of hypoxia activation (n = 220) against those with a lower degree of hypoxia activation (n = 220; Supplementary Figure S2C ).…”

“…For HTA 2.0 triple-negative breast cancer patient profile (GSE76250) ( 23 ), CEL files for 165 tumor samples were downloaded from GEO, and the gene expression levels were obtained using oligo package in R ( 24 ). The HYPOXIA_HALLMARK signature from Molecular Signature Database ( 25 ) was used to stratify the patient samples into hypoxic or normoxic using a similar strategy as described previously ( 26 ). The sum of expression levels corresponding to genes contained in the HYPOXIA_HALLMARK signature was calculated (hypoxia activation score) for all the samples.…”

“…To check the enrichment of hypoxia pathway genes (using the WINTER_HYPOXIA_UP gene signature) in normal versus tumor samples, gene set enrichment analysis was performed using GSEA software ( 27 ). Stratification of tumor samples was then done, as described previously ( 26 ). Hypoxia activation score was calculated by summing up the expression of genes contained in the WINTER_HYPOXIA_UP signature for each tumor sample, and then the samples were ranked on the basis of this score.…”

An IDH-independent mechanism of DNA hypermethylation upon VHL inactivation in cancer

“…In breast cancers, Mediated by members of the HIF family of transcription factors, hypoxia leads to a more aggressive tumor phenotype by transactivation of several genes as well as reprogramming of pre-mRNA splicing. The expression level of one of the exons of DHX32 and BICD2 significantly correlated with the methylation levels and even made it possible to predict patient survival [12] .…”