Osteoprotegerin (OPG), a critical regulator of osteoclastogenesis, is expressed by prostate cancer cells, and OPG levels are increased in patients with prostate cancer bone metastases. The objective of this study was to investigate the effects of OPG overexpression on prostate cancer cells and prostate cancer/bone cell interactions in vitro and in vivo. OPGtransfected C4-2 cells expressed 8.0 ng OPG per mL per 10 6 cells, whereas no OPG was detected in the media of C4-2 cells transfected with a control plasmid. OPG overexpressed by C4-2 cells protected these cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis and decreased osteoclast formation. Subcutaneous OPG-C4-2 and pcDNA-C4-2 tumors exhibited similar growth and take-rate characteristics. However, when grown in bone, tumor volume was decreased in OPG-C4-2 versus pcDNA-C4-2 (P = 0.0017). OPG expressed by C4-2 cells caused increases in bone mineral density (P = 0.0074) and percentage of trabecular bone volume (P = 0.007), and decreases in numbers of osteoblasts and osteoclasts when compared with intratibial pcDNA-C4-2 tumors (P = 0.003 and P = 0.019, respectively). In summary, our data show that increased expression of OPG in C4-2 cells does not directly affect proliferation of prostate cancer cells but indirectly decreases growth of C4-2 tumors in the bone environment. Our data also show that OPG expressed by C4-2 cells inhibits bone lysis associated with C4-2 bone metastasis, which results in net increases in bone volume. We therefore hypothesize that OPG expressed in prostate cancer patient bone metastases may be at least partially responsible for the osteoblastic character of most prostate cancer bone lesions. (Cancer Res 2005; 65(5): 1710-8)

BACKGROUND The mitogen-activated protein kinases (MAPKs) regulate cell growth, differentiation, and stress responses, and many critical signaling pathways are subject to cross-regulation by MAPK signaling. Previous studies have yielded evidence of cross-talk between the MAPK pathways and androgen receptor (AR) signaling, which plays a critical role in growth control of both normal prostate and prostate cancer (PCa). Objective of this study was to evaluate the expression of MAPK-like protein nemo-like kinase (NLK) in PCa and its effects on AR-mediated transcription. METHODS Real-time PCR and IHC were used to evaluate levels of NLK in prostatic samples. Effects of over-expression of NLK on apoptosis and proliferation were determined using Western blot and flow cytometry. Effects on AR signaling were evaluated using over-expression and knockdown of NLK in PCa cells in combination with PCR, Western blotting and reporter assays. RESULTS Our results show that the expression of NLK is decreased in PCa metastases in comparison to normal prostate epithelium and primary PCa. Our results also show that over-expression of NLK resulted in induction of apoptosis, which was more pronounced in AR-expressing LNCaP versus AR-negative PC-3 cells. Higher levels of NLK decreased levels of AR mRNA and protein as well as inhibited AR-mediated transcription. CONCLUSIONS NLK expression is altered during PCa progression and it is involved in regulation of AR signaling in these cells. A deeper understanding of the roles of NLK in regulation of AR-mediated transcription and control of PCa progression may point the way to new modes of therapeutic intervention in this disease. Prostate 69: 1481–1492, 2009.

INTRODUCTION-mTOR activity is increased in advanced prostate cancer (CaP) as a result of a high rate of PTEN mutations. RAD001 (Everolimus) is a new orally available mTOR inhibitor. The objective of our study was to evaluate the effects of RAD001 on the growth of CaP in the bone, both alone and in combination with docetaxel and zoledronic acid.

Bone morphogenetic protein (BMP) signaling is important in prostate development and prostate cancer (PCa) progression. However, because of the multiple effects of different BMPs, no final conclusions have been made as to the role of BMPs in PCa. In our studies, we have focused on BMP-7 because it is involved in prostate morphogenesis, and its expression is regulated by androgens. The objective of our study was to determine BMP-7 expression in PCa metastases and investigate the effects of BMP-7 on PCa cells. Our results show that BMP-7 is expressed in metastatic PCa and its levels are increased in castration-resistant PCa versus androgen-dependent PCa, whereas the expression of BMP-7 is decreased in primary PCa versus normal prostate. Our in vitro results show that BMP-7 inhibits proliferation of androgen-sensitive LNCaP cells, stimulates androgen receptor signaling, increases the expression of differentiation-associated genes, and decreases the levels of some wingless-regulated transcripts. Interestingly, these effects were not detected in C4-2 castration-resistant PCa cells. In vivo expression of BMP-7 in castration-resistant C4-2 cells did not alter proliferation when these cells were grown subcutaneously, but their growth was inhibited in the bone environment. In summary, our results show that BMP-7 is expressed at the highest level in advanced castration-resistant PCa cells and that the inhibitory effects of BMP-7 are dependent on the differentiation status of PCa cells and the tumor microenvironment. Further studies are needed to identify changes in BMP-7 signaling that lead to the loss of its control of proliferation during PCa progression.

Treatments for advanced prostate cancer (CaP) typically involve androgen deprivation therapy. However, most patients eventually develop castration-resistant CaP (CRPC) for which highly effective therapies are limited. We explored the efficacy of a novel agent, HE3235, in inhibiting growth of CRPC in preclinical models. Castrated male mice were implanted subcutaneously with LuCaP35V CaP xenografts in the presence and absence of 5'-androstenediol (AED) and treated with HE3235. To investigate the effect of HE3235 on CaP tumor in the bone, castrated mice were injected intratibially with C4-2B CaP cells and treated with HE3235. Serum prostate-specific antigen (PSA) levels, tumor volume, immunohistochemistry, gene expression, and levels of intratumoral androgens were analyzed. HE3235 significantly prolonged the tumor doubling time of LuCaP35V, decreased androgen receptor expression, and lowered levels of intratumoral testosterone by approximately 89% and dihydrotestosterone by approximately 63% in both the presence and the absence of AED. HE3235 inhibited tumor growth in the bone environment. Weights of tumored tibiae of HE3235-treated animals were lower than those of control (P = .031), and normalized PSA levels were also significantly decreased at the end of study by HE3235 treatment (P = .0076). HE3235 inhibits the growth of subcutaneous CRPC as well as CRPC in the bone environment. Our data show that HE3235 exhibits a wide range of effects, including alteration of androgen receptor signaling and reductions in levels of intratumoral androgens. Our results support ongoing clinical investigations into the effectiveness of HE3235 in the setting of CRPC and warrants further studies into the mechanisms behind the effects of HE3235.