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Title: Tumour metabolism in squamous cell carcinoma of the head & neck : consequences & potential therapeutic implications of TP53 mutation
Author: Wilkie, Mark David
ISNI:       0000 0004 7428 5176
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Survival outcomes for traditional Human papillomavirus (HPV)-negative squamous cell carcinoma of the head and neck (SCCHN) have not improved significantly over the last 20-30 years. In contrast, HPV-positive oropharyngeal SCC is associated with favourable survival outcomes, but patients often suffer long-term functional ramifications from the toxicity of their treatment. Therefore, in the context of HPV-negative SCCHN there is a need to improve treatment efficacy to enhance survival outcomes, while for HPVpositive oropharyngeal SCC there is a need to minimise treatment-associated toxicity. Fundamental to this is to identify novel radiosensitising therapeutic approaches given that radiotherapy (RT) remains a mainstay of treatment for all SCCHN patients. Cancer cell metabolism is an attractive putative target in this regard, yet has received relatively little attention in the context of SCCHN. p53 is known to be a potentially important metabolic mediator, while TP53 mutation is central to SCCHN oncogenesis and is associated with poorer clinical outcomes. Consequently, the primary of aims of thesis these were to examine the metabolic phenotype exhibited by SCCHNs, whether this was related to TP53 status, and to determine whether tailored anti-metabolic treatment might have potential therapeutic value, specifically in enhancing the effects of ionising radiation (IR). Secondary aims were to explore the mechanistic basis underlying any metabolic alterations and any observed anti-metabolic therapeutic effects. Microplate-based extra-cellular flux analysis revealed that mutant TP53 SCCHN cell lines exhibited a distinct metabolic phenotype to that of wild-type TP53 cell lines. Wildtype TP53 cells maintained metabolic diversity, while mutant TP53 cells exhibited a specific survival dependence on glycolysis. This correlated with radiation response following glycolytic inhibition with 2-deoxy-D-glucose (2-DG), which potentiated IR effects in mutant TP53 cells only. In line with their more diverse metabolic phenotype, in wild-type TP53 cells (HPV-positive SCCHN cells included) a broader anti-metabolic approach, comprising both 2-DG and metformin (inhibitor of mitochondrial respiration), was required to achieve a similar effect. The potentiating effects of glycolytic inhibition on IR in mutant TP53 SCCHN cells were reversed by the addition of N-acetylcysteine (free radical scavenger). Consistent with this, flow cytometry demonstrated a marked increase in reactive oxygen species (ROS) following IR + 2-DG, and also increased levels of apoptosis, implicating oxidative stress-mediated activation of apoptotic signalling as the mechanism underlying the radiosensitising effects of 2-DG. That oxidative stress was centrally involved in the mechanism underlying glycolytic inhibition in SCCHN also suggested that the impetus driving mutant TP53 SCCHN cells towards glycolysis was at least partly to regulate cellular redox status and evade excessive ROS accumulation. In accordance with this, pentose phosphate pathway (PPP) enzyme expression and intracellular NADPH/NADP ratios were greater in mutant TP53 SCCHN cells, indicative of increased PPP flux. This appeared to be mediated by de-regulated TIGAR overexpression in those cells with loss of p53 function. Therapeutically, the addition of the PPP inhibitor 6-aminonicotinamide further potentiated IR effects in mutant TP53 cells over and above glycolytic inhibition. Ultimately, the findings described in this thesis present the opportunity for a novel, tailored anti-metabolic therapeutic approach in SCCHN, which not only carries a selective therapeutic index, but is also informed by TP53 status as a predictive biomarker. Specifically, we propose that in mutant TP53 SCCHNs glycolytic inhibition with 2-DG, possibly in combination with 6-AN-induced PPP inhibition, would result in significant radiosensitisation. This strategy would be applicable in upwards of 60-85% of SCCHN tumours and would be preferentially effective in targeting the treatmentresistant disease typically associated with TP53 mutation. For SCCHNs harbouring wildtype TP53 (HPV-positive disease included) we suggest the combination of 2-DG with metformin. This strategy may also provide an attractive platform for the treatment deintensification of carefully selected HPV-positive cases by facilitating RT dose reduction to minimise the impact of treatment on long-term function. The efficacy and safety of these strategies will require further validation in pre-clinical models prior to translation into the clinical setting, and future work will be directed in this regard.
Supervisor: Vlatkovic, Nikolina ; Jones, Terence ; Boyd, Mark Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral