SUN-001 Analysis of Isoform Specific Differential Splicing Effects in Prostate Cancer

Abstract

The androgen receptor (AR) is a hormone activated transcription factor which is important for the growth and progression of prostate cancer (PCa). Metastatic PCa is treated with androgen deprivation therapy (ADT) based regimen but eventually, these tumours develop resistance through multiple mechanisms that reactivate AR. These include expression of constitutively active AR splice variants that lack a ligand binding domain such as AR-V7. To characterize the common and unique activities of AR and AR-V7, we engineered LNCaP and VCaP cell lines that express AR-V7 in response to doxycycline and performed RNA-Seq. Many genes were found to be regulated by both AR and AR-V7, but there were also genes regulated by only one isoform. Moreover, both isoforms altered mRNA splicing of numerous target genes, in some cases without altering overall levels of mRNA. In many cases, the alterations in splicing are AR isoform specific. To understand the differential splicing by AR isoforms, CD44 mini-gene splicing assay and studies of endogenous target genes are being done. The CD44 mini-gene contains an MMTV promoter and is a multi-functional molecule containing two variable exons, v4 and v5 that can be either included or excluded during the splicing process. Both AR and AR-V7 induce transcription from an MMTV promoter. Co-transfection of AR or AR-V7 with the CD44 mini-gene in Hela cells demonstrated that CD44 splicing was altered by AR and its variant AR-V7. R1881 treated samples showed a shift to RNA products resulting from the skipping of both variable exons. Treatment with R1881 showed a decrease in the PSI (Percent Spliced Inclusion) as compared to obtained in the absence of R1881 or AR-V7. In contrast, AR-V7 minimally induced single exon skipping as compared to vehicle. These observations suggest that AR and its variants can have marked effects on RNA splicing to produce radically different proteins with different splicing/inclusion ratio. Analysis of the RNA-Seq data revealed that multiple types of splicing events are affected by the AR isoforms. For instance, whether an isoform favours exclusion or inclusion of a particular exon is event specific. A comparison of splicing in VCaP and LNCaP models showed that only a subset of events were common to the two models. One of the most differentially regulated events in both lines is an exon inclusion/exclusion event in PGAP2. Both isoforms increase the inclusion/exclusion ratio. However, further analysis showed that AR-V7 preferentially increased the levels of inclusion, but did not affect the basal levels of skipping. Also, AR increased the inclusion/skipping ratio through repression of expression of the skipped product. Similar analyses for other splicing events are ongoing. Our studies will provide a better understanding for the molecular basis for isoform specific actions and the role of AR/AR-V7 in the regulation of mRNA transcription and splicing.