Expression of androgen receptor splice variants in prostate cancer bone metastases is associated with castration-resistance and short survival

Abstract

Background: Constitutively 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 findings: Hormone-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/significance: Expression 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.

Figure 1. Normalized mRNA levels of the A) full length androgen receptor (ARfl), B) AR-V1 , and C) AR-V7 in a total of 66 tissue samples from prostate cancer patients.

All mRNA levels were adjusted for corresponding housekeeping gene (RPL13a) mRNA levels, normalized to the median value for the primary tumors samples, and transformed by the natural logarithm. Levels in non-malignant prostate tissue (n = 13), primary prostate tumor (n = 13), hormone-naïve bone metastases (n = 10), and castration-resistant bone metastases (n = 30) samples are shown in white, light grey, dark grey, and black, respectively. Undetectable levels are not indicated, but represented by columns symbolizing the lowest measurable level in RT-PCR analysis of each transcript variant.

Figure 2. Western blot analysis of the full length androgen receptor (ARfl) and ligand binding domain (LBD) truncated AR variants in castration resistant prostate cancer (CRPC) bone metastasis samples.

A) Antibodies (N-20 and PG-21) targeting the N-terminal domain (NTD) of the AR detected the ARfl and AR variants (AR-V; presumingly AR-V1, AR-V7, and/or AR-V567es). Antibody C-19 targeting the LBD detected the ARfl of ∼110 kDa, but not the LBD-truncated AR variants of approximately ∼80 kDa. B) Analysis of CRPC bone metastases representing samples with high levels of AR-V7 mRNA (levels in the highest quartile, Q4, according to RT-PCR analysis), as well as samples with AR-V7 mRNA levels in Q1, Q2, and Q3, by using the N-20 antibody. 22Rv1 cell extract served as positive control for ARfl and ∼80 kDa AR variants and a primary prostate cancer sample (P) served as negative control for the AR variants.

Figure 3. Kaplan-Meier analysis of androgen receptor (AR) variant transcript levels in bone metastasis samples from castration-resistant prostate cancer (CRPC) patients versus cancer-specific survival after metastasis surgery.

Patients with A) AR-V7 mRNA levels in the highest quartile (Q4), B) detectable AR-V567es mRNA levels, and C) detectable AR-V567 and/or AR-V7 Q4 (AR-V high) mRNA levels had shorter survival than other patients with CRPC disease.

Figure 4. Network of directly interacting gene products of differentially expressed genes between the AR-V high (detectable AR-V567 and/or AR-V7 in the highest quartile, Q4) and other castration-resistance prostate cancer (CRPC) bone metastases (P≤0.05 and fold change ≥1.5), generated by Metacore™ (Genego Inc, USA) bioinformatics analysis.

Red circles indicate higher and blue circles indicate lower transcript levels in the AR-V high than in other CRPC bone metastases. Note that many of the differentiating gene products are directly interacting via AR, C-MYC and CDK1.