Amphiregulin reflects brain metastasis progression and leads to PD-L1 expression in non-small cell lung cancer cells.

BACKGROUND: The Hippo kinase Nuclear Dbf2-related kinase 2 (NDR2) promotes brain metastasis (BM) in non-small cell lung cancer (NSCLC) by the disrupting Yes-associated protein 1 (YAP-1), suggesting a role in circulating tumor cells and/or brain colonization. The underlying mechanism remains to be clarified.

METHODS: Human bronchial epithelial tumor cells (A549, H1975, H2030 and the brain-tropic H2030-BrM3 line) with or without NDR2 depletion (via siRNA or shRNA), were exposed to shear stress (up to 40 dyn/cm² for 3 h) using an Ibidi® pump system, in the presence or absence of exogenous Amphiregulin (AREG), and subsequently reseeded or not. Viability, apoptosis, proliferation, and YAP-1-dependent gene expression were analyzed. H2030-BrM3 cells (shControl or shNDR2) were injected intracardially into nude athymic mice (n = 10/group) to evaluate plasma AREG levels correlation with BM. AREG expression was assessed in human NSCLC tumor samples from primary and/or brain metastatic sites.

RESULTS: NSCLC cells tolerated shear stress and showed reduced apoptosis after reseeding when expressing NDR2. Shear stress induced a dedifferentiation (via Sox2/9 variation) and increased AREG expression in most NSCLC cell lines. AREG enhanced NSCLC cells survival and proliferation under shear stress. In mice, plasma AREG levels correlated with BM volume. In human NSCLC samples, AREG-positive tumors displayed elevated Programmed Death-Ligand 1 (PD-L1), suggesting immune escape. Exogenous AREG treatment in H2030-BrM3 cells induced PD-L1 expression in vitro.

CONCLUSION: AREG supports tumor adaptation to mechanical stress and may drive immune tolerance via PD-L1. Its correlation with BM burden highlights its potential as a biomarker and therapeutic target in NSCLC.