Restoration of Radiosensitivity by Downregulation of Ribosomal Protein S6

Radiation Therapy (RT) is a curative treatment for localized prostate cancer (PCA), but is limited by dose‐related toxicities and radioresistance. Ribosomal protein S6 (rpS6), a downstream effector of PI3K/Akt/mTOR signaling, can mediate radioresistance by tempering with protein translation, survival, cell cycle progression and DNA repair. Analysis from the Oncomine® database showed that prostate carcinoma and prostatic intraepithelial neoplasia had higher rpS6 expression compared to normal prostate. Similarly, PCA cell lines had a higher level of total and p‐rpS6 compared to non‐tumorigenic prostate cell lines. Previously, we have shown that administration of Nexrutine® (Nx), an inexpensive herbal supplement from Phellodendron amurense bark extract for 6 weeks prior to RT inhibited progression to poorly differentiated carcinoma in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice and did not show any prominent toxicity. Remarkably, p‐mTOR, p70S6K, NF k B, Ki67 and Cyclin D1 were also decreased in the prostate tissue of mice receiving the combination treatment compared to RT alone. However, the precise molecular mechanism underlying Nx‐mediated tumor growth inhibitory effects in combination with RT is unclear. We hypothesize that downregulation of rpS6 sensitizes prostate tumor to overcome radioresistance. 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 and p53 null) and PC‐3 AR (AR expressing and p53 null) cells. Androgen dependent LNCaP cells were more sensitive to RT compared to androgen‐independent PC‐3 cells, while PC‐3 AR cells were the most resistant. The combinatorial effect of Nx and low dose RT depicted strong synergism and had similar effects as high dose RT. Notably, we observed an increase in activation of Akt/mTOR/NF k B signaling molecules after treatment of PC‐3 cells with RT, which was either abrogated (p‐rpS6, p‐NF k B and 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 the downstream targets of rpS6. Additionally, immunofluorescence analysis revealed that Nx pretreatment combined with 2 Gy increased the p‐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 increase in cleaved PARP levels, subG1 analysis 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. Overall, our data suggests that Nx has a radiosensitizing effect in a range of PCA cell lines and could prevent progression to advanced PCA by downregulating rpS6. Interestingly, a phase 0/1 clinical trial of Nx in PCA patients receiving RT or undergoing radical prostatectomy decreased PSA levels and was found to be well‐tolerated. Thus, Nx shows enormous potential to improve the current PCA management warranting further investigation for use its use as an adjuvant to RT. Support or Funding Information Supported by NCCAM (R01 AT‐007448) and VA‐MERIT Award (I01 BX 000766; APK)