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Background <p>Hepatocellular carcinoma (HCC) is characterized by profound metabolic reprogramming, including altered nitrogen metabolism that supports tumor survival. phosphoribosyl pyrophosphate amidotransferase (ppAT) is the rate-limiting enzyme in purine biosynthesis, catalyzing the incorporation of glutamine-derived nitrogen. However, clinical significance of ppAT in HCC remain largely undefined.</p> Methods <p>Transcriptomic data from TCGA-LIHC and paired clinical HCC samples were analyzed to assess ppAT expression and its prognostic value. Functional assays including CCK-8, EdU, colony formation, wound healing, Transwell invasion, flow cytometry, and Western blotting were conducted in HCC cell lines with ppAT knockdown. Glutamine deprivation, amino acid supplementation, and LC-MS–based metabolomics were employed to investigate metabolic dependencies. IMPDH inhibition and a xenograft mouse model were used to evaluate therapeutic implications in vitro and in vivo.</p> Results <p>ppAT was significantly upregulated in HCC tissues and correlated with advanced TNM stage and shortened progression-free survival. Silencing ppAT impaired proliferation, migration, invasion, and colony formation, induced G1/S arrest and apoptosis, and heightened glutamine dependency. Under glutamine-free conditions, asparagine—but not glutamate—partially rescued the growth of PPAT-deficient cells, revealing a substrate-specific asparagine rescue mechanism for nitrogen stress adaptation. Metabolomic profiling revealed that ppAT knockdown reduced intracellular levels of purine intermediates (IMP, AMP, GMP), ATP, and NADPH, thereby defining a glutamine–purine–energy axis in which PPAT couples glutamine-derived nitrogen to both nucleotide supply and bioenergetic/redox homeostasis. This was accompanied by compensatory upregulation of GLS1 and ASNS. Combined treatment with ppAT knockdown and IMPDH inhibitor mycophenolic acid (MPA) showed synergistic antitumor effects. In vivo, ppAT depletion significantly suppressed tumor growth in a xenograft model.</p> Conclusions <p>PPAT drives HCC via a glutamine–purine–energy metabolic axis and a substrate-specific asparagine rescue mechanism under glutamine stress. These features make PPAT a key metabolic vulnerability and support PPAT–IMPDH co-targeting as a potential HCC therapy.</p>

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ppAT drives glutamine-dependent purine biosynthesis and malignant progression in hepatocellular carcinoma

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月21日

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  • Junbin Zhou,
  • Cainiao Dong,
  • Shengqian Xu,
  • Jun Wang,
  • Hailin Ye,
  • Xiaopeng Fan,
  • Shimiao Li,
  • Shi Wang

摘要

Background

Hepatocellular carcinoma (HCC) is characterized by profound metabolic reprogramming, including altered nitrogen metabolism that supports tumor survival. phosphoribosyl pyrophosphate amidotransferase (ppAT) is the rate-limiting enzyme in purine biosynthesis, catalyzing the incorporation of glutamine-derived nitrogen. However, clinical significance of ppAT in HCC remain largely undefined.

Methods

Transcriptomic data from TCGA-LIHC and paired clinical HCC samples were analyzed to assess ppAT expression and its prognostic value. Functional assays including CCK-8, EdU, colony formation, wound healing, Transwell invasion, flow cytometry, and Western blotting were conducted in HCC cell lines with ppAT knockdown. Glutamine deprivation, amino acid supplementation, and LC-MS–based metabolomics were employed to investigate metabolic dependencies. IMPDH inhibition and a xenograft mouse model were used to evaluate therapeutic implications in vitro and in vivo.

Results

ppAT was significantly upregulated in HCC tissues and correlated with advanced TNM stage and shortened progression-free survival. Silencing ppAT impaired proliferation, migration, invasion, and colony formation, induced G1/S arrest and apoptosis, and heightened glutamine dependency. Under glutamine-free conditions, asparagine—but not glutamate—partially rescued the growth of PPAT-deficient cells, revealing a substrate-specific asparagine rescue mechanism for nitrogen stress adaptation. Metabolomic profiling revealed that ppAT knockdown reduced intracellular levels of purine intermediates (IMP, AMP, GMP), ATP, and NADPH, thereby defining a glutamine–purine–energy axis in which PPAT couples glutamine-derived nitrogen to both nucleotide supply and bioenergetic/redox homeostasis. This was accompanied by compensatory upregulation of GLS1 and ASNS. Combined treatment with ppAT knockdown and IMPDH inhibitor mycophenolic acid (MPA) showed synergistic antitumor effects. In vivo, ppAT depletion significantly suppressed tumor growth in a xenograft model.

Conclusions

PPAT drives HCC via a glutamine–purine–energy metabolic axis and a substrate-specific asparagine rescue mechanism under glutamine stress. These features make PPAT a key metabolic vulnerability and support PPAT–IMPDH co-targeting as a potential HCC therapy.