A pyroptosis-related molecular signature stratifies prognosis and highlights GSDMB-mediated malignancy in intrahepatic cholangiocarcinoma

BACKGROUND: Pyroptosis, a form of inflammatory programmed cell death, plays a dual role in tumor progression and anti-tumor immunity. Its comprehensive clinical and biological significance in intrahepatic cholangiocarcinoma (iCCA) remains to be elucidated. METHODS: Utilizing bulk transcriptome, single-cell transcriptome, proteome, phosphoproteome, spatial transcriptomic profiling and genomic mutation data, we first identified three distinct iCCA molecular subtypes based on the expression patterns of prognostic pyroptosis-related genes (PRGs). Subsequently, we developed and validated an eight-PRG (CASP3, TIRAP, GPX4, NOD2, GSDMB, GSDMC, CASP9, IL6) prognostic signature using regression analyses. The tumor immune microenvironment was characterized, and metabolic pathways were analyzed. Single-cell RNA sequencing (scRNA-seq) of 165,236 cells from iCCA tissues was performed to investigate cellular communication. Spatial transcriptomic profiling was used to visualize cell distribution. In vitro functional assays, including TGF-β treatment, GSDMB and CDH3 knockdown, were conducted to validate mechanistic insights. RESULTS: The eight-PRG signature effectively stratified iCCA patients into high- and low-risk groups with significant survival differences. The high-risk subtype correlated with aggressive clinicopathological features, an immunosuppressive microenvironment, and dysregulated metabolism. scRNA-seq analysis revealed malignant epithelial cells expressing GSDMB showed heightened responsiveness to TGF-β signaling. Functional studies demonstrated that GSDMB knockdown inhibited iCCA cell proliferation, migration, and invasion, potentially through downregulation of CDH3. Further mechanistic studies validated that TGF-β promotes CDH3 transcription in a GSDMB-dependent manner. GSDMB knockdown reduced basal CDH3 expression and abolished TGF-β-induced CDH3 upregulation at both mRNA and protein levels. CONCLUSIONS: We established a novel and clinically applicable pyroptosis-based classifier for iCCA prognosis. Our findings hint at a possible connection of the TGF-β-GSDMB-CDH3 axis with enhanced tumor aggressiveness, providing new insights for patient risk stratification and revealing potential therapeutic targets for iCCA.