Genetic Variations in the Folate Regulatory Enzyme Aldh1l1 and Their Impact on Cancer Cell Metabolism, Proliferation, and Motility

Folate metabolism supports essential biosynthetic pathways that are frequently upregulated in cancer cells. The regulatory mechanisms linking folate metabolism to cancer are complex and depend on the coordinated activities across multiple enzymes. Among these enzymes is ALDH1L1 (10-formyltetrahydrofolate dehydrogenase), which exhibits strong antiproliferative effects. ALDH1L1 is often underexpressed or silenced in several cancer types, supporting its role as a tumor suppressor. Notably, ALDH1L1 contains many common nonsynonymous exonic single-nucleotide polymorphisms (SNPs) that may further influence downstream metabolic pathways and cellular growth.To investigate ALDH1L1’s impact across cancer types, this dissertation examines how genetic variation and altered expression affect cancer cell metabolism, proliferation, migration, and tumorigenicity. Two complementary approaches were used: (1) knock-in of ALDH1L1 into ALDH1L1-deficient cancer cell lines (A549; lung adenocarcinoma, and PC3; prostate adenocarcinoma), and (2) knockdown of ALDH1L1 in RT4 bladder cancer cells, which naturally express high levels of the protein.Lentiviral expression systems were used to generate panels of A549 and PC3 cells expressing the 10 major ALDH1L1 genetic variants. Untargeted metabolomics revealed cell-specific metabotype differences associated with these variants. Certain variants showed reduced ability to regulate one-carbon metabolism and related pathways, including serine-to-glycine conversion, nucleotide biosynthesis, glutathione metabolism, and the TCA cycle. Functionally, these variants exhibited reduced inhibition of proliferation and motility compared to the main haplotype, which showed the strongest suppressive effects. Comparative analyses further indicated that ALDH1L1-mediated metabolic regulation depends on cell-specific capacity to manage one-carbon metabolic pathways.Using an shRNA-based approach, RT4 clones with varying ALDH1L1 expression (high, low, and undetectable) were generated and evaluated in vitro and in vivo using a mouse xenograft model. ALDH1L1 downregulation reprogrammed metabolism across pathways, including fatty acid, amino acid, glutathione, and the TCA cycle. ALDH1L1-deficient clones showed increased proliferation, motility, and tumor growth. Interestingly, partial loss conferred a greater growth advantage than complete loss in vivo, suggesting that limited ALDH1L1 expression may optimize tumor-promoting metabolic conditions.Overall, these findings demonstrate that ALDH1L1 is a key regulator of metabolic pathways linked to proliferation and motility. Both genetic variation and altered expression influence cancer cell behavior, suggesting that ALDH1L1 SNPs contribute to tumorigenesis and cancer-type-specific metabolic regulation.