To investigate the expression characteristics and clinical significance of STE20 family genes in lung squamous cell carcinoma (LUSC), and to explore their association with immune infiltration and their potential as prognostic biomarkers and therapeutic targets.

Transcriptomic data from TCGA, GTEx, and GEO databases (GSE229509, GSE268175) comprising 85 normal lung tissues and 559 LUSC tissues were integrated to analyze the differential expression of STE20 family members. ESTIMATE and TIMER databases were used to evaluate the correlation between STE20s and immune cell infiltration. LASSO regression was employed to construct a prognostic risk model, and random forest model was used to assess the prognostic value of MST4. GO and KEGG enrichment analyses were performed to explore the potential functions of MST4. qRT-PCR and Western blotting were used to validate the expression of key genes in LUSC cell lines (NCIH520, H-1703, SK MES 1).

Among STE20 family members, SPAK, PAK1, PAK6, and MST4 were significantly upregulated in LUSC tissues (P<0.05), while MST1, OSR1, TAO2, MINK, TNIK, and LOK were significantly downregulated (P<0.05). Immune infiltration analysis revealed that MST3, MYO3B, and TNIK were positively correlated with immune scores, whereas MST1, MST4, PAK1, and PAK6 were negatively correlated. TIMER analysis showed that MST4 expression was significantly negatively correlated with infiltration of CD8⁺ T cells (r=-0.23, P<0.001), CD4⁺ T cells (r=-0.19, P<0.01), and macrophages (r=-0.21, P<0.001). LASSO regression identified five prognosis-related genes (PAK1, PAK6, MST1, OSR1, MST4). The risk score model indicated that the high-risk group had significantly shorter overall survival than the low-risk group (HR=2.34, 95%CI: 1.78~3.08, P<0.001). The random forest model predicting 1-, 3-, and 5-year survival achieved AUC values of 0.71, 0.75, and 0.75, respectively. High MST4 expression was associated with poor prognosis (HR=1.89, 95%CI: 1.42~2.51, P<0.001). GO/KEGG enrichment analysis revealed that MST4 is involved in cell cycle, ECM-receptor interaction, and EMT-related pathways. qRT-PCR and Western blotting confirmed that mRNA and protein levels of PAK1, PAK6, and MST4 were significantly higher in LUSC cell lines than in normal lung epithelial cells (BEAS-2B) (P<0.01), while MST1 and OSR1 were significantly lower (P<0.01).

STE20 family genes are aberrantly expressed in LUSC and are closely associated with immune infiltration and prognosis. MST4, as a key member, may promote LUSC progression by regulating cell cycle and ECM remodeling, highlighting its potential as a prognostic biomarker and therapeutic target.