Tumour suppressor role of Serine hydroxymethyltransferase in Drosophila melanogaster

Serine hydroxymethyltransferase (SHMT) works in the one carbon (1C) pathway to regulate essential cellular processes such as nucleotide synthesis and epigenetic maintenance. SHMT catalyses the reversible interconversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methyleneTHF (meTHF). The one carbon units produced in this reaction are used by thymidylate synthase enzyme (TS) to produce Thymidylate (dTMP) an essential precursor of DNA. Therefore, it is expected that a depletion of SHMT could reduce the biosynthesis of dTMP, thus increasing the genome instability. To test this hypothesis, we depleted SHMT in Drosophila melanogaster, using the RNA interference strategy, which reduced the catalytic activity of SHMT of about 40%. We showed that SHMT depletion resulted in chromosome damage that was also produced by RNAi-mediated TS silencing. Interestingly, dTMP supplementation reduced the chromosome aberrations suggesting the SHMT depletion impacts on genome integrity by impairing the folate cycle. More interestingly, we found that the administration of pyridoxal 5’-phosphate (PLP), the enzymatic cofactor of SHMT was able to rescue the chromosome damage. In contrast, the administration of the PLP antagonist 4-deoxypyridoxine (4DP) exacerbated the CAB phenotype, thus unveiling a gene-nutrient relationship between SHMT and vitamin B6 which impacts on genome stability. Several studies associated the overexpression of SHMT2 and, at lesser extent SHMT1, to cancer indicating that this activity takes part to metabolic reprograming by supplying cancer cells with molecular building blocks essential for their rapid growth. On the other hand, only a few studies have focused on the consequence of reduced SHMT levels in cancer. Dottorato di ricerca in Genetica e Biologia Molecolare Pag. 9 In this thesis we exploited the RasV12DlgRNAi Drosophila cancer model to test whether the RNAi-induced silencing of SHMT could increase the malignancy of tumours induced by the expression of the RasV12 oncogene combined to silencing of Disc large (Dlg) polarity gene in larval eye discs. From this analysis it emerged that SHMT depletion exacerbates cancer phenotype of RasV12DlgRNAi tumours and causes genome instability by synergizing with the tumour background. CABs induced by SHMT silencing were rescued by dTMP supplementation that likewise rescued the malignant phenotype of RasV12DlgRNAi larvae, thus linking genome instability to tumours. Moreover, we provided evidence that the levels of PLP can modulate the tumour phenotypes as well as the genome instability resulting from SHMT silencing, thus suggesting that the gene-nutrient interaction between SHMT and vitamin B6 is able to impact on cancer through the genome damage. We found that SHMT depletion in RasV12DlgRNAi cells promotes the formation reactive oxygen species (ROS) due to the overexpression of the NAPDH oxidase enzyme, suggesting that ROS may contribute to CABs which, in turn, might be responsible for promoting tumour phenotypes in the RasV12DlgRNAi cells depleted of SHMT. Taken together, our studies highlighted a clear tumour suppressor role of SHMT that acts as a safeguard against the genome damage and also showed, for the first time, how the interplay between SHMT activity and the vitamin B6 availability can modulate the cancer risk by impacting on genome integrity.