Positive Feedback Regulation of Glycolysis by H4K5 Lactylation Promotes Malignant Progression of Hepatocellular Carcinoma [ChIP-seq]

[Background & Aims] Histone lactylation is a lactate-dependent epigenetic modification driven by glycolysis. Although elevated histone lactylation has been reported in various cancer, the biological relevance of histone H4 lactylation in HCC remains unclear. This study aimed to investigate the clinicopathological relevance of H4K5 lactylation (H4K5la) and its role in regulating lactate metabolism to promote HCC progression. [Methods] The expression levels of H4K5la, H3K18la, and PanKla were evaluated in HCC tissues by immunohistochemistry. RNA-seq and ChIP-PCR analyses were performed on HCC cells treated with lactate to identify the downstream target genes mediated by H4K5la. Functional validation of these targets was conducted through knockdown and overexpression experiments, and biological effects were assessed both in vitro and in vivo. [Results] Elevated levels of H4K5la, H3K18la, and PanKla were associated with a poor prognosis, with H4K5la identified as an independent prognostic factor of overall survival. Upon lactate treatment, five genes including glycolytic enzyme ENO2 were consistently identified in three HCC cell lines, showing enrichment of H4K5la at their promoter regions. ENO2-KD reduced intracellular lactate and H4K5la levels, as well as in vivo tumorigenicity in HCC cells, while ENO2 overexpression exerted opposite functions. These findings establish a positive feedback loop: H4K5la increases ENO2 expression, which in turn amplifies glycolysis and lactate production, further elevating H4K5la. Moreover, ENO2 expression was tightly correlated with H4K5la levels in HCC tissues. [Conclusions] High H4K5la expression is an independent prognostic biomarker in HCC. H4K5la promotes HCC aggressiveness by enhancing glycolysis and tumor growth, forming a positive feedback loop with ENO2. Targeting this pathway may represent a promising therapeutic strategy for HCC.