Abstract
Head and neck squamous cell carcinomas (HNSCCs) are genetically complex, difficult to treat and quick to metastasize. Due to their anatomical locations, treatment is associated with damage to normal functioning and subsequent severe reductions in quality of life. Adding to this, radiotherapy, the main treatment modality for these tumours, is ineffective for 50% of patients. An understanding of the underlying tumour biology is necessary to inform clinical advancements, as has been proven by the recent introductions of immunotherapies and the hypoxia-activated prodrug nimorazole into patient therapy. In line with this, comprehensive genetic profiling of these tumours has been performed, allowing for a greater appreciation of HNSCC genetics and signalling. These molecular profiles have identified biomarkers associated with oncogenesis, prognosis and therapy response in HNSCC.In one such study, data generated by this lab identified a signature of microRNAs differentially expressed in patient classed as good or bad responders to radiotherapy. Of this signature, miR-196a was found to be highly expressed in patients with recurrent disease. In vitro, HNSCC cell lines modulated for expression of miR-196a were more oncogenic and radioresistant. However, changes in expression of previously validated targets could not account for the radioresistant phenotype.
To further investigate these mechanisms, RNA-sequencing was performed on HNSCC cell lines modulated for expression of miR-196a. Analysis of this and publicly available data using bioinformatics techniques identified a number of potential targets that could be implicated in radioresistance. The network of targets identified had associations with PI3K/Akt signalling, the cell cycle and hypoxia, where low expression of these genes in The Cancer Genome Atlas samples was associated with high hypoxia scores.
Following on from these associations, it was hypothesised that re-investigation of existing targets of miR-196a in the hypoxic context could illuminate previously obscured mechanisms. Annexin A1 (ANXA1) is a stress-response protein and a target of miRNA-196a. In this work, HNSCC cell lines bearing knockdown of ANXA1 (ANXA1-kd) were shown to be highly radioresistant, particularly when incubated in hypoxia (1% O2). Hypoxic incubation and irradiation were both shown to promote nuclear translocation of ANXA1 in wildtype cells, which was shown to be in part regulated by EGFR. To assess whether this radioresistant phenotype could be subverted, the hypoxia-activated prodrug tirapazamine was used, and shown to eradicate the survival benefit of ANXA1 depletion in hypoxia. This is the first time that ANXA1 has been implicated in hypoxia and radioresistance.
Another recent study by our lab linked hypoxic volume in patients to a genetic signature correlating to progression-free survival. To further investigate the role of hypoxia in poor survival of HNSCCs, a member of this signature, Lysyl oxidase (LOX), was chosen for further study. LOX has been previously implicated in poor prognosis and hypoxia-induced metastasis in other cancers but has not been extensively studied in the context of radioresistance. LOX was found to be differentially expressed in a metastatic pair of HNSCC cell lines. Using this model, LOX was shown to induce proliferation and migration. Overexpression of LOX in an ultra-low expressing breast cancer cell line was shown to reduce E-cadherin and promote proliferation. However, LOX overexpression did not significantly alter response to irradiation.
To conclude, these findings offer glimpses into the mechanisms behind radiotherapy resistance, forming the basis for future work that could lead to clinically translatable advancements.
| Date of Award | 1 Jul 2021 |
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| Original language | English |
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| Supervisor | Mahvash Tavassoli (Supervisor) |