METTL3-Driven m6A Modification of Cpt1a Gene in High Fat Diet Related Liver Cancer Tumor Macrophages Facilitates Type II Macrophage Differentiation.

Abstract

Objective: Obesity induces chronic inflammation and hormonal imbalances that contribute to tumor growth. This study explores the less understood dynamics of tumor-related macrophages under a high-fat diet and its consequent impact on tumor growth, with a focus on elucidating the role of high-fat diets on macrophage behavior in liver cancer.

Methods: We established a mouse obesity model using a high-fat diet, combined with a liver cancer implantation approach. Tumor-infiltrating macrophages were isolated for analysis. We investigated the specific effects of a high-fat diet on macrophages through transcriptomic and metabolomic studies and further explored the influence of N6-methyladenosine (m6A) RNA modification on macrophage differentiation using in vitro and in vivo models.

Results: Our findings reveal that a high-fat diet significantly accelerates in-situ liver cancer growth and fosters type II differentiation of tumor-associated macrophages. RNA sequencing indicated upregulation of Cpt1a and Mettl3 genes, which are crucial for m6A modification in macrophages. Using human and mouse macrophage cell lines with either elevated Mettl3 expression or Cpt1a gene knockout, we demonstrated that methyltransferase-like 3 (METTL3) enhances fatty acid metabolism in macrophages, a process reversible by Cpt1a gene knockout. These effects were corroborated in vivo. Further, macrophages infused with high Mettl3 expression, when combined with an in-situ implantation model and adoptive cell therapy, markedly promoted liver cancer growth and increased type II macrophage differentiation (p < 0.001). Knockout of the Cpt1a gene counteracted the METTL3 effect compared to the control group (p > 0.05). METTL3 and m6A RNA Immunoprecipitation (RIP) assays confirmed that METTL3 stabilizes Cpt1a mRNA. Additionally, multispectral staining of clinical specimens revealed a positive correlation between METTL3 protein levels in liver cancer tumor-associated macrophages and M2 macrophage prevalence, inversely correlating with M1 macrophages (p < 0.01). High Mettl3 expression in macrophages was associated with poor prognosis in liver cancer patients, correlating significantly with tumor size and tumor node metastasis (TNM) classification stage.

Conclusion: Our research identifies that a high-fat diet elevates METTL3-driven m6A modification of carnitine palmitoyltransferase 1A (CPT1A) in tumor macrophages, fostering type II macrophage differentiation, and exacerbating liver cancer growth and immune evasion.