Abstract

Targeting protein for Xklp2 (TPX2) is a known mitotic regulator overexpressed in lung adenocarcinoma (LUAD), yet its role in cancer stemness and autophagy remains unclear. Through integrative bioinformatic analysis of TCGA and GTEx datasets, we discovered that TPX2 overexpression in LUAD correlates not only with poorer overall, disease-free, disease-specific, and progression-free survival but also significantly associates with elevated stemness scores (RNAss/DNAss) and increased expression of CSC markers SOX2 and c−MYC. Functionally, CRISPR-Cas9-mediated TPX2 knockout in PC9 and H1975 cells led to marked reductions in sphere-forming capability and CSC marker expression, while TPX2 overexpression had the opposite effect. Mechanistic exploration through Co-immunoprecipitation coupled with mass spectrometry (IP-MS) and transcriptome analysis revealed a correlation between TPX2 and autophagy regulation in LUAD cells. TPX2 knockdown inhibited autophagic flux, as evidenced by decreased Beclin−1 and accumulation of p62 and LC3-II levels, together with increased GFP+/mRFP+ (yellow) LC3 puncta. Conversely, TPX2 overexpression enhanced autolysosome formation. Importantly, treating TPX2-overexpressing cells with the autophagy inhibitor chloroquine significantly reversed their enhanced migration, proliferation, and stemness marker expression. In vivo, TPX2 silencing in xenograft models reduced tumor growth and altered autophagy marker profiles. These findings unveil a novel mechanism whereby TPX2 promotes CSC properties in LUAD by driving autophagy, offering a promising therapeutic avenue targeting TPX2-mediated autophagy.