WP1: Biomarker Discovery
TRACT will carry out discovery research into biomarkers associated with OOC to develop state-of-the-art diagnostic assays for 1) earlier, more reliable detection, and 2) therapeutic response prediction. Overall, research carried out under WP1 will improve OOC survival rates by developing biomarker-based assays for earlier, more reliable disease diagnosis and stratification of individual patients to more effective chemotherapeutic regimes. This comprehensive patient profiling provides essential information to the clinician, enabling earlier and more accurate diagnosis and the potential to administer more appropriate treatment. This approach will not only benefit the patient, with the potential for improved quality of life, disease control and minimise side-effects by administering more targeted treatment, but will also potentially lead to reduced costs for healthcare providers.
The most effective approach to reducing OOC morbidity is early detection, yet no effective diagnostic procedures for OOC currently exist. At present, definitive diagnosis of OOC relies upon tissue biopsy, a procedure that often yields false-negative diagnoses and results in the recovery of non-diagnostic tissue. ESR 1, Valentina Dikova, (UVEG recruit; NIBRT secondment) will explore the development of a biomarker-based diagnostic approach as an alternative to tissue biopsy. To date, the search for biomarkers associated with the early onset of oral squamous cell carcinoma (OSCC) has focused on a single proteomic approach and has met with only limited success. ESR 1 will adopt an alternative approach using state-of-the-art multidisciplinary techniques to determine novel salivary biomarkers at protein, glycan and molecular levels. Saliva collection has distinct advantages over tissue and blood collection as the collection procedure is non-invasive and does not require specialised resources, and the resulting samples are safer to handle and easier to store. Saliva is currently being used in many diagnostic procedures, including screening for HIV, hepatitis and ebola. Currently the O’Sullivan lab in TCD has built up a biobank of saliva samples from patients exhibiting a fixed stage of oral pre-cancer/cancer along with positive pilot studies demonstrating the use of saliva as an oral cancer diagnostic tool. As inflammation has previously been linked to the pathogenesis of OSCC8, saliva from OSCC patients will be analysed to determine the relationship between pro-inflammatory cytokine markers (TNF-D, IL-1E, IL-6, and IL-8), salivary glycan profiles and early disease progression. Results from this profiling will ultimately lead to the development of a clinical test for early diagnosis of OSCC.
The application of pre-operative, neo-adjuvant chemotherapy or chemoradiotherapy has delivered significant improvements in disease free and overall survival in oesophageal cancer. However, not all patients respond equally well to all treatments. ESR 2, Éilis Sutton, (QUB recruit; TCD, ALMAC secondments) will undertake whole genome sequencing and microarray-based gene expression profiling of biopsies from early stage OAC to identify molecular signatures predictive of response to chemotherapy. Results from this profiling will ultimately lead to the development of a clinical diagnostic test to predict responders and non-responders.
The typically late diagnosis of oral cancer patients usually necessitates radiotherapy and surgical intervention. Lower inflammatory responses post-intervention are associated with successful recovery from cancer treatment. Monitoring the healing rate and control of inflammation is essential to aid in successful recovery. However, researchers and clinicians are currently unable to determine the effect of inflammation on wound healing, as the associated inflammatory profile is unknown. Thus, inflammatory profiles have the potential to be utilised as a measure of patient recovery, but there are currently no clinical assays for monitoring inflammation during treatment. ESR 3, Sara Principe, (UVEG recruit; IME-SP secondment) will evaluate the levels of local inflammatory markers as an indicator of positive response to treatment in order to determine specific inflammatory profiles linked to wound healing and develop assays to monitor toxicity, tumour control and patient recovery. This project builds on the use of diagnostic tools developed in the Bagan lab which identified EGF as a discriminating factor in oral cancer.
WP2: Resistance Mechanisms
In WP2, TRACT will uncover the molecular basis of drug resistance mechanisms in OOC with the aim of 1) improving the efficacy of existing therapies, 2) identifying new drug targets, and 3) developing novel therapies. Initial pre-clinical testing will be carried out to support future translation to clinical studies.
Current treatment strategies for OOC include a combination of surgery, radiotherapy and chemotherapy. Chemotherapeutic treatment is currently impeded by drug resistance and a lack of selectivity. A greater understanding of the cellular mechanisms that contribute to chemotherapeutic resistance in OOC will enable the development of combination therapies with greater efficacy than current chemotherapeutic regimes. Targeted combination therapies hold the promise of improved response rates, decreased chemotherapeutic toxicity and enhanced survival rates.
ESR 4, Niamh McCabe (QUB recruit; ALMAC, IME-SP, TCD secondments) will perform RNA-seq and microarray-based gene expression profiling on matched pre-chemotherapy endoscopic biopsies of early stage oesophageal adenocarcinomas and normal tissue resections. The resulting profiles will allow the identification of differentially expressed/frequently mutated genes and associated molecular pathways in pathological responders and nonresponders, informing the design of new therapies for OAC. Preliminary data analysis has identified the MAPK and glycolytic pathways as potential targetable pathways. Determinants of drug resistance may also lead to the development of a potential diagnostic test to classify non-responders versus responders.
Recent research by Creagh in TCD has implicated inflammatory caspases as key mediators of intestinal inflammation and as biomarkers for colon cancer. As OAC is an inflammation-associated cancer, ESR 5, Ewelina Flis, (TCD recruit; QUB, OPSONA secondments) will conduct a study to establish whether inflammatory caspases may also represent biomarkers for early stage OAC. The involvement of inflammatory caspases in OAC development and resistance will also be examined using siRNA and inflammation/caspase inhibitors in OAC cell lines & in vivo models, ultimately leading to the development of novel diagnostics for OAC, and assays for enhanced patient stratification, enabling more effective therapeutic choices.
Drug resistance and a lack of selectivity impede current chemotherapeutics for OOC. Thus, new therapeutic options for the treatment of recurrent OOCs are urgently needed. Cancer biology research has led to the selective inhibition of rate-limiting targets in the progression of many chemotherapy resistant cancers (e.g. Cetuximab (anti-EGFR) - colorectal cancer; Bortezomib (Proteasome inhibitor) - multiple myeloma). Resistance to cell death is a common hallmark of cancer and is often mediated by the Bcl-2 family of proteins. Among all anti-apoptotic Bcl-2 members, Mcl-1 functions as a major survival factor, particularly in solid cancers. In the last year Mcl-1 has been identified as an important therapeutic target for OOC. However, no specific Mcl-1 inhibitors exist. ESR 6, Prashant Saraswati,(UNISI recruit; Exosomics, TCD secondments) will computationally design and synthesise Mcl-1 inhibitors with the ability to sensitise OOC cells to apoptosis with appropriate pharmacokinetic properties. The agents will be tested in models of OSCC and efficacy of the novel compounds will also be determined by means of exosome content evaluation.
Pre-operative reduction of OOC tumour masses greatly improves patient survival post surgery. However, current chemo- and radio-therapeutic strategies have undesirable long-term effects. Full length and peptide fragments of the natural human milk protein D-lactalbumin non-covalently bound to oleic acid (a.k.a HAMLET) have proven effective in treating bladder and intestinal tumors with no observable side effects. The tumoricidal mechanism is multi-faceted (work by Mok in TCD and others, providing further opportunities to design targeted therapeutics. ESR 7, Magda Ghanim, (TCD recruit; Oroboros & UNISI secondments) will generate neo-adjuvant HAMLET therapy of enhanced efficacy by chemically coupling oleic acid to a variety of D-lactalbumin peptides (UNISI). Hamlet derivatives will be tested on OAC cell lines, and the mode of action studied by genome-scale CRISPR knockoutand next generation sequencing (TCD). Metabolic changes will also be analysed by respirometry (OROBOROS).
Genetic and pharmacological screens have identified autophagic mediators as effective adjuvant and neoadjuvant targets. However, results from a recent phase 1 trial of the autophagy inhibitor hydroxychloroquine to treat newly diagnosed glioma patients demonstrated dose limiting toxicity. Therefore, in order to exploit autophagic mediators as therapeutic targets, lower toxicity compounds are required. ESR 8, Tuhina Khan, (UNISI recruit; Exosomics, TCD secondments) will carry out bioinformatic screening to identify and develop new targets. Novel compounds against promising targets will be rationally designed and synthesised, and efficacy screening will be carried out in OSCC models. Efficacy of the novel compounds will also be evaluated by means of exosome content evaluation.
Tumour resistance to therapy is related to the cell survival properties of autophagy and this pathway is frequently activated by chemotherapies in patients with various types of cancer. However, the role of autophagy in OSCC remains unclear, although preliminary studies in TCD have demonstrated that OSCC cell lines undergo autophagy in response to chemotherapy treatment. ESR 9, Stefania Magnano (TCD recruit; UVEG and Andor secondments) will investigate the expression of key autophagic regulatory proteins in OSCC patient samples and correlate expression with clinicopathologic factors and overall patient survival. ESR 9 will also determine whether combining existing OSCC chemotherapy strategies with autophagy inhibition represents a better treatment strategy for the benefit of OSCC patients
WP3: Metabolic Transformation
In WP3 TRACT will examine metabolic transformation mechanisms in OOC with the aim of identifying new drug targets for future therapeutic development. Metabolic transformation is a universal property of tumour formation and is a rich source of targets for development of therapeutic interventions.
Pilot studies performed in QUB using a pathways based approach to identify determinants of drug resistance in OOC have identified the glycolytic pathway as a potential targetable pathway. ESR 10, (Oroboros recruit; TCD secondment) will further characterise the bioenergetic and metabolic differences in normal, dysplastic and cancerous oral cancer cells using the Oroboros Respirometer Multisensor system and state-of-the-art Seahorse analysis. This approach will identify differential novel drug targets and means to enhance the chemotherapeutic sensitivity of cancer cells.
Factors that control mitochondrial dynamics in cancer cells have also emerged as possible therapeutic targets. The dynamic structure of the mitochondria in mammalian cells is defined by the opposing forces of fission and fusion, but the regulation of these mitochondrial processes is poorly understood. This is an emerging area in cancer research where cutting-edge imaging technologies are merging with molecular and cellular biology techniques. Pilot studies performed by Porter in TCD have identified a key molecule involved in controlling mitochondrial abundance (namely SIRT3) as a determinant of drug resistance in some solid cancers (manuscript in preparation). ESR 11, Marilena Karavyraki, (TCD recruit; Oroboros secondment) will establish the relationship between mitochondrial abundance, morphology, functional proteins involved in mitochondrial dynamics and metabolic differences in normal, dysplastic and oral cancer cells. This new knowledge will lead to the identification of novel therapeutic targets