Sensitization Of Prostate Cancer To Chemo-Radiation By Ribosomal Protein S6 Inhibition

Radiation Therapy (RT) is curative for most localized prostate cancer (PCA) patients, but is limited by dose-related toxicities and radioresistance. Administration of Nexrutine® (Nx), an inexpensive supplement from Phellodendron amurense bark extract prior to RT inhibited progression to poorly differentiated carcinoma in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. However, the precise molecular mechanism underlying Nx-mediated tumor growth inhibitory effects in combination with RT is unclear. Using global transcriptome profiling coupled with GeneSpring analysis, we have identified significant modulation of number of signaling pathways including genes involved in mTOR and NFκB signaling. Based on these data, we hypothesized that downregulation of ribosomal protein S6 (rpS6) sensitizes prostate tumor to overcome radioresistance. PCA cell lines had higher levels of total and p-rpS6 compared to non-tumorigenic prostate cell lines. Using clonogenic assays, we observed that pretreatment of Nx for 8h could potentiate the effect of low dose RT in LNCaP (AR positive with WT p53), PC-3 (AR negative u0026 p53 null) and PC-3 AR (AR expressing u0026 p53 null) cells. The combinatorial effect depicted strong synergism and had similar effects as high dose RT. Notably, we observed increased activation of Akt/mTOR/NFκB signaling molecules after treatment of PC-3 cells with RT, which was either abrogated (p-rpS6, p-NFκB u0026 p-p70S6K) or decreased (p-Akt) in cells pretreated with Nx prior to RT. Also, Nx pretreatment inhibited the levels of HIF1-α and CyclinD1, which are downstream targets of rpS6. Using immunofluorescence assay, we saw that the combination treatment increased the γH2AX foci compared to 2 Gy alone, indicating increased DNA damage. This is associated with increased protein levels of key G2/M regulatory proteins including Wee-1, p-cdc2 levels and decreased Chk1, p-cdc25C resulting in prolonged RT-induced G2/M arrest. The combination treatment also caused a significant increase in apoptosis, which was evidenced by increased cleaved PARP levels, subG1 levels and Annexin/PI staining. Strikingly, knockdown of rpS6 sensitized PC-3 cells to RT and reversed the observed effects of Nx, indicating the importance of rpS6 in mediating these changes. Remarkably, p-mTOR, p70S6K, NFκB, Ki67 u0026 Cyclin D1 were decreased in the prostate tissue of mice receiving the combination compared to RT alone. Recently, a phase 0/1 clinical trial of Nx in PCA patients receiving RT decreased PSA levels and was well-tolerated. Interestingly, tumor tissue from Nx treated patients showed decreased rpS6 staining intensity compared to the normal tissue. Collectively, our data suggests that Nx has radiosensitizing effect in a range of PCA cell lines and could prevent progression to advanced PCA by inhibiting rpS6. Supported by NCCAM (R01 AT-007448) u0026 VA-MERIT Award (I01 BX 000766) Citation Format: Suleman S. Hussain, Carolina B. Livi, Nikos Papanikolaou, Daniel C. Chan, Rita Ghosh, Addanki P. Kumar. Sensitization of prostate cancer to chemo-radiation by ribosomal protein S6 inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3047.