Abstract
Despite recent advances in targeted therapeutics, administration of 5-fluorouracil (5-FU) remains a common clinical strategy for post-surgical treatment of solid tumors. Although it has been proposed that RNA metabolism is disturbed by 5-FU treatment, the key cytotoxic response is believed to be enzymatic inhibition of thymidylate synthase resulting in nucleotide pool disproportions. An operating p53 tumor suppressor signaling network is in many cases essential for the efficiency of chemotherapy, and malfunctions within this system remain a clinical obstacle. Since the fate of chemotherapy-insensitive tumor cells is rarely described, we performed a comparative analysis of 5-FU toxicity in p53-deficient cells and conclude that p53 acts as a facilitator rather than a gatekeeper of cell death. Although p53 can act as a regulator of several cellular stress responses, no rerouting of cell death mode was observed in absence of the tumor suppressor. Thus, the final death outcome of 5-FU-treated p53-/- cells is demonstrated to be caspase-dependent, but due to a slow pace, accumulation of mitochondrial reactive oxygen species contributes to necrotic characteristics. The oligomerization status of the p53 target gene DR5 is determined as a significant limiting factor for the initiation of caspase activity in an intracellular TRAIL-dependent manner. Using several experimental approaches, we further conclude that RNA-rather than DNA-related stress follows by caspase activation irrespectively of p53 status. A distinct 5-FU-induced stress mechanism is thereby functionally connected to a successive and discrete cell death signaling pathway. Finally, we provide evidence that silencing of PARP-1 function may be an approach to specifically target p53-deficient cells in 5-FU combinatorial treatment strategies. Together, our results disclose details of impaired cell death signaling engaged as a consequence of 5-FU chemotherapy. Obtained data will contribute to the comprehension of factors restraining 5-FU efficiency, and by excluding DNA as the main stress target in some cell types they propose alternatives to currently used and suggested synergistic treatment regimens.
Highlights
Administration of 5-fluorouracil (5-FU, fluorouracil) is a common post-surgical treatment regimen used for several categories of solid tumors, and especially for patients suffering from colorectal cancers (CRC)
In contrast to the analysis of functional stress pathways where the silencing of key regulatory elements mostly serves as controls, we have explored in detail the kinetics and underlying mechanisms of p53-independent cell death by using parental and genetically-modified HCT116 cells, one of the most common in vitro systems for 5-FU toxicity analyses
To verify that caspase-8-activation occurs at the level of DISC formation, we investigated apoptotic markers appearing in response to 5-FU in HCT116 p53-/- cells stably overexpressing either the regulatory cellular caspase-8 (FLICE)-like inhibitory protein (c-FLIPL) or a truncated version of the Fas-associated death domain (FADD) adaptor protein (FADD-DN)
Summary
Administration of 5-fluorouracil (5-FU, fluorouracil) is a common post-surgical treatment regimen used for several categories of solid tumors, and especially for patients suffering from colorectal cancers (CRC). The therapeutic potential of 5-FU was primarily described as an effect of its metabolic conversion into fluorodeoxyuridine monophosphate (FdUMP), a suicide substrate for thymidylate synthase (TS), and as such able to starve www.impactjournals.com/oncotarget cells from deoxythymidine monophosphate (dTMP) [1]. By this means, a rate-limiting step obligatory for DNA replication and repair is eliminated and a link to cell death mechanisms provided. Misincorporation of 5-FU metabolites into RNA transcripts and genomic DNA appears to occur simultaneously in tumor cells, but no significant correlation between the magnitude of integration into either species and the response to 5-FU therapy has yet been found [3]. Despite the extensive clinical use of 5-FU, the relative importance of each stress target is not clearly established
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