EMT-Dependent Downregulation of Folate Pathway Induces 5-FU Chemoresistance in Cancer

The folate-mediated one-carbon metabolism is a central component of nucleotide biosynthesis. Indeed, several chemotherapeutic drugs, such as 5-fluorouracil (5-FU) and methotrexate (MTX), target major enzymes of this metabolic pathway and are used in cancer treatment. However, despite their high efficacy, patients gradually develop resistance necessitating by yet undefined mechanisms. Here, we set to systematically determine the cellular mechanisms by which cancers develop resistance to antifolates-based drugs. First, by employing dose-response cell viability assays, we identified that resistance to 5-FU treatment is higher in cells expressing mesenchymal rather than epithelial markers. In addition, gene expression analysis in both cancer patients and cell lines identified a negative correlation between folate pathway genes and mesenchymal markers. Next, we induced the epithelial-mesenchymal transition (EMT) program in lung cancer A549 and pancreatic cancer PANC-1 epithelial cells using transforming growth factor β (TGFβ). This stimulation resulted in 5-FU resistance and the downregulation of the folate pathway genes as well as enzymes. Finally, we detected a significant TGFβ - dependent reduction in adenosine biosynthesis by applying LC-MS-based tracing experiments. Furthermore, EMT-induced cells exhibit reduced proliferation rate that was rescued upon supplementing with 5-mTHF (folate pathway intermediate metabolite) and hypoxanthine (purine salvage pathway nucleotide precursor), individually. Overall, we demonstrate EMT-dependent metabolic remodeling wherein, due to the folate pathway downregulation, the cells are less dependent on this pathway and subsequently become resistant to antifolate-based drugs.