H4K5 lactylation - ENO2 loop drives glycolysis and HCC progression.

Histone lactylation is a lactate-dependent epigenetic modification driven by glycolysis. Although elevated histone lactylation occurs in various cancers, the biological relevance of histone H4 lactylation in hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to investigate the clinicopathological relevance of histone H4 lysine 5 lactylation (H4K5la) and its role in regulating lactate metabolism to promote HCC progression. H4K5la, H3K18la, and panKla (global lactylated lysine) expression levels were evaluated by immunohistochemistry in HCC tissues (n = 104). RNA sequencing and chromatin immunoprecipitation qPCR analyses were performed in HCC cells treated with lactate to identify downstream target genes regulated by H4K5la. Functional validation of these targets was conducted using knockdown and overexpression experiments, and biological effects were assessed in vitro and in vivo. Elevated H4K5la, H3K18la, and panKla levels were associated with poor prognosis, and H4K5la was identified as an independent prognostic factor of overall survival (p = 0.0064). Upon lactate treatment, four genes including the glycolytic enzyme enolase 2 (ENO2) were identified in three HCC cell lines, showing enrichment of H4K5la in their promoter regions. ENO2 knockdown reduced intracellular lactate and H4K5la levels, and in vivo tumorigenicity in HCC cells (p <0.001), whereas ENO2 overexpression exerted the opposite effects. These findings indicated a positive feedback loop: H4K5la increased ENO2 expression, which in turn amplified glycolysis and lactate production, further elevating H4K5la levels. Moreover, ENO2 expression closely correlated with H4K5la levels in HCC tissues. High histone H4K5la serves as an independent prognostic biomarker in HCC and promotes tumor progression via a positive feedback loop with ENO2. Targeting this H4K5la-ENO2 axis may represent a novel therapeutic strategy for patients with HCC and the hyper-lactylation phenotype. Despite limited understanding of the role of histone H4 lactylation in cancer, this study identifies H4K5la as a critical epigenetic regulator of hepatocellular carcinoma (HCC) progression. Lactate-induced elevation of H4K5la enhances ENO2 expression, which in turn increases H4K5la levels, establishing a positive H4K5la-ENO2 feedback loop in HCC. These data highlight that elevated H4K5la and ENO2 expression are potential prognostic biomarkers and therapeutic targets in patients with HCC.