IADR Abstract Archives
mEAK-7 Regulates Clonogenic Self-renewal and Radiation Resistance in Cancer Cells
Objectives: Recently, we demonstrated that mammalian EAK-7 (mEAK-7) is an alternative activator of mTOR signaling. However, the molecular role of mEAK-7 under genotoxic stressors such as radiation therapy 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. X-irradiation produced by Orthovoltage. Clonogenic assays performed in 2D and 3D. Comet assays prepared according to Cell Biolabs. Flow cytometry utilized for cell sorting of 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. H1299 and H1975 cells treated with control or mEAK-7 siRNA, and treated with X-irradiation resulted in a statistically significant decrease in 2D colony formation 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 signaling promotes the formation of radiation-resistant CSCs through aberrantly enhanced DNA-damage response mechanisms. 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.
Conclusions: mEAK-7 regulates alternative mTOR signaling under nutrient stimulation. However, the role of mEAK-7-mediated mTOR signaling in human cancer, was unknown. Thus, this study demonstrates that mEAK-7 is highly expressed in the CSC population, is required for clonogenic self-renewal, enhances the DNA-damage response, and promotes DNA-PK-mTOR cross-talk signaling in human cancer cells.
Methods: Cell lines were derived from ATCC. Multiple unique siRNAs were utilized. X-irradiation produced by Orthovoltage. Clonogenic assays performed in 2D and 3D. Comet assays prepared according to Cell Biolabs. Flow cytometry utilized for cell sorting of 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. H1299 and H1975 cells treated with control or mEAK-7 siRNA, and treated with X-irradiation resulted in a statistically significant decrease in 2D colony formation 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 signaling promotes the formation of radiation-resistant CSCs through aberrantly enhanced DNA-damage response mechanisms. 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.
Conclusions: mEAK-7 regulates alternative mTOR signaling under nutrient stimulation. However, the role of mEAK-7-mediated mTOR signaling in human cancer, was unknown. Thus, this study demonstrates that mEAK-7 is highly expressed in the CSC population, is required for clonogenic self-renewal, enhances the DNA-damage response, and promotes DNA-PK-mTOR cross-talk signaling in human cancer cells.