Androgen receptor (AR) and estrogen receptor-β (ERβ) have been implicated in urothelial tumor outgrowth as promoters, while underlying mechanisms remain poorly understood. Our transcription factor profiling previously performed identified FOXO1 as a potential downstream target of AR in bladder cancer cells. We here investigated the functional role of FOXO1 in the development and progression of urothelial cancer in relation to AR and ERβ signals. In non-neoplastic urothelial SVHUC cells or bladder cancer lines, AR/ERβ expression or dihydrotestosterone/estradiol treatment reduced the expression levels of FOXO1 gene and induced those of a phosphorylated inactive form of FOXO1 (p-FOXO1). In chemical carcinogen-induced models, FOXO1 knockdown via shRNA or inhibitor treatment resulted in considerable induction of the neoplastic transformation of urothelial cells or bladder cancer development in mice. Similarly, FOXO1 inhibition considerably induced the viability, migration, and invasion of bladder cancer cells. Importantly, in FOXO1 knockdown sublines, an anti-androgen hydroxyflutamide or an anti-estrogen tamoxifen did not significantly inhibit the neoplastic transformation of urothelial cells, while dihydrotestosterone or estradiol did not significantly promote the proliferation or migration of urothelial cancer cells. In addition, immunohistochemistry in surgical specimens showed that FOXO1 and p-FOXO1 expression was down-regulated and up-regulated, respectively, in bladder tumor tissues, which was further associated with worse patient outcomes. AR or ERβ activation is thus found to correlate with inactivation of FOXO1 which appears to be their key downstream effector. Moreover, FOXO1, as a tumor suppressor, is likely inactivated in bladder cancer, which contributes in turn to inducing urothelial carcinogenesis and cancer growth.
Supplementary Figure S1. The profiling assay in UMUC3 cells treated with ethanol (mock) or 1 nM R1881 for 24 hours. There are a total of 14 transcription factors whose activity is down-regulated by androgen treatment.
Supplementary Figure S2. Western blot of ERβ in 647V, SVHUC, UMUC3, and 5637, as well as a prostate cancer line LNCaP which is known to be ERβ-negative (A), and ERα in SVHUC, UMUC3, 5637, and 647V (B). GAPDH served as a loading control.
Supplementary Figure S3. Effects of androgen or estrogen on the expression of FOXO1 gene in urothelial cells. Quantitative real-time RT-PCR of FOXO1 in AR-positive or AR-negative cells treated with 0-10 nM DHT (A) and ERβ-positive or ERβ-negative cells treated with 0-100 nM E2 (B) for 24 hours. The expression of FOXO1 gene was normalized to that of GAPDH, and transcription amount is presented relative to that of mock treatment. Each value represents the mean (+SD) from 3 independent experiments. *P<0.05 (vs. mock treatment).
Supplementary Figure S4. Western blot of p-FOXO1 in SVHUC-AR (A) or UMUC3 (B) treated with 10 nM DHT or 10 nM E2 for 0-24 hours, as indicated. GAPDH served as a loading control.
Supplementary Figure S5. AR or ERβ binding to the FOXO1 promoter in urothelial cells. (A) Sequence of FOXO1 promoter. Online search (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) identified 6 and 3 putative binding sites of AR and ERβ, respectively, in the 2 kb FOXO1 promoter region. (B) ChIP assay using cell lysates of UMUC3 immunoprecipitated with an anti-ERβ antibody or mouse IgG as a negative control. The DNA fragments were PCR amplified with a FOXO1 promoter-specific primer set, and PCR products (780 bp) were electrophoresed on 1% agarose gel.
Supplementary Figure S6. FOXO1 silencing in urothelial cell lines. Western blot and quantitative RT-PCR of FOXO1 in SVHUC-control-shRNA/SVHUC-FOXO1-shRNA, UMUC3-control-shRNA/UMUC3-FOXO1-shRNA, 5637-AR-control-shRNA/5637-AR-FOXO1-shRNA, and 647V-AR-control-shRNA/647V-AR-FOXO1-shRNA. In western blot, GAPDH served as a loading control. In RT-PCR analysis, the expression of FOXO1 was normalized to that of GAPDH, and transcription amount is presented relative to that of each control line [each value representing the mean (+SD) from 3 independent experiments; *P<0.05 (vs. control-shRNA cells)].
Supplementary Table S1. List of antibodies.
Supplementary Table S2. Sequences of primers for real-time PCR.
Supplementary Table S3. The relative ratio of phosphorylated to total FOXO1 expression.