Presentation Authors: Satoshi Inoue*, Taichi Mizushima, Rochester, NY, Hiroki Ide, Tokyo, Japan, Guiyang Jiang, Takuro Goto, Yujiro Nagata, Rochester, NY, George Netto, Birmingham, AL, Hiroshi Miyamoto, Rochester, NY
Introduction: Activating transcriptional factor 2 (ATF2) is normally activated via its phosphorylation in response to phospho-ERK (p-ERK) signals, leading to promotion of tumor outgrowth demonstrated in several types of malignancies. However, biological significance of ATF2 in bladder cancer remains poorly understood. This study aimed to determine the functional role of ATF2 in urothelial carcinogenesis as well as cancer progression in relation to androgen receptor (AR) signaling.
Methods: First, we immunohistochemically stained for ATF2, phospho-ATF2 (p-ATF2), and p-ERK in 129 bladder cancer and paired non-neoplastic bladder tissue specimens. Second, in human bladder cancer lines, we assessed the effects of ATF2 knockdown via shRNA virus infection on cell proliferation, migration, and invasion. Third, in human normal urothelial SVHUC cells with exposure to a chemical carcinogen 3-methylcholanthrene (MCA), we assessed the effects of ATF2 knockdown on neoplastic transformation.
Results: ATF2/p-ATF2/p-ERK were positive in 84%/32%/26% of bladder tumors, which were significantly elevated, compared with non-neoplastic urothelial tissues [64% (P=0.001)/2% (P < 0.001)/10% (P=0.008)], respectively. Multivariate analysis showed that moderate/strong ATF2 expression and p-ATF2 positivity were independent predictors for recurrence of low grade tumors (HR=2.956, P=0.045) and cancer-specific mortality of muscle-invasive tumors (HR=5.317, P=0.012), respectively. In both neoplastic and non-neoplastic urothelial cells, the expression levels of AR were associated with those of p-ATF2. Dihydrotestosterone treatment in AR(+) bladder cancer cells also induced the expression of p-ATF2/p-ERK and nuclear translocation/transcriptional activity of ATF2. ATF2 knockdown in AR(+) bladder cancer lines resulted in significant decreases in cell viability/migration/invasion, as well as tumor growth in xenograft-bearing mice, and significant increases in apoptosis and G0/G1 cell cycle phase. Finally, ATF2 knockdown in SVHUC cells stably expressing AR resulted in significant inhibition in MCA-induced neoplastic transformation and the expression of several oncogenes. By contrast, ATF2 modulation did not exhibit significant inhibitory effects in AR(-) bladder cancer or SVHUC lines.
Conclusions: ATF2 activity appears to correlate with bladder cancer outgrowth in the presence of an activated AR. Accordingly, ATF2 inhibition, together with AR inactivation, has the potential of being an effective chemopreventive and/or therapeutic approach for urothelial carcinoma.