AbstractsMedical & Health Science

Radiosensitization of bladder cancer cells by the mTOR inhibitor, RAD001: a novel strategy for treatment

by Roland Nassim

Institution: McGill University
Department: Department of Surgery
Degree: PhD
Year: 2015
Keywords: Health Sciences - Medicine and Surgery
Record ID: 2059735
Full text PDF: http://digitool.library.mcgill.ca/thesisfile130285.pdf


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. 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 and Western blotting. In the in vivo study, nude mice were subcutaneously inoculated 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 kinetic was measured and tumor weight at the experimental endpoint. Knockdown of p21 was performed using shRNA followed by clonogenic assays in order to assess the role of p21 in radiosensitization. Autophagy and apoptosis were assessed by LC3 immunofluorescence and Western blotting for caspase-3, respectively.In vitro, a significant decrease in colony formation was observed in the combined treatment when compared to RAD001 or radiation alone (p<0.05) in all cell lines. A G0/G1 as well as a significant increase in G2 arrests 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 inhibited mTOR downstream signaling as shown by the inhibition of the S6 protein phosphorylation. 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: a significant decrease in tumor weight was observed in the combined treatment arm (90% decrease, p<0.001) compared to either treatment alone (60% decrease for RAD001, p<0.05; 77% decrease for IR, p<0.05). In the absence of p21, cells were rendered more sensitive to IR, and an increase in autophagy and apoptosis was observed. These findings point to additive beneficial effects of the combined therapy on bladder cancer, mediated by various pathways including p21.The inhibition of mTOR signaling appears promising as a therapeutic modality for bladder cancer, especially in the context of combination with radiation therapy. La radiothérapie pour le cancer invasif de la vessie permet la préservation de l'organe, mais la toxicité systémique et le contrôle local restent problématiques. En tant que tel, il existe un besoin d'augmenter la radiosensibilisation des cellules tumorales afin d'améliorer l'efficacité du traitement. Le but de cette étude était d'examiner si la protéine mTOR (cible de la rapamycine chez les mammifères), une…