Systematic and functional characterization of novel androgen receptor variants arising from alternative splicing in the ligand-binding domain

Abstract

The presence of intact ligand-binding domain (LBD) ensures the strict androgen-dependent regulation of androgen receptor (AR): binding of androgen induces structural reorganization of LBD resulting in release of AR from HSP90, suppression of nuclear export which otherwise dominates over import and nuclear translocation of AR as a transcription factor. Thus, loss or defects of the LBD abolish constraint from un-liganded LBD as exemplified by constitutively active AR variants (AR-Vs), which are associated with emerging resistance mechanism to anti-AR therapy in castration-resistant prostate cancer (mCRPC). Recent analysis of the AR splicing landscapes revealed mCRPC harboring multiple AR-Vs with diverse patterns of inclusion/exclusion of exons (exons 4-8) corresponding to LBD to produce namely exon-skipping variants. In silico construction for these AR-Vs revealed four novel AR-Vs having unique features: Exclusion of specified exons introduces a frameshift in variants v5es, v6es and v7es. ARv56es maintains the reading frame resulting in the inclusion of the C-terminal half of the LBD. We systematically characterized these AR-Vs regarding their subcellular localization, affinity for HSP90 and transactivation capability. Notably, ARv5es was free from HSP90, exclusively nuclear, and constitutively active similarly as previously reported for v567es. In contrast, v6es and v7es were similar in that they are cytoplasmic, transcriptionally inactive and bind HSP90, ARv56es was present in both nucleus and cytoplasm, does not bind HSP90 and is transcriptionally inactive. Converting these transcriptionally inactive AR-Vs into active forms, we identified the two separate elements that allosterically suppress otherwise constitutively active AR-Vs; one in exon 5 for v6es and v7es and the other in exon 8 for v56es. Our findings identify a novel constitutively active AR-V, ARv5es and establish a method to predict potential activities of AR-Vs carrying impaired LBD.

Allosteric alterations in the androgen receptor and activity in prostate cancer

“…The bipartite nuclear localization signal (NLS) is encoded by exon 3 and exon 4 (Centenera et al 2008). Moreover, a proposed ligand-regulated nuclear export signal (NES), which operates independently of CRM1, resides in the region corresponding to the latter half of exon 5 through exon 6 (Saporita et al 2003; Wang et al 2004; Gong et al 2012; Uo et al 2017). Androgen binding to AR suppresses NES activity.…”

“…Initial findings were made by biased traditional molecular biology approaches, including 3’ rapid amplification of cDNA and targeted PCR in PCa cell lines and xenografts (Dehm et al 2008; Hu et al 2009). Thereafter, enormous progress was made in finding multiple species of AR-Vs owing to the advancement of next-generation sequencing technologies such as the establishment of integrated landscape studies of whole genome and transcriptome sequencing as well as splicing patterns in clinical samples (Robinson et al 2015; Uo et al 2017). Collectively, more than twenty different AR-Vs have been detected and identified in human samples (Marcias et al 2010; Cao et al 2016; Coutinho et al 2016).…”

“…With limited exceptions, AR-Vs generally harbor exons 1 and 2, undergo either (i) alterative inclusion/exclusion of exon 3 in tandem with incorporation of a cryptic exon after exon 2 or 3 or (ii) alternative inclusion/exclusion of exons 4–8 resulting in a deletion in the LBD (Robinson et al 2015). As a definition, the former and the latter groups are termed truncated and exon-skipping variants, respectively (Uo et al 2017).…”

“…The most common AR-V, AR-V7, represents the members of this group and displays constitutive transactivation activity and includes cryptic exon 3 (CE3)(Guo et al 2009; Hu et al 2009). CE3 is able to reconstitute NLS activity thus allowing AR-V7’s predominant nuclear localization (Sprenger and Plymate 2014; Uo et al 2017). In sharp contrast, AR-V1, -V4 and -V6 are produced as a result of inclusion of CE1, CE4, and CE2, respectively (Hu et al 2011; Chan et al 2012; Zhan et al 2017)}.…”

“…However, as the microtubule-binding domain of AR is retained, which encompasses the DBD and the HR, ARv567es but not AR-V7 undergoes taxane-sensitive trafficking to the nucleus by microtubules (Darshan et al 2011; Carbonaro et al 2012; Thadani-Mulero et al 2014). The discovery of ARv567es prompted us to systematically and comprehensively analyze the presence of exon-skipping variants in the Cancer Genome Atlas (TCGA) and Stand Up 2 Cancer (SU2C) data sets (Uo et al 2017). AR-Vs that undergo alternative inclusion/exclusion of exons 5–8 were expressed at varying levels.…”