Integrative bulk and single-cell transcriptomics link EZH2 to immunosuppressive programs and tumor-Treg crosstalk in castration-resistant prostate cancer.

Background: Enhancer of zeste homolog 2 (EZH2) is frequently upregulated in prostate cancer (PCa) and further increased in castration-resistant prostate cancer (CRPC), a lethal state characterized by profound immune dysfunction. However, EZH2-associated immune programs in bulk cohorts and their corresponding cell-type-specific features in single-cell RNA sequencing (scRNA-seq) data have not been systematically delineated in advanced PCa. Methods: We integrated bulk RNA-seq and scRNA-seq to map EZH2-associated transcriptional and immune features in PCa. In bulk cohorts (TCGA-PRAD and an independent metastatic CRPC cohort), we quantified EZH2 expression, clinical outcomes, and immune-signature enrichment. Immune-modulated differentially expressed genes (IMDEGs) were defined by intersecting EZH2-associated differential expression with correlations to Treg/TAM-related signature scores, and were used for NMF-based immune subtyping and penalized Cox modeling with validation. In scRNA-seq cohorts (GSE264573 and an independent CRPC cohort), malignant epithelial cells were inferred by copy-number alteration profiles, EZH2^high versus EZH2^low malignant programs were characterized, T-cell subsets were quantified, and tumor-Treg communication was inferred using CellPhoneDB as hypothesis-generating predictions. For perturbation, the EZH2 inhibitor tazemetostat was evaluated in the CRPC-relevant C42 cell line with H3K27me3 readouts and transcriptomic profiling, with key changes validated by RT-qPCR. Results: Across bulk cohorts, higher EZH2 expression was associated with adverse clinical outcomes and increased enrichment of immunosuppressive signatures, including Treg- and TAM-related programs. IMDEG-based NMF subtyping identified patient groups with distinct immune states, and an IMDEG-derived risk score stratified prognosis. Single-cell profiling revealed elevated EZH2 in CRPC malignant cells and Tregs; EZH2^high malignant cells exhibited a proliferative transcriptional state accompanied by reduced expression of immune-related programs. Predicted tumor-Treg interaction patterns were stronger in CRPC and positively associated with EZH2 expression. In C42 cells, tazemetostat reduced H3K27me3 and induced coordinated transcriptional changes, including upregulation of immune- and inflammation-associated genes such as TIMP3, PLCG2, and SOCS3, validated by RT-qPCR. Conclusion: This multi-layer integrative analysis suggests that EZH2 is associated with proliferative malignant states and immunosuppressive microenvironment features in advanced PCa, including Treg-linked crosstalk. Transcriptomic profiling following EZH2 inhibition supports modulation of these programs by EZH2-targeted perturbation, while functional and causal mechanisms warrant further investigation.