Abstract A032: miR-205 reconstitution sensitizes prostate cancer cells and xenografts to radiotherapy, through PKCε suppression

Abstract

Abstract Radiotherapy is a standard treatment for organ-confined prostate cancer (PCa). Although treatment technical optimization has greatly improved local tumor control, a significant percentage of patients still undergo recurrence. The onset of radioresistance is a complex phenomenon involving several genetic and epigenetic abnormalities, including the deregulation of microRNAs (miRNAs), endogenous small noncoding RNA molecules that negatively regulate gene expression. In this study, we investigated the ability of miR-205 to radiosensitize PCa cells. The possible relevance of the miRNA in PCa radiation response is based on our previous observation that miR-205 reconstitution in PCa cells i) exerts a tumor-suppressive effect in human PCa cells by counteracting epithelial-mesenchymal transition (EMT) and reducing cell migration/invasion, at least in part through the downregulation of its target PKCε, shown to play a role also in the nuclear translocation of EGFR, and ii) counteracts CAF-induced EMT, thus impairing enhancement of tumor cell invasion, acquisition of stem cell traits, tumorigenicity, and metastatic dissemination. The biologic role of miR-205 was elucidated through reconstitution in DU145 PCa cells by using a miRNA mimic. Radiation response in miRNA-reconstituted and control cells was assessed by the clonogenic assay. Transfected cells were exposed to 2-8 Gy delivered as a single dose using the 137Cs γ-irradiator IBL-437. Xenografts generated in mice following s.c. transplantation of DU145 cells stably expressing miR-205 received 5 Gy single-dose irradiation using a micro-CT/microirradiator (225Cx, Precision X-ray). We found that transient or stable reconstitution of miR-205 in DU145 cells consistently increased in vitro radiosensitivity. Among miR-205 direct targets, we were able to attribute a functional role in the radiosensitizing effect to PKCε. Indeed, siRNA-mediated silencing of PKCε in DU145 cells fully reproduced the enhanced radiation response induced by the miRNA. The effect was mediated, at least in part, by a decreased radiation-induced EMT and a reduced Non-Homologous End-Joining-based repair, as a consequence of the inhibited EGFR nuclear translocation. Based on our previous evidence indicating the ability of miR-205 to improve the activity of DNA damaging drugs through the downregulation of the lysosome-associated protein LAMP3 and autophagy impairment, we assessed the effect of LAMP3 knockdown on DU145 cell radiosensitivity and observed an enhanced radiation response, though at a lower extent compared to that induced by miR-205 reconstitution. The miRNA-mediated in vitro radiosensitizing effect was also appreciable in vivo. Indeed, xenografts generated by DU145 cells stably expressing the miRNA showed a significantly enhanced tumor growth inhibition following 5 Gy single-dose irradiation, as compared to control xenografts. Moreover, miR-205 reconstitution significantly increased the time for DU145 xenografts to reach 1,000 mm3 tumor burden. Consistent with in vitro findings, immunohistochemistry data obtained in tumor sections from miR-205-reconstituted xenografts revealed decreased PKCε expression. Overall, our results suggest miR-205 as a possible relevant determinant of ionizing radiation response, supporting the clinical interest in developing a novel therapeutic approach based on miR-205 reconstitution to increase PCa sensitivity to radiotherapy. Citation Format: Paolo Gandellini, Rihan El Bezawy, Marco Folini, Denis Cominetti, Stella Tinelli, Tiziana Rancati, Riccardo Valdagni, Nadia Zaffaroni. miR-205 reconstitution sensitizes prostate cancer cells and xenografts to radiotherapy, through PKCε suppression [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A032.