Multi-Dimensional Data Integration Reveals the Molecular Mechanism of Metabolic Reprogramming Associated With Low Asparaginase Expression in Hepatocellular Carcinoma

BACKGROUND: To investigate the impact of low expression of asparaginase (ASPG) in hepatocellular carcinoma (HCC) on tumor metabolic reprogramming, tumor microenvironment interactions, and drug sensitivity, and to assess the potential of ASPG as a tumor suppressor gene through multi-dimensional functional mechanisms. METHODS: Thus, ASPG expression in HCC was systemically evaluated by integrating global multi-center mRNA datasets, including RNA-seq and microarray data from 3967 HCCs and 2645 non-HCC samples, single-cell RNA sequencing (scRNA-seq) data from 10 HCCs and 8 adjacent normal tissues, spatial transcriptomics (STs), and internal immunohistochemistry data from 301 HCCs and matched adjacent liver tissues. A CRISPR-mediated gene knockout model was employed to examine the effects of ASPG deletion on HCC cell proliferation. ASPG-associated regulatory pathways were analyzed using Gene Set Enrichment Analysis (GSEA), GeneMANIA, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Changes in ligand-receptor interactions between low-expression ASPG (ASPG-low) and the tumor microenvironment were examined using CellChat, based on scRNA-seq data. Single-cell metabolism (scmetabolism) and single-cell flux estimation analysis (scFEA) were applied to infer metabolic activity and metabolite conversion pathways under ASPG-low conditions. Finally, correlations between ASPG expression and the IC50 values of anticancer drugs were assessed using OncoPredict. RESULTS: Multi-dimensional analyses consistently showed that both ASPG mRNA and protein levels were significantly downregulated in HCC. CRISPR-mediated ASPG knockout was associated with significantly increased cell proliferation. Mechanistically, HCC cells with low ASPG expression were enriched in pathways related to alanine, aspartate, and glutamate metabolism, as well as amino acid biosynthesis, gluconeogenesis, and lipid metabolism. Cell-cell communication analysis revealed strong interactions between ASPG-low malignant hepatocytes and myeloid cells, with significant activation of the MIF-(CD74+CXCR4) and MIF-(CD74+CD44) signaling axes. Metabolic analysis demonstrated that the ASPG-low state was associated with alanine, aspartate, and glutamate metabolism, as well as the citric acid (TCA) cycle, thereby regulating the conversion of the aspartate-asparagine and glutamate-2OG metabolites. ASPG-low HCC was associated with resistance to drugs such as cisplatin, oxaliplatin, and gemcitabine, but increased sensitivity to lapatinib and paclitaxel. CONCLUSION: Low ASPG expression in HCC may drive aspartate metabolism and reprogramming of the TCA cycle, thereby influencing sensitivity to drug treatment.