Identification of Novel Androgen-Regulated Pathways and mRNA Isoforms through Genome-Wide Exon-Specific Profiling of the LNCaP Transcriptome

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.

雄激素通过调控雄激素受体（androgen receptor, AR）的转录活性，驱动前列腺癌（prostate cancer, PCa）的发生与进展。尽管已有多项基于微阵列的研究鉴定出雄激素调控基因，但本研究通过对LNCaP细胞转录组进行外显子水平分析，同步鉴定了基因表达与可变mRNA剪接异构体表达的全局雄激素依赖性变化。虽然全基因组基因表达变化与已发表研究结果契合度较高，但本研究额外发现了226个新的雄激素调控基因子集。对该子集进行基因表达通路分析后发现，存在与酪氨酸激酶LYN相关且包含LYN的基因簇，同时还包含在癌症中常发生失调的雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）通路的相关组分。本研究还鉴定出1279个推定的雄激素调控可变剪接事件，其中325个（约25%）对应于已报道的可变剪接事件或可变首/末外显子。本研究选取了30个雄激素依赖性可变剪接事件进行逆转录聚合酶链式反应（Reverse Transcription-Polymerase Chain Reaction, RT-PCR）验证，其中包含源自肿瘤抑制基因与细胞周期调控基因的mRNA。在7个验证阳性的事件（约23%）中，有5个事件的转录本源自已知AR靶基因的可变启动子。具体而言，本研究发现了一种源自TSC2肿瘤抑制基因内部可变启动子的新型雄激素依赖性mRNA剪接异构体，该异构体编码的蛋白质缺失了mTOR抑制所需的相互作用结构域。本研究证实，该可变TSC2 mRNA剪接异构体的表达直接受雄激素调控；染色质免疫沉淀（chromatin immunoprecipitation, ChIP）实验显示，在雄激素刺激后的早期时间点，AR会被招募至该可变启动子区域，这与可变转录本的表达水平呈正相关。综上，本研究数据表明，可变mRNA剪接异构体的表达可能介导细胞对雄激素的应答，并可能在临床前列腺癌中发挥作用。