Targeting LRPPRC lactylation disrupts metabolic-immune crosstalk and restores antitumor immunity in hepatocellular carcinoma.

Abstract

Background: The Warburg effect drives lactate accumulation in the tumor microenvironment (TME), where it functions as a signaling molecule. Lactate-derived lysine lactylation (Kla) is a novel post-translational modification (PTM) implicated in regulating immune cell function. Leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) is overexpressed in hepatocellular carcinoma (HCC) and plays key roles in mitochondrial metabolism and immune evasion. However, whether and how LRPPRC is regulated by lactylation to coordinate metabolic-immune crosstalk in HCC remains unknown. This study aims to investigate the role and mechanism of LRPPRC lactylation in linking tumor glycolysis to macrophage polarization in HCC.

Methods: Bioinformatics analysis identified lactate metabolism-related genes and hub nodes in HCC datasets. LRPPRC lactylation was detected via immunoprecipitation and western blot using pan-Kla antibody. The specific lactylation site was mapped by prediction database and validated by site-directed mutagenesis (K326R). Functional impacts of LRPPRC-Kla³²⁶ on HCC cell proliferation, invasion, and glycolysis were assessed using Cell Counting Kit-8 (CCK-8), Transwell, wound-healing, and Seahorse assays. The role of LRPPRC-Kla³²⁶ in macrophage polarization was examined in bone marrow-derived macrophages (BMDMs) from LRPPRC^WT and LRPPRC^K326R knock-in mice using flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), and cytokine measurement. An in vivo tumor admix model co-injecting Lewis lung carcinoma (LLC) cells with polarized BMDMs was used to evaluate tumor growth and immune cell infiltration.

Results: LRPPRC was identified as a hub gene among lactate metabolism-related genes in HCC and was upregulated in tumor tissues, correlating with poor prognosis. LRPPRC undergoes lactylation in a lactate-dependent manner, with K326 being the major modification site. The LRPPRC^K326R mutation impaired HCC cell proliferation, invasion, and glycolytic flux. In macrophages, lactylation at LRPPRCK326was required for lactate-induced M2 polarization and glycolytic reprogramming; the K326R mutation skewed polarization towards an M1 phenotype with reduced glycolysis. In the tumor admix model, co-injection of LRPPRC^K326R M2 macrophages significantly suppressed tumor growth compared to LRPPRC^WT M2 macrophages, which was associated with increased infiltration of activated IFN-γ⁺ CD8⁺ and CD4⁺ T cells.

Conclusions: Lactate-induced lactylation of LRPPRC at K326 serves as a critical metabolic-immune switch in HCC. It enhances tumor glycolysis and simultaneously drives M2-like macrophage polarization, fostering an immunosuppressive TME conducive to tumor progression. Targeting the LRPPRC-Kla³²⁶ axis may represent a promising therapeutic strategy to disrupt the metabolic symbiosis between tumor cells and immune cells in HCC.