Differential Expression of One-Carbon Pathway Enzyme ALDH1L1 Is Linked to Tumorigenicity of Low-Grade Bladder Cancer Cells Through Metabolic Reprogramming.

BACKGROUND

RT4 bladder cancer cell line, derived from a nonmuscle-invasive low-grade subtype, is one of the few neoplastic cell lineages that maintain high expression of the candidate tumor suppressor ALDH1L1. Here, we investigated how differential ALDH1L1 expression affects cellular characteristics and tumorigenicity of RT4 cells as well as tumor metabotypes.

METHODS

We characterized RT4 cells and two shRNA clones (sh506/low ALDH1L1 expression; sh572/ALDH1L1 is lost) for proliferation, migration, clonogenic capacity, and mitochondrial respiration. We have further evaluated the tumorigenic potential of RT4 cells and the two clones in nude mice and compared metabotypes of derived tumors using untargeted metabolomics.

RESULTS

Both clones with diminished ALDH1L1 expression exhibited increased proliferation rates with doubling times of 19.4 h (sh506) and 23.2 h (sh572) versus 36.3 h for RT4 cells. Downregulation of ALDH1L1 expression also enhanced motility and clonogenic capacity. Proliferation and clonogenic capacity were highest for the sh506 clone (low ALDH1L1 expression), while motility was strongest for the sh572 clone (complete ALDH1L1 loss). Both clones showed altered energy metabolism, as indicated by a reduced basal oxygen consumption rate and enhanced maximal respiration rate following oligomycin treatment. Mouse xenograft tumors derived from ALDH1L1-deficient RT4 clones were significantly larger than RT4 cell-derived tumors. Of note, complete ALDH1L1 loss (sh572 clone) was less advantageous for tumor growth than the partial loss of the protein (sh506 clone). Untargeted metabolomics has shown that tumors with downregulated ALDH1L1 have altered the metabolism of fatty acids, amino acids, CoA, and acylcarnitines. Alterations in several key pathways, including glutathione metabolism (sh506), and TCA cycle (sh572), depend on the extent of ALDH1L1 downregulation.

CONCLUSIONS

Our study underscores ALDH1L1 as a key metabolic regulator of proliferation, migration, and tumorigenicity in RT4 bladder cancer cells, suggesting that retaining low ALDH1L1 expression can provide a metabolic advantage for growth of aggressive tumors.