Integrative analysis of HMGB3 in esophageal carcinoma: expression profile, prognostic significance, and immune microenvironment associations

Background: Esophageal carcinoma (ESCA) remains highly lethal and lacks robust biomarkers for early detection and risk stratification. High mobility group box 3 (HMGB3) has been implicated as an oncogene in several cancers, but its role in ESCA has not been fully defined. To address these clinical and biological gaps, this study systematically characterized the expression profile, diagnostic and prognostic value, biological functions, and immune-related features of HMGB3 in ESCA through integrative bioinformatics analyses and in vitro validation.

Methods: HMGB3 expression across multiple cancer types was analyzed using the Tumor Immune Estimation Resource (TIMER) database. For ESCA, transcriptomic data, clinicopathological characteristics, and survival information were examined using The Cancer Genome Atlas (TCGA), University of Alabama at Birmingham Cancer (UALCAN), and Gene Expression Profiling Interactive Analysis (GEPIA). Functional enrichment analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA), were performed to identify HMGB3-associated pathways. Immune cell infiltration was assessed using Microenvironment Cell Populations-counter (MCP-counter), Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT), Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE), and TIMER. Gene Set Cancer Analysis (GSCA) was employed to evaluate the associations between HMGB3 expression and drug sensitivity. HMGB3 protein expression was further validated by immunohistochemistry in ESCA tissues and adjacent non-tumorous tissues and by western blotting in ESCA cell lines. The functional effects of HMGB3 knockdown in ESCA cells were evaluated using Cell Counting Kit-8 (CCK-8) proliferation assays, Transwell invasion assays, and flow cytometric analysis.

Results: HMGB3 was broadly upregulated across multiple cancer types and was significantly overexpressed in ESCA, in which its high expression was correlated with advanced clinicopathological stage and poor prognosis. Enrichment analyses indicated that HMGB3-associated genes were involved in immune regulation, cell adhesion, DNA replication, and cell cycle control, as well as Wnt and Hedgehog signaling pathways. Immune profiling revealed negative correlations between HMGB3 expression and CD8⁺ and CD4⁺ T cells, dendritic cells, and natural killer (NK) cells, but a positive correlation with M2 macrophages. HMGB3 expression was also associated with multiple immune cell-related markers and key immune checkpoint molecules, including PD-1, CTLA-4, CD47, and CD276. Drug sensitivity analyses suggested that HMGB3 expression might be linked to responses to several small-molecule inhibitors. In vitro experiments indicated that HMGB3 knockdown inhibited ESCA cell proliferation and invasion and altered cell cycle progression.

Conclusions: HMGB3 is markedly overexpressed in ESCA and is associated with unfavorable clinicopathological features and prognosis. It promotes malignant phenotypes and is linked to an immunosuppressive tumor microenvironment. These findings support the potential utility of HMGB3 as a biomarker for ESCA diagnosis and prognosis.