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As loss of DNA-repair proteins is common in urothelial carcinoma (UC), a rationale can be made to evaluate the activity of poly (ADP-ribose) polymerase (PARP) inhibitors to exploit synthetic lethality. We aimed to preclinically evaluate a PARP inhibitor, CEP-9722, and its active metabolite, CEP-8983, in UC. The activity of CEP-8983 was evaluated using a 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay against human UC cell lines. Flow cytometry, COMET assay, and western blot were performed to assess apoptosis, DNA damage, and DNA-repair proteins, respectively. RT4 xenografts received placebo or CEP-9722 (100 or 200 mgkgday) orally. Xenografts were subjected to immunohistochemistry for apoptosis cleaved caspase (cc)-3 and angiogenesis (CD31). CEP-8983 (1 moll) reduced the viability of RT4 and T24 cells by 20, but did not reduce the viability of 5637 and TCC-SUP cells. Apoptosis and necrosis occurred in 9.7 and 9.1 of RT4 and 5637 cells, respectively. RT4 cells showed greater DNA damage compared with 5637 cells. Increased DNA damage occurred with combination versus CEP-8983 or cisplatin alone in RT4 and 5637 cells. T24 and RT4 showed the least RAD51 foci 8 h following radiation, whereas TCC-SUP and 5637 robustly induced RAD51 foci. CEP-9722 showed dose-dependent antitumor activity in RT4 xenografts; 200 mgkg daily was better than control (P=0.04) and 100 mgkg was not (P=0.26). Immunohistochemistry of xenografts showed a significant increase in cc-3 and decrease in CD31 with both doses (P<0.05). Biomarker-driven evaluation of PARP inhibitors in UC is justified as the activity of CEP-9722 correlated inversely with homologous recombination repair response to DNA damage.

Bladder cancer is the fifth most frequent tumor in men and ninth in women in the United States. Due to a high likelihood of recurrence, effective chemoprevention is a significant unmet need. Estrogen receptors (ERs), primarily ERβ, are expressed in normal urothelium and urothelial carcinoma, and blocking ER function with selective ER modulators such as tamoxifen inhibits bladder cancer cell proliferation in vitro. Herein, the chemoprotective potential of tamoxifen was evaluated in female mice exposed to the bladder-specific carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Carcinogen treatment resulted in a 76% tumor incidence and increased mean bladder weights in comparison to controls. In contrast, mice receiving tamoxifen concurrent (8–20 weeks) or concurrent and subsequent (8–32 weeks) to BBN administration had no change in bladder weight and only 10% to 14% incidence of tumors. Non-muscle-invasive disease was present in animals treated with tamoxifen before (5–8 weeks) or after (20–32 weeks) BBN exposure, while incidence of muscle-invasive bladder carcinoma was reduced. ERβ was present in all mice and thus is a potential mediator of the tamoxifen chemoprotective effect. Surprisingly, ERα expression, which was detected in 74% of the mice exposed to BBN alone but not in any controlmice, was correlated with tumor incidence, indicating a possible role for this receptor in carcinogen-induced urothelial tumorigenesis. Thus, these data argue that both ERα and ERβ play a role in modulating carcinogen-induced bladder tumorigenesis. Administration of tamoxifen should be tested as a chemopreventive strategy for patients at high risk for bladder cancer recurrence.

Estrogen receptors (ERs) are known to mediate important physiologic responses as well as the growth of some tumors in response to estradiol stimulation. In a previous study the selective ER modulator raloxifene was shown to induce apoptosis in an ERβ‐positive bladder cancer cell line. However, the expression of ERβ in human bladder cancer has not been thoroughly investigated.ERα and ERβ expression in 224 bladder tumor samples was evaluated using tissue microarray and immunohistochemistry. Levels of ERα and ERβ protein and mRNA expression were determined in several bladder cancer cell lines using quantitative reverse‐transcriptase polymerase chain reaction (RT‐PCR) and Western blot analysis. The effect of estradiol and antiestrogen treatments on RT4 bladder cancer cell growth was determined by cell proliferation assays.Analyses revealed that only 2 human bladder cancers weakly expressed ERα. In contrast, the expression of ERβ was detected in 141 tumors (63%). ERβ was expressed in 58% of WHO Grade 1 and 2 tumors, whereas 70% of Grade 3 tumors demonstrated expression (P=.085). Importantly, although only 53% and 55% of Ta and T1 tumors demonstrated ERβ expression, 80% of T2, 81% of T3, and 75% of T4 tumors showed ERβ expression. The differences in ERβ expression between Ta/T1 and T2/T3/T4 tumors were found to be highly significant (P<.001). Metastatic transitional cell carcinomas had ERβ expression (80%) comparable to that of muscle invasive bladder cancers. Western blot analysis detected ERβ protein expression in each of the 5 bladder cancer cell lines tested, whereas no or very low levels of ERα were found. Quantitative RT‐PCR revealed that higher levels of ERβ than ERα mRNA were present in 5637, T‐24, TSU‐Pr1, and TCC‐Sup bladder cancer cells, whereas ER‐α mRNA levels were greater than ERβ in RT4 cells. Treatment with 17β‐estradiol modestly increased RT4 cell growth, whereas the antiestrogens, 4‐hydroxtamoxifen, raloxifene, or ICI 182,780 inhibited the growth of RT4 cells.ERβ is the dominant receptor expressed in bladder cancer cell lines and in the majority of human bladder tumors. Moreover, the degree of ERβ expression increases with increasing stage and grade of differentiation. Antiestrogens have an inhibitory effect on the growth of bladder cancer cells in vitro. Cancer 2006. © 2006 American Cancer Society.

We investigated the relationship between cyclooxygenase-2 (COX-2) expression and molecular alterations commonly found in transitional cell carcinoma (TCC) of the bladder and determined whether COX-2 immunoreactivity is associated with cancer stage, progression and survival in patients undergoing radical cystectomy.