Expression of Androgen Receptor Splice Variants in Prostate Cancer Bone Metastases is Associated with Castration-Resistance and Short Survival

Abstract

BackgroundConstitutively active androgen receptor variants (AR-V) lacking the ligand binding domain (LBD) may promote the development of castration-resistant prostate cancer (CRPC). The expression of AR-Vs in the clinically most important metastatic site, the bone, has, however, not been well documented. Our aim was therefore to compare levels of AR-Vs in hormone-naive (HN) and CRPC bone metastases in comparison to primary PC and non-malignant prostate tissue, as well as in relation to AR protein expression, whole-genome transcription profiles and patient survival.Methodology/Principal FindingsHormone-naïve (n = 10) and CRPC bone metastases samples (n = 30) were obtained from 40 patients at metastasis surgery. Non-malignant and malignant prostate samples were acquired from 13 prostatectomized men. Levels of full length AR (ARfl) and AR-Vs termed AR-V1, AR-V7, and AR-V567es mRNA were measured with RT-PCR and whole-genome transcription profiles with an Illumina Beadchip array. Protein levels were examined by Western blotting and immunohistochemistry. Transcripts for ARfl, AR-V1, and AR-V7 were detected in most primary tumors and metastases, and levels were significantly increased in CRPC bone metastases. The AR-V567es transcript was detected in 23% of the CRPC bone metastases only. A sub-group of CRPC bone metastases expressed LBD-truncated AR proteins at levels comparable to the ARfl. Detectable AR-V567es and/or AR-V7 mRNA in the upper quartile, seen in 1/3 of all CRPC bone metastases, was associated with a high nuclear AR immunostaining score, disturbed cell cycle regulation and short survival.Conclusions/SignificanceExpression of AR-Vs is increased in CRPC compared to HN bone metastases and associated with a particularly poor prognosis. Further studies are needed to test if patients expressing such AR-Vs in their bone metastases benefit more from drugs acting on or down-stream of these AR-Vs than from therapies inhibiting androgen synthesis.

Identification of Androgen Receptor Splice Variant Transcripts in Breast Cancer Cell Lines and Human Tissues

“…This data is also consistent with observations of ARV expression in non-cancerous prostate tissues [10,12,30]. While such low expression of the ARVs challenges their physiological significance, a recent study of prostate cancer bone metastases shows that mRNA levels may not accurately reflect protein levels [18]. We did not assess protein levels of the ARVs identified in the breast cancer cell lines because our primary aim was to examine the frequency and diversity of variant transcript expression.…”

“…Most ARVs have been shown to be constitutively (AR-V3, -V4, -V7, and -V12) [9,10,12,17] or conditionally (AR-V1 and -V9) [10,12] active androgen-independent transcription factors in a prostate cancer context. Pre-clinical and clinical studies have supported the concept that ARVs are biologically relevant in prostate cancer, particularly in the genesis of a CRPC [11,12,[17][18][19][20].…”

Androgen receptor signaling in castration-resistant prostate cancer: a lesson in persistence

“…Although the associations between AR-Vs (mRNA and protein) with clinical parameters and drug resistance are compelling, the relevance of these findings has been called into question by the observation that AR-V expression at the mRNA level may only be approximately 0.03-7% of the full-length transcript in CRPC metastases (Hornberg et al 2011, Robinson et al 2015. However, we believe that this is a poor reason to discount the biological relevance of AR-Vs. First, as full-length AR (AR-FL) expression is increased by approximately an order of magnitude in CRPC compared to hormone-sensitive disease (Hu et al 2009), the aformentioned ratio is misleading in terms of absolute expression.…”

“…Rapid, reversible induction of AR-V expression can be achieved by alternative splicing (Watson et al 2010, Hu et al 2012, Gillis et al 2013, Liu et al 2013, Yu et al 2014b, whereas genomic rearrangements within the AR gene can mediate a fully androgen-independent phenotype through a mechanism of switching AR expression from full-length AR to ARv567es (Nyquist et al 2013). AR-V mRNAs have been identified in prostate cancer cell lines, xenografts, mouse models and, most importantly, patient specimens (Dehm et al 2008, Guo et al 2009, Hu et al 2009, Watson et al 2010, Hornberg et al 2011, McGrath et al 2013, Robinson et al 2015 (Table 2). The most recent genomic data, based on the detection of unique exon junctions in RNAseq data, estimates that at least 12 distinct AR-V mRNA species are detectable in primary PCa (Cancer Genome Atlas Research 2015) and 23 in mCRPC (Robinson et al 2015) (for a comprehensive summary of the AR splicing landscape, Supplementary Table 2).…”

“…Expression of AR-Vs in clinical samples has provided evidence for clinically relevant functions. For example, AR-V7 is elevated in castrate-resistant vs hormoneresponsive tumor tissues (Guo et al 2009, Hu et al 2009), in CRPC vs benign or localized PCa (McGrath et al 2013) and in CRPC bone metastases compared to benign tissues, primary tumors or hormone-naïve bone metastases (Hornberg et al 2011). Although therapydriven increases in AR-V7 expression could be due to ADT-mediated alterations to AR splicing factors, RNAbinding proteins or miRNAs (Liu et al 2014b, Shi et al 2015, Stockley et al 2015, it must be noted that ligand depletion can result in increased expression of the AR gene due to transcriptional autoregulation (described above) (Cai et al 2011).…”