Metabolic reprogramming of colorectal cancer cells resistant to 5-FU

Metabolic rearrangements are essential to satisfy the different needs of cancer cells during tumorigenesis. Recent studies highlighted a role for such metabolic reprogramming in adaptation to therapies and chemo-resistance development. 5-fluorouracil (5-FU) is an antimetabolite drug widely used as a first-line treatment for colorectal cancer. Despite several advantages of 5-FU, its clinical application is still greatly limited, due to the acquisition of drug resistance. In the first part of this thesis, we illustrate the role of micro RNAs (miRNAs) in reprogramming colon cancer cells toward a resistant phenotype as well as their involvement in the response of resistant cells to acute treatment with 5-FU. We performed a global gene expression profile for the entire miRNA genome, and we found a change in the expression of four miRNAs following acute treatment with 5-FU in cells resistant to this drug. Among them, we focused on miR-210-3p, previously described as a key regulator of DNA damage repair mechanisms and mitochondrial metabolism. Here we show that miR-210-3p downregulation enables resistant cells to counteract the toxic effect of the drug increasing the expression of RAD-52 protein, involved in DNA damage repair. Moreover, miR-210-3p downregulation enhances mitochondrial oxidative metabolism, increasing the expression levels of succinate dehydrogenase subunits D, decreasing intracellular succinate levels and inhibiting HIF-1α expression. These results suggest that miR-210-3p downregulation following 5-FU treatment sustains DNA damage repair and metabolic adaptation to counteract drug treatment, thus supporting the resistant phenotype. In the second part of this thesis, we reveal important adaptations in serine and one-carbon metabolism associated with the acquisition of 5-FU resistance in colorectal cancer cells. 5-FU resistant cells showed an increase in both serine up-take from extracellular medium and de novo serine synthesis pathway. Together with increased serine availability, dynamic labeling experiment after 13C-serine incubation underlined a different utilization of serine-derived carbons in resistant cells with a sustained flux into the mitochondrial compartment supporting increased purine nucleotides synthesis. Accordingly, we found a strong decrease in the expression of the cytosolic isoform of the enzyme serine hydroxy-methyltransferase (SHMT1) and a concomitant increase in the expression of the mitochondrial isoform (SHMT2) in 5-FU resistant cells compared to parental cells, confirming the shift toward mitochondrial one-carbon branch activity. Accordingly, higher expression levels of the mitochondrial serine transporter SFXN1 have been observed in resistant cells with respect to the sensitive ones. Silencing SHMT2 in 5-FU resistant cells increases the efficacy of the treatment with 5-FU against resistant cells confirming the importance of the reported adaptation in the acquisition of resistance to 5-FU. In conclusion, the data shown in this thesis underline different adaptations related to both miRNAs expression and nutrient metabolism carried out by 5-FU resistant cells. This reprogramming supports the response of 5-FU resistant cells to overcome the toxic effect of the drug. The identification of such alterations opens the possibility of new therapeutic approaches to tackle resistant cells and overcome colon cancer relapse.