Tumor cells in the bone interact with the microenvironment to promote tumor cell survival and proliferation, resulting in a lethal phenotype for patients with advanced prostate cancer. Monocyte chemoattractant protein 1 (CCL2) is a member of the CC chemokine family and is known to promote monocyte chemotaxis to sites of inflammation. Here we have shown that human bone marrow endothelial (HBME) cells secrete significantly higher levels of CCL2 compared to human aortic endothelial cells and human dermal microvascular endothelial cells. Furthermore, we demonstrate that CCL2 is a potent chemoattractant of prostate cancer epithelial cells, and that stimulation of PC-3 and VCaP cells resulted in a dose-dependent activation of PI3 kinase/Akt signaling pathway. Activation of the PI3 kinase/Akt pathway was found to be vital to the proliferative effects of CCL2 stimulation of both PC-3 and VCaP cells. Additionally, CCL2 stimulated the phosphorylation of p70-S6 kinase (a downstream target of Akt) and induced actin rearrangement, resulting in a dynamic morphologic change indicative of microspike formation. These data suggest that bone marrow endothelial cells are a major source of CCL2, and that an elevated secretion of CCL2 recruits prostate cancer epithelial cells to the bone microenvironment and regulates their proliferation rate.

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Prostate epithelial cell growth is dependent on the presence of androgens and the transition of prostate cancer to an androgen independent phenotype results in a highly aggressive, currently incurable cancer. We have developed a new preclinical model of androgen independent prostate cancer derived from the VCaP prostate cancer epithelial cell line. VCaP cells were subcutaneously implanted and serially passaged in castrated male SCID mice. Androgen independence was confirmed by WST-1 (a tetrazolium salt) cell proliferation assay in the absence or presence of dihydrotesterone (DHT) (1 -100 nM). VCaP androgen sensitive (VAS) cells responded dose dependently to DHT whereas VCaP androgen independent (VAI) cells did not alter their proliferation in response to DHT. Gene expression of androgen receptor, Bcl-2, DD3, prostate acid phosphatase, STEAP, and survivin was determined by PCR amplification. Bcl-2 expression was upregulated in the VAI lines compared to the VAS suggesting a possible mechanism of androgen independence. Futhermore, tumorassociated -angiogenesis was assessed by immunofluorescence confocal microscopy of CD31. VAI tumors demonstrated enhanced angiogenesis compared to VAS tumors. These results demonstrate the development of a novel model of prostate cancer androgen independence and provide a new system to study angiogenesis and the transition to androgen independence.