5-fluorouracil
: mechanisms of action (or resistance) in p53-proficient and -deficient colorectal cancer

  • Fiammetta Falcone

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Colorectal cancer (CRC) is the second most common cancer for women and the third for men with an estimated 690,000 death per year worldwide. 5- fluorouracil (5-FU) remains the backbone of treatment of primary CRC, which is most frequently combined with Oxaliplatin in stage III and high-risk stage II patients. However, 20-30% of these patients relapse with a treatment- refractory disease, likely correlated to their molecular background. Mutations in the TP53 tumour suppressor are observed in at least 50% of all primary CRC, with frequency increasing in the metastatic and recurrent settings. While TP53 status has been shown to influence Oxaliplatin response, the correlation between 5-FU and p53 status and its interaction with Oxaliplatin are still not well understood.

Using a panel of p53 isogenic models of colorectal cancer, we conducted extensive phenotypic and functional genomic analyses of the effects of 5-FU and its combination with Oxaliplatin. Treatment of these models with clinically relevant doses of chemotherapy revealed that, in contrast to Oxaliplatin, 5- FU induced cell death is largely p53-independent. Interestingly, 5-FU treatment resulted in a dramatic increase in S-phase in p53-deficient cells, which coincides with increased DNA damage. This effect is not observed in p53-proficient cells due to the cell cycle inhibitory effects of p21. Remarkably, we found that DNA damage and S-phase arrest are accompanied by a significant increase in the expression of the immune-regulator Programmed death-ligand 1 (PD-L1).

Integrative analysis of results of a genome-wide 5-FU GeCKO CRISPR screen in HCT116 p53 isogenic models, identified and validated a number of clinically tractable genes, which influence the sensitivity to 5-FU. These genes include the Caspase-8 inhibitor CFLARtFLIP (Fas-associated death domain (FADD)-like IL1 (3-converting enzyme inhibitory protein), which we identified as a negative regulator of both p53-dependent and -independent death. Interestingly, FLIP expression can be attenuated by Entinostat (Class I HDAC inhibitor). Importantly, we observed that the combination of Entinostat with 5-FU significantly impacted long-term survival in a p53-independent manner, while its combination with Oxaliplatin enhanced p53-dependent effects on cell death and long-term survival.

In conclusion, our investigation supports two novel combination treatments for 5-FU-based therapies correlated to p53-status. First, in p53-deficient patients, we propose the use of 5-FU-based treatments (5-FU, FOLFOX) in combination with anti-PD-L1 treatment. Second, in p53-proficient patients, we propose the combination of 5-FU or FOLFOX with class I Entinostat. Further investigations are necessary to support these proposals, particularly in light of the recently described transcriptomic sub-groups of CRC and emergent immunomodulatory effects of Entinostat.
Date of AwardJul 2019
Original languageEnglish
Awarding Institution
  • Queen's University Belfast
SponsorsThe Queen's University of Belfast Foundation
SupervisorDaniel Longley (Supervisor) & Simon McDade (Supervisor)

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