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.

In humans, six common non-synonymous exonic single-nucleotide polymorphisms in ALDH1L1 lead to ten enzyme haplotypes. Since ALDH1L1 is a metabolic regulator and a candidate tumor suppressor, we studied how haplotype variants affect metabotypes, proliferation and motility. Using lentiviral delivery to A549 and PC3 cells, we have generated two cell panels, with each cell line in the panel carrying one of ten ALDH1L1 haplotype variants. Untargeted metabolomics revealed strong differences in metabotypes between cells carrying ALDH1L1 variants. Certain ALDH1L1 variants had decreased ability to regulate one-carbon metabolism and downstream pathways, including amino acid and glutathione metabolism, nucleotide biosynthesis, and the TCA cycle. The direct comparison of A549 and PC3 cells using an untargeted metabolomic dataset also showed that the two cell lines have vastly different metabotypes. In agreement with this finding, the analysis of A549 and PC3 ALDH1L1 haplotype panels suggested that the ALDH1L1-linked metabolic regulation is strongly influenced by specific cellular metabotypes and genetic background. ALDH1L1 variants also exhibited reduced inhibition of cellular proliferation and motility compared to the most common haplotype, which supports further investigation of their potential relevance to individual risk of metabolic disorders.