Studying the role of E2F1 and E2F2 in the metabolism of nucleotides: therapeutic implications for the treatment of prostate cancer.

Abstract

E2F-dependent transcriptional network ensures the timely entry of cells into S-phase through the regulation of a vast array of genes involved in DNA replication and cell cycle progression. In this Thesis, we aimed to decipher the role of E2F1 and E2F2 in the metabolism of nucleotides and the implications that E2F activity might have in tumor progression and resistance against chemotherapy. We show that in non-tumoral cells, targeted depletion of E2f1 and E2f2 in mice leads to a downregulation of genes encoding Tk1 and Dck (rate-limiting enzymes of the salvage pathway for nucleotide production), as well as to the activation of DNA damage. Importantly, this phenotype could be rescued with an exogenous supply of nucleotides.In tumor cells, enhanced E2F activity is thought to supply the nucleotides required for their exacerbated DNA replication. We found that the expression of E2F1/E2F2 correlates with malignancy in human prostate cancer and is necessary to maintain steady-state levels of TK1, DCK and TYMS and to prevent genomic instability during S phase. Silencing of E2F1/E2F2 in metastatic prostate cancer (mPCa) cells leads to DNA damage and compromises cellular viability, whereas addition of nucleosides, or the products of TK1 and DCK enzymatic activities improves cellular viability of E2F1/E2F2 knockdown cells, suggesting that the E2F/TK1-DCK-TYMS axis is crucial to preserve DNA integrity in mPCa cells. Furthermore, we show that mPCa cells are resistant to the apoptosis induced by the nucleoside analog 5-fluorouracil (5-FU) through the induction of TK1, DCK and TYMS expression. Remarkably, combined silencing of E2F1/E2F2 prevents 5-FU-induced upregulation of TK1, DCK and TYMS and reverses resistance of mPCa cells to 5-FU, whereas adding nucleosides or the products of TK1 and DCK activities increases the viability of E2F1/E2F2 knockdown cells treated with 5-FU. Interestingly, treatment of mPCa cells with the E2F pathway inhibitor Palbociclib in combination with 5-FU dramatically reduced their viability.Collectively, our findings emphasize the relevance of the E2F pathway for providing nucleotide intermediates necessary to preserve DNA integrity in normal and cancer cells, and for driving resistance to 5-FU of mPCa cells. Inhibition of E2F activity could be a promising strategy to fully prevent nucleotide biosynthesis and sensitize tumor cells to cell death after treatment with 5-FU.