Inhibition of Glycogen Synthase Kinase-3 Increases the Cytotoxicity of Enzastaurin

Cutaneous T-cell lymphomas (CTCL) represent a spectrum of several distinct non-Hodgkin's lymphomas that are characterized by an invasion of the skin by malignant, clonal lymphocytes. Our laboratory has previously demonstrated that the protein kinase C (PKC) β inhibitor Enzastaurin increases apoptosis in malignant lymphocytes of CTCL. These results directly led to a clinical trial for Enzastaurin in CTCL in which it was well tolerated and showed modest activity. To ascertain a means of improving the efficacy of Enzastaurin, we investigated complementary signaling pathways and identified glycogen synthase kinase-3 (GSK3) as important in survival signaling in CTCL. Enzastaurin combined with GSK3 inhibitors demonstrated an enhancement of cytotoxicity. Treatment with a combination of Enzastaurin and the GSK3 inhibitor AR-A014418 resulted in upregulation of β-catenin total protein and β-catenin-mediated transcription. Inhibition of β-catenin-mediated transcription or small hairpin RNA (shRNA) knockdown of β-catenin decreased the cytotoxic effects of Enzastaurin plus AR-A014418. In addition, treatment with Enzastaurin and AR-A014418 decreased the mRNA levels and surface expression of CD44. shRNA knockdown of β-catenin also restored CD44 surface expression. Our observations provide a rationale for the combined targeting of PKC and GSK3 signaling pathways in CTCL to enhance the therapeutic outcome

Prostate cancer cells regulate growth and differentiation of bone marrow endothelial cells through TGFΒ and its receptor, TGFΒRII

BACKGROUND The underlying mechanisms permitting prostate cancer bone metastasis are poorly understood. We previously showed that the highly metastatic prostate cancer cell line, PC-3, inhibits bone marrow endothelial (HBME-1) cell growth in collagen gels and induces them to differentiate into cords, resembling angiogenesis in vivo. METHODS cDNA microarray analysis was performed to identify cytokines responsible for the effects of PC-3 cells on HBME-1 cells. Cytokine and neutralizing antibody studies were done to further investigate specific angiogenic factors, such as transforming growth factor Β (TGFΒ). TGFΒ RNA and protein were detected by real-time RT-PCR and enzyme-linked immunosorbent assay (ELISA) analysis to measure their production by prostate cancer cell lines. Conditioned media experiments using TGFΒ neutralizing antibodies were used to analyze TGFΒ activation by prostate cancer cells. RESULTS PC-3 conditioned media altered the expression of several TGFΒ-regulated or -associated genes in HBME-1 cells. Low concentrations of TGFΒ cytokines inhibited HBME-1 cell growth to a similar level as PC-3 conditioned media and partially induced differentiation. Inhibitors and neutralizing antibodies directed against TGFΒ isoforms and TGFΒ receptor type 2 (TGFΒRII) reversed the growth inhibition of HBME-1 cells conferred by PC-3 conditioned media. Yet, only TGFΒRII neutralizing antibodies significantly inhibited HBME-1 differentiation. Also, prostate cancer cell lines produced low levels of TGFΒ RNA and protein, and were shown to activate serum-derived TGFΒ. CONCLUSIONS These results suggest that prostate cancer cells mediate growth inhibition and differentiation of bone marrow endothelial cells both through production and activation of TGFΒ as well as alteration of TGFΒRII-mediated signal transduction. This could contribute to the establishment and growth of bone metastases. Prostate 66:632–650, 2006. © 2005 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50648/1/20370_ftp.pd