Abstract 4466: GULP1 is a potential tumor suppressor that modulates chemo-resistance through regulation of Nrf2-Keap1 signaling axis in urothelial carcinoma
Authors: Guida Elisa , Hayashi Masamichi , Baras Alexander , Maldonado Leonel , Brait Mariana , Reis Leonardo , oki akira , Izumchenko Evgeny , bivalaqua trinity , Netto george J. , Koch Wayne , Sidransky David , Hoque mohammad O. 
Published: July 2016

By an integrated genomic approach, we recently identified engulfment adaptor PTB-domain-containing 1 (GULP1) as a potential tumor suppressor gene (TSG), selectively silenced in different types of solid tumors by promoter methylation (PM). Here, we aimed to test GULP1 PM in urothelial carcinoma (UC) and to functionally characterize GULP1 during UC initiation, progression and acquisition of chemo-resistance.

Initially, using a panel of cell lines, we observed that silencing of GULP1 expression is mediated by PM in UC cell lines. Furthermore, to test if this phenomenon is clinically relevant, we first employed quantitative methylation specific PCR on 20 primary tumors and matched normal samples. We observed higher frequency of GULP1 PM in tumors (35.0%) than in matched normal tissues (10.0%). Secondly, additional 76 tumors of different stages and grades of UC were tested and the methylation frequency were similar in UCs (27.1%) and significantly higher than in normal urothelial tissues (9.5%). Interestingly, UC cases with GULP1 PM had a tendency of lymphatic/venous invasion (P = 0.066) when compared with cases without GULP1 PM. Moreover, to explore the correlation of GULP1 expression with different clinicopathological parameters, we performed immunohistochemical analyses on 2 different UC Tissue-Micro-Arrays. Overall GULP1 silencing was observed in 85% of UC cases and silencing is significantly more in muscle invasive UC (MIUCs) than in non-muscle invasive UC (NMIUCs) (P&lt;0.001, Fisher's exact test).

Numerous cell based assays revealed that GULP1 silencing confers growth advantage to tumor cells. Further mechanistic analysis revealed that GULP1 has a crucial role in the regulation of Nrf2-KEAP1 axis, maintaining actin cytoskeleton architecture and assisting KEAP1 to prevent Nrf2 nuclear translocation. By using RT2 pathway expression array analysis of ShGULP1 T24 cells and T24 cells, we discovered that GULP1 silencing induces activation of Nrf2 targets, such as heme oxygenase 1 (HMOX1). We further confirmed the inverse correlation between GULP1 and HMOX1 expression in numerous UC cell lines by forcefully modulating GULP1 expression. Since constitutive activation of Nrf2 signature has been defined as responsible for chemoresistance, we analyzed GULP1 PM and expression in cisplatin based therapy responsive and resistant primary UC samples; and in isogenic cisplatin sensitive and resistant T24 cell lines. Interestingly, GULP1 expression is lower both in resistant primary UC samples and in resistant T24 cell line.

Altogether, our findings determined that GULP1 is an epigenetically silenced potential TSG in UC and its inactivation lead to malignant progression by constitutive activation of NRF2 pathway in UC carcinogenesis. Furthermore, GULP1 expression and/or PM may guide in selecting candidate patients for cisplatin based neo-adjuvant therapy.

Citation Format: Elisa Guida, Masamichi Hayashi, Alexander Baras, Leonel Maldonado, Mariana Brait, Leonardo Reis, akira oki, Evgeny Izumchenko, trinity bivalaqua, george J. Netto, Wayne Koch, David Sidransky, mohammad O. Hoque. GULP1 is a potential tumor suppressor that modulates chemo-resistance through regulation of Nrf2-Keap1 signaling axis in urothelial carcinoma. [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 4466.