WTAP-mediated m6A modification of JUNB contributes to poor prognosis of HCC patients through the NLRP3-GSDMD pathway

To the Editor: Hepatocellular carcinoma (HCC) is a prominent contributor to cancer-related mortality on a global scale. While therapeutic approaches, such as surgical resection, liver transplantation, and radiofrequency ablation, have demonstrated effectiveness for specific patient populations, the 5-year overall survival (OS) rate in HCC is discouragingly low, estimated at only 18%. This unfavorable outcome can primarily be attributed to the high recurrence and metastasis rates.[1] Therefore, there is an urgent need for novel therapeutic strategies or targets to surmount the challenges in treating HCC. It has been postulated that tumor resistance to pyroptosis and an immunosuppressive tumor microenvironment significantly contribute to a suboptimal therapeutic response in various types of cancer interventions.[2] Pyroptosis, a form of programmed cell death triggered by inflammation, is distinguished by the formation of membrane pores mediated by gasdermin (GSDM) proteins. This process results in the release of cellular contents and cytokines, thus inciting a robust inflammatory response.[3] The intricate connections between pyroptosis and HCC have been unveiled. On the one hand, pyroptosis demonstrates the ability to inhibit tumorigenesis and tumor progression, presenting a favorable effect on anticancer therapy.[4] On the other hand, the release of inflammatory molecules prompted by pyroptosis creates environments conducive to the proliferative, invasive, metastatic, and drug-resistance potential of tumor cells.[5] Key regulators of pyroptosis have also been implicated in the establishment of an immunosuppressive microenvironment and linked to poorer prognosis. Given the conflicting roles of pyroptosis in HCC, investigating the underlying molecular mechanistic actions of pyroptosis in tumorigenesis and progression holds paramount importance in unearthing novel biomarkers and therapeutic targets. Epigenetic abnormalities play pivotal roles in HCC development and progression, and alterations in RNA N6-methyladenosine (m6A) modifications are involved in pyroptosis.[6] However, the potential connection between m6A modification and pyroptosis in HCC has not yet been extensively investigated. Epigenetic abnormalities play crucial roles in HCC development and progression. Additionally, alterations in RNA m6A modifications have been proposed to be linked with pyroptosis. In our analysis of the The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset, we examined the expression patterns of m6A writers and erasers in both cancerous and adjacent normal tissues. Interestingly, we discovered that among all the modifying enzymes, Wilms' tumor 1-associated protein (WTAP) exhibited the highest expression specifically in tumor tissues [Supplementary Figure 1A, https://links.lww.com/CM9/C298]. Furthermore, patients exhibiting high expression of WTAP had a poorer prognosis compared to those with low expression levels [Supplementary Figure 1B, https://links.lww.com/CM9/C298]. WTAP can serve as an independent prognostic factor, as suggested by both univariate and multivariate clinical analyses [Supplementary Figures 1C, D, https://links.lww.com/CM9/C298]. Additionally, there is a strong correlation between WTAP and the malignancy degree of HCC [Supplementary Figure 1E, https://links.lww.com/CM9/C298]. We assessed the expression level of WTAP in both normal hepatocytes and HCC cell lines, revealing markedly higher levels in cancer cells [Supplementary Figure 2A, https://links.lww.com/CM9/C298]. Following the transfection of Hep3B and PLC cells with synthetic small interfering RNA (siRNA) targeting WTAP [Supplementary Figure 2B, https://links.lww.com/CM9/C298], we observed a consistent reduction in the m6A level of HCC cells when the expression of WTAP was silenced [Supplementary Figure 2C, https://links.lww.com/CM9/C298]. We categorized the expression of WTAP and generated a heatmap of the differentially expressed genes [Supplementary Figure 2D, https://links.lww.com/CM9/C298]. Pathway enrichment analysis revealed that WTAP was involved in various inflammation and immune pathways. Notably, the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) signaling pathway caught our attention due to its involvement in the caspase-1-gasdermin D (GSDMD) pyroptosis pathway [Supplementary Figure 2E, https://links.lww.com/CM9/C298]. To further confirm the involvement of WTAP in the apoptosis-related pathway in HCC, we conducted experiments where Hep3B cells were treated with lipopolysaccharide (LPS) and nigericin individually. Intriguingly, knocking down WTAP exhibited a remarkable reduction in cell pyroptosis [Figure 1A]. Additionally, we observed a noticeable downregulation in the protein levels of caspase-1 and GSDMD [Figure 1B]. Although WTAP has been reported to function in m6A modification through its interaction with methyltransferase-like 3 (METTL3) and METTL14, WTAP knockdown in HCC cells did not lead to a significant change in the expression levels of METTL3 and METTL14. This suggests that there might be an alternative pathway through which WTAP influences m6A modification [Supplementary Figure 3A, https://links.lww.com/CM9/C298]. Afterward, we utilized Cytoscape to construct the regulatory network of WTAP and its target genes.[7] Intriguingly, we found a correlation between WTAP and four target genes: JUNB, HK2, CCNB1, and ENO1 [Figure 1C]. The mRNA levels of HK2, JUNB, and ENO1 were significantly reduced after WTAP knockdown [Figure 1D]. However, through the methylation immunoprecipitation combined with quantitative real-time polymerase chain reaction (MeRIP-qPCR) assay, a notable reduction in m6A modification was observed only in JUNB after WTAP knockdown [Figure 1E]. Consequently, we predicted potential m6A sites on JUNB (http://www.cuilab.cn/sramp; Supplementary Figures 3B, C, https://links.lww.com/CM9/C298) and subsequently confirmed them through a RNA immunoprecipitation (RIP) assay. Finally, we established that WTAP could bind to a novel m6A site at position + 1456 on JUNB [Figure 1F], thereby maintaining the stability of JUNB mRNA [Figure 1G]. JUNB enhances the mRNA and protein expression of NLRP3.[8] To further investigate whether WTAP could regulate HCC pyroptosis through JUNB, we established a stable Hep3B cell line expressing JUNB-short hairpin RNA (shRNA) to analyze the role of JUNB in WTAP-involved pyroptosis [Supplementary Figure 3D, https://links.lww.com/CM9/C298]. Our findings demonstrated that the reduction of JUNB significantly inhibited the pyroptotic pathway of NLRP3-caspase-1-GSDMD, and this activity could not be restored by WTAP overexpression [Figures 1H, I]. This finding indicates that JUNB is required for WTAP-involved pyroptosis.Figure 1: WTAP promotes HCC cell pyroptosis through the JUNB-NLRP3-GSDMD pathway. (A) Microscope images (scale bar: 100 μm) and histogram showing relative pyroptotic cells in Hep3B cells transfected with si-NC or si-WTAP1 and treated with 0.1% LPS and 10 μmol/L nigericin. (B) Western blot analysis of WTAP, caspase-1, and GSDMD protein levels in Hep3B cells transfected with si-NC or si-WTAP1 after treatment with 10 μmol/L nigericin and 0.1% LPS. (C) Networks depicting the relationship between WTAP and its targets. (D) qRT-PCR results indicating the relative mRNA levels of WTAP, HK2, CCNB1, JUNB, and ENO1 in Hep3B cells transfected with si-NC or si-WTAP1. (E) MeRIP-seq showing m6A enrichment of HK2, ENO1, JUNB, and CCNB1 in Hep3B cells after si-NC or si-WTAP1 transfection. (F) RIP-RT-PCR assays detecting m6A sites in Hep3B cell lysates immunoprecipitated with WTAP antibody. (G) qPCR results showing JUNB mRNA levels at different time points after treatment with actinomycin D in Hep3B cells transfected with si-NC or si-WTAP. (H) Western blot analysis of WTAP, JUNB, NLRP3, Caspase-1, and GSDMD protein levels in Hep3B cells transfected with shNC, shJUNB, or shJUNB plus WTAP after treatment with 0.1% LPS and 10 μmol/L nigericin. (I) Microscope images (scale bar: 100 μm) and histogram depicting relative pyroptotic cells in Hep3B cells transfected with shNC, shJUNB, or shJUNB plus WTAP and treated with 0.1% LPS and 10 μmol/L nigericin. "ns" indicates not significant, * P <0.05, † P <0.01, ‡ P <0.001. HCC: Hepatocellular carcinoma; LPS: Lipopolysaccharide; m6A: N6-methyladenosine; meRIP-seq: Methylated RNA immunoprecipitation sequencing; qPCR: Quantitative polymerase chain reaction; qRT-PCR: Quantitative reverse transcription polymerase chain reaction; WTAP: Wilms' tumor 1-associated protein.To investigate the clinical significance of WTAP, we analyzed tissue sections from 56 patients with HCC. immunohistochemical (IHC) staining and the resulting IHC scores suggested a higher expression level of WTAP in tumor tissues than in adjacent non-tumor tissues [Supplementary Figures 4A, B, https://links.lww.com/CM9/C298]. Using IHC scoring of WTAP, the tissue sections of the 56 patients were categorized into two groups. Although no correlation existed between WTAP expression and patient age, sex, tumor size, or clinical stage, a negative association was observed between WTAP and HCC differentiation [Supplementary Figure 4C, https://links.lww.com/CM9/C298]. Furthermore, patients with lower WTAP expression levels exhibited better OS [Supplementary Figure 4D, https://links.lww.com/CM9/C298]. To explore potential connections between WTAP and the immune microenvironment, we analyzed the correlation of WTAP with JUNB, CD8, and FOXP3. Patients with high expression of WTAP showed increased levels of JUNB and FOXP3, as well as decreased CD8 staining. This indicates an immunosuppressive microenvironment characterized by enhanced infiltration of regulatory T cells and a reduced number of cytotoxic T cells in patients with high expression of both WTAP and JUNB [Supplementary Figure 4E, https://links.lww.com/CM9/C298]. In conclusion, we validated the pivotal role of WTAP in HCC pyroptosis. WTAP enhances the NLRP3-associated GSDMD pyroptosis pathway by binding to and stabilizing JUNB mRNA, creating an immune-suppressive microenvironment that fosters HCC development. As a crucial m6A writer involved in pyroptosis, WTAP emerges as a critical determinant of immune suppression in HCC and holds promise as a potential target for HCC immunotherapy. Acknowledgments The authors thank Professor Guangjin Guo from the Laboratory of Air Force Medical Center for his assistance with the immunohistochemistry experiments. Funding This work was supported by grants from the Capital's Funds for Health Improvement and Research (No. 2022-25121) and Project of the Eighth Medical Center of PLA General Hospital (No. 2021MS003). Conflicts of interest None.