Bladder Cancer (BC) is the second common genitourinary cancer with high recurrence and mortality rate due to metastatic muscle invasive BC (MIBC). Since majority of BCs are non-invasive at diagnosis, developing agents that effectively block BC progression may be beneficial for clinical translation. In this study, clinically approved agents, everolimus, mTOR inhibitor, [0.19mg/kg;7x/week (low dose, LD) or 1.33mg/kg;1x or 2X/week (high dose, HD)] at various dosing schedules alone or in combination with naproxen, an NSAID (30mg/kg body weight) intermittent dosing (3 Wks ON/OFF) were tested for efficacy in an N-butyl-N-(4- hydroxybutyl)nitrosamine (BBN)-induced BC rat model. Female F344 rats (8 weeks age; N=30) were gavaged 16 doses of BBN (150mg/dose). Either one-week (early intervention) or 12-weeks (late intervention) after BBN treatment, rats in each group received respective drug treatments by gavage. At 50 weeks age, rats were euthanized, and tissues were analyzed. Results suggest that BBN-exposed rats developed high number of Non-MIBC (NMIBC) and MIBC and had significantly large bladders (430±57mg, Mean±SEM; p<0.0001) compared to normal bladders in vehicle group (68.8±1.3mg). Importantly, there was significant reduction in tumor growth and progression of hyperplasia/papilloma with naproxen alone (3 weeks intermittent dosing) by 70% (129.7±7.7mg; p<0.0001) and 58% (180.8±27.6mg; p<0.001) at early and later interventions respectively compared to untreated BBN-exposed rats. Continuous LD or HD everolimus regimens during early intervention showed significant inhibitory effect on papilloma progression, whereas its delayed administration had modest to insignificant inhibitory effect of papilloma progression to NMIBC/MIBC. Intermittent dosing of naproxen alone inhibited large tumors (>200mg) incidence by 90% (p<0.0001) and by 75% (p<0.0001) with early and late interventions respectively. Early treatment with combinations led to >72% reduction in tumor weight (120.8±7.8mg, 121.8±11.5mg, and 118.6±5.2mg; p<0.0001) while late intervention had 58%-65% tumor reductions (179.6±14.4mg, 150.6±12.6mg, and 172.5±15.6mg; p<0.0005) with the combination dosing regimens. Notably, all 3 regimens of naproxen plus everolimus combinations significantly inhibited large tumor incidence at both early intervention (90%-100%; p<0.0001) and late intervention (60%-70%; p<0.0005), with significant suppression of papilloma progression to NMIBC and MIBC, including squamous cell carcinoma (SCC). Protein biomarker analysis suggested decrease in markers of proliferation, inflammation and mTOR signaling with an increased apoptosis. In summary, our study indicated that naproxen and everolimus combination can prevent bladder cancer progression and warrants further evaluation. (Supported by NCI-PREVENT program 75N91019D00020-75N91020F00003)

Citation Format: Venkateshwar Madka, Gopal Pathuri, Surya P. Singh, Anil Singh, Anh Bao, Nicole Stratton, Stanley Lightfoot, Clinton J. Grubbs, Jennifer Fox, John L. Clifford, Brian Cholewa, Shizuko Sei, CV Rao. Chemopreventive efficacy of everolimus and naproxen combination against carcinogen induced bladder cancer in F344 rats. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5260.