Abstract 4066: Loss of Sh3gl2/endophilin A1 is an early event in urothelial carcinoma that regulates malignant behavior.

Abstract

Abstract Urothelial carcinoma (UC) causes substantial morbidity and mortality worldwide. However, the molecular mechanisms underlying urothelial cancer development and tumor progression are still largely unknown. Using informatics analysis we identified Sh3gl2 (endophilin A1) as a transcript and protein highly enriched in bladder epithelium (urothelium). The gene encoding Sh3gl2 is located on chromosome 9p, a genomic region frequently altered in UC. Sh3gl2 is known to regulate endocytosis of receptor tyrosine kinases implicated in oncogenesis, such as the epidermal growth factor receptor (EGFR) and c-Met. However, its role in UC pathogenesis is unknown. Informatics analysis of expression profiles in Oncomine™, as well as immunohistochemical staining of tissue microarrays revealed Sh3gl2 expression to be decreased in UC specimens compared to non-tumor tissues. Loss of Sh3gl2 was associated with increasing tumor grade and with muscle invasion, a reliable predictor of metastatic disease and cancer-derived mortality. Sh3gl2 expression was undetectable in 15 of 16 human UC cell lines, but preserved in the low grade, well- differentiated cell line RT4. In UC cell lines lacking expression of Sh3gl2, treatment with the demethylating agent 5-aza-deoxycytidine led to upregulation of Sh3gl2 mRNA levels. Methylation-specific restriction analysis and PCR of genomic DNA revealed hypermethylation of a CpG island in the Sh3gl2 promoter in cells lacking Sh3gl2 expression, but no promoter methylation in RT4 cells that express Sh3gl2, suggesting that lack of Sh3gl2 expression results, at least in part, from methylation-induced gene silencing. Stable knockdown of Sh3gl2 in RT4 cells by RNA interference (i) enhanced proliferation and colony formation in vitro; (ii) inhibited EGF-induced EGFR internalization and increased EGFR phosphorylation; (iii) stimulated phosphorylation of Src family kinases and STAT3; (iv) altered sensitivity to chemotherapeutic agents, including the dual-specificity EGFR/ERBB2 inhibitor lapatinib; and (v) promoted formation of RT4 xenografts in subrenal capsule tissue recombination experiments. Together, these findings identify loss of Sh3gl2 as an early event in UC development that promotes disease progression. Sh3gl2 status in UC biopsies may provide a potential marker of risk for UC progression or recurrence and may enable determination of which patients require more aggressive intervention in the form of surgery or chemotherapy in order to improve prognosis. Citation Format: Shyama Majumdar, Edward Gong, Dolores Di Vizio, Jonathan Dreyfuss, David DeGraff, Martin Hager, Peter Park, Joaquim Bellmunt, Robert Matusik, Jonathan Rosenberg, Rosalyn Adam. Loss of Sh3gl2/endophilin A1 is an early event in urothelial carcinoma that regulates malignant behavior. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4066. doi:10.1158/1538-7445.AM2013-4066