Delineating mechanisms which underlie differential cell fates induced by p53 activation and HDAC inhibition in colorectal cancer

Student thesis: Doctoral ThesisDoctor of Philosophy


The tumour suppressor p53 is the most frequently mutated gene in human cancer with loss or suppression of wild-type function thought to be a prerequisite for the development of most malignancies. In colorectal cancer (CRC), approximately 50% of tumours harbour mutations in the TP53 gene, whilst the remaining wild-type tumours suppress or circumvent p53 activation via non-mutational mechanisms. This includes dysregulation of the p14ARF/MDM2 axis which constitutes the major mechanism for inducing p53 stabilisation within the cell. Strategies aimed at reactivating latent wild-type p53 in such tumours therefore hold enormous clinical potential. This led to the development of small molecule inhibitors of the E3 ubiquitin ligase, MDM2, which normally targets p53 for degradation and disrupts its transcription factor activity. Blocking this negative MDM2-p53 interaction results in rapid stabilisation of p53 protein, however, this most often leads to the activation of p53-induced cell cycle arrest rather than cell death. Understanding the mechanisms which are responsible for this decision making process are therefore of great importance in order to utilise these compounds and augment the efficacy of other therapeutic agents that activate p53.

Recent work in our lab has demonstrated that combination of direct (MDM2 inhibition) or indirect (DNA-damaging chemotherapy) p53 activation with inhibitors of nuclear Class-I Histone deacetylases (HDACi) is effective in enhancing p53- dependent apoptotic cell death in multiple models of CRC. Interestingly, despite a notable switch in phenotype from cell cycle arrest to cell death, few changes in the mRNA and protein expression of pro-apoptotic p53 targets were observed following combined treatment with the MDM2 inhibitor, Nutlin-3A, and the Class I specific HDACi, Entinostat, when compared to Nutlin-3A treatment alone. Indeed, the addition of Entinostat was instead found to decrease the expression of p53 induced anti-apoptotic proteins which most notably included the only known pseudo-caspase and cell death regulatory protein, FLIPL.

The work presented in this thesis builds upon these previous observations and delves into the complex mechanisms and pathways responsible for the synergistic induction of cell death following combined MDM2- and HDAC-inhibition in p53 wild-type models of CRC. Using both functional genomics and molecular techniques, this work identifies FLIPL as a direct, p53-induced transcriptional target which is potently upregulated by Nutlin-3A and suppressed by Entinostat. Importantly, both pharmacological and mutational inhibition of the NFκB pathway reveal that the Nutlin-3A-induced upregulation of FLIPL occurs independently of its canonical regulation by NFκB, further supporting the p53-dependent nature of this response.

Phenotypic analyses conducted by Annexin V/PI flow cytometry reveal that whilst treatment with Nutlin-3A or Entinostat alone fail to induce cell death, combining these agents significantly increases the induction of apoptotic cell death in a p53- dependent manner. In order to delineate the role of Entinostat mediated FLIPL downregulation in the cell death resulting from the combined Nutlin-3A/Entinostat treatment, siRNA-mediated FLIPL depletion was used to successfully phenocopy this result. Subsequently, further mechanistic analyses demonstrated that p53-mediated FLIPL upregulation blocks the induction of apoptosis by inhibiting caspase-8 activation at a TRAIL-R2/DR5 death inducing signalling complex. Notably, the activation of this p53-induced complex occurs independently of canonical TRAIL ligand binding.

In addition to the early induction of caspase-8 dependent apoptotic cell death, this work reveals that depleting FLIPL in combination with p53 activation can also result in the induction of caspase-8 independent cell death at later timepoints. Herein, the p53 transcriptional target and caspase-8 paralog, caspase-10, is demonstrated to compensate for the loss of caspase-8 in order to induce apoptosis, albeit to a lesser extent than in caspase-8 proficient cells. Moreover, FLIPL is also revealed to modulate the expression of p53 transcriptional targets such that in the absence of both caspase-8 and -10 cell death can still proceed. Depleting FLIPL is shown to suppress the p53-induced expression of the cell-cycle inhibitor, p21, whilst simultaneously enhancing the p53-induced expression of the pro-apoptotic protein, PUMA. Indeed, this upregulation of PUMA significantly contributes to the cell death induced by FLIPL depletion and p53 activation at later timepoints. Thus, the results presented in this thesis identify novel, clinically-relevant biology in which FLIPL acts to determine cell fate following p53 activation. Therapeutically targeting FLIPL with Entinostat therefore represents a viable means of overcoming FLIPL-mediated resistance to MDM2-inhibitors in tumours retaining wild-type p53.
Date of AwardDec 2021
Original languageEnglish
Awarding Institution
  • Queen's University Belfast
SponsorsNorthern Ireland Department for the Economy
SupervisorSimon McDade (Supervisor) & Daniel Longley (Supervisor)


  • p53
  • Cell death
  • Nutlin-3A
  • Entinostat
  • Apoptosis
  • FLIP
  • HDAC inhibitors

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