Identifying Genes That Regulate Bladder Cancer Progression And Invasion

Transitional cell carcinoma (TCC) is the most common type of bladder cancer and can be categorized as either non-muscle invasive (Ta-T1) or muscle invasive (≥T2). Approximately 50% of bladder cancers are T1 at initial diagnosis; however, the recurrence rate for these tumors is high and they may progress into T2. In this study, we aimed to determine if there are specific gene expression differences between T1 vs. T2 bladder cancer that can help identify key regulators in bladder cancer progression and invasion. T1 and T2 bladder cancer tissues were subjected to RNA-Seq to evaluate for differences among these stages. Additionally, the Oncomine database was examined to further narrow down potential candidates that differentiate T1 from T2. These efforts led to the identification of an extracellular matrix glycoprotein, fibulin-3 (FBLN3), as being highly expressed in T2 compared to T1 tissues. To validate these findings, FBLN3 expression was measured using qRT-PCR from formalin fixed and paraffin embedded tissues from patient bladder samples ranging from stages Ta-T4. These studies confirmed that FBLN3 expression was elevated in muscle-invasive compared to non-muscle invasive bladder cancer. Consistent with these findings, FBLN3 expression level correlated with the invasive ability of several bladder cancer cell lines. Specifically, FBLN3 expression was determined using both qRT-PCR and western blotting amongst the T24, UMUC-13, UMUC-3, RT4, and 5637 bladder cancer cell lines. The most invasive cell lines, T24 and UMUC-13, demonstrated the highest FBLN3 expression. In contrast, the least invasive cells, RT4 and 5637, demonstrated the least FBLN3 expression. To determine a functional role for FBLN3 in bladder cancer invasion, we knocked down or increased FBLN3 expression in bladder cancer cell lines using lentiviral transduction. Knockdown of FBLN3 expression in the T24 and UMUC-13 cells inhibited the invasion and migration of these bladder cancer cells; whereas, FBLN3 overexpression in the 5637 cells promoted the invasiveness of the bladder cancer cells. Furthermore, cell viability and growth rates were not affected by manipulation of FBLN3 expression. Our results indicate that FBLN3 serves as a pro-invasive factor in bladder cancer. These findings suggest that FBLN3 could serve as (1) a biomarker to differentiate T1 from T2 bladder cancers and (2) a promising therapeutic target. Citation Format: Amy L. Han, Brendan Veeneman, Scott A. Tomlins, Evan T. Keller. Identifying genes that regulate bladder cancer progression and invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 692.