LIMS2 downregulation is associated with tumor progression and immune landscape alterations in lung adenocarcinoma

Figures

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  Fig. 1. Expression pattern of LIMS2 across normal tissues and multiple cancer types. A RNA tissue specificity analysis of LIMS2 across normal human tissues, showing a tissue-enhanced expression pattern with relatively higher expression in intestinal tissues. B Comparison of LIMS2 expression between tumor and normal tissues across multiple cancer types based on TCGA datasets. C Relative mRNA expression levels of LIMS2 were measured by qRT-PCR in LUAD cell lines (NCI-H358, A549, PC9, and H1299) and compared with the normal bronchial epithelial cell line BEAS-2B. LIMS2 expression was significantly decreased in all LUAD cell lines. D Paired analysis of LIMS2 expression in tumor tissues and matched adjacent normal tissues in representative cancer types, including lung adenocarcinoma (LUAD). E Pan-cancer distribution of LIMS2 expression levels across TCGA tumor cohorts. Expression values are shown as log₂ (TPM + 1). Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 2. Association of LIMS2 expression with prognosis and clinical characteristics in lung adenocarcinoma. A Forest plot showing the association between LIMS2 expression and disease-specific survival (DSS) across multiple cancer types based on TCGA cohorts. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using univariate Cox regression analysis. B Forest plot illustrating the relationship between LIMS2 expression and progression-free interval (PFI) across TCGA cancer types. C Receiver operating characteristic (ROC) curve evaluating the discriminatory performance of LIMS2 expression between tumor and normal tissues in lung adenocarcinoma (LUAD). The area under the curve (AUC) and corresponding 95% confidence interval are shown. D Comparison of LIMS2 expression levels among different pathological T stages in LUAD. Patients with advanced stages (T2–T4) exhibited significantly lower LIMS2 expression compared with those at early stage (T1). Statistical significance was assessed using the Wilcoxon rank-sum test. ***P < 0.001

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  Fig. 3. Genomic and epigenetic alterations of LIMS2 across cancers and in LUAD. A Pan-cancer mutation frequency of LIMS2 across TCGA tumor types. B Distribution and types of LIMS2 mutations along the protein structure, with LIM domains indicated. C Landscape of copy number variations (CNV) of LIMS2 across multiple cancers, including amplification, mutation, and deep deletion events. D Promoter methylation levels of LIMS2 in LUAD tumor tissues and normal lung samples based on TCGA data

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  Fig. 4. Potential association between m6A-related regulation and LIMS2 expression. A Distribution of predicted m6A modification scores along the LIMS2 transcript, with indicated confidence thresholds and candidate regions. B Predicted RNA secondary structures of LIMS2 highlighting putative m6A-related sites. C, D qRT-PCR analysis of METTL3 and LIMS2 mRNA levels in NCL-H358 (C) and A549 (D) cells following METTL3 knockdown. Data are presented as mean ± SD from three independent experiments. *P < 0.05, ***P < 0.001

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  Fig. 5. Effects of LIMS2 overexpression on proliferation and colony formation in LUAD cells. A, B qRT-PCR analysis of LIMS2 mRNA expression in NCL-H358 (A) and A549 (B) cells following LIMS2 overexpression. C, D Cell proliferation curves measured by CCK-8 assay in NCL-H358 (C) and A549 (D) cells transfected with negative control (NC) or LIMS2 overexpression (oe-LIMS2) plasmids. E, F Representative images and quantitative analysis of colony formation assays in NCL-H358 (E) and A549 (F) cells. Data are presented as mean ± SD from three independent experiments. **P < 0.01, ***P < 0.001

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  Fig. 6. LIMS2 overexpression is associated with reduced migration of LUAD cells. A–B Representative images and quantitative analysis of Transwell migration assays in NCL-H358 (A) and A549 (B) cells transfected with negative control (NC) or LIMS2 overexpression (oe-LIMS2) plasmids. Migrated cells were stained and counted after 24 h. C, D Representative images and quantification of wound healing assays in NCL-H358 (C) and A549 (D) cells at 0 h and 24 h following scratch formation. Migration ability was evaluated by the percentage of wound closure. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

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  Fig. 7. Protein interaction network and functional enrichment analysis of LIMS2. A Protein–protein interaction (PPI) network of LIMS2 and its interacting partners constructed using the STRING database. B Core interaction network extracted from the PPI network, highlighting key LIMS2-associated proteins involved in cytoskeleton and focal adhesion regulation. C Subcellular localization enrichment analysis showing predominant enrichment in focal adhesion- and cytoskeleton-related compartments. D Gene Ontology (GO) cellular component enrichment analysis of LIMS2-associated proteins. E GO biological process enrichment analysis indicating involvement in cell–substrate adhesion, actin cytoskeleton organization, and integrin-mediated signaling. F GO molecular function enrichment analysis highlighting actin binding and cytoskeletal protein binding activities. G KEGG pathway enrichment analysis revealing significant association with focal adhesion, MAPK signaling pathway, Hippo signaling pathway, and pathways in cancer. H GO enrichment overview showing broader cellular context of LIMS2-associated proteins

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  Fig. 8. LIMS2 overexpression is associated with modulation of MAPK- and Hippo-related signaling in vitro and in vivo. A, B Representative Western blot images and quantitative analysis of LIMS2, phosphorylated p38 (p-P38), phosphorylated JNK (p-JNK), phosphorylated YAP (p-YAP), total YAP, and GAPDH in NCI-H358 (A) and A549 (B) cells transfected with negative control (NC) or LIMS2 overexpression (oe-LIMS2) plasmids. Band intensities were normalized to GAPDH or total YAP where appropriate. C–E Representative immunohistochemical staining of LIMS2 (C), phosphorylated YAP (p-YAP) (D), and total YAP (E) in xenograft tumor tissues derived from NC and oe-LIMS2 groups. (F) Semi-quantitative analysis of immunohistochemical staining intensity. Data are presented as mean ± SD from three independent experiments. **P < 0.01; ***P < 0.001; ns, not significant

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  Fig. 9. Association between LIMS2 expression and immune infiltration, immune checkpoint features, and immunotherapy-related signatures. A–F Correlation analysis between LIMS2 expression and immune cell infiltration in TCGA-LUAD samples using TIMER, CIBERSORT, EPIC, MCPcounter, xCELL, and QUANTISEQ algorithms. G Correlations between LIMS2 expression and immune checkpoint–related genes, including inhibitory and stimulatory molecules, in the TCGA-LUAD cohort. H Correlation between LIMS2 expression and immune score in TCGA-LUAD samples. I Lims2 expression in responder and baseline samples from the CT26_ERP114266 dataset following combined anti–CTLA-4 and anti–PD-1 treatment. J–K ELISA analysis of CXCL10 and CCL5 levels in culture supernatants of NCL-H358 and A549 cells transfected with negative control (NC) or LIMS2 overexpression (oe-LIMS2) plasmids. Data are presented as mean ± SD. *P < 0.05, **P < 0.01