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.

We studied heterogeneities at a molecular level in bone metastasis samples obtained from men with castration-resistant prostate cancer. We found differences of possible importance for therapy selection in individual patients.

BackgroundIntra-tumoral steroidogenesis and constitutive androgen receptor (AR) activity have been associated with castration-resistant prostate cancer (CRPC). This study aimed to examine if CRPC bone metastases expressed higher levels of steroid-converting enzymes than untreated bone metastases. Steroidogenic enzyme levels were also analyzed in relation to expression of constitutively active AR variants (AR-Vs) and to clinical and pathological variables.Methodology/Principal FindingsUntreated, hormone-naïve (HN, n = 9) and CRPC bone metastases samples (n = 45) were obtained from 54 patients at metastasis surgery. Non-malignant and malignant prostate samples were acquired from 13 prostatectomy specimens. Transcript and protein levels were analyzed by real-time RT-PCR, immunohistochemistry and immunoblotting. No differences in steroidogenic enzyme levels were detected between CRPC and HN bone metastases. Significantly higher levels of SRD5A1, AKR1C2, AKR1C3, and HSD17B10 mRNA were however found in bone metastases than in non-malignant and/or malignant prostate tissue, while the CYP11A1, CYP17A1, HSD3B2, SRD5A2, and HSD17B6 mRNA levels in metastases were significantly lower. A sub-group of metastases expressed very high levels of AKR1C3, which was not due to gene amplification as examined by copy number variation assay. No association was found between AKR1C3 expression and nuclear AR staining, tumor cell proliferation or patient outcome after metastases surgery. With only one exception, high AR-V protein levels were found in bone metastases with low AKR1C3 levels, while metastases with high AKR1C3 levels primarily contained low AR-V levels, indicating distinct mechanisms behind castration-resistance in individual bone metastases.Conclusions/SignificanceInduced capacity of converting adrenal-gland derived steroids into more potent androgens was indicated in a sub-group of PC bone metastases. This was not associated with CRPC but merely with the advanced stage of metastasis. Sub-groups of bone metastases could be identified according to their expression levels of AKR1C3 and AR-Vs, which might be of relevance for patient response to 2nd line androgen-deprivation therapy.

Bone metastasis is the lethal end‐stage of prostate cancer (PC), but the biology of bone metastases is poorly understood. The overall aim of this study was therefore to explore molecular variability in PC bone metastases of potential importance for therapy. Specifically, genome‐wide expression profiles of bone metastases from untreated patients ( n = 12) and patients treated with androgen‐deprivation therapy (ADT, n = 60) were analyzed in relation to patient outcome and to morphological characteristics in metastases and paired primary tumors. Principal component analysis and unsupervised classification were used to identify sample clusters based on mRNA profiles. Clusters were characterized by gene set enrichment analysis and related to histological and clinical parameters using univariate and multivariate statistics. Selected proteins were analyzed by immunohistochemistry in metastases and matched primary tumors ( n = 52) and in transurethral resected prostate (TUR‐P) tissue of a separate cohort ( n = 59). Three molecular subtypes of bone metastases (MetA‐C) characterized by differences in gene expression pattern, morphology, and clinical behavior were identified. MetA (71% of the cases) showed increased expression of androgen receptor‐regulated genes, including prostate‐specific antigen (PSA), and glandular structures indicating a luminal cell phenotype. MetB (17%) showed expression profiles related to cell cycle activity and DNA damage, and a pronounced cellular atypia. MetC (12%) exhibited enriched stroma–epithelial cell interactions. MetB patients had the lowest serum PSA levels and the poorest prognosis after ADT. Combined analysis of PSA and Ki67 immunoreactivity (proliferation) in bone metastases, paired primary tumors, and TUR‐P samples was able to differentiate MetA‐like (high PSA, low Ki67) from MetB‐like (low PSA, high Ki67) tumors and demonstrate their different prognosis. In conclusion, bone metastases from PC patients are separated based on gene expression profiles into molecular subtypes with different morphology, biology, and clinical outcome. These findings deserve further exploration with the purpose of improving treatment of metastatic PC.

Advanced prostate cancer frequently metastasizes to bone and induces a mixed osteoblastic/osteolytic bone response. Standard treatment for metastatic prostate cancer is androgen-deprivation therapy (ADT) that also affects bone biology. Treatment options for patients relapsing after ADT are limited, particularly in cases where castration-resistance does not depend on androgen receptor (AR) activity. Patients with non-AR driven metastases may, however, benefit from therapies targeting the tumor microenvironment. Therefore, the current study specifically investigated bone cell activity in clinical bone metastases in relation to tumor cell AR activity, in order to gain novel insight into biological heterogeneities of possible importance for patient stratification into bone-targeting therapies. Metastasis tissue obtained from treatment-naïve (n = 11) and castration-resistant (n = 28) patients was characterized using whole-genome expression analysis followed by multivariate modeling, functional enrichment analysis, and histological evaluation. Bone cell activity was analyzed by measuring expression levels of predefined marker genes representing osteoclasts (ACP5, CTSK, MMP9), osteoblasts (ALPL, BGLAP, RUNX2) and osteocytes (SOST). Principal component analysis indicated a positive correlation between osteoblast and osteoclast activity and a high variability in bone cell activity between different metastases. Immunohistochemistry verified a positive correlation between runt-related transcription factor 2 (RUNX2) positive osteoblasts and tartrate-resistant acid phosphatase (TRAP, encoded by ACP5) positive osteoclasts lining the metastatic bone surface. No difference in bone cell activity was seen between treatment-naïve and castration-resistant patients. Importantly, bone cell activity was inversely correlated to tumor cell AR activity (measured as AR, FOXA1, HOXB13, KLK2, KLK3, NKX3-1, STEAP2, and TMPRSS2 expression) and to patient serum prostate-specific antigen (PSA) levels. Functional enrichment analysis indicated high bone morphogenetic protein (BMP) signaling in metastases with high bone cell activity and low tumor cell AR activity. This was confirmed by BMP4 immunoreactivity in tumor cells of metastases with ongoing bone formation, as determined by histological evaluation of van Gieson-stained sections. In conclusion, the inverse relation observed between bone cell activity and tumor cell AR activity in prostate cancer bone metastasis may be of importance for patient response to AR and/or bone targeting therapies, but needs to be evaluated in clinical settings in relation to serum markers for bone remodeling, radiography and patient response to therapy. The importance of BMP signaling in the development of sclerotic metastasis lesions deserves further exploration.