ASH2L-K312-Lac Stimulates Angiogenesis in Tumors to Expedite the Malignant Progression of Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a common malignant tumor. However, the role of lactic acid-modified proteins in its pathogenesis is unclear. This study determines the distribution of a novel post-translational modification-protein lactylation-in HCC to identify potential targets and obtain mechanistic insights into this disease. Using high-throughput proteomics, lysine 312 lactylation (K312-lac) of the Set1/Ash2 histone methyltransferase complex subunit (ASH2L) is revealed as a candidate for further investigation. Subsequently, alanyl-tRNA synthetase 1 (AARS1) and histone deacetylase 1 (HDAC1) are shown to mediate lactylation modification of ASH2L. In vivo experiments demonstrate that ASH2L-K312-lac promotes HCC malignant progression and is positively correlated with tumor microvessel density, and vascular endothelial growth factor A (VEGFA) is identified as the key mediator in ASH2L-K312-lac-induced angiogenesis. High-throughput sequencing reveals ASH2L-K312-lac enrichment in the genome regions encoding VEGFA, facilitating targeted recruitment of the mixed lineage leukemia complex to these loci and enhancing VEGFA expression through synergistic activation of enhancers and promoters. Finally, clinical sample analyses and robust in vivo preclinical experiments identify ASH2L-K312-lac as a promising therapeutic target for clinical application. These findings provide a theoretical foundation for the clinical translation of ASH2L-K312-lac-based treatment approaches, offering potential advancements in HCC diagnosis and treatment.

Keywords: ASH2L; H3K4me3; HCC; VEGFA; angiogenesis; lactylation.

Figure 1

Lysine‐312 is the key residue that mediates ASH2L lactylation. a) High‐throughput lactylation‐modified omics analysis identified lysine‐312 as a potential lactylation site on the ASH2L protein. b) Development of ASH2L‐WT and ASH2L‐K312R cellular models. First, we used CRISPR‐Cas9 technology to eliminate ASH2L from genomic DNA. Subsequently, we separately introduced wild‐type (WT) and mutant ASH2L plasmids to establish ASH2L‐WT and ASH2L‐K312R cell lines, respectively. c) Western blotting revealed successful establishment of ASH2L‐WT and ASH2L‐K312R cell lines. d) Custom antibody developed to target lysine‐312 lactylation of the ASH2L protein (provided by Huaan Biotechnology Co., Ltd., Hangzhou, China). e) Following the direct addition of lactic acid (a final concentration of 100 mm) to the culture medium for 12 h, western blotting, and immunoprecipitation (IP) assays revealed a significant increase in the total lactylation and corresponding lysine‐312 lactylation levels in ASH2L. f) Western blotting and IP assays revealed a significant reduction in total lactylation and lysine‐312 autosomal lactylation levels in ASH2L in ASH2L‐K312R cells compared with ASH2L‐WT cells. g) Co‐IP screening assay performed to identify potential writers responsible for ASH2L lactylation. Only AARS1 interacted with ASH2L. h) Co‐IP assay revealed physical interaction between AARS1 and ASH2L. i) Western blotting and co‐IP assays revealed significantly elevated levels of total and lysine‐312‐specific lactylation of ASH2L following AARS1 overexpression. j) Western blotting and co‐IP assays revealed that HDAC1 was a delactylase that catalyzes the elimination of lactylation modification from ASH2L. k) Co‐IP assay revealed the physical interaction between HDAC1 and ASH2L. l) Schematic illustrating the synergistic regulation of ASH2L lactylation in hepatocellular carcinoma cells by AARS1 and HDAC1.

Figure 2

Lactylation of ASH2L facilitates the malignant progression of hepatocellular carcinoma (HCC) by promoting vascular endothelial cell proliferation. a) Adeno‐associated virus (AAV) tail vein injection in Ash2l^{flox/flox, Alb‐cre} mice and chemically induced (DEN+CCl₄) HCC mouse models were used to analyze the effect of ASH2L‐K312‐lac on HCC progression (n = 5/group). b) Compared with Ash2l‐WT mice, mutant mice exhibited a significant reduction in tumor size and number following chemical induction of HCC (n = 5/group). c) Single‐cell RNA sequencing (scRNA‐seq) was performed on mouse liver cancer tissues obtained from the aforementioned experiments, followed by cell annotation and clustering. d) Cell annotation and clustering revealed a significant reduction in the proportion of endothelial cells in the mutant group compared with that in the Ash2l‐WT group. e) Multiplex immunohistochemical (mIHC) assays validated the observations derived from the scRNA‐seq‐based clustering analysis. f) Targeted analysis of the scRNA‐seq liver cell subpopulation data revealed that compared with the WT group, the mutant group contained a large number of genes with significant changes, including Vegfa. g) The differentially expressed genes (DEGs) identified above underwent GO enrichment and KEGG signaling pathway analysis, revealing their involvement in facilitating angiogenesis within HCC.

Figure 3

ASH2L lactylation affects angiogenesis in HCC tumor cells via VEGFA. a) The developed Huh7‐ASH2L‐WT and Huh7‐ASH2L‐K312R cells were subjected to transcriptome sequencing (bulk mRNA‐seq). b) Bulk mRNA‐seq data analysis showed that ASH2L‐K312‐lac effectively facilitated the upregulation of VEGFA expression to promote angiogenesis in HCC. c) Analysis using a Proteome Profiler Human Angiogenesis Antibody Array Detection Kit revealed reduced VEGFA secretion in Huh7‐ASH2L‐K312R cells. d) Quantitative polymerase chain reaction (qPCR) and enzyme‐linked immunosorbent assay (ELISA) results revealed reduced VEGFA expression and secretion in Huh‐7 cells with the K312R mutation. e) Tube formation assays revealed that the angiogenic potential of HUVECs to form capillary‐like structures in the supernatant of Huh7‐ASH2L‐K312R cells was significantly lower than that of Huh7‐ASH2L‐WT group. f) The supernatants of Huh7‐ASH2L‐WT and Huh7‐ASH2L‐K312R cells were separately combined with Matrigel for use in Matrigel plug assays in mice. g) Images of the retrieved Matrigel plugs. Compared with the Matrigel plug containing supernatant from the control group, those containing supernatant from the ASH2L‐K312R group showed more vascular infiltration (left), supporting the IHC results (right).

Figure 4

ASH2L‐K312‐lac exhibited increased affinity for VEGFA, facilitating its transcription. a) ChIP‐seq was used to investigate the genomic distribution of ASH2L in Huh7‐ASH2L‐WT and Huh7‐ASH2L‐K312R cells and to analyze its localization patterns. b) Analysis of the distribution disparities of ASH2L‐WT and ASH2L‐K312R across various chromatin elements revealed a significant reduction in the abundance of ASH2L‐K312R within the proximal promoter region. c) ATAC‐seq was used to examine alterations in chromatin accessibility in Huh7‐ASH2L‐WT and Huh7‐ASH2L‐K312R cells. d) The ATAC‐seq results indicated a significant reduction in open chromatin regions within the proximal promoter region of Huh7‐ASH2L‐K312R cells compared with Huh7‐ASH2L‐WT cells. e) Comparison of the disparities in open chromatin regions between Huh7‐ASH2L‐WT and Huh7‐ASH2L‐K312R cells. The overall openness decreased in the ASH2L‐K312R group, with approximately 10% of the peaks representing unique open regions. f) Comprehensive analysis of the unique open chromatin regions in Huh7‐ASH2L‐K312R cells indicated that they influenced various biological processes in HCC, including angiogenesis. g) Integrated analysis of ChIP‐seq, ATAC‐seq, and mRNA‐seq data revealed that ASH2L‐K312R regulates chromatin accessibility by directly binding to genomic DNA, thereby influencing the transcriptional regulation of specific genes, including VEGFA. h) IGV visualization validated the direct binding of ASH2L‐K312‐lac to the transcription start site (TSS) of the VEGFA‐encoding region, facilitating accessibility to this region and influencing VEGFA mRNA expression in HCC.

Figure 5

ASH2L‐K312‐lac facilitates angiogenesis in HCC by modulating H3K4 methylation in the VEGFA‐encoding region. a) Schematic illustrating the role of ASH2L and its associated COMPASS complex in regulating H3K4 methylation by the MLL complex. b) CUT&Tag (anti‐MLL1) was performed in Huh7‐ASH2L‐WT and Huh7‐ASH2L‐K312R cells. c) Statistical analysis of disparities in MLL1 distribution across the genome before and after ASH2L lactylation loss. d) A high level of concordance was observed between the differentially bound sites on genomic DNA due to ASH2L lactylation and the disparate distribution of MLL1 on the chromatin. e) CUT&Tag (anti‐H3K4me1/2/3) analyses were performed using Huh7‐ASH2L‐WT and Huh7‐ASH2L‐K312R cells. f) Separate analysis of the distribution of H3K4me1/3 in Huh7‐ASH2L‐WT and Huh7‐ASH2L‐K312R cells and clustering analysis showed that angiogenesis was among the top regulated biological processes. g) IGV visualization revealed that ASH2L‐K312‐lac regulated the distribution of MLL1 on the VEGFA‐encoding region and modulated H3K4 methylation distribution in that chromatin segment, enhancing VEGFA expression in collaboration with promoters and enhancers.

Figure 6

ASH2L‐K312‐lac is a potential target for the future clinical diagnosis and treatment of HCC. a) Previously prepared tissue microarrays (TMAs) used in conjunction with immunohistochemical (IHC) staining revealed increased ASH2L‐K312‐lac levels in HCC, which were negatively correlated with patient prognosis (P = 0.0028). Scale bar: 500/100 µm. b) Using an identical set of TMAs as described above, IHC staining revealed elevated MVD (indicated by CD31 expression) in HCC tumors, along with a positive correlation with ASH2L‐K312‐lac levels (R = 0.4293, P < 0.0001). Scale bar: 500/100 µm. c) Preliminary investigation procedure using a nude mouse subcutaneous tumor model in conjunction with bevacizumab as a pretreatment strategy. d) The pretreatment experiment demonstrated a significantly slower growth rate of tumors with low ASH2L‐K312‐lac levels, accompanied by a notable enhancement in bevacizumab responsiveness. e) Schematic of the model used in this study. ASH2L undergoes lactylation at Lys312. The high expression levels of ASH2L‐K312‐lac in HCC, in conjunction with the associated COMPASS complex, modulate MLL1 distribution on chromatin, facilitating the methylation of the H3K4 residue in the nearby VEGFA‐encoding region and leading to enhanced VEGFA expression and secretion in HCC. This process promotes angiogenesis within HCC and accelerates disease progression.