TRIM21-mediated METTL3 degradation promotes PDAC ferroptosis and enhances the efficacy of Anti-PD-1 immunotherapy

Abstract

Pancreatic cancer remains the most lethal human malignancy with limited clinical benefits from currently available anticancer treatments. Ferroptosis has recently attracted great attention as a potential antineoplastic strategy. However, the study of ferroptosis in PDAC remains insufficient. This study revealed that Methyltransferase like 3 (METTL3), as a key oncogenic factor, is frequently upregulated and inhibits ferroptosis by stabilizing SLC7A11 mRNA in PDAC. In addition, we identified a novel post-translational modification of METTL3 and characterized specific regulatory mechanisms of METTL3 protein degradation. The E3 ligase TRIM21 mediated K48-linked polyubiquitination of METTL3 at the K459 site, leading to the proteasomal degradation of METTL3, which prevented tumor progression by promoting ferroptosis. Interestingly, the TRIM21-METTL3 axics mediated ferroptosis effectively increased the expression of immune checkpoint PD-L1 and strengthened antitumor immunity in pancreatic cancer. Together, our findings first elucidated the detailed molecular mechanism of METTL3 degradation and revealed the pivotal role of the TRIM21-METTL3 axis in regulating ferroptosis and antitumor immunity, which may serve as a potential target for pancreatic cancer treatment.

Fig. 1. METTL3 is upregulated in pancreatic cancer.

A, B METTL3 expression between pancreatic cancer tissues and normal tissues in GEO database. C The protein expression of METTL3 in cell lines was detected by western blotting. D–F Representative images of METTL3 IHC staining in paired pancreatic cancer and adjacent normal tissues with different malignancy histologic grades (G1–G3). Scale bars, 100 μm. G The degree of METTL3 immunohistochemical staining was evaluated in pancreatic cancer and adjacent normal tissues according to the immunoreactive score (IRS). H IRS of METTL3 IHC staining in tumors with different malignancy histological grades (G1–G3). I Kaplan–Meier survival plots (log-rank test, P values displayed) demonstrated the prognostic impact of METTL3 expression in pancreatic cancer. J Kaplan–Meier survival plots exhibited the effect of METTL3 expression on the prognosis of pancreatic cancer in GEO database GSE183795. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 2. TRIM21 directly interacts with METTL3.

A, B Flag-tagged METTL3 was transfected into AsPC-1 and MIA PaCa-2 cells for 48 h, followed by immunoprecipitation with the anti-Flag antibody and immunoblotting analysis with anti-TRIM21 antibody. C Immunoprecipitation analysis with anti-Flag antibody was used to detect the interaction between exogenous Flag-METTL3 and His-TRIM21 in MIA PaCa-2 cells. D, E Immunoprecipitation with anti-METTL3 antibody verified the interaction between endogenous METTL3 and TRIM21 in pancreatic cancer cells. F Confocal immunofluorescence of endogenous METTL3 colocalized with TRIM21 stained by anti-METTL3 (red) and anti-TRIM21 (green) antibodies in pancreatic cancer cells. Scale bars, 10 μm. G Western Blot analysis for pulldown of purified His-TRIM21 incubated with purified GST or GST-METTL3 fusion protein. H Schematic of TRIM21 and different domain-deletion mutants. I Immunoprecipitation analysis with anti-Flag antibody to explore the interaction domain of TRIM21.

Fig. 3. TRIM21 promotes the proteasomal degradation of METTL3.

A, B The protein expression levels of METTL3 were detected by western blot assays in AsPC-1 and MIA PaCa-2 cells transfected with sh-TRIM21. C, D The protein expression levels of METTL3 were detected by western blot assays in AsPC-1 and MIA PaCa-2 cells transfected with TRIM21-WT or TRIM21-LD mutant. E, F In the presence of CHX (100 μg/ml), cell proteins were detected at the indicated time to analyze the content of METTL3 protein in the lysate. G, H Western blot analysis for METTL3 protein expression in TRIM21 overexpression cells followed by the treatment with MG132 (10 μM) or chloroquine (10 μM) for 6 h. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns not significant).

Fig. 4. TRIM21 mediates the K48-linked polyubiquitination of METTL3 at K459 site.

A IP-IB of ubiquitinated METTL3 in AsPC-1 cells treated with MG132 (10 μM) for 8 h. B IP-IB of ubiquitinated METTL3 in AsPC-1 cells treated with MG132 for different time. C IP-IB of ubiquitinated METTL3 using AsPC-1 cells transfected with Flag-METTL3, HA-ubiquitin and/or sh-TRIM21, followed by treatment with MG132. D IP-IB of ubiquitinated METTL3 using MIA PaCa-2 cells transfected with Flag-METTL3, HA-ubiquitin and/or His-TRIM21, followed by treatment with MG132. E IP-IB of ubiquitinated METTL3 using MIA PaCa-2 cells transfected with Flag-METTL3, HA-ubiquitin and/or His-TRIM21 WT or His-TRIM21-LD, followed by treatment with MG132. F IP-IB of ubiquitinated METTL3 using 293 T cells transfected with Flag-METTL3, HA-Ub (WT, K48 only or K63 only) and/or His-TRIM21 and treated with MG132. G Flag-METTL3 or arginine substitution mutants were expressed in 293 T cells and Lysates were prepared for IP-IB.

Fig. 5. TRIM21 promotes ferroptosis by targeting METTL3 in vitro.

A Flow cytometry analysis was used to determine cell death rates in subgroups of METTL3-knockdown cells treated with various cell death inhibitors. B The electron microscopy images show changes in mitochondria following treatment with shMETTL3 in MIA PaCa-2 cells. Scale bar, 2.0μm and 500 nm. C RIP-qPCR revealed the binding enrichment of SLC7A11 to METTL3 in MIA PaCa-2 cells. Western blot was used to verify the immunoprecipitation efficiency of METTL3 protein. D, E MeRIP-qPCR analysis demonstrated the impact of METTL3 on mediating m6A methylation modification of SLC7A11 mRNA in pancreatic cancer cells. F, G The SLC7A11 mRNA expression was measured by RT-qPCR in pancreatic cancer cells with the intervention of TRIM21 and METTL3. H–K The Altered protein expression of SLC7A11 was measured by western blot in subgroups. L The cell death induced by TRIM21 overexpression was effectively rescued by METTL3 overexpression or treatment with ferroptosis inhibitors ferrostatin-1 (1 μM). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns not significant).

Fig. 6. TRIM21 promotes ferroptosis by targeting METTL3 in vivo.

A Treatment scheme for subcutaneous tumor models. B Tumors were excised and photographed. C Differences in tumor volume in response to different treatments (n = 5). D Quantification of immunofluorescence tissue staining with anti-4-HNE antibody. E Hematoxylin and eosin (H&E) staining and immunofluorescence staining with anti 4-HNE antibody. Scale bar, 50 µm. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns not significant).

Fig. 7. TRIM21-METTL3 axis promotes the therapeutic efficacy of PD-1 antibodies.

A Schematic showing the treatment schedule to evaluate anti–mPD-1 treatment in different groups. B Tumors were dissected and photographed. C Differences in tumor volume in response to different treatments (n = 5). D, E IF staining and quantification showing the expression of PD-L1 in different groups. Scale bar, 50 µm. F Flow cytometry analysis exhibiting the proportions of infiltrating CD4⁺ and CD8⁺ cells in tumor tissues in response to different groups. G, H Granzyme B and IFN-γ expression in CD8⁺ T cells from anti PD-1–treated tumors. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns not significant).

Fig. 8. A proposed molecular model.

TRIM21-mediated METTL3 degradation promotes PDAC ferroptosis and enhances the efficacy of Anti-PD-1 immunotherapy.