Abstract 1510: The sonic hedgehog pathway as a therapeutic target in bladder cancer

Abstract

Abstract Introduction: The sonic hedgehog (SHH) signaling pathway regulates embryonic developmental processes such as pattern formation, differentiation, proliferation, and organogenesis and has been hypothesized to play an integral role in the maintenance and progression of bladder cancer. Blocking this pathway may be an efficacious molecular target against high-risk bladder cancer. Methods: SHH pathway protein expression was assessed in an in-house human bladder cancer tissue array comprising superficial, invasive and lymph node metastasized transitional cell carcinoma. In addition, a panel of bladder cancer cell lines was assessed for SHH pathway activation and two lines, UM-UC3 and 253J-BV, were further evaluated for SHH pathway function and responsiveness. Messenger RNA and protein expression, proliferation, apoptosis and cell invasion were measured in response to the addition of SHH ligand, a SHH overexpressing construct, ligand and receptor blockers, small-molecule pathway inhibitors, and antisense oligonucleotides (ASO) targeted to the mRNA sequence of the down-stream transcription factors Gli family zinc fingers 1 and 2 (Gli1, Gli2). In-vitro ASO results were confirmed in an in-vivo experiment on UM-UC3 cells intravesically instilled into the bladder of nude mice. Results: Bladder tissues show a positive and variable pattern of SHH pathway expression within and between tumors. The bladder cancer cell lines UM-UC3 and 253J-BV both express the main components of the SHH pathway, notably the transcription factors GLi1 and Gli2. The most effective inhibitors of UM-UC3 are an indirect ciliagenesis inhibitor and direct Gli knock-down through ASO treatment. Both cell lines show decreased growth and increased apoptosis with the smoothened protein inhibitor cyclopamine but 253J-BV does not respond in proliferation or apoptosis to Gli1 ASO suggesting a difference in pathway function between the lines. UM-UC3 secretes approximately 10 fold higher amounts of SHH than 253J-BV but does not respond to exogenous SHH treatment. 253J-BV does respond with an increase in proliferation, invasion, and SHH and Forkhead Box M1 expression. Robotnikinin and the SHH antibody 5E1 show a modest decrease in cell viability at high concentrations in both lines. The addition of SHH through plasmid transfection has no effect. In-vivo, Gli2 ASO significantly decreases UM-UC3 tumour growth. Conclusions: Bladder cancer cells are differentially sensitive to specific regions of SHH pathway modulation, as seen by a greater reliance on GLi2 in 253J-BV. Direct knock-down of the down-stream transcription factors Gli1 and Gli2 with ASOs is the most effective strategy and may be a promising treatment for bladder cancer. Citation Format: Peter A. Raven, Summer Lysakowski, Yoshiyuki Matsui, Shintaro Narita, Alan I. So. The sonic hedgehog pathway as a therapeutic target in bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1510. doi:10.1158/1538-7445.AM2015-1510