Metabolic Plasticity and Combinatorial Radiosensitisation Strategies in Human Papillomavirus-Positive Squamous Cell Carcinoma of the Head and Neck Cell Lines

SIMPLE SUMMARY: A subset of head and neck cancers (SCCHN) are caused by human papillomavirus (HPV). As these tumours tend to affect younger patients and are associated with favourable survival, there is a pressing need to find ways to reduce long-term treatment toxicity while maintaining oncological efficacy. We studied utilisation of metabolic pathways in HPV-positive SCCHN cells with the aim of exploiting such for potential therapeutic benefit. We found that these tumours maintained metabolic diversity, in contrast to what we have observed in traditional SCCHN cells associated with mutations in the TP53 gene. This, in turn, correlated with susceptibility to metabolic inhibitors, insofar as a combination of these agents acting on different metabolic pathways was required to augment the effects of ionising radiation (a mainstay of treatment for SCCHN). Notionally, this may provide a means of treatment de-intensification by facilitating radiation dose reduction to minimise the impact of treatment on long-term function. ABSTRACT: Background: A major objective in the management of human papillomavirus (HPV)-positive squamous cell carcinoma of the head and neck (SCCHN) is to reduce long-term functional ramifications while maintaining oncological outcomes. This study examined the metabolic profile of HPV-positive SCCHN and the potential role of anti-metabolic therapeutics to achieve radiosensitisation as a potential means to de-escalate radiation therapy. Methods: Three established HPV-positive SCCHN cell lines were studied (UM-SCC-104, UPCI:SCC154, and VU-SCC-147), together with a typical TP53 mutant HPV-negative SCCHN cell line (UM-SCC-81B) for comparison. Metabolic profiling was performed using extracellular flux analysis during specifically designed mitochondrial and glycolytic stress tests. Sensitivity to ionising radiation (IR) was evaluated using clonogenic assays following no treatment, or treatment with: 25 mM 2-deoxy-D-glucose (glycolytic inhibitor) alone; 20 mM metformin (electron transport chain inhibitor) alone; or 25 mM 2-deoxy-D-glucose and 20 mM metformin combined. Expression levels of p53 and reporters of p53 function (MDM2, p53, Phospho-p53 [Ser15], TIGAR and p21 [CDKN1A]) were examined by western blotting. Results: HPV-positive SCCHN cell lines exhibited a diverse metabolic phenotype, displaying robust mitochondrial and glycolytic reserve capacities. This metabolic profile, in turn, correlated with IR response following administration of anti-metabolic agents, in that both 2-deoxy-D-glucose and metformin were required to significantly potentiate the effects of IR in these cell lines. Conclusions: In contrast to our recently published data on HPV-negative SCCHN cells, which display relative glycolytic dependence, HPV-positive SCCHN cells can only be sensitised to IR using a complex anti-metabolic approach targeting both mitochondrial respiration and glycolysis, reflecting their metabolically diverse phenotype. Notionally, this may provide an attractive platform for treatment de-intensification in the clinical setting by facilitating IR dose reduction to minimise the impact of treatment on long-term function.