Subcellular localization of AKT and mTOR after radiation. A, Western blots of mTOR, phospho-AKT S473 and AKT 2 h after 3 fractions of 2 Gy in 3D-grown PC3 cells after CRISPR/Cas9-mediated depletion of mTOR (crMTOR) using different targeting sequences. A non-targeting sequence (NTS) was used as control. β-actin expression was evaluated to ensure equal sample loading. B, Colony formation data of 3D colony formation assays. Cells were irradiated either with 6 Gy single dose or with 3 fractions of 2 Gy (2 Gy per day). Results show mean {plus minus} SD (n = 3, ** P < 0.01, Student''s t test). (See also Figure 6)
ARTICLE ABSTRACT
Implementing targeted drug therapy in radio-oncologic treatment regimens has greatly improved the outcome of cancer patients. However, the efficacy of molecular targeted drugs such as inhibitory antibodies or small molecule inhibitors essentially depends on target expression and activity, which both can change during the course of treatment. Radiotherapy has previously been shown to activate prosurvival pathways, which can help tumor cells to adapt and thereby survive treatment. Therefore, we aimed to identify changes in signaling induced by radiation and evaluate the potential of targeting these changes with small molecules to increase the therapeutic efficacy on cancer cell survival. Analysis of “The Cancer Genome Atlas” database disclosed a significant overexpression of AKT1, AKT2, and MTOR genes in human prostate cancer samples compared with normal prostate gland tissue. Multifractionated radiation of three-dimensional–cultured prostate cancer cell lines with a dose of 2 Gy/day as a clinically relevant schedule resulted in an increased protein phosphorylation and enhanced protein–protein interaction between AKT and mTOR, whereas gene expression of AKT, MTOR, and related kinases was not altered by radiation. Similar results were found in a xenograft model of prostate cancer. Pharmacologic inhibition of mTOR/AKT signaling after activation by multifractionated radiation was more effective than treatment prior to radiotherapy. Taken together, our findings provide a proof-of-concept that targeting signaling molecules after activation by radiotherapy may be a novel and promising treatment strategy for cancers treated with multifractionated radiation regimens such as prostate cancer to increase the sensitivity of tumor cells to molecular targeted drugs. Mol Cancer Ther; 17(2); 355–67. ©2017 AACR.See all articles in this MCT Focus section, “Developmental Therapeutics in Radiation Oncology.”
