Abstract B52: Targeting epigenetic dysregulation in a canine model of bladder cancer

Bladder cancer, which consists mainly of Transitional Cell Carcinoma (TCC), is the fifth most common cancer in the United States with over 70,000 cases and 15,000 deaths expected in 2013. Despite the successful development of molecularly targeted therapeutics for many cancers, there has been little change in the standard of care for TCC patients over the last few decades. New studies have revealed that aberrant epigenetic events, such as DNA hypermethylation and altered histone modifications, are common in TCC and are thought to promote tumorigenesis. Epigenetic therapies targeting these dysregulated pathways may reverse these effects and can have a profound influence on tumor growth. One such epigenetic modifier, EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2), is overexpressed in a variety of cancers and was found to enhance tumor progression by silencing several tumor suppressor genes. Recently, we have verified that EZH2 is upregulated in human TCC through an FGF2-NDY1/KDM2B-dependent suppression of miR-101. These data suggest that FGF2-NDY1/KDM2B-EZH2 pathway may be important in transitional cell carcinogenesis and may provide relevant epigenetic targets for the treatment of TCC. Spontaneous canine TCC also exhibits activation of FGF2 signaling and has been suggested as a model for bladder cancer. Therefore, we hypothesized that the development of aberrant epigenetic mechanisms downstream of FGF2 would be conserved between humans and dogs and that canine TCC could serve as a relevant large animal model for the clinical targeting of the FGF2-NDY1-EZH2 axis. To test our hypothesis, we employed canine TCC cell lines that we developed from dogs of various breeds and found that both EZH2 and NDY1/KDM2B are upregulated in canine TCC when compared to normal tissue. In order to target the pathway, we inhibited EZH2 both pharmacologically, using the commercially available inhibitor DZNep (3-deazaneplanocin), and through targeted knockdown. We determined that both DZNep and shRNA knockdown of EZH2 inhibited proliferation and induced apoptosis in canine TCC cells in anchorage-dependent (2D) and anchorage-independent (3D) cultures. By conducting these experiments in parallel with human TCC cell lines, we showed that sensitivity to DZNep is comparable between human and canine TCC cell lines. Western blot analysis and qRT-PCR demonstrated that, upon DZNep treatment, there is reduction of EZH2 at the protein level and inhibition of its activity, as evidenced by decreased global trimethylation of Histone H3 at lysine 27 (H3K27me3). Furthermore, inhibition of EZH2 was associated with upregulation of a set of genes that are commonly silenced by H3K27me3. Human and canine bladder cancer share histopathologic characteristics and clinical behavior. With this study, we confirm that the molecular underpinnings of spontaneous TCC are also shared between dogs and humans. Therefore canine TCC provides a means to understand epigenetic dysregulation in the urothelium as well as to investigate its clinical targeting. Citation Format: Parthena Foltopoulou, Monica Betancur-Boissel, Philip N. Tsichlis, Elizabeth A. McNiel. Targeting epigenetic dysregulation in a canine model of bladder cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B52.