Prostate cancer frequently metastasizes to bone where it forms osteoblastic lesions through unknown mechanisms. Bone morphogenetic proteins (BMP) are mediators of skeletal formation. Prostate cancer produces a variety of BMPs, including BMP-6. We tested the hypothesis that BMP-6 contributes to prostate cancer-induced osteosclerosis at bone metastatic sites. Prostate cancer cells and clinical tissues produced BMP-6 that increased with aggressiveness of the tumor. Prostate cancer-conditioned medium induced SMAD phosphorylation in the preosteoblast MC3T3 cells, and phosphorylation was diminished by anti-BMP-6 antibody. Prostate cancer-conditioned medium induced mineralization of MC3T3 cells, which was blocked by both the BMP inhibitor noggin and anti-BMP-6. Human fetal bones were implanted in severe combined immunodeficient mice and after 4 weeks, LuCaP 23.1 prostate cancer cells were injected both s.c. and into the bone implants. Anti-BMP-6 or isotype antibody administration was then initiated. Anti-BMP-6 reduced LuCaP 23.1-induced osteoblastic activity, but had no effect on its osteolytic activity. This was associated with increased osteoblast numbers and osteoblast activity based on bone histomorphometric evaluation. As endothelin-1 has been implicated in bone metastases, we measured serum endothelin-1 levels but found they were not different among the treatment groups. In addition to decreased bone production, anti-BMP-6 reduced intraosseous, but not s.c., tumor size. We found that BMP-2, BMP-4, BMP-6, and BMP-7 had no direct effect on prostate cancer cell growth, but BMP-2 and BMP-6 increased the in vitro invasive ability of prostate cancer cell. These data show that prostate cancer promotes osteoblastic activity through BMP-6 and that, in addition to its bone effects, suggest that BMPs promote the ability of the prostate cancer cells to invade the bone microenvironment. (Cancer Res 2005; 65(18): 8274-85)

Prostate cancer (PCa) metastasizes selectively to bone through unknown mechanisms. In the current study, we identified exosome-mediated transfer of pyruvate kinase M2 (PKM2) from PCa cells into bone marrow stromal cells (BMSCs) as a novel mechanism through which primary tumor-derived exosomes promote premetastatic niche formation. We found that PKM2 up-regulates BMSC CXCL12 production in a HIF-1α-dependent fashion, which subsequently enhances PCa seeding and growth in the bone marrow. Furthermore, serum-derived exosomes from patients with either primary PCa or PCa metastasis, as opposed to healthy men, reveal that increased exosome PKM2 expression is associated with metastasis, suggesting clinical relevance of exosome PKM2 in PCa. Targeting the exosome-induced CXCL12 axis diminished exosome-mediated bone metastasis. In summary, primary PCa cells educate the bone marrow to create a premetastatic niche through primary PCa exosome-mediated transfer of PKM2 into BMSCs and subsequent up-regulation of CXCL12. This novel mechanism indicates the potential for exosome PKM2 as a biomarker and suggests therapeutic targets for PCa bone metastasis.

Prostate cancer (PCa) is frequently accompanied by osteosclerotic (i.e., excessive bone production) bone metastases. Although bone morphogenetic proteins (BMPs) and Wnts are mediators of PCa-induced osteoblastic activity, the relation between them in PCa bone metastases is unknown. The goal of this study was to define this relationship. Wnt3a and Wnt5a administration or knockdown of (DKK-1), a Wnt inhibitor, induced BMP4 and 6 expression and promoter activation in PCa cells. DKK-1 blocked Wnt activation of the BMP promoters. Transfection of C4-2B cells with axin, an inhibitor of canonical Wnt signaling, blocked Wnt3a, but not Wnt5a induction of the BMP promoters. In contrast, Jnk inhibitor I blocked Wnt5a, but not Wnt3a induction of the BMP promoters. Wnt3a, Wnt5a, and conditioned-media (CM) from C4-2B or LuCaP23.1 cells induced osteoblast differentiation in vitro. The addition of DKK-1 and Noggin, a BMP inhibitor, to CM diminished PCa CM-induced osteoblast differentiation in a synergistic fashion. However, pretreatment of PCa cells with DKK-1 prior to collecting CM blocked osteoblast differentiation; whereas, pretreatment with Noggin only partially reduced osteoblast differentiation and pre-treatment with both DKK-1 and Noggin had no greater effect than pretreatment with DKK-1 alone. Additionally, knockdown of BMP expression in C4-2B cells inhibited Wnt-induced osteoblastic activity. These results demonstrate that PCa promotes osteoblast differentiation through canonical and non-canonical Wnt signaling pathways that stimulate both BMP-dependent and independent osteoblast differentiation. These results demonstrate a clear link between Wnts and BMPs in PCa-induced osteoblast differentiation and provide novel targets, including the non-canonical Wnt pathway, for therapy of PCa.

Purpose To investigate the efficacy and mechanisms of Notch signaling inhibition as an adjuvant to docetaxel in castration-resistant prostate cancer (CRPC) using a γ-secretase inhibitor (GSI), PF-03084014. Experimental Design The effect of PF-03084014 on response to docetaxel was evaluated in docetaxel-sensitive and -resistant CRPC cell lines in vitro and in murine models. Both soft tissue and bone sites were evaluated in vivo. Impacts on cell proliferation, apoptosis, cancer stem cells and angiogenesis were evaluated. Results The combination of PF-03084014 plus docetaxel reduced both docetaxel-sensitive and -resistant CRPC tumor growth in soft tissue and bone greater than either agent alone. Antitumor activity was associated with PF-03084014-induced inhibition of Notch pathway signaling; decreased survival signals (Cyclin E; MEK-ERK, PI3K-AKT, EGFR and NF-κB pathway; BCL-2, BCL-XL); increased apoptotic signals (BAK, BAX; cleaved-caspase 3); reduction of microvessel density; reduced epithelial mesenchymal transition; and reduction of cancer stem-like cells in the tumor. Conclusion These results reveal that PF-03084014 enhances docetaxel-mediated tumor response and provides a rationale to explore GSIs as adjunct therapy in conjunction with docetaxel for men with CRPC.

Prostate cancer frequently metastasizes to bone resulting in the formation of osteoblastic metastases through unknown mechanisms. Vascular endothelial growth factor (VEGF) has been shown recently to promote osteoblast activity. Accordingly, we tested if VEGF contributes to the ability of prostate cancer to induce osteoblast activity. PC-3, LNCaP, and C4-2B prostate cancer cell lines expressed both VEGF-165 and VEGF-189 mRNA isoforms and VEGF protein. Prostate cancer cells expressed the mRNA for VEGF receptor (VEGFR) neuropilin-1 but not the VEGFRs Flt-1 or KDR. In contrast, mouse preosteoblastic cells (MC3T3-E1) expressed Flt-1 and neuropilin-1 mRNA but not KDR. PTK787, a VEGFR tyrosine kinase inhibitor, inhibited the proliferation of human microvascular endothelial cells but not prostate cancer proliferation in vitro. C4-2B conditioned medium induced osteoblast differentiation as measured by production of alkaline phosphatase and osteocalcin and mineralization of MC3T3-E1. PTK787 blocked the C4-2B conditioned medium-induced osteoblastic activity. VEGF directly induced alkaline phosphatase and osteocalcin but not mineralization of MC3T3-E1. These results suggest that VEGF induces initial differentiation of osteoblasts but requires other factors, present in C4-2B, to induce mineralization. To determine if VEGF influences the ability of prostate cancer to develop osteoblastic lesions, we injected C4-2B cells into the tibia of mice and, after the tumors grew for 6 weeks, administered PTK787 for 4 weeks. PTK787 decreased both intratibial tumor burden and C4-2B-induced osteoblastic activity as measured by bone mineral density and serum osteocalcin. These results show that VEGF contributes to prostate cancer-induced osteoblastic activity in vivo. (Cancer Res 2005; 65(23): 10921-9)