Abstract
Androgens drive the onset and progression of prostate cancer (PCa) by modulating androgen receptor (AR) transcriptional activity. Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome. While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes. Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin) pathway, which is commonly dysregulated in cancer. We also identified 1279 putative androgen-regulated alternative events, of which 325 (∼25%) mapped to known alternative splicing events or alternative first/last exons. We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators. Of seven positively-validating events (∼23%), five events involved transcripts derived from alternative promoters of known AR gene targets. In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition. We confirmed that expression of this alternative TSC2 mRNA isoform was directly regulated by androgens, and chromatin immunoprecipitation indicated recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts. Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical PCa.
Highlights
The growth, development and function of the prostate gland are dependent on androgens, which affect gene expression via the androgen receptor (AR), a steroid hormone receptor transcription factor
We identified androgen-dependent up-regulation of the LYN-associated kinase mammalian target of rapamycin (mTOR). mTOR kinase activity has been previously found to be up-regulated in prostate cancer (PCa) cells in response to androgens [35], and the mTOR pathway is important in clinical PCa, in tumours lacking expression of PTEN
Since AR gene targets can remain active in CRPCa, the mTOR complex may be a possible therapeutic target for CRPCa [35,36]
Summary
The growth, development and function of the prostate gland are dependent on androgens, which affect gene expression via the androgen receptor (AR), a steroid hormone receptor transcription factor. Alternative mRNA isoforms with different exon contents can be generated through variable alternative splicing patterns, and selection of alternative transcriptional initiation and termination sites. Using these mechanisms, mRNAs with different exon combinations are transcribed from most (,70 to 90%) human genes [6,7,8], and can encode proteins with different sub-cellular localisations and functions. In addition to transcriptional effects, androgens and other steroid hormones are implicated in the posttranscriptional regulation of gene expression [9,10,11,12,13]. In PCa, there is emerging evidence to suggest that expression of specific splice isoforms derived from cancerrelevant genes may underlie tumour biology and contribute to clinical disease progression [15]
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