Cytosolic 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) is commonly downregulated in human cancers through promoter methylation. We proposed that ALDH1L1 loss promotes malignant tumor growth. Here, we investigated the effect of the Aldh1l1 mouse knockout (Aldh1l1^−/−) on hepatocellular carcinoma using a chemical carcinogenesis model. Fifteen-day-old male Aldh1l1 knockout mice and their wild-type littermate controls (Aldh1l1^+/+) were injected intraperitoneally with 20 μg/g body weight of DEN (diethylnitrosamine). Mice were sacrificed 10, 20, 28, and 36 weeks post-DEN injection, and livers were examined for tumor multiplicity and size. We observed that while tumor multiplicity did not differ between Aldh1l1^−/− and Aldh1l1^+/+ animals, larger tumors grew in Aldh1l1^−/− compared to Aldh1l1^+/+ mice at 28 and 36 weeks. Profound differences between Aldh1l1^−/− and Aldh1l1^+/+ mice in the expression of inflammation-related genes were seen at 10 and 20 weeks. Of note, large tumors from wild-type mice showed a strong decrease of ALDH1L1 protein at 36 weeks. Metabolomic analysis of liver tissues at 20 weeks showed stronger differences in Aldh1l1^+/+ versus Aldh1l1^−/− metabotypes than at 10 weeks, which underscores metabolic pathways that respond to DEN in an ALDH1L1-dependent manner. Our study indicates that Aldh1l1 knockout promoted liver tumor growth without affecting tumor initiation or multiplicity.

Folate (vitamin B9) is involved in one-carbon transfer reactions and plays a significant role in nucleic acid synthesis and control of cellular proliferation, among other key cellular processes. It is now recognized that the role of folates in different stages of carcinogenesis is complex, and more research is needed to understand how folate reactions become dysregulated in cancers and the metabolic consequences that occur as a result. ALDH1L1 (cytosolic 10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism expressed in many tissues, is ubiquitously downregulated in cancers and is not expressed in cancer cell lines. The RT4 cell line (derived from papillary bladder cancer) which expresses high levels of ALDH1L1 represents an exception, providing an opportunity to explore the metabolic consequences of the loss of this enzyme. We have downregulated this protein in RT4 cells (shRNA driven knockdown or CRISPR driven knockout) and compared metabolomes of ALDH1L1-expressing and -deficient cells to determine if metabolic changes linked to the loss of this enzyme might provide proliferative and/or survival advantages for cancer cells. In this study, cell extracts were analyzed using Ultra High Performance Liquid Chromatography High Resolution Mass Spectrometry (UHPLC-HR-MS). A total of 13,339 signals were identified or annotated using an in-house library and public databases. Supervised and unsupervised multivariate analysis revealed metabolic differences between RT4 cells and ALDH1L1-deficient clones. Glycine (8-fold decrease) and metabolites derived from S-adenosylmethionine utilizing pathways were significantly decreased in the ALDH1L1-deficient clones, compared with RT4 cells. Other changes linked to ALDH1L1 downregulation include decreased levels of amino acids, Krebs cycle intermediates, and ribose-5-phosphate, and increased nicotinic acid. While the ALDH1L1-catalyzed reaction is directly linked to glycine biosynthesis and methyl group flux, its overall effect on cellular metabolism extends beyond immediate metabolic pathways controlled by this enzyme.

ALDH1L1 (10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism, is highly expressed in the liver. It regulates the overall flux of folate-bound one-carbon groups by converting 10-formyltetrahydrofolate to tetrahydrofolate and CO₂ in a NADP⁺-dependent reaction. Our previous study revealed that Aldh1l1 knockout (KO) mice have an altered liver metabotype with metabolic symptoms of folate deficiency when fed a standard chow diet containing 2 ppm folic acid. Here we performed untargeted metabolomic analysis of liver and plasma of KO and wild-type (WT) male and female mice fed for 16 weeks either standard or folate-deficient diet. OPLS-DA, a supervised multivariate technique that was applied to 6595 and 10,678 features for the liver and plasma datasets, respectively, indicated that genotype and diet, alone or in combination, gave distinct metabolic profiles in both types of biospecimens. A more detailed analysis of affected metabolic pathways based on most confidently identified metabolites in the liver and plasma (OL1 and OL2a ontology level) indicated that the dietary folate restriction itself does not fully recapitulate the metabolic effect of the KO. Of note, dietary folate withdrawal enhanced the metabolic perturbations linked to the ALDH1L1 loss only for a subset of metabolites. Importantly, both the ALDH1L1 loss and dietary folate deficiency produced sex-specific metabolic effects.

We read with interest the article by Lee et al

ALDH1L1 (10‐formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism, is highly expressed in liver. This enzyme metabolizes 10‐formyltetrahydrofolate to produce tetrahydrofolate (THF) and is involved in the regulation of reduced folate pools, de novo purine biosynthesis, and the flux of folate‐bound methyl groups. The goal of this study was to survey the metabolic impact of the Aldh1l1 gene loss in mouse liver. Aldh1l1 ^{− / −} (KO) mice were generated using gene‐targeted ES cell clone (C57BL/6N background) from KOMP repository. KO mice were viable and did not show obvious phenotypic differences from the wild type animals. Flash frozen hepatic tissues from 12–14 weeks old Aldh1l1 ^+/+ and Aldh1l1 ^{− / −} mice were profiled to identify metabolite changes and define associated pathways. In this study, mice were kept on standard AIN‐93 diet with 2.2 ppm of folic acid. Global metabolomic analysis was performed by Metabolon Inc. (Durham, NC) using Ultrahigh Performance Liquid Chromatography‐Tandem Mass Spectroscopy (UPLC‐MS/MS) for five samples from each group. This analysis has identified 628 known metabolites and principal component analysis demonstrated good segregation between the two genotypes. The difference for 91 metabolites was statistically significant between the two groups, with 31 compounds being decreased and 60 being elevated in Aldh1l1 ^{− / −} compared to the Aldh1l1 ^+/+ mice. This analysis indicated that KO mice might experience functional folate deficiency. Specifically, the intermediate of the histidine degradation pathway serving as a marker of folate deficiency, formiminoglutamate, was increased more than 15‐fold in KO mice. Furthermore, tissue folic acid and dihydrofolate were strongly depleted (more than 2‐fold and 5‐fold, respectively) in these animals, which would be in agreement with folate deficiency. In addition, a 2‐fold decrease in glycine was detected in KO mice. Strong drop in several glycine conjugates was also observed in the KO genotype, which is apparently associated with the deficiency of glycine. The depletion of glycine was likely caused by the decrease of the THF‐dependent synthesis of glycine from serine, with the underlying basis for this phenomenon being the decrease of the THF pool observed in Aldh1l1 ^{− / −} mice. The depletion of glycine and/or folate could be the cause of several secondary effects on metabolism including the decrease in glutathione, lower availability of ascorbate, impaired mitochondrial lipid metabolism, oxidative stress, and less efficient remediation of toxic products. Our data also imply a compromised liver function in the KO mice and associated changes in gut microbiome activity. Overall, our study indicated significant metabolic perturbations caused by the Aldh1l1 gene knockout. Support or Funding Information NIH R01DK 54388 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .