CMTM7 Knockout Decreases The Radiosensitivity Of A549 Non-Small Cell Lung Cancer Cells By Increasing Akt Signaling

Abstract

Radiotherapy is the main treatment for patients with early non-surgical and locally advanced non-small cell lung cancer (NSCLC). However, radiotherapy resistance is the main factor that leads to treatment failures such as local recurrence and distant metastasis in patients with NSCLC. Studies have demonstrated that radioresistance is associated with the constitutive activity of Akt-signaling. We previously demonstrated that CMTM7 as a tumor suppressor decreased the activation of Akt in the NSCLC cells. However, whether CMTM7 plays a role in radiosensitivity is still unknown. The purpose of this study is to elucidate the effect of CMTM7 on the radiosensitivity of A549 NSCLC cells and its mechanism. CRISPER/Cas9-mediated genome editing was used to knock out CMTM7 in A549 cells. CCK8 and clone formation assays were used to detect the sensitivity of A549 to ionizing radiation (IR). Flow cytometry was used to test the cell cycle and apoptosis. Western blotting and immunofluorescent assay were used to detect the number of γH2AX foci and the level of Akt-phosphorylation. A xenograft model in nude mice was used to test the in vivo radiosensitivity. We knocked out CMTM7 in A549 NSCLC cells using CRISPR/Cas9 genome editing strategy. Through a series of screens, two Cas9-CMTM7 clones were selected and CMTM7 was undetectable by RT-PCR and Western blotting. CCK8 and clone formation assays showed that knockout of CMTM7 decreased the sensitivity of A549 cells to radiotherapy compared with WT control group. Mechanically, CMTM7 knockout increased DNA damage repair, prolonged G2/M arrest, and enhanced the Akt phosphorylation upon IR, and the PI3K inhibitor LY294002 promoted the radiosensitivity in CMTM7knockout cells to the levels similar as WT control cells. In addition, CMTM7 knockout also markedly decreased the inhibition of tumor xenograft growth by IR in nude mice. Together, we found that knockout of CMTM7 decreased the radiosensitivity of A549 cells in vitro and in vivo. The mechanism of radioresistance might be that the constitutive activation of Akt decreased the repair of DNA damage induced by IR, consequently leading to radiotherapy resistance.