Abstract B05: mEAK-7 supports self-renewal and radioresistance in metastatic cancer
Authors: Nguyen Joe T. , Haidar Fatima S. , Fox Alexandra L. , Ray Connor , Mendonça Daniela B. , Kim Jin K. , Krebsbach Paul H. 
Published: December 2020

Abstract
Mammalian EAK-7, or MTOR associated protein, eak-7 homolog, (mEAK-7) functions as a crucial regulator of mTOR signaling. Dysregulation of mTOR signaling contributes to the pathogenicity of human carcinomas, so we sought to clarify the role of mEAK-7 in tumorigenesis and metastasis. Bioinformatics analysis revealed that MEAK7 mRNA or gene copy number amplification was observed in a multitude of human carcinomas, including non-small cell lung carcinoma (NSCLC). Tissue microarrays containing sections of primary NSCLC tumors, noncancerous adjacent tissue, and metastatic lymph tissue were used to quantify mEAK-7 and mTOR signaling. Our analyses revealed high mEAK-7 expression and mTOR signaling in metastasized lymph nodes. NSCLC cell lines H1299 and H1975 were used for all in vitro experiments. To examine the role mEAK-7 may play in cancer stem cell (CSC) self-renewal, NSCLC cells were FACS sorted into two groups: CD44+/CD90+ CSCs and non-CSCs. CSCs expressing CD44+/CD90+ demonstrated higher mEAK-7 expression and mTOR activation than non-CSCs, suggesting a role for mEAK-7 in cancer self-renewal and radiation resistance. Following X irradiation-induced DNA damage, 2D clonogenicity and 3D spheroid assays were used to further examine mEAK-7's role in clonogenic potential and self-renewal, respectively. These assays revealed that mEAK-7 is essential for the clonogenic potential and self-renewal of cancer cells in response to DNA damage. Comet assays were used to quantify the DNA damage response, and immunoblot and immunoprecipitation analysis confirmed that mEAK-7 is required for promoting radiation stress. Similarly, siRNA-mediated knockdown of mEAK-7 resulted in reduced DNA damage repair and enhanced cellular apoptosis following genotoxic stress. To do so, mEAK-7 may be interacting with components of the DNA damage response, such as DNA-PK. Immunoprecipitation and mass spectrometry analyses identified that mEAK-7 interacts with DNA-PK, and this interaction is strengthened following treatment with genotoxic stress. Further, DNA-PK interacts with mEAK-7 to regulate S6K2, a crucial target of the mTOR. This suggests crosstalk between the DNA-PK and PI3K pathways, since mEAK-7 interacts with DNA-PK to impact the DNA damage response in human cancer cells. Therefore, DNA-PK inhibitors may be utilized to regulate mEAK-7 and mTOR signaling in specific human carcinomas. Thus, mEAK-7 is a novel marker for NSCLC metastasis; overexpressed in both NSCLC and CSCs; and required for clonogenic potential, spheroid formation, and radiation resistance.
Citation Format: Joe T. Nguyen, Fatima S. Haidar, Alexandra L. Fox, Connor Ray, Daniela B. Mendonça, Jin K. Kim, Paul H. Krebsbach. mEAK-7 supports self-renewal and radioresistance in metastatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B05.