ATF4/MYC Regulates MTHFD2 to Promote NSCLC Progression by Mediating Redox Homeostasis.

Purpose: Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) has been reported to be overexpressed in non-small-cell lung cancer (NSCLC) and to correlate with malignant proliferation. However, the mechanism of high MTHFD2 expression in NSCLC has not been clarified. Methods: qPCR, western blot, and immunofluorescence experiments were used to measure the expression of related mRNAs and proteins. Cell apoptosis was measured by flow cytometry and TUNEL assays. The CCK-8 assay was used to determine cell viability. Flow cytometry was used to analyze the cell cycle. ROS, H2O2, MDA, SOD, and NADPH/NADP+ were evaluated by relevant assay kits. Transfection of siRNA or vectors was used to downregulate or upregulate gene expression. Dual-luciferase reporter gene assays were used to evaluate the regulated relationship between MTHFD2 and ATF4 or MYC. Results: MTHFD2 was highly expressed in NSCLC cells. Knockdown of MTHFD2 inhibited proliferation and increased apoptosis. Furthermore, oxidative factors significantly increased, while antioxidant factors significantly decreased in NSCLC cells with MTHFD2 knockdown, indicating that MTHFD2 was involved in NSCLC progression through the redox pathway. Although MTHFD2 was downregulated with ATF4 silencing, the dual-luciferase reporter assay suggested that ATF4 did not directly mediate MTHFD2 transcription. Further studies revealed that MYC had a transcriptional effect on MTHFD2 and was also regulated by ATF4. PCR, and western blotting experiments with ATF4 knockdown and MYC overexpression as well as ATF4 overexpression and MYC knockdown proved that ATF4 stimulated MTHFD2 through MYC mediation. Conclusions: ATF4 promoted high expression of MTHFD2 in NSCLC dependent on MYC.