Regulation of Cell Death Pathways by Folate Metabolism

Antifofates hinder cellular division and replication by disrupting metabolic processes necessary for DNA synthesis. This is due to the role of folate coenzymes in the transfer of one carbon units in the enzymatic reactions of thymidine, purine, and amino acid biosynthesis. Understanding the biochemical basis of folate antagonists is still a major focal point in anti-cancer research. In addition to synthetic compounds that inhibit enzymes of folate metabolism, other means of folate stress exist. The major folate enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is of special interest due to its strong down-regulation in several tumor types and its growth inhibitory effects in cancer cell lines deficient in the enzyme. The aim of this project was to investigate cell death mediators in the antiproliferative response to the disruption of folate metabolism by FDH. Earlier studies in our lab demonstrated that FDH expression causes G1 cycle arrest and apoptosis. We also established that these effects were p53-dependent in p53 positive cancer cells lines. In addition, expression of FDH in A549 cells resulted in the elevation of p53 transcriptional targets p21 and PUMA. The first study confirmed our previous notions that FDH expression does not induce DNA damage; instead it causes a considerable decrease in intracellular folates. Most notably, A549 and HCT116 cells showed a significant decrease in 10-formyl-THF, a substrate for two reactions of de novo purine synthesis. Examination of ATP levels demonstrated that FDH results in purine depletion, which was in agreement with our previous findings. We also determined that p21 activation was anti-apoptotic and specifically responsible for the G1 and G2 cell cycle arrest resulting from the metabolic effects of FDH activity. However, knockdown of PUMA prevented FDH-induced apoptosis, indicating that this protein is a crucial mediator of this process. Furthermore, we have demonstrated that accumulation of ceramide is also a key component in the cellular response to metabolite deprivation, as FDH expression does not result in apoptosis when ceramide pathways are inhibited. The increased de novo synthesis of C16-ceramide was found to be dependent on the up-regulation of ceramide synthase 6 (CerS6). While FDH-induced ceramide accumulation was shown to be downstream of p53, we have demonstrated that the knockdown of PUMA in A549 cells prevents ceramide elevation in response to FDH expression. This data suggests a regulatory role for PUMA as an upstream activator of ceramide generation. Overall, this project provides additional insight to the upstream and downstream mechanisms involved in the cellular response to the disruption folate metabolism by FDH.