Altered L-Arginine Metabolic Pathways in Gastric Cancer: Potential Therapeutic Targets and Biomarkers

Abstract

There is a pressing need for molecular targets and biomarkers in gastric cancer (GC). We aimed at identifying aberrations in L-arginine metabolism with therapeutic and diagnostic potential. Systemic metabolites were quantified using mass spectrometry in 293 individuals and enzymes' gene expression was quantified in 29 paired tumor-normal samples using qPCR and referred to cancer pathology and molecular landscape. Patients with cancer or benign disorders had reduced systemic arginine, citrulline, and ornithine and elevated symmetric dimethylarginine and dimethylamine. Citrulline and ornithine depletion was accentuated in metastasizing cancers. Metabolite diagnostic panel had 91% accuracy in detecting cancer and 70% accuracy in differentiating cancer from benign disorders. Gastric tumors had upregulated NOS2 and downregulated ASL, PRMT2, ORNT1, and DDAH1 expression. NOS2 upregulation was less and ASL downregulation was more pronounced in metastatic cancers. Tumor ASL and PRMT2 expression was inversely related to local advancement. Enzyme up- or downregulation was greater or significant solely in cardia subtype. Metabolic reprogramming in GC includes aberrant L-arginine metabolism, reflecting GC subtype and pathology, and is manifested by altered interplay of its intermediates and enzymes. Exploiting L-arginine metabolic pathways for diagnostic and therapeutic purposes is warranted. Functional studies on ASL, PRMT2, and ORNT1 in GC are needed.

Keywords: arginine auxotrophy; argininosuccinate lyase; argininosuccinate synthase; dimethylarginine; dimethylarginine dimethylaminohydrolase; metabolic reprogramming; nitric oxide synthase; ornithine decarboxylase; ornithine translocase; protein arginine methyltransferase.

Figure 1

Overview of metabolic pathways of L-arginine. Pathway players not investigated in the current study are indicated by the grey color, while the analyzed pathway metabolites are depicted in rectangular frames, and enzymes in elliptical frames. Inhibitory effects are marked by black blunt-ended arrows—dashed if the effect is weak. ADMA, asymmetric dimethylarginine; SDMA, symmetric dimethylarginine; DMA, dimethylamine; ASL, argininosuccinate lyase; ARG1,2, arginase 1 and 2; ASS1, argininosuccinate synthase 1; CATs, cationic amino acid transporters; DDAH1,2, dimethylarginine dimethylaminohydrolase 1 and 2; NO, nitric oxide; NOS2, nitric oxide synthase 2; ODC1, ornithine decarboxylase 1; PRMT, protein arginine methyltransferase.

Figure 2

Serum concentrations of metabolites associated with arginine metabolism in patients with gastric cancers or benign disorders: (a) Arginine (Arg); (b) Citrulline (Cit); (c) Ornithine (Orn); (d) Asymmetric dimethylarginine (ADMA); (e) Symmetric dimethylarginine (SDMA); (f) Dimethylamine (DMA). Data presented as dot-plots with means (Arg and ADMA), geometric means (Orn and DMA) or medians (Cit and SDMA), accompanied by 95% confidence interval (orange squares with whiskers). Data were analyzed using one-way analysis of variance with Scheffe post-hoc test (Arg, Orn, ADMA, and DMA) or Kruskal–Wallis H test with Conover post-hoc test (Cit and DMA). Groups differing significantly in a post-hoc analysis (p < 0.05) are indicated by the same type of symbol: *, #, ^, +, or ∍. CTRL, controls; BN, benign gastric disorders; CA, cardia subtype of gastric adenocarcinoma; GC, non-cardia subtype of gastric adenocarcinoma.

Figure 3

Effect of anatomical site on enzyme expression: (a) NOS2 in cardia subtype of gastric adenocarcinoma (CA; n = 11); (b) NOS2 in non-cardia subtype of gastric adenocarcinoma (GA; n = 16); (c) ORNT1 in CA (n = 12); (d) ORNT1 in GA (n = 17); (e) ASL in CA (n = 12); (f) ASL in GA (n = 17); (g) PRMT2 in CA (n = 11); (h) PRMT2 in GA (n = 11). Data analyzed using t-test for paired samples (NOS2) or Wilcoxon test and presented as geometric means or medians with 95% confidence interval. T/N, tumor-to-normal expression ratio; N/T, normal-to-tumor expression ratio; NOS2, inducible nitric oxide synthase; ORNT1, ornithine translocase 1; ASL, argininosuccinate lyase; PRMT2, protein arginine methyltransferase; NRQ, normalized relative quantities.

Figure 3

Effect of anatomical site on enzyme expression: (a) NOS2 in cardia subtype of gastric adenocarcinoma (CA; n = 11); (b) NOS2 in non-cardia subtype of gastric adenocarcinoma (GA; n = 16); (c) ORNT1 in CA (n = 12); (d) ORNT1 in GA (n = 17); (e) ASL in CA (n = 12); (f) ASL in GA (n = 17); (g) PRMT2 in CA (n = 11); (h) PRMT2 in GA (n = 11). Data analyzed using t-test for paired samples (NOS2) or Wilcoxon test and presented as geometric means or medians with 95% confidence interval. T/N, tumor-to-normal expression ratio; N/T, normal-to-tumor expression ratio; NOS2, inducible nitric oxide synthase; ORNT1, ornithine translocase 1; ASL, argininosuccinate lyase; PRMT2, protein arginine methyltransferase; NRQ, normalized relative quantities.

Figure 4

Effect of anatomical site on ARG1 expression in: (a) macroscopically normal tumor-adjacent tissues; (b) tumors. Data presented as geometric means with 95% confidence interval and analyzed using t-test for independent samples. ARG1, arginase-1; NRQ, normalized relative quantities; CA, cardia subtype of gastric adenocarcinoma; GA, non-cardia subtype of gastric adenocarcinoma.

Figure 5

Effect of metastasis on NOS2 and ASL expression: (a) Lymph node metastasis and tumor NOS2 expression; (b) Distant metastasis and tumor NOS2 expression; (c) Distant metastasis and ASL expression ratio. Data presented as geometric means and analyzed using t-test for independent samples (NOS2) or medians and analyzed using Mann–Whitney U test (ASL). NOS2, inducible nitric oxide synthase; ASL, argininosuccinate lyase; NRQ, normalized relative quantities; N/T, normal-to-tumor expression ratio.