Abstract 4676: In silico and in vitro analyses of androgen receptor splicing variants with a special focus on human patient samples
Authors: Takuma Uo, Heidi Dvinge, Cynthia C. Sprenger, Shihua Sun, Robert K. Bradley, Peter S. Nelson, Stephen R. Plymate
Journal: Cancer Research (2015)

Abstract The androgen receptor (AR) is arguably the critical driver of not only early but also advanced prostate cancer. AR is a transcription factor which comprises the four functional domains with the N-terminal transactivation domain encoded by exon 1, the DNA-binding domain by exons 2 and 3, the hinge domain by exon 4, and the ligand-binding domain (LBD) by exons 4-8. The functional LBD provides a strict ligand-dependency of AR's release from cytoplasmic HSP90 and nuclear translocation to act as a transcription factor. Conversely, disruption of LBD can be caused by some point mutations or alternative splicing within exon 4-8, thus allowing AR to exert ligand-independent nuclear actions. Numerous AR splicing variants (AR-Vs) have been identified and predicted, but these are as yet less characterized regarding their biochemical functions and clinical relevance. This prompted us to perform systematic analysis of the clinical samples in relation to expression patterns of AR-Vs and disease manifestation. AR-Vs compositions were evaluated on a group of deeply RNA-sequenced metastases from patients treated with androgen deprivation. By strictly selecting and counting the reads spanning junctions of exons defined by Hu et al (1), novel AR-Vs were found to be substantially expressed: v5es is exon 5 skipped variant whereas v7es skips exon 7. According to the preliminary results, the v5es and v7es variants were detected in 20% and 26% of total patient samples, respectively. The v78es and v8es variants, which had been previously identified but less characterized, were found to be 28% and 6%, respectively, in the same comparison. It is of note that AR V7 was detected in 63% of the samples. While v5es structurally lacks the most part of LBD, the v7es, v78es, and v8es variants possess LBD with a short C-terminal truncation. Both v7es and v8es variants showed diffuse distribution in both cytoplasm and nucleus of the AR-negative M12 cells. These localization patterns were not largely affected by the presence or absence of the ligand dihydroxytestosterone. The above observations were consistent with the results from the probasin ARE-ARR3-luciferase reporter assay showing that these two variants possessed weak transcriptional activities regardless of ligand status. The bioinformatics project is now being updated by including the data from additional patient samples. The biochemical characterization of AR-Vs (v5es, v7es, v8es, and v78es variants) will be detailed and reported regarding their transcriptional activities, subcellular localization, as well as their potential protein-protein interactions with the full-length AR, HSP90 and co-regulators. Ligand-independent nuclear actions of AR have been mostly believed to confer resistance to anti-androgens enzalutamide and androgen biosynthesis inhibitors abiraterone acetate. This study thus may provide a foundation for novel AR-targeted therapies. 1. Hu R et al. Prostate (2011) 7, 1656 Citation Format: Takuma Uo, Heidi Dvinge, Cynthia C. Sprenger, Shihua Sun, Robert K. Bradley, Peter S. Nelson, Stephen R. Plymate. In silico and in vitro analyses of androgen receptor splicing variants with a special focus on human patient samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4676. doi:10.1158/1538-7445.AM2015-4676