Role of PI3K/Akt pathway in cancer stem cell mediated radioresistance

Radiation therapy is applied alone or in combination with surgery and/or chemotherapy for therapy of most solid tumors. However, despite many advances in radiation oncology, radioresistance and cancer recurrence are still major problems. There are various mechanisms, which trigger radioresistance and tumor relapse and cancer stem cells (CSCs) have been identified as the major cause of radioresistance. Several mechanisms are described in CSCs to mediate radioresistance including stimulated DNA repair, hyperactivated survival pathways, like the PI3K/Akt as well as expression and stimulated activity of the CSC marker protein ALDH. As of yet, the precise mechanisms of how the CSC marker ALDH triggers therapy resistance are not fully understood. In the present study, the role of PI3K/Akt pathway and other CSC markers in the regulation of ALDH activity and radiation response of human cancer cell lines in vitro was investigated. By the use of FACS-selected highly ALDH-positive subpopulations of breast cancer cell lines in vitro, it could be demonstrated that ALDH-positive cancer cells presented a significantly elevated radioresistance profile when compared to ALDH-negative subpopulations. Knockdown of Akt isoforms, Akt1, Akt2, and Akt3 led to downregulated ALDH activity in HBL-100 and HCT116 cancer cells. Yet, downregulation of Akt isoforms mediated radiosensitization of HBL-100 but not of MCF-7 breast cancer cells and inhibition of ALDH activity with DEAB treatment did not show any additional effect on post-irradiation cell survival after knockdown of Akt isoforms. Assessing the protein expression profile of ALDH-positive cells revealed an upregulation of the stem cell markers Nanog, Bmi1 and Notch proteins as well stimulated P-Akt levels when compared to ALDH negative cells. In line with these data it could be demonstrated that ALDH-positive and radioresistant in contrast to ALDH-negative and radiosensitive cancer cells presented a stimulated DNA-DSB repair capacity. Moreover, knockdown of Nanog in ALDH-positive cancer cells resulted in an abrogation of the stimulated DNA-DSB repair capacity and consequently in a radiosensitization when compared to control cells. Additionally, knockdown of Nanog led to downregulation of Notch expression and Akt activity. In line with this data, inhibition of Notch and Akt activity resulted in abrogation of stimulated post-irradiation cell survival as well as ALDH activity in Nanog overexpressing cancer cells. Further and most interestingly, it could be observed that nuclear translocation of Nanog is stimulated in a time dependent manner after radiation exposure of cancer cells. Accordingly, Nanog knockout DU145 cells present an impaired DNA-DSB repair and are more radiosensitive than Nanog expressing parental cells. These data confirmed the results obtained from Nanog knock down cells and thus supported the conclusion concerning the role of Nanog in post radiation response of cancer cells. Altogether, the results presented in this study provide evidence that the high expression of ALDH in potential breast cancer stem cells is a consequence of upregulation of Nanog and Notch protein expression as well as stimulated Akt activity, which results in an improved DNA-DSB repair capacity. Compared to breast cancer cells with low ALDH activity these molecular alterations most likely result in the observed radioresistant phenotype of ALDH-positive breast cancer cells