KNL1 Regulates Ferroptosis Resistance and Migration in Lung Adenocarcinoma Cells via AMPK-mTOR Signaling

Background: Lung adenocarcinoma (LUAD), the most prevalent histological subtype of lung cancer, remains a leading cause of cancer-related mortality due to late diagnosis, metastasis, and therapy resistance. The aim of the study is to investigate the role of Kinetochore Scaffold 1 (KNL1) in promoting LUAD progression and its underlying molecular regulatory mechanisms. Methods: KNL1 mRNA expression levels across 33 cancer types were analyzed using bioinformatics analysis based on the TCGA database. Immunohistochemistry (IHC) was used to assess KNL1 expression in LUAD and normal tissues. Stable KNL1-knockdown and KNL1-overexpressing LUAD cell lines were established using lentiviral infection. Western blotting (WB) was used to measure epithelial-mesenchymal transition (EMT) markers and ferroptosis-related protein expression. Cell migration was evaluated via scratch wound healing assays. The thiobarbituric acid (TBA) method was employed for the detection of malondialdehyde. a fluorescent probe was utilized to determine ferrous ion content. WB determined the phosphorylation ratios of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) proteins. Results: 1. KNL1 was highly expressed in 31 cancer types, including LUAD. Kaplan-Meier curves showed significantly shorter median survival in patients with high KNL1 expression. IHC confirmed upregulated KNL1 expression in LUAD tissues. 2. KNL1 overexpression significantly promoted LUAD cell migration and increased mesenchymal marker expression, whereas KNL1 knockdown exerted opposite effects. 3. KNL1 overexpression significantly reduced MDA content and Fe2+ levels in RSL3-treated LUAD cells while increasing the expression of key ferroptosis defense proteins; conversely, it markedly increased the accumulation of MDA and Fe2+ and downregulated these proteins. KNL1 overexpression significantly increased phosphorylated AMPK (p-AMPK) expression but decreased phosphorylated mTOR (p-mTOR) expression in RSL3-treated LUAD cells; conversely, it inhibited p-AMPK expression and activated p-mTOR. Conclusion: KNL1 promotes lung adenocarcinoma progression by suppressing ferroptosis through regulation of the AMPK-mTOR signaling pathway.