TIMELESS promotes the proliferation and migration of lung adenocarcinoma cells by activating EGFR through AMPK and SPHK1 regulation

Background: Lung adenocarcinoma (LUAD) has high morbidity and is prone to recurrence. TIMELESS (TIM), which regulates circadian rhythms in Drosophila, is highly expressed in various tumors. Methods: ： We used tumor samples from patients with lung carcinoma and LUAD patient data from public databases to confirm the relationship of TIM expression with lung cancer. We used NSCLC cell lines and siRNA to knock down TIM expression, and further analyzed cell proliferation, migration and colony formation. By using western blot and qPCR, we detected the influence of TIM on EGFR, Sphk1 and AMPK. With proteomics analysis, we comprehensively inspected the different changed proteins influenced by TIM and did global bioinformatic analysis. Results: In this study, we found that TIM expression was elevated in LUAD and that this high expression was positively correlated with more advanced tumor pathological stages and shorter overall and disease-free survival. Moreover, gefitinib efficacy in patients with LUAD could be influenced by TIM expression, and the antitumor effect of gefitinib was significantly improved with TIM knockdown. TIM knockdown inhibited epidermal growth factor receptor (EGFR) activation and phosphorylation of its downstream AKT/mTOR and ERK1/2 pathways. We also clarified that TIM regulated the activation of sphingosine kinase 1 (SPHK1) in LUAD cells, while SPHK1 knockdown inhibited EGFR activation. Quantitative proteomics techniques combined with bioinformatics analysis were adopted to clarify the global molecular mechanisms regulated by TIM in LUAD. The results of proteomics suggested that mitochondrial translation elongation and termination were altered, which were closely related to the process of mitochondrial oxidative phosphorylation. Knockdown of TIM reduced the ATP content and promoted AMP-activated protein kinase (AMPK) activation. Conclusions: Our study revealed that TIM could regulate EGFR activation through AMPK and SPHK1, as well as influence mitochondrial function and alter the ATP level; thus, TIM is a key factor in LUAD.