We examined whether mTOR inhibition by RAD001 (Everolimus) could be therapeutically efficacious in the treatment of bladder cancer. RAD001 markedly inhibited proliferation of nine human urothelial carcinoma cell lines in dose- and sensitivity-dependent manners in vitro. FACS analysis showed that treatment with RAD001 for 48 h induced a cell cycle arrest in the G(0)/G(1) phase in all cell lines, without eliciting apoptosis. Additionally, RAD001 significantly inhibited the phosphorylation of S6 downstream of mTOR and VEGF production in all cell lines. We also found tumor weights from nude mice bearing human KU-7 subcutaneous xenografts treated with RAD001 were significantly reduced as compared to placebo-treated mice. This tumor growth inhibition was associated with significant decrease in cell proliferation rate and angiogenesis without changes in cell death. In conclusion inhibition of mTOR signaling in bladder cancer models demonstrated remarkable antitumor activity both in vitro and in vivo. This is the first study showing that RAD001 could be exploited as a potential therapeutic strategy in bladder cancer.

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PurposeRadiation therapy for invasive bladder cancer allows for organ preservation but toxicity and local control remain problematic. As such, improving efficacy of treatment requires radiosensitization of tumor cells. The aim of study is to investigate if the mammalian Target of Rapamycin (mTOR), a downstream kinase of the phosphatidylinositol 3-kinase (PI3K)/AKT survival pathway, may be a target for radiation sensitization.Experimental DesignClonogenic assays were performed using 6 bladder cancer cell lines (UM-UC3, UM-UC5, UM-UC6, KU7, 253J-BV, and 253-JP) in order to examine the effects of ionizing radiation (IR) alone and in combination with RAD001, an mTOR inhibitor. Cell cycle analysis was performed using flow cytometry. In vivo, athymic mice were subcutaneously injected with 2 bladder cancer cell lines. Treatment response with RAD001 (1.5 mg/kg, daily), fractionated IR (total 9Gy = 3Gy×3), and combination of RAD001 and IR was followed over 4 weeks. Tumor weight was measured at experimental endpoint.ResultsClonogenic assays revealed that in all bladder cell lines tested, an additive effect was observed in the combined treatment when compared to either treatment alone. Our data indicates that this effect is due to arrest in both G1 and G2 phases of cell cycle when treatments are combined. Furthermore, our data show that this arrest is primarily regulated by changes in levels of cyclin D1, p27 and p21 following treatments. In vivo, a significant decrease in tumor weight was observed in the combined treatment compared to either treatment alone or control.ConclusionsAltering cell cycle by inhibiting the mTOR signaling pathway in combination with radiation have favorable outcomes and is a promising therapeutic modality for bladder cancer.

BackgroundTwo signalling molecules that are attractive for targeted therapy are the epidermal growth factor receptor (EGFR) and the peroxisome proliferator-activated receptor gamma (PPARγ). We investigated possible crosstalk between these 2 pathways, particularly in light of the recent evidence implicating PPARγ for anticancer therapy.Principal FindingsAs evaluated by MTT assays, gefitinib (EGFR inhibitor) and DIM-C (PPARγ agonist) inhibited growth of 9 bladder cancer cell lines in a dose-dependent manner but with variable sensitivity. In addition, combination of gefitinib and DIM-C demonstrated maximal inhibition of cell proliferation compared to each drug alone. These findings were confirmed in vivo, where combination therapy maximally inhibited tumor growth in contrast to each treatment alone when compared to control (p<0.04). Induction of PPARγ expression along with nuclear accumulation was observed in response to increasing concentrations of gefitinib via activation of the transcription factor CCAT/enhancer-binding protein-β (CEBP-β). In these cell lines, DIM-C significantly sensitized bladder cancer cell lines that were resistant to EGFR inhibition in a schedule-specific manner.ConclusionThese results suggest that PPARγ agonist DIM-C can be an excellent alternative to bladder tumors resistant to EGFR inhibition and combination efficacy might be achieved in a schedule-specific manner.

Background: Radiation therapy for invasive bladder cancer allows for organ preservation but systemic toxicity and local control remain problematic. As such, there is a need to increase radiosensitization of tumor cells to improve efficacy. The aim of this study was to investigate if mTOR (mammalian target of rapamycin), a downstream kinase of the PI3K/Akt survival pathway, may be a target for combined bladder cancer therapy. Methods: Clonogenic assays were performed using 6 bladder cancer cell lines in order to address the effects of ionizing radiation (IR) on growth, when tested alone and in combination with RAD001, a potent mTOR inhibitor. Cell cycle analysis was performed using flow cytometry. In the in vivo study, nude mice were subcutaneously injected with KU7 and 253J-BV cells. Treatment with RAD001 (1.5 mg/kg, daily), fractionated IR (total 9 Gy), and the combination of RAD001 and IR was followed over 4 weeks. Tumor growth kinetics was measured and tumor weight at the experimental endpoint. Results: In vitro, a significant decrease in colony formation was observed in the combined treatment when compared to RAD001 or radiation alone (p&lt;0.05) in all cell lines. A G0/G1 as well as a significant increase in G2 arrest was observed in the combined treatment compared to either treatment alone. Changes in the levels of Cyclin D1, p27 and p21 correlated with the observed changes in the cell cycle. Moreover, IR rapidly activated AKT whereas RAD001 effectively inhibited the mTOR downstream signaling as shown by the inhibition of the phosphorylation of S6. Furthermore, autophagy was induced following the treatment with RAD001 and in combination as indicated by the conversion of LC3-I to LC3-II, a protein marker for autophagy. Our in vivo data confirmed our in vitro data, wherein a significant decrease in tumor weight was observed in the combined treatment arm (90% decrease, p&lt;0.001) compared to either treatment alone (60% decrease for RAD001, p&lt;0.05; 77% decrease for IR, p&lt;0.05). These findings point to additive and possibly synergistic beneficial effects of the combined on bladder cancer. Conclusions: The inhibition of mTOR signaling appears promising as a therapeutic modality for bladder cancer, especially in the context of combination with radiation therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1452. doi:1538-7445.AM2012-1452