Abstract 2071: Identification of FOXO1 as a tumor suppressor modulated by androgen receptor/estrogen receptor-βsignals: Implications for bladder cancer promotion and chemoresistance
Authors: Ide Hiroki , Inoue Satoshi , Mizushima Taichi , Jiang Guiyang , Goto Takuro , Nagata Yujiro , Teramoto Yuki , Netto George J. , Oya Mototsugu , Miyamoto Hiroshi 
Published: September 2020

Introduction and Objective: The androgen receptor (AR) and estrogen receptor-β (ERβ) pathways have been implicated in urothelial tumor outgrowth, as well as chemoresistance, for which underlying mechanisms are poorly understood. Therefore, the present study aims to identify downstream effectors of AR and ERβ signals and further investigate their role in bladder cancer promotion and chemosensitivity.

Methods: We first performed a transcription factor (TF) profiling assay in an AR/ERβ-positive and ERα-negative bladder cancer line, UMUC3. We then performed in vitro and in vivo assays, using cell line and mouse models, as well as immunohistochemical staining in two sets of bladder cancer tissue microarray.

Results: The TF array data in UMUC3 cells treated with mock versus synthetic androgen R1881 displayed that 14 out of 96 were down-regulated. Specifically, the expression levels of FOXC1, STAT4, and FOXO1 were reduced by &gt;60%. Subsequent RT-PCR showed that dihydrotestosterone and estradiol treatment significantly down-regulated the expression of FOXO1 (58% and 28% decreases), but not that of STAT4 (22% and 2% decreases) or FOXC1 (10% and 2% decreases), respectively. Of note, our online search identified 6 and 3 putative binding sites of AR and ERβ, respectively, in the FOXO1 promoter region. In chemical carcinogen-induced models, FOXO1 knockdown via shRNA virus infection or inhibitor treatment resulted in considerable induction of the neoplastic transformation of urothelial cells or bladder cancer development in mice. Similarly, FOXO1 inhibition significantly induced the viability/migration/invasion of bladder cancer cells and reduced the cytotoxic effects of cisplatin. Finally, immunohistochemistry in surgical specimens showed down-regulation and up-regulation of the expression of FOXO1 and its phosphorylated inactive form (p-FOXO1), respectively, in bladder cancer tissues, which was further associated with patient outcomes. In a separate tissue microarray consisting of muscle-invasive bladder cancer specimens from patients who received cisplatin-based neoadjuvant chemotherapy, p-FOXO1 expression tended to correlate with chemoresistance (i.e. 41% of responders vs. 69% of non-responders; P=0.068).

Conclusions: FOXO1 is found to be a potential tumor suppressor modulated by both AR and ERβ signals. FOXO1 signaling may thus represent a novel chemopreventive and/or therapeutic target for bladder cancer. Its inactivation may also be useful for overcoming chemoresistance.

Citation Format: Hiroki Ide, Satoshi Inoue, Taichi Mizushima, Guiyang Jiang, Takuro Goto, Yujiro Nagata, Yuki Teramoto, George J. Netto, Mototsugu Oya, Hiroshi Miyamoto. Identification of FOXO1 as a tumor suppressor modulated by androgen receptor/estrogen receptor-βsignals: Implications for bladder cancer promotion and chemoresistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2071.