Abstract 5002: Functional significance and mechanisms of inactivation of Secreted Wnt antagonist DICKKOPF-3 (DKK-3) gene in human renal cell carcinoma

Abstract

Abstract Introduction: The Wnt signaling pathway is activated in most cancers while Wnt antagonist genes are inactivated. It has been reported that mRNA expression of Wnt antagonists is regulated by promoter methylation in many cancers. However the functional significance and mechanisms of inactivation of Secreted Wnt antagonist DKK-3 gene in kidney cancer has not been reported. Methods : DKK-1, DKK-2 and DKK-3 mRNA expression levels were detected by using real-time PCR. RCC cells were incubated with 5-aza-2′-deoxycytidine or with TSA. The mRNA expression of apoptosis and cell cycle genes analyzed by real-time PCR 72 hr after transfection. Chromatin DNA was immunoprecipitated with antibodies for histone H3 (H3), and tri-methyl-histone H3 (Lys4) (H3K4). RCC cell lines were transiently cotransfected with either mock or DKK-3, TOPflash reporter plasmid, and pRL-TK plasmid encoding Renilla luciferase as an internal control for transfection efficiency. Cell lysates were measured for relative luciferase activities. Cell viability was analyzed by the MTS assay after transfection. Stable transfectants were used for cell cycle and apoptosis analysis using a Cell Lab Quanta SC Flow Cytometer. Mock and DKK-3 stable cells were injected subcutaneously into the left and right back side flanks of nu/nu mice, respectively. Results : DKK-3 mRNA expression in Caki-2, A498 and 769-P RCC cells was lower than that of normal kidney HK-2 cells. The DKK-3 promoter was methylated in RCC cells, but unmethyalted in normal kidney HK-2 cells. Expression of DKK-1 and DKK-2 mRNA was upregulated after treating RCC cells with 5-Aza-2′-deoxycytidine, but DKK-3 mRNA levels were not affected. In contrast, the level of DKK-3 mRNA was upregulated after TSA treatment, whereas these of DKK-1 and DKK-2 were not. The level of H3K4 in A-498 and 769-P cells was higher compared with HK-2 cells after TSA treatment, while that of H3 in A-498 and 769-P cells were not changed. Expression of cell cycle and apoptosis related genes showed that p21, MDM-2, Puma and Bik genes were increased after transfecting RCC cell lines with a DKK-3 expression plasmid. DKK-3 did not inhibit the Tcf-reporter transcriptional activity in RCC cells, but decreased the cell viability of A-498 and 769-P cells. DKK-3 stable transfectants increased the rate of apoptosis and induced G2/M arrest together with an increase in p21 expression. Growth of stable DKK-3 transfected cells in nude mice was decreased compared to controls. Conclusions : Our data suggests for the first time that mRNA expression of DKK-3 is regulated by histone modification and that DKK-3 inhibits cell growth and induces apoptosis in RCC cell. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5002.