Single-cell and clinical analyses reveal elevated HAX1 expression and cell-cycle-associated programs during metastatic progression of lung adenocarcinoma, particularly in brain metastasis

Introduction Metastatic dissemination is a major determinant of poor outcome in lung adenocarcinoma (LUAD). However, molecular features associated with distinct metastatic states, particularly lymph node and brain metastases, remain incompletely defined. We investigated the relationship between HAX1 and LUAD metastatic progression. Methods We reanalyzed publicly available single-cell RNA-sequencing data from GSE131907 to characterize HAX1 expression and associated transcriptional programs across normal lung, primary LUAD, advanced tumors, metastatic lymph nodes (mLN), and brain metastases. Findings were validated using additional public datasets from GEO and TCGA, including the independent single-cell dataset GSE69405. Clinical LUAD specimens were analyzed to assess HAX1 protein expression and its clinicopathological associations. Functional validation was performed in A549 and H1975 cells, and tumor growth was assessed using an A549-derived subcutaneous xenograft model. Results HAX1 expression and the proportion of HAX1-positive epithelial cells increased across LUAD disease states and were significantly enriched in mLN and brain-metastatic epithelial populations compared with primary tumors. In a 125-patient clinical cohort, high HAX1 expression was associated with lymph node metastasis, advanced stage, and shorter overall survival, and remained independently associated with lymph node metastasis after adjustment for T stage (OR = 2.581, 95% CI 1.161–5.742; P = 0.020). An independent single-cell dataset further confirmed elevated HAX1 expression in brain-metastatic LUAD cells. HAX1-associated transcriptional programs converged on E2F, MYC, MTORC1, DNA replication, and cell-cycle signatures, whereas brain-metastatic epithelial cells additionally exhibited mitochondrial and translational programs. Integrated analyses identified UBE2S, PITX1, GGH, and PDCD2L as HAX1-associated candidates.EPIC and TIMER analyses consistently showed inverse correlations between HAX1 expression and estimated B-cell, CD4+ T-cell, CD8+ T-cell, and macrophage abundance. HAX1 knockdown also reduced cell viability, increased the G0/G1-phase fraction and decreased the S-phase fraction, reduced G1/S-related protein levels and PI3K/AKT signaling, and inhibited subcutaneous xenograft growth. Conclusion HAX1 is closely associated with metastatic progression of LUAD, with strong clinical evidence for lymph node involvement and reproducible enrichment in brain-metastatic epithelial cells. Its associations with cell-cycle programs and immune cell infiltration, together with its functional role in G1/S progression, support further investigation of HAX1 in metastatic adaptation and tumor–immune interactions.