mEAK-7 Regulates Clonogenic Potential and Radiation Resistance in Metastatic Cancer

Objectives: Recently, we demonstrated that MTOR associated protein, eak-7 homolog or mammalian EAK-7 (mEAK-7) is an alternative activator of mTOR signaling. However, the role of mEAK-7 under genotoxic stressors, such as radiation damage, is unknown. The purpose of this study was to identify the role of mEAK-7 in regulating radiation resistance in non-small cell lung carcinoma cell lines. Methods: Cell lines were derived from ATCC. Multiple unique siRNAs were utilized. Tissue Microarrays were obtained by US Biomax. X-irradiation produced by Orthovoltage. Clonogenic assays performed in 2D and spheroid assays in 3D. Comet assays prepared according to Cell Biolabs. Flow cytometry utilized for cell sorting of cancer stem cells (CSCs). mTOR signaling analyzed via immunoblot assays. Results: Bioinformatics analysis demonstrated that a subset of human cancers exhibits significant mEAK-7 gene copy number amplification or mRNA upregulation in certain human cancers. Tissue microarrays analysis demonstrates significant expression of mEAK-7 and mTOR signaling in metastatic cancer. Kaplan Meier analysis demonstrated poorer prognoses for NSCLC patients with higher mEAK-7 expression. H1299 and H1975 cells treated with control or mEAK-7 siRNA, and treated with X-irradiation resulted in a statistically significant decrease in 2D clonogenic potential and 3D spheroid formation. Flow cytometry demonstrated that CSCs derived from the H1299 cell line, defined by CD44+/CD90+, translate more mEAK-7 protein and this correlates with elevated levels of mTOR signaling. mEAK-7 knockdown resulted in a reduction in DNA damage repair, as analyzed by comet assay, and enhanced cell apoptosis after X-irradiation treatment. Immunoprecipitation/mass spectrometry analysis demonstrates that mEAK-7 associates with DNA-PK, and this interaction increases, in response to X-irradiation. DNA-PK interacts with mEAK-7 and S6K2 to regulate mTOR signaling. We demonstrated that mEAK-7 is required for S6K2 activity under IR damage. Finally, we demonstrate that DNA-PK inhibitors resulted in further suppressed mTOR signaling in the presence of DNA damage, suggesting that these two signaling pathways cross-talk (Figure 1).Conclusions: mEAK-7 regulates mTOR signaling under nutrient stimulation. However, the role of mEAK-7-mediated mTOR signaling in human cancer, was unknown. This study demonstrates that mEAK-7 is highly expressed in the NSCLC metastasized lymph nodes in vivo, is highly expressed in CSC population in vitro, is required for clonogenic potential, enhances the DNA-damage response, and promotes DNA-PK-mTOR cross-talk signaling in human cancer cells. Thus, mEAK-7 likely forms a novel mTOR complex that requires DNA-PK, mTOR, and S6K2 (Figure 2).