Abstract 3862: Functional characterization of sFRP1 expression loss in human papillary bladder cancer

Abstract

Abstract Introduction: Secreted frizzled-related protein 1 (sFRP1) is an inhibitor of the Wnt-signaling pathway and expression loss is associated with e.g. colon and breast cancer. Our previous studies revealed an involvement of sFRP1 loss in progression of papillary bladder cancer. We therefore performed functional studies to characterise the role of sFRP1 in papillary bladder cancer cell lines. Material and methods: Human papillary bladder cancer cell lines RT112 and BFTC 905 were used for the analyses. mRNA-expression status of sfrp1 and Wnt-target genes c-myc and cyclin D1 was determined using qRT-PCR. Methylation status of sfrp1 promoter was analysed with MS-PCR. sFRP1 knockdown was performed using sFRP1-specific siRNAs. 5’ Aza-2’deoxycytidine (5’Aza) treatment was used for promoter demethylation and restoration of sFRP1 expression. To determine Wnt pathway activity a luminescence-based TOP flash/FOP flash assay was used. Proliferation level was investigated with ELISA-based BrdU Cell Proliferation assay. Results: RT112 is strongly expressing sfrp1 on mRNA level, whereas BFTC905 showed no sfrp1 expression. MS-PCR revealed sfrp1-promoter methylation in BFTC905. This methylation could be reversed after 5’-Aza-treatment and led to re-expression of sfrp1 on mRNA level. sfrp1 siRNA transfection in RT112 led to ∼80% gene silencing. sfrp1 knockdown resulted in an upregulation of c-myc (515%) and cyclin D1 (51,5%) on mRNA level, however TCF/LEF-presence could not be detected, neither in untreated nor in siRNA transfected RT112 cells. Untreated BFTC905 cells showed a very weak Wnt signalling activitiy, compared to positive control cell line SW480, but 7-fold higher than in untreated and siRNA treated RT112. sfrp1 knowdown in RT112 had no influence on cell proliferation. Conclusion: sfrp1 knockdown in RT112 cells is associated with an up-regulation of proliferation-associated genes c-myc and cyclin D1, whereas this increased expression showed no effects on proliferation behaviour of the cells. Knockdown of sFRP1 did not result in significant Wnt-signaling activity in RT112 cells. Reduced sFRP1 expression might increase the proliferative potential of the cells; however additional genetic events might have to occur to induce increased proliferation of the cells. Ongoing analyses of BFTC905 with transient restoration of sFRP1 expression will give further insights into the role of sFRP1 in papillary bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3862. doi:10.1158/1538-7445.AM2011-3862