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.
•CAFs are heterogenous cells but only subtypes expressing α-SMA are known.•CAF-secreted exosomes carry microRNA that change cancer cell behaviour.•CAF-metabolism is context-dependent and rely on the ...interaction with cancer cells.•CAFs affect immune cells through direct and indirect mechanisms.•There is little information about CAFs derived from HPV positive tumours.
Complex interactions take place during cancer formation and progression. In this regard, there has been increasing focus on the non-malignant cells that make up the tumour microenvironment (TME), and how they interact with malignant tumour cells. TME is highly heterogeneous and has a major influence on tumour behaviour and therapy response. Cancer-associated fibroblasts (CAFs), one of the main components of the TME, establish dangerous liaisons with cancer cells and other components of the TME to shape a tumour-supportive environment in many types of cancer. Head and neck squamous cell carcinoma (HNSCC) encompass the malignant neoplasms arising from the mucosal lining of the oral cavity, pharynx and larynx. The TME of HNSCC contributes to tumour progression and this stromal compartment may be an interesting target for treatment. There is an emerging picture of the behaviour of CAFs in HNSCC; how they affect and are affected by the TME. We aim to summarise and discuss the current understanding of CAFs in head and neck cancer, exploring CAF activation and heterogeneity, and interaction with cancer cells and other cells within the TME.
•Hypoxia has long been recognised as a major cause of treatment failure in solid tumour and is a crucial feature of aggressive tumours.•Head and neck cancers have limited treatment options and remain ...dependent on radiotherapy, hypoxic adaption is particularly restrictive to RT success.•To reverse, correct or diminish hypoxia has important benefits in treatment sensitization of head and neck cancers to RT, chemo- and immunotherapy.•We provide an overview of all main strategies to target tumor hypoxia: HIF-inhibitors, hypoxia-activated prodrugs, and anti-angiogenic agents.•We underline emerging strategies towards efficient delivery of drugs to combat restrictive microenvironment of hypoxic tumour tissue.
Most solid tumors, such as head and neck cancers, feature a hypoxic microenvironment due to angiogenic dysregulation and the consequent disruption of their vascular network. Such nutrient-deprived environment can induce genomic changes in several tumor cell populations, conferring survival and proliferative advantages to cancer cells through immunosuppression, metabolic switches and enhanced invasiveness. These transcriptional changes, together with the selective pressure hypoxia exerts on cancer cells, leads to the propagation of more aggressive and stress-resistant subpopulations increasing therapy resistance and worsening patient outcomes. Although extensive preclinical and clinical studies involving hypoxia-targeted drugs have been performed, most of these drugs have failed late-stage clinical trials and only a few have managed to be implemented in clinical practice. Here, we provide an overview of three main strategies to target tumor hypoxia: HIF-inhibitors, hypoxia-activated prodrugs and anti-angiogenic agents; summarizing the clinical advances that have been made over the last decade. Given that most hypoxia-targeted drugs seem to fail clinical trials because of insufficient drug delivery, combination with anti-angiogenic agents is proposed for the improvement of therapy response via vascular normalization and enhanced drug delivery. Furthermore, we suggest that using novel nanoparticle delivery strategies might further improve the selectivity and efficiency of hypoxia-targeted therapies and should therefore be taken into consideration for future therapeutic design. Lastly, recent findings point out the relevance that hypoxia-targeted therapy is likely to have in head and neck cancer as a chemo/radiotherapy sensitizer for treatment efficiency improvement.
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EGFR upregulation is an established biomarker of treatment resistance and aggressiveness in head and neck cancers (HNSCC). EGFR-targeted therapies have shown benefits for HPV-negative HNSCC; ...surprisingly, inhibiting EGFR in HPV-associated HNSCC led to inferior therapeutic outcomes suggesting opposing roles for EGFR in the two HNSCC subtypes. The current study aimed to understand the link between EGFR and HPV-infected HNSCC particularly the regulation of HPV oncoproteins E6 and E7. We demonstrate that EGFR overexpression suppresses cellular proliferation and increases radiosensitivity of HPV-positive HNSCC cell lines. EGFR overexpression inhibited protein expression of BRD4, a known cellular transcriptional regulator of HPV E6/E7 expression and DNA damage repair facilitator. Inhibition of EGFR by cetuximab restored the expression of BRD4 leading to increased HPV E6 and E7 transcription. Concordantly, pharmacological inhibition of BRD4 led to suppression of HPV E6 and E7 transcription, delayed cellular proliferation and sensitised HPV-positive HNSCC cells to ionising radiation. This effect was shown to be mediated through EGFR-induced upregulation of microRNA-9-5p and consequent silencing of its target BRD4 at protein translational level, repressing HPV E6 and E7 transcription and restoring p53 tumour suppressor functions. These results suggest a novel mechanism for EGFR inhibition of HPV E6/E7 oncoprotein expression through an epigenetic pathway, independent of MAPK, but mediated through microRNA-9-5p/BRD4 regulation. Therefore, targeting EGFR may not be the best course of therapy for certain cancer types including HPV-positive HNSCC, while targeting specific signalling pathways such as BRD4 could provide a better and potentially new treatment to improve HNSCC therapeutic outcome.
Radiotherapy is a major treatment modality for head and neck squamous cell carcinoma (HNSCC). Up to 50% of patients with locally advanced disease relapse after radical treatment and there is ...therefore a need to develop predictive bomarkers for clinical use that allow the selection of patients who are likely to respond. MicroRNA (miRNA) expression profiling of a panel of HNSCC tumours with and without recurrent disease after surgery and radiotherapy detected miR‐196a as one of the highest upregulated miRNAs in the poor prognostic group. To further study the role of miR‐196a, its expression was determined in eight head and neck cancer cell lines. Overexpression of miR‐196a in HNSCC cells, with low endogenous miR‐196a expression, significantly increased cell proliferation, migration and invasion, and induced epithelial to mesenchymal transition. Conversely, miR‐196a knockdown in cells with high endogenous expression levels significantly reduced oncogenic behaviour. Importantly, overexpression of miR‐196a increased radioresistance of cells as measured by gamma H2AX staining and MTT survival assay. Annexin A1 (ANXA1), a known target of miR‐196a, was found to be directly modulated by miR‐196a as measured by luciferase assay and confirmed by Western blot analysis. ANXA1 knockdown in HNSCC exhibited similar phenotypic effects to miR‐196a overexpression, suggesting the oncogenic effect of miR‐196a may at least be partly regulated through suppression of ANXA1. In conclusion, this study identifies miR‐196a as a potential important biomarker of prognosis and response of HNSCC to radiotherapy. Furthermore, our data suggest that miR‐196a and/or its target gene ANXA1 could represent important therapeutic targets in HNSCC.
What's new?
There is accumulating evidence that miR‐196a plays an important role in the pathogenesis of a number of cancers. Through functional studies, here the authors demonstrate that miR‐196a confers an oncogenic phenotype in head and neck cancer cells, through the targeting of ANXA1. The results also show that MiR‐196a modulation is associated with response to radiation. MiR‐196a may therefore represent both a prognostic and a predictive biomarker in head and neck cancer. Furthermore, the data suggest that miR‐196a and/or its target gene ANXA1 could represent important therapeutic targets in head and neck cancer.
Head and neck squamous cell carcinomas (HNSCC) are associated with poor morbidity and mortality. Current treatment strategies are highly toxic and do not benefit over 50% of patients. There is ...therefore a crucial need for predictive and/or prognostic biomarkers to allow treatment stratification for individual patients. One class of biomarkers that has recently gained importance are microRNA (miRNA). MiRNA are small, noncoding molecules which regulate gene expression post‐transcriptionally. We performed miRNA expression profiling of a cohort of head and neck tumours with known clinical outcomes. The results showed miR‐9 to be significantly downregulated in patients with poor treatment outcome, indicating its role as a potential biomarker in HNSCC. Overexpression of miR‐9 in HNSCC cell lines significantly decreased cellular proliferation and inhibited colony formation in soft agar. Conversely, miR‐9 knockdown significantly increased both these features. Importantly, endogenous CXCR4 expression levels, a known target of miR‐9, inversely correlated with miR‐9 expression in a panel of HNSCC cell lines tested. Induced overexpression of CXCR4 in low expressing cells increased proliferation, colony formation and cell cycle progression. Moreover, CXCR4‐specific ligand, CXCL12, enhanced cellular proliferation, migration, colony formation and invasion in CXCR4‐overexpressing and similarly in miR‐9 knockdown cells. CXCR4‐specific inhibitor plerixafor abrogated the oncogenic phenotype of CXCR4 overexpression as well as miR‐9 knockdown. Our data demonstrate a clear role for miR‐9 as a tumour suppressor microRNA in HNSCC, and its role seems to be mediated through CXCR4 suppression. MiR‐9 knockdown, similar to CXCR4 overexpression, significantly promoted aggressive HNSCC tumour cell characteristics. Our results suggest CXCR4‐specific inhibitor plerixafor as a potential therapeutic agent, and miR‐9 as a possible predictive biomarker of treatment response in HNSCC.
MicroRNA‐9 is frequently downregulated in head and neck cancer. MiR‐9 directly targets and downregulates CXCR4 in HNSCC. CXCR4 activation by its ligand CXCL12 induces growth and invasion. Loss of miR‐9 induces CXCR4 overexpression and oncogenic activation. Plerixafor specifically inhibits CXCR4 activity, abrogating the invasive cancer phenotype, and therefore, miR‐9 may be a potential predictive biomarker of response to plerixafor treatment.
Malignant melanoma (MM) is known to be intrinsically chemoresistant, even though only ~20% of MM carry mutations of the tumor suppressor p53. Despite improvement of systemic therapy the mortality ...rate of patients suffering from metastatic MM is still ~70%, highlighting the need for alternative treatment options or for the re-establishment of conventional therapeutic approaches, including chemotherapy. Screening the p53 mutation status in a cohort of 19 patient-derived melanoma samples, we identified one rarely described missense mutation of p53 leading to E285K amino acid exchange (mutp53(E285K)). Employing structural and computational analysis we revealed a major role of E285 residue in maintaining stable conformation of wild-type p53 (wtp53). E285K mutation was predicted to cause interruption of a salt-bridge network affecting the conformation of the C-terminal helix of the DNA-binding domain (DBD) thereby preventing DNA interaction. In this context, a cluster of frequently mutated amino acid residues in cancer was identified to putatively lead to similar structural effects as E285K substitution (E285 cluster). Functional analysis, including knockdown of endogenous p53 and reconstitution with diverse p53 missense mutants confirmed mutp53(E285K) to have lost transcriptional activity, to be localized in the cytosol of cancer cells, by both means conferring chemoresistance. Re-sensitization to cisplatin-induced cell death was achieved using clinically approved compounds aiming to restore p53 wild-type function (PRIMA1-Met), or inhibition of AKT-driven MAPK survival pathways (afuresertib), in both cases being partially due to ferroptosis induction. Consequently, active ferroptosis induction using the GPX4 inhibitor RSL3 proved superior in tumorselectively fighting MM cells. Due to high prevalence of the E285-cluster mutations in MM as well as in a variety of other tumor types, we conclude this cluster to serve an important function in tumor development and therapy and suggest new implications for ferroptosis induction in therapeutic applications fighting MM in particular and cancer in general.
High-dimensional cytometry is an innovative tool for immune monitoring in health and disease, and it has provided novel insight into the underlying biology as well as biomarkers for a variety of ...diseases. However, the analysis of large multiparametric datasets usually requires specialist computational knowledge. Here, we describe
(https://github.com/kordastilab/ImmunoCluster), an R package for immune profiling cellular heterogeneity in high-dimensional liquid and imaging mass cytometry, and flow cytometry data, designed to facilitate computational analysis by a nonspecialist. The analysis framework implemented within
is readily scalable to millions of cells and provides a variety of visualization and analytical approaches, as well as a rich array of plotting tools that can be tailored to users' needs. The protocol consists of three core computational stages: (1) data import and quality control; (2) dimensionality reduction and unsupervised clustering; and (3) annotation and differential testing, all contained within an R-based open-source framework.
The clinical and histologic features alone cannot accurately predict whether potentially malignant disorders of the oral mucosa remain stable, regress or progress to malignancy. Some of them, with or ...without epithelial dysplasia, may transform to invasive oral squamous cell carcinomas (OSCC). Identification of molecular markers which can predict disease progression is necessary to improve the management of these disorders. Many genes and signaling pathways have been shown to be involved in the development of OSCC. This review summarizes some molecular markers researched in the detection of pre‐cancer. We highlight selected markers that are reported to be significantly associated with progression of potentially malignant disorders to OSCC. These include alterations in genes/pathways which control cellular signaling, cell cycle, apoptosis, genomic stability, cytoskeleton, angiogenesis, etc. However, these genetic tumor markers have so far not gained any use in routine diagnosis and their utility in the prediction of risk of malignant transformation remains unknown. It is, however, clear from the large number of studies, some described in this review, that multiple genes/pathways are involved in the progression from normal to metaplastic/dysplastic, and subsequently to cancer. It is therefore necessary to study those significant alterations in multiple genes simultaneously in biopsy samples from large cohorts of subjects.
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