Head and neck squamous cell carcinomas (HNSCCs) are an aggressive, genetically complex and difficult to treat group of cancers. In lieu of truly effective targeted therapies, surgery and radiotherapy represent the primary treatment options for most patients. But these treatments are associated with significant morbidity and a reduction in quality of life. Resistance to both radiotherapy and the only available targeted therapy, and subsequent relapse are common. Research has therefore focussed on identifying biomarkers to stratify patients into clinically meaningful groups and to develop more effective targeted therapies. However, as we are now discovering, the poor response to therapy and aggressive nature of HNSCCs is not only affected by the complex alterations in intracellular signalling pathways but is also heavily influenced by the behaviour of the extracellular microenvironment. The HNSCC tumour landscape is an environment permissive of these tumours’ aggressive nature, fostered by the actions of the immune system, the response to tumour hypoxia and the influence of the microbiome. Solving these challenges now rests on expanding our knowledge of these areas, in parallel with a greater understanding of the molecular biology of HNSCC subtypes. This update aims to build on our earlier 2014 review by bringing up to date our understanding of the molecular biology of HNSCCs and provide insights into areas of ongoing research and perspectives for the future.

The hypoxic tumour is a chaotic landscape of struggle and adaption. Against the adversity of oxygen starvation, hypoxic cancer cells initiate a reprogramming of transcriptional activities, allowing for survival, metastasis and treatment failure. This makes hypoxia a crucial feature of aggressive tumours. Its importance, to cancer and other diseases, was recognised by the award of the 2019 Nobel Prize in Physiology or Medicine for research contributing to our understanding of the cellular response to oxygen deprivation. For cancers with limited treatment options, for example those that rely heavily on radiotherapy, the results of hypoxic adaption are particularly restrictive to treatment success. A fundamental aspect of this hypoxic reprogramming with direct relevance to radioresistance, is the alteration to the DNA damage response, a complex set of intermingling processes that guide the cell (for good or for bad) towards DNA repair or cell death. These alterations, compounded by the fact that oxygen is required to induce damage to DNA during radiotherapy, means that hypoxia represents a persistent obstacle in the treatment of many solid tumours. Considerable research has been done to reverse, correct or diminish hypoxia’s power over successful treatment. Though many clinical trials have been performed or are ongoing, particularly in the context of imaging studies and biomarker discovery, this research has yet to inform clinical practice. Indeed, the only hypoxia intervention incorporated into standard of care is the use of the hypoxia-activated prodrug Nimorazole, for head and neck cancer patients in Denmark. Decades of research have allowed us to build a picture of the shift in the DNA repair capabilities of hypoxic cancer cells. A literature consensus tells us that key signal transducers of this response are upregulated, where repair proteins are downregulated. However, a complete understanding of how these alterations lead to radioresistance is yet to come.

Joint morphogenesis requires mechanical activity during development. Loss of mechanical strain causes abnormal joint development, which can impact long-term joint health. Although cell orientation and proliferation are known to shape the joint, dynamic imaging of developing joints in vivo has not been possible in other species. Using genetic labelling techniques in zebrafish we were able, for the first time, to dynamically track cell behaviours in intact moving joints. We identify that proliferation and migration, which contribute to joint morphogenesis, are mechanically controlled and are significantly reduced in immobilised larvae. By comparison with strain maps of the developing skeleton, we identify canonical Wnt signalling as a candidate for transducing mechanical forces into joint cell behaviours. We show that, in the jaw, Wnt signalling is reduced specifically in regions of high strain in response to loss of muscle activity. By pharmacological manipulation of canonical Wnt signalling, we demonstrate that Wnt acts downstream of mechanical activity and is required for joint patterning and chondrocyte maturation. Wnt16, which is also downstream of muscle activity, controls proliferation and migration, but plays no role in chondrocyte intercalation.

Joint morphogenesis requires mechanical activity during development. Loss of mechanical strain causes abnormal joint development, which can impact long term joint health. While cell orientation and proliferation are known to shape the joint, dynamic imaging of developing joints in vivo have not been possible in other species. Using genetic labelling techniques in zebrafish we were able, for the first time, to dynamically track cell behaviours in intact moving joints. We identify that proliferation and migration, which contribute to joint morphogenesis, are mechanically controlled and are significantly reduced in immobilised larvae. By comparison to strain maps of the developing skeleton we identify canonical Wnt signalling as a candidate to transduce mechanical forces into joint cell behaviours. We show that in the jaw Wnt signalling is reduced specifically in regions of high strain in response to loss of muscle activity. By pharmacological manipulation of canonical Wnt signalling we demonstrate that Wnt acts downstream of mechanical activity and is required for joint patterning and chondrocyte maturation. Wntl6, independent of muscle activity, controls proliferation and migration, but plays no role in chondrocyte intercalation.

Introduction Hypoxia occurs when tumours out-grow the vasculature. It can induce therapy resistance, contribute to metastasis and is a potential biomarker of radioresistance (RR) in Head and Neck Squamous Cell Carcinoma (HNSCC). However, neither identification of patients who might benefit from hypoxia-modification, nor a full understanding of the molecular changes that contribute to metastasis/RR, has been achieved. A recent study by our lab was able to link hypoxic volume in patients with a genetic signature correlating to poor progression-free survival. Lysyl oxidase (LOX) was chosen for further investigation as it has been previously implicated in poor prognosis and hypoxia-induced metastasis. LOX has not however been extensively studied in the context of RR. Additionally, factors other than hypoxia have been shown to contribute to LOX's regulation, but, their relative contributions to metastasis and RR have not been defined. Material and methods LOX was found to be differentially expressed in a metastatic pair of HNSCC cell lines: HSC3 (primary tumour, low expression) and HSC3-M3 (metastatic daughter line, high expression). Using this model, the role of LOX in hypoxia-induced metastasis was studied using invasion, migration and anoikis-resistance assays. LOX was also overexpressed and inhibited (using active site inhibitor BAPN) to determine LOX-specificity. Currently, we are investigating LOX as an inducer of RR by clonogenic assays, comet assay and gH2AX staining. Pathways including epithelial-to-mesenchymal transition (EMT) have so far been investigated as mechanisms. Next we will use bioinformatics to correlate follow up data from our original study and existing datasets, to LOX's involvement in treatment failure and survival. Results and discussions Our results have shown that high expression of LOX, endogenously in metastatic HSC3-M3 cells or by overexpression, can induce a metastatic phenotype resulting in increased invasion, migration and anoikis resistance. Inhibition of LOX reverses these effects, exaggerated in hypoxia where LOX is upregulated. LOX upregulation has also been linked to a change in expression/localisation of EMT regulators such as E-Cadherin and Vimentin. Early results show a potential link to RR. Ongoing studies aim to determine if this effect is a result of LOX's enzymatic activity, or potential transcription factor functioning. Conclusion This work will be the first to highlight LOX as an important biomarker not only in hypoxia-induced metastasis of HNSCC, but also in therapy resistance.