Introduction

Recent preclinical evidence suggests the involvement of ER signaling in urothelial tumorigenesis, although its underlying mechanisms remain unclear. Meanwhile, cross-talk between FOXO1, a transcriptional factor known to induce apoptosis through the PI3K-Akt pathway, and ERβ has been demonstrated in prostate cancer cells. We therefore investigated the role of FOXO1 in neoplastic transformation of urothelial cells in relation to ERβ signaling.

Methods

We immunohistochemically stained for ERβ and phospho-FOXO1 (p-FOXO1), an inactive form of FOXO1, in the tissue microarrays consisting of 99 cases of upper urinary tract urothelial carcinoma and paired non-neoplastic urothelium. Then, in ERα(-)/ERβ(+) human normal urothelial SVHUC sublines stably expressing control- or FOXO1-shRNA with or without exposure to a chemical carcinogen 3-methylcholanthrene (MCA), we assessed the expression of FOXO1 and other known tumor suppressors (via RT-PCR and western blot) as well as neoplastic transformation (via MTT assay and mouse xenograft model).

Results

ERβ and p-FOXO1 were positive in 63% and 100% of urothelial tumors, which were significantly lower and higher than in non-neoplastic urothelial tissues [85% (P=0.001) and 94% (P=0.018)], respectively. In addition, there was a significant correlation between strong (3+) p-FOXO1 expression and ERβ positivity (P=0.002). In SVHUC cells, estradiol reduced FOXO1 expression, which was abolished by an anti-estrogen tamoxifen. FOXO1 knockdown considerably accelerated neoplastic transformation of MCA-SVHUC cells. Moreover, the expression levels of several genes known to inhibit urothelial carcinogenesis (e.g. p21, p27, UGT1A) were significantly lower in MCA-SVHUC-FOXO1-shRNA than in control cells. Notably, tamoxifen treatment resulted in inhibition of neoplastic transformation of control cells, which was abolished by a FOXO1 inhibitor AS1842856. However, no significant effects of tamoxifen on neoplastic transformation were seen in MCA-SVHUC-FOXO1-shRNA cells.

Conclusions

FOXO1 appeared to function as a tumor suppressor and could strongly prevent urothelial tumorigenesis. In contrast, ERβ signals were found to promote it presumably via down-regulation of FOXO1 expression. Accordingly, FOXO1 stimulation via an activator, together with ERβ inactivation via an anti-estrogen, has the potential of being an effective chemopreventive approach for urothelial carcinoma.

Funding

None