BACKGROUND: Renal cell carcinoma (RCC) is highly resistant to chemotherapy because of a high apoptotic threshold. Recent evidences suggest that GSK-3b positively regulates human pancreatic cancer and leukaemia cell survival in part through regulation of nuclear factor (NF-kB)-mediated expression of anti-apoptotic molecules. Our objectives were to determine the expression pattern of GSK-3b and to assess the anti-cancer effect of GSK-3b inhibition in RCC. METHODS: Immunohistochemistry and nuclear/cytosolic fractionation were performed to determine the expression pattern of GSK-3b in human RCCs. We used small molecule inhibitor, RNA interference, western blotting, quantitative RT -PCR, BrDU incorporation and MTS assays to study the effect of GSK-3b inactivation on renal cancer cell proliferation and survival. RESULTS: We detected aberrant nuclear accumulation of GSK-3b in RCC cell lines and in 68 out of 74 (91.89%) human RCCs. We found that pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of renal cancer cells. We observed that inhibition of GSK-3 results in decreased expression of NF-kB target genes Bcl-2 and XIAP and a subsequent increase in renal cancer cell apoptosis. Moreover, we show that GSK-3 inhibitor and Docetaxel synergistically suppress proliferation and survival of renal cancer cells. CONCLUSIONS: Our results show nuclear accumulation of GSK-3b as a new marker of human RCC, identify that GSK-3 positively regulates RCC cell survival and proliferation and suggest inhibition of GSK-3 as a new promising approach in the treatment of human renal cancer.

Purpose: Although recent studies have shown glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase, as a positive regulator of pancreatic, colon, and kidney cancer cell survival and proliferation, the role of GSK-3 in bladder cancer remains unknown. Our objectives were to determine the subcellular localization of GSK-3β and to evaluate the effect of GSK-3 inhibition in bladder cancer.Experimental Design: We used immunohistochemical staining and nuclear/cytosolic fractionation to determine the expression pattern of GSK-3β in human urothelial carcinomas. To study the effect of GSK-3 inhibition on bladder cancer cell proliferation and survival, we used pharmacologic inhibitors of GSK-3, RNA interference, MTS assay, bromodeoxyuridine incorporation assay, quantitative reverse transcriptase-PCR, and Western blotting.Results: We found aberrant nuclear accumulation of GSK-3β in 62% (43 of 69) and 91% (21 of 23) of noninvasive and invasive human urothelial carcinomas, respectively. GSK-3β nuclear staining was significantly associated with high-grade tumors (P < 0.001), advanced stage of bladder cancer (P < 0.05), metastasis (P < 0.05), and worse cause-specific survival (P < 0.05) in bladder cancer patients. Moreover, we found that pharmacologic inhibition or genetic depletion of GSK-3β resulted in decreased viability of bladder cancer cells.Conclusions: Our results suggest nuclear accumulation of GSK-3β as a novel prognostic marker in bladder cancer, show that GSK-3 contributes to urothelial cancer cell proliferation and survival, and identify GSK-3 as a potential therapeutic target in human bladder cancer.

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Renal cell carcinoma (RCC) is known to be resistant to chemo-and radiotherapy due to a high apoptotic threshold. Smac and XIAP (X-linked inhibitor of apoptosis protein) proteins were detected in all RCC cell lines and tissue samples examined. We modulated the function of XIAP, either through its constitutional downregulation with an shRNA vector or by applying a Smac-mimicking peptide. Among RCC cell lines, Caki1 expresses the highest levels of XIAP. We transfected Caki1 with XIAP-targeting shRNA vector and generated stable clones. XIAP was knocked down by RNA interference in clone no. 14 by 81.6% and in clone no. 19 by 85.3%. Compared to the parental and mock-transfected cells, neither clone was more sensitive to conventional chemotherapeutic agents, but both clones were more susceptible to Fas stimulation (Po0.0001) and to pharmacological Bcl-2 inhibition (Po0.0001), as well as to a combination of the two (Po0.0001). Mature Smac binds to XIAP via the N-terminal residues, disrupting its interaction with caspases and promoting their activity. We determined that exposure of Caki1 cells to Smac-N7 peptide (AVPIAQK) resulted in a slight but significant decrease in viability (P ¼ 0.0031) and potentiated cisplatin's effect (P ¼ 0.0027). In contrast with point targeting of XIAP by shRNA, Smac-N7 peptide is active against several IAP (inhibitor of apoptosis protein) family members, which can explain its role in sensitising cells to cisplatin. Our results suggest that multiple targeting of both Bcl-2 and XIAP or, alternatively, of several IAP family members by the Smac-N7 peptide is a potent way to overcome resistance of RCC to apoptosis-triggering treatment modalities, and might be a new tool for molecular targeted therapy.

We investigated a prognostic significance and the mechanism of aberrant nuclear expression of EZH2, a histone methyltransferase, in human renal cell carcinoma (RCC). We found nuclear EZH2 in 48 of 100 RCCs and it was significantly correlated with worse survival in RCC patients. We detected a decreased expression of miR-101 in 15 of 54 RCCs. We found that re-expression of miR-101 resulted in EZH2 depletion and decreased renal cancer cell proliferation. Our results show nuclear EZH2 as a prognostic marker of worse survival in human RCC, and identify miR-101 as a negative regulator of EZH2 expression and renal cancer cell proliferation.

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X-linked inhibitor of apoptosis (XIAP) is a suppressor of apoptosis that supports an increased survival and resistance to chemotherapy of human prostate cancer (PCa) cells. Effects of transient (24 h) and chronic (beyond 1 month) downregulation of XIAP in DU145 hormone refractory prostate cancer (HRPC) cells were studied. We found that transient downregulation of XIAP by siRNAs resulted in an increase of apoptosis and a decrease in Bcl-2 levels and sensitized PCa cells to cisplatin. XIAP downregulation by shRNA vector stable transfection led to upregulation of Bcl-2 protein. Our results identify the adaptability of PCa cells to chronic loss of XIAP in part through upregulation of Bcl-2 and indicate that multitargeting approach is the most effective application in the chemotherapy of human HRPC.

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