Effect Of MCP-1/CCR2 Axis On Prostate Cancer Skeletal Metastasis

Bone is the most common metastatic site for several tumor typesincluding prostate, breast, lung, thyroid and renal cancers. When the tumormetastasizes to distant organ, such as bone, the mortality rate is significantlyhigh and all patients with bone metastasis eventually die of their disease.Therefore, new therapeutic strategies are urgently needed. When prostatecancer (PCa) cells metastasize to bone, they interact with the bonemicroenvironment. These interactions result in enhanced growth of tumorcells and osteolysis that is followed by an osteoblastic response. Bidirectionalinteractions between PCa and the bone microenvironment increaseexpression of cytokines, chemokines and adhesion molecules by both bonecells and PCa cells, including monocyte chemotactic protein-1 (MCP-1) andRANKL, contributing to the bone destruction. MCP-1 plays a critical role inrecruitment and activation of monocytes during acute inflammation andangiogenesis. Recent report of increased MCP-1 serum levels in patients withvarious cancers were correlated with advanced stage. Parathyroid hormone-related protein (PTHrP), produced by bone cells and PCa, binds to receptorson osteoblasts and stimulates bone formation and resorption. PTHrP is highlyexpressed on metastatic bone lesions compared to moderate expression on localized PCa. PTHrP has been shown to enhance bone metastasis in animalmodels of PCa and breast cancer. However, the mechanisms through PTHrPpromote PCa induced-bone lesion and the role of MCP-1 in PCa bonemetastasis remain poorly understood.Our hypothesis is that MCP-1, produced in the tumor and bonemicroenvironment, stimulates the growth and migration of prostate cancercells, as well as osteoclastogenesis. These effects result in the increasedbone destruction associated with PCa and enhance tumor growth. Thus, theMCP-1/CCR2 signaling pathway plays a critical role in the skeletal metastasisof PCa.First, a human cytokine antibody array was used to measure cytokineexpression in conditioned medium collected from primary prostate epithelialcells (PrEC), prostate cancer LNCaP and its derivative C4-2B, and PC3 cells.All PCa cells produced high amounts of MCP-1 compared with PrEC cells.Furthermore, levels of interleukin IL-6, IL-8, GROα, ENA-78, and CXCL-16were higher in PC3 than LNCaP. These results were confirmed by ELISA.Second, human bone marrow mononuclear cells (HBMC) were cultured withPC3 conditioned medium. We found that conditioned medium stimulatedHBMC differentiation into osteoclast-like cells. This effect was inhibited byMCP-1 neutralizing antibody. Third, effects of MCP-1 on PCa cellsproliferation and invasion were determined by MTS and invasion assays. Weshowed that MCP-1 induced proliferation and invasion of PCa cells and thiswas abolished partially either by CCR2 antagonist or PI3 Kinase inhibitor. As the cellular effects of MCP-1 are mediated by CC chemokinereceptor 2 (CCR2), we hypothesized that CCR2 may contribute PCaprogression. Accordingly, we determined CCR2 mRNA and proteinexpression in various cancer cell lines, including PCa and other cancer types.All cells expressed CCR2 mRNA and protein, but in PCa, more aggressivecancer cells such as C4-2B, DU145, and PC3 expressed a higher amount ofCCR2 compared with the less aggressive cancer cells such as LNCaP ornon-neoplastic PrEC and RWPE-1 cells. A positive correlation was observedbetween CCR2 expression and PCa progression by analyzing anONCOMINE gene array database. We confirmed that CCR2 mRNA washighly expressed in PCa metastatic tissues compared with the localized PCaor benign prostate tissues by realtime RT-PCR. CCR2 protein expression wasexamined by immunohistochemical staining on tissue microarray specimens.We found that CCR2 expression correlated with Gleason score and clinicalpathologic stages.To investigate the mechanism of PTHrP on PCa-induced bone lesionand the role of PTHrP in regulating MCP-1 expression in bonemicroenvironment, specifically by HBME and osteoblasts (hFOB), as well asby PCa cells, we determined the effect of PTHrP on MCP-1 expression bybone cells and PCa cells. PTHrP induced both MCP-1 protein and mRNAexpression by HBME and hFOB cells, but not by PCa LNCaP and PC3 cells.Finally, when a constitutively-active PTH receptor construct was transfectedinto HBME and hFOB cells, MCP-1 production was increased. The conditioned media collected from these cells induced osteoclast differentiationand PC3 proliferation and invasion in vitro. These inductions were partiallyinhibited by MCP-1 neutralizing antibody.In conclusion, MCP-1 acts as a paracrine and autocrine factor for CaPgrowth and invasion; MCP-1 mediates PCa metastasis-induced bone lesion;CCR2 may contribute to PCa development; and PTHrP-induced MCP-1production by HBME and hFOB cells promotes osteoclast differentiation andPCa cells proliferation and invasion in vitro. This study may provide a novelmolecular model of the mechanism of tumor skeletal metastasis. TargetingMCP-1/CCR2 axis may yield important outcomes which will impacttherapeutic targeting of prostate cancer skeletal metastasis.