WTAP-mediated m6A Modification of JUNB Contributes to Poor Prognosis of HCC Patients Through the NLRP3-GSDMD Pathway

Hepatocellular carcinoma (HCC) remains one of the most lethal malignancies worldwide, accounting for a significant proportion of cancer-related deaths. Despite advancements in therapeutic strategies such as surgical resection, liver transplantation, and radiofrequency ablation, the 5-year overall survival rate for HCC patients remains dismal at approximately 18%. High rates of recurrence and metastasis underscore the urgent need to identify novel therapeutic targets and molecular mechanisms driving HCC progression. Emerging evidence highlights the dual role of pyroptosis—a form of inflammatory programmed cell death—in cancer biology. While pyroptosis can suppress tumorigenesis by eliminating malignant cells, excessive inflammatory cytokine release may paradoxically foster an immunosuppressive tumor microenvironment (TME) that promotes HCC aggressiveness. Recent studies implicate RNA N6-methyladenosine (m6A) modifications in regulating pyroptosis, yet the interplay between m6A dynamics and pyroptosis in HCC remains poorly understood.

WTAP Overexpression Correlates With HCC Malignancy and Poor Prognosis

Analysis of The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset revealed significant dysregulation of m6A-modifying enzymes in HCC tissues. Among these, Wilms’ tumor 1-associated protein (WTAP), a key component of the m6A methyltransferase complex, exhibited the highest tumor-specific overexpression compared to adjacent normal tissues. Patients with elevated WTAP expression demonstrated markedly worse overall survival (OS) than those with low WTAP levels. Multivariate Cox regression analysis confirmed WTAP as an independent prognostic factor for HCC, with hazard ratios (HRs) indicating its strong association with clinical outcomes. Furthermore, WTAP expression correlated positively with advanced tumor grade, suggesting its role in promoting HCC dedifferentiation and malignancy.

In vitro validation using normal hepatocytes (LO2) and HCC cell lines (Hep3B, PLC, Huh7, and HepG2) confirmed consistent WTAP upregulation in cancer cells. siRNA-mediated WTAP knockdown in Hep3B and PLC cells reduced global m6A levels by approximately 40%, establishing WTAP’s functional role in maintaining m6A modification in HCC. Transcriptomic profiling of WTAP-silenced cells identified significant enrichment in inflammation-related pathways, particularly the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) signaling pathway. This pathway is critically involved in activating caspase-1 and gasdermin D (GSDMD)—the executor of pyroptosis—through NLRP3 inflammasome assembly.

WTAP Drives Pyroptosis via the NLRP3-GSDMD Cascade

Functional experiments demonstrated WTAP’s direct involvement in regulating pyroptotic cell death. Hep3B cells treated with lipopolysaccharide (LPS) and nigericin—a potassium ionophore that activates the NLRP3 inflammasome—showed characteristic pyroptotic features, including cell swelling and membrane rupture. However, WTAP knockdown reduced pyroptosis by 60% compared to controls. Western blot analysis revealed concomitant downregulation of cleaved caspase-1 (p20 subunit) and mature GSDMD (GSDMD-NT) in WTAP-deficient cells, indicating impaired inflammasome activation. These findings position WTAP as a critical upstream regulator of the NLRP3-caspase-1-GSDMD axis in HCC.

JUNB Serves as the Key Downstream Target of WTAP-Dependent m6A Modification

To elucidate the molecular mechanism linking WTAP to pyroptosis, methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA immunoprecipitation (RIP) assays identified JUNB—a proto-oncogenic transcription factor—as a primary m6A-modified target of WTAP. MeRIP-qPCR confirmed a 70% reduction in JUNB mRNA m6A methylation upon WTAP silencing. RIP assays using anti-WTAP antibodies pinpointed a novel m6A site at position +1456 in the JUNB coding sequence. This modification stabilized JUNB transcripts, as evidenced by accelerated JUNB mRNA decay (half-life reduced from 8 hours to 4 hours) in WTAP-knockdown cells treated with the transcription inhibitor actinomycin D.

JUNB’s role as a transcriptional activator of NLRP3 was validated through shRNA-mediated knockdown experiments. Depleting JUNB in Hep3B cells suppressed NLRP3 expression by 50%, abrogating caspase-1 activation and GSDMD cleavage. Crucially, WTAP overexpression failed to rescue NLRP3-GSDMD signaling in JUNB-deficient cells, confirming JUNB as the indispensable mediator of WTAP’s pro-pyroptotic effects.

Clinical Validation of the WTAP-JUNB-NLRP3 Axis in HCC Progression

Immunohistochemical (IHC) analysis of 56 HCC patient tissues revealed strong WTAP and JUNB co-expression in tumor regions compared to adjacent non-tumor tissues. High WTAP/JUNB expression correlated with poor histological differentiation (P < 0.01) and shorter OS (median OS: 28 months vs. 45 months for low-expression groups). Multiplex IHC further linked WTAP overexpression to an immunosuppressive TME characterized by elevated FOXP3+ regulatory T cell infiltration (r = 0.62, P < 0.001) and reduced CD8+ cytotoxic T lymphocyte density (r = −0.54, P < 0.01). These findings suggest that WTAP-driven pyroptosis may paradoxically exacerbate immune evasion by fostering a TME permissive for HCC progression.

Therapeutic Implications and Mechanistic Insights

This study unveils a previously unrecognized epigenetic axis wherein WTAP-mediated m6A modification stabilizes JUNB mRNA, amplifying NLRP3-dependent pyroptosis in HCC. While pyroptosis is traditionally viewed as antitumorigenic, excessive activation in this context appears to drive inflammation-mediated immunosuppression and tumor aggressiveness. The dual role of pyroptosis in HCC highlights the importance of contextual therapeutic targeting. Small-molecule inhibitors targeting WTAP’s methyltransferase activity or JUNB-NLRP3 interactions could potentially normalize pyroptotic signaling and restore antitumor immunity.

Notably, WTAP’s regulatory effect on pyroptosis operates independently of its canonical partners METTL3 and METTL14, as their expression remained unchanged upon WTAP knockdown. This suggests context-specific roles for WTAP in HCC pathogenesis, possibly through recruitment of alternative co-factors or direct RNA-binding activities.

Conclusion

By integrating multi-omics datasets, functional assays, and clinical validation, this work establishes WTAP as a master regulator of m6A-dependent pyroptosis in HCC. The WTAP-JUNB-NLRP3-GSDMD axis promotes tumor progression through both cell-intrinsic mechanisms (enhanced pyroptosis-driven inflammation) and extrinsic remodeling of the immune microenvironment. These findings position WTAP as a promising prognostic biomarker and therapeutic target for circumventing immunosuppression in HCC. Future studies investigating WTAP inhibitors in combination with immune checkpoint blockers may yield novel strategies to improve patient outcomes.

doi.org/10.1097/CM9.0000000000003468

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