Modulation of paracrine interactions between prostate cancer cells and bone marrow stromal cells by transforming growth factor-beta signaling during colonization of bone

Sikes, Robert A.Prostate cancer (PCa) is the most prevalent cancer and the second leading cause of cancer related death for men in the United States. Death is primarily due to bone metastasis with more than 80% of men who die of PCa having bone involvement at autopsy. The complex bone microenvironment may initially resist the newly resident PCa cells but PCa cells acquire adaptive changes that allow them to survive and grow in the "hostile" new microenvironment as they co-evolve in their genotypic and phenotypic characters with bone cells. We have previously shown that soluble factors released from immortalized human bone marrow stromal cells (BMSC) induce apoptosis of PCa cells, and the surviving cells undergo neuroendocrine differentiation (NED), characterized by morphological changes consistent with a neuroendocrine phenotype. The presence of neuroendocrine tumor cells in PCa is associated with aggressiveness, resistance to hormonal therapy, and poor prognosis. Using the LNCaP progression model of increasingly metastatic and castrate-resistant prostate cancer (CRPC) cell lines, I examined the influence of BMSC factors on PCa survival, using the HS-5 and HS-27a BMSC lines, which were characterized previously for their ability to support different stages of hematopoiesis. Neuroendocrine markers were elevated in PCa cells surviving HS-5 BMSC conditioned medium (CM) treatment, while differentiation markers such as androgen receptor (AR) and prostate-specific antigen (PSA) were decreased. PCa cells that undergo NED using HS-5 BMSC CM or serum withdrawal had elevated phosphorylated-Smad2 levels. Furthermore, NED of PCa cells prevented HS-5 BMSC-induced apoptosis. Cell death induced by BMSC CM was analyzed using live/dead analysis while the effect on cell growth was examined in soft agarose colony formation assays in the presence and absence of intact TGF-Beta signaling. Using immunoblotting and luciferase reporter assays, I measured levels and activity of phosphorylated-Smad2 in PCa cells surviving treatment with HS-5 BMSC CM. Treatment of PCa cells with the ALK-4, 5, and 7 kinase inhibitor, SB-431542, resulted in a significant reduction in HS-5-mediated cell death. Correspondingly, pre-treatment of HS-5 BMSC with TGF-Beta1 yielded a CM that elicited a marked reduction in PCa cell death. The ancillary TGF-Beta receptor endoglin levels were also decreased upon TGF-Beta1 stimulation of HS-5 cells suggesting the importance of endoglin in mediating BMSC-induced PCa cell death. Small interfering RNA-mediated knockdown of endoglin in HS-5 cells verified that the effect on PCa cell death was a direct result of the attenuation of endoglin. Futhermore, the loss of the cytoplasmic domain of endoglin in HS-5 cells attenuated BMSC-induced PCa cell death indicating the importance of the cytoplasmic domain in maintaining endoglin function and expression of the factor(s) responsible for PCa cell death. Collectively, my findings indicate that 1) TGF-Beta signaling in PCa cells is induced during stimulation of NED, 2) TGF-Beta family cytokines secreted from HS-5 BMSC mediate PCa cell death, 3) TGF-Beta signaling in HS-5 BMSC alters paracrine signaling to promote PCa survival, 4) Endoglin is required for HS-5 BMSC-induced PCa cell death, 5) The cytoplasmic domain of endoglin is required for the expression of the factor(s) responsible for PCa cell death.University of Delaware, Department of Biological SciencesPh.D