Abstract
BackgroundOver the past two decades more than fifty thousand unique clinical and biological samples have been assayed using the Affymetrix HG-U133 and HG-U95 GeneChip microarray platforms. This substantial repository has been used extensively to characterize changes in gene expression between biological samples, but has not been previously mined en masse for changes in mRNA processing. We explored the possibility of using HG-U133 microarray data to identify changes in alternative mRNA processing in several available archival datasets.ResultsData from these and other gene expression microarrays can now be mined for changes in transcript isoform abundance using a program described here, SplicerAV. Using in vivo and in vitro breast cancer microarray datasets, SplicerAV was able to perform both gene and isoform specific expression profiling within the same microarray dataset. Our reanalysis of Affymetrix U133 plus 2.0 data generated by in vitro over-expression of HRAS, E2F3, beta-catenin (CTNNB1), SRC, and MYC identified several hundred oncogene-induced mRNA isoform changes, one of which recognized a previously unknown mechanism of EGFR family activation. Using clinical data, SplicerAV predicted 241 isoform changes between low and high grade breast tumors; with changes enriched among genes coding for guanyl-nucleotide exchange factors, metalloprotease inhibitors, and mRNA processing factors. Isoform changes in 15 genes were associated with aggressive cancer across the three breast cancer datasets.ConclusionsUsing SplicerAV, we identified several hundred previously uncharacterized isoform changes induced by in vitro oncogene over-expression and revealed a previously unknown mechanism of EGFR activation in human mammary epithelial cells. We analyzed Affymetrix GeneChip data from over 400 human breast tumors in three independent studies, making this the largest clinical dataset analyzed for en masse changes in alternative mRNA processing. The capacity to detect RNA isoform changes in archival microarray data using SplicerAV allowed us to carry out the first analysis of isoform specific mRNA changes directly associated with cancer survival.
Highlights
Over the past two decades more than fifty thousand unique clinical and biological samples have been assayed using the Affymetrix HG-U133 and HG-U95 GeneChip microarray platforms
Alternative processing defines a range of events, including alternative splicing and alternative polyadenylation, which result in distinct mRNA species
These metrics were taken from conventional gene expression analysis
Summary
Over the past two decades more than fifty thousand unique clinical and biological samples have been assayed using the Affymetrix HG-U133 and HG-U95 GeneChip microarray platforms. The recent use of splicing sensitive microarrays has allowed quantification of changes in alternative processing between individual samples (reviewed in [1]). These arrays have been used to detect changes in alternative processing between normal human tissues and in breast, brain, colon, prostate, and bladder carcinomas [12,13,14,15,16] using various splicing algorithms (reviewed in [17]). Large scale clinical analyses of changes in alternative processing; remain sparse, and there are no highthroughput analyses of changes in mRNA processing associated with poor patient prognosis Such studies require years of patient follow-up and have not been reported using the new splicing arrays
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