Oral squamous cell carcinoma (OSCC) is a highly aggressive cancer that is characterized by a high rate of invasion and destruction to the surrounding tissues, with patients showing poor 5-year survival rate. A pre-malignant stage of cellular atypia and loss of stratification within the epithelium (dysplasia) occurs prior to the establishment of OSCC, which is manifested as white (leukoplakia) or red (erythroplakia) lesions. Treatment of dysplasia/OSCC involves surgical intervention with the removal of an adequate safety margin. However, high grade dysplasia and OSCC exhibit high recurrence rates. To date the only method used to diagnose oral epithelial dysplasia (OED) and OSCC is haematoxylin and eosin staining of biopsies and examination by an experienced pathologist. Furthermore, the mechanisms of dysplasia formation, transition to OSCC, and high recurrence rates are little understood. Recent data from the Ward lab suggests that the cell surface tumour suppressor protein E-cadherin plays an important role in regulating many cellular functions in epithelial cells. Loss of E-cadherin in carcinomas has been well studied and is linked with tumour invasion, metastasis and poorer patient outcomes. In this thesis, I have investigated expression of E-cadherin in low grade (LG) and high grade (HG) dysplasia and T1 and T4 OSCC patient biopsies to determine whether loss of this protein occurs prior to tumour cell invasion. Furthermore, microarray data analysis identified Epithelial Membrane Protein-1 (EMP-1) as a putative early marker of tumorigenesis, with alterations of N-cadherin, CD44 and 5T4 oncofoetal antigen expression during tumorigenesis inferred from our studies in embryonic stem cells. Immunofluorescence microscopy (IFM) analysis revealed that normal oral epithelium exhibits cell surface E-cadherin, EMP-1 and 5T4 expression but generally lacks N-cadherin and CD44 reactivity. The statistically significant loss of both E-cadherin and EMP-1 was observed in low and high grade dysplastic tissue and OSCC biopsies (p < 0.001). 5T4 expression was decreased in HG dysplasia (p < 0.001), suggesting this may be a useful assay for discriminating between LG and HG dysplastic tissues. N-cadherin or CD44 did not show any statistical significance in expression between normal, LG/HG dysplasia and T1/T4 OSCC, although there was a trend for increased N-cadherin expression in T4 OSCC. Significantly, Ecadherin expression was decreased or absent from surgical margins of LG/HG dysplasia and T1 OSCC, and EMP-1 and 5T4 were absent from the margins of LG and HG dysplastic tissue, indicating that these margins are abnormal. Therefore, loss of Ecadherin and EMP-1 are early events associated with dysplastic tissue and may be a useful method to confirm the removal of sufficient surgical safety margin. The OSCC cell line BICR56 was assessed for marker expression and exhibited a phenotype consistent with normal oral epithelium. Inhibition of E-cadherin protein in BICR56 cells using a neutralising antibody led to decreased cell surface localisation of 5T4 antigens, suggesting that E-cadherin expression is critical for maintenance of a normal epithelial phenotype. In conclusion, both E-cadherin and EMP-1 are useful markers for detecting early cancerous changes and 5T4 expression can discriminate between LG and HG dysplasia. This study has also shown significant abnormalities within the surgical safety margins of LG/HG dysplasia and T1 OSCC biopsies, which requires further investigation, and may explain the high recurrence rates seen in such patients.

Oral cancer in early stages (I and II) may be curable by surgery or radiation therapy alone but advanced stage disease (III and IV) has a relatively low survival rate. The pathogenic pathways that contribute to Oral Squamous Cell Carcinoma (OSCC) remain poorly characterised and the critical factor in the lack of prognostic improvement is that a significant proportion of cancers initially are asymptomatic lesions and are not diagnosed or treated until they reach an advanced stage. Hence, a clinically applicable gene expression signature is in high demand and improved characterization of the OSCC gene expression profile would constitute substantial progress. For OSCC, possible themes that might be addressed using microarray data include distinguishing the disease from normal at the molecular level; determining whether specific biomarkers or profiles are predictive for tumour behaviour; and identifying biologic pathways necessarily altered in tumourigenesis, potentially illuminating novel therapeutic targets. However, OSCC is a heterogeneous disease, making diagnostic biomarker development difficult. Although this phenotypic variation is striking when one compares OSCC from different geographic locales, little is known about the underpinning biological mechanisms. Cancer may be accompanied by the production and release of a substantial number of proteins, metabolites and/or hormones into the blood, saliva, and other body fluids that could also serve as useful markers for assessing prognosis, metastasis, monitoring treatment, and detecting malignant disease at an early stage. The primary aim of this thesis is to investigate metabolomic and transcriptomic profiles using multiple bioinformatics approaches and biological annotation tools in an attempt to identify specific biomarkers and prediction models for OSCC from each profile as well as from the interface outcomes of integrating the two platforms. Additional aims of the thesis go further to identify the mechanisms underlying the biological changes during tumorigenic transformation of OSCC, as well as to determine biological processes underpinning the heterogeneity of the disease among populations. Two review studies were carried out in this thesis. The review study of published transcriptomic profiles of OSCC specified several genes and pathways exhibiting substantially altered expression in cancerous versus noncancerous states across studies. However, the result of the review suggests not relying on the final set of genes published by the individual studies, but to access the raw data of each study and start subsequent analysis from that stage using unified bioinformatics approaches to acquire useful and complete understanding of the systems biology. The other review study focused on the metabolic profiles of OSCC and revealed a systemic metabolic response to cancer, which bears great potential for biomarker development and diagnosis of oral cancer. However, the metabolic signature still needs to improve specificity for OSCC from other types of cancer. In an attempt to detect a robust gene signature of OSCC overcoming the limitation of the transcriptomic review in accessing the raw data from the previous works, four public microarray raw datasets (comprising 365 tumour and normal samples) of OSCC were successfully integrated using ComBat data integration method in R software, determining the common set of genes, biomarkers, and the relative regulatory pathways possibly accountable for tumour transformation and growth in OSCC. Examination of the meta-analysis datasets showed several discriminating gene expression signatures for OSCC relative to normal oral mucosa; with a signature of 8 genes (MMP1, LAMC2, PTHLH, TPBG, GPD1L, MAL, TMPRSS11B, and SLC27A6) exhibiting the best discriminating performance and show potential as a diagnostic biomarker set. In addition, 32 biomarkers specific to OSCC and HNSCC were identified with the majority involved in extracellular matrix (ECM), interleukins, and peptidase activity where around 2/3 of them are located in the extracellular space and plasma membrane. Additionally, investigation of the interactive network created by merging metabolic and transcriptomic profiles highlighted the significant molecular and cellular biofunctions, pathways, and biomarkers distinguishing OSCC from normal oral mucosa. The results highlighted interactions of significantly altered expression of Dglucose, ethanol, glutathione, GABA, taurine, choline, creatinine, and pyruvate metabolites with the expressed PTGS2, IL1B, IL8, IL6, MMP1, MMP3, MMP9, SERPINE1, COL1A1, COL4A1, LAMC2, POSTN, ADAM12, CDKN2A, PDPN, TGM3, SPINK5, TIMP4, KRT19, and CRYAB biomarkers of OSCC. Such a pattern may represent a clinically useful surrogate for the presence of OSCC which might help in deciphering some of the obscure multifaceted mechanisms underlying carcinogenesis of OSCC which emerged from dysregulated genetic and metabolic system of the body. In an attempt to define pathways of importance in two phenotypically different forms of OSCC, transcriptomic analysis of OSCC from UK and Sri Lankan patients was undertaken. The development of OSCCs in UK and Sri Lankan populations appears largely mediated by similar biological pathways despite the differences related to race, ethnicity, lifestyle, and/or exposure to environmental carcinogens. However, results revealed a highly activated “Cell-mediated Immune Response” in Sri Lankan tumour and normal samples relative to UK cohorts which may reflects a role in resistance of patients to invasiveness, metastasis, and mortality observed in Sri Lankan relative to UK patients. In conclusion, multiple molecular profiles were able to identify a unique transcriptomic profile for OSCC and could further discriminate the tumour from normal oral mucosa on the basis of 8 genes. Altered expression of several metabolic and transcriptomic biomarkers specific for OSCC were identified, along with several dysregulated pathways and molecular processes found common in patient with oral cancer. Integrating both metabolomic and transcriptomic signatures revealed a promising strategy in analysing the concurrent perturbation in both genetic and metabolic systems of the body. Additional results revealed possible impact of specific supplementary dietary components in boosting the immune system of the body against invasion, progression, and metastasis of the disease. Further clinical studies are required to confirm and validate the current results.

Head and neck cancer (HNC) makes up 6 ¢H of the cancer patients in the world every year. This disease usually occurs in males and the incidence is increasing year by year. According to statistical analysis, HNC has less than 50 ¢H five-year survival rate. Therefore, the research of HNC seems imminent so that may lead to the development of new approaches of diagnosis and therapy. Recent research had shown that expression of podoplanin caused cellular proliferation, and may be associated with tumor invasion, metastasis and malignant prognosis. Podoplanin, a mucin-type transmembrane sialoglycoprotein, is highly expressed in lymphatic endothelial cells but not expressed in vascular endothelial cells. The purpose of this study was to determine the clinical and pathological significance of podoplanin in oral squamous cell carcinoma (OSCC). Therefore, we collected clinical specimen and associated patient history of OSCC. Further, we used the human cell lines of HNSCC (Fadu, Hep2) to investigate the molecular regulation of podoplanin. Podoplanin expression was analyzed by RT-PCR and Western blot assay firstly. As shown, podoplanin was found to be overexpressed in tumors compared with normal adjacent tissues. Further, immunohistochemical analysis revealed the location of podoplanin expression in OSCC tissues. The results showed that podoplanin had higher expression in T4 stage tumor section than in normal adjacent tissues of OSCC samples, or in T1 stage. Here, podoplanin was highly expressed in the OSCC tumor cell and lymphatics of stage T4 OSCC tissue. Furthermore, we found that overexpression of podoplanin in OSCC patients was associated with decreased five-years survival rate. In the univariate analysis, several factors were statistically significantly associated with disease-specific survival rate, including Tumor stage, Nodal stage, and podoplanin expression. In the subsequent multivariate analysis, only Tumor stage and Nodal stage showed a trend toward worse disease-specific survival. To further investigate the regulatory mechanism of podoplanin and its position of expression within the cell, immunofluorescence and transfection were utilized to assay. The results showed that podoplanin was expressed in the nuclear membrane of the Fadu and Hep2 cell lines, and the PI3K/AKT signaling pathway was involved. We suggest that the role of podoplanin in OSCC should be further investigated for potential future treatment.

Migration Stimulating Factor (MSF) is an oncofoetal protein which is constitutively produced by both epithelial and stromal cells during foetal development, not expressed by the majority of their normal adult counterparts, but re-expressed during pathological processes such as cancer and wound healing. Scotland has the highest occurrence of oral cancers in the UK; the incidence is still increasing, but patient survival remains very poor. The expression of MSF in oral tumours has not been previously reported. The aims of this study were: • To determine the effects of MSF on the migration of oral tumour cell lines and normal stromal cells in culture (chapter 3), • To ascertain the possible presence, diagnostic and prognostic value of MSF in oral squamous cell carcinoma (OSCC; chapter 4), and salivary gland tumours (SGT; chapter 5). • To identify the putative MSF receptors in oral tumour cell lines (chapter 6). For tissue culture studies, the effects of rhMSF (wild type and mutant proteins) were examined on human cell lines TYS, HSG, Endo 742 and FSF44. These cells were derived from OSCC, SGT, microvascular endothelial cells and skin fibroblasts, respectively. For ex-vivo studies, paraffin embedded archival specimens of OSCC and SGT were stained with specific MSF antibodies and the level of staining was assessed by consensus of 2-4 independent observers. The association between MSF expression and patient survival was determined by Kaplan-Meier and log-rank tests. Results presented in this thesis indicate that TYS and HSG cells secrete bioactive MSF in culture. rhMSF stimulated the migration of these tumour cells. The use of mutant proteins demonstrated marked differences among the cells examined: Five bioactive motifs (4x IGD and 1x HEEGH) were required for MSF bioactivity on TYS and HSG cells, whereas only one of these motifs was required for Endo 742 and two for FSF44. MSF+aa and MSF-aa showed the same migration-stimulating activity, but differ in their interaction with the MSF-inhibitor Neutrophil Gelatinase-Asscciated Lipocalin (NGAL). NGAL was shown to bind to and inhibit MSF+aa, but not MSF-aa. The bioactivity of MSF+aa and MSF-aa was inhibited by Insulin-like Growth Factor Binding Protein-7 (IGFBP7), MSF-function-neutralising antibody and antibody to the integrin avß3. This integrin was identified in the cell membrane material bound to MSF, suggesting that avß3 is a receptor for MSF.

Estimates for 2017 indicate that 20% of cancers globally are gastrointestinal tract (GIT) cancers, with oesophageal cancer being the 8th most common cancer. Oesophageal squamous cell carcinoma (OSCC) occurs in the upper to mid oesophagus and is present at high incidence in developing countries including South Africa. There are no early symptoms, resulting in late diagnosis and poor prognosis. In this study, tumour and blood DNA was obtained from 35 OSCC patients and subjected to whole genome sequencing (WGS). Bioinformatics analysis pipelines were designed to identify the possibility of novel viral insertions, investigating Human Endogenous Retroviruses (HERV's) insertions alongside the presence of somatic mutations in patient samples. The aims being to identify integration of any foreign DNA, to investigate if there is any linkage between HERV insertion and somatic mutations, and to identify any somatic mutations of potential interest in the OSCC cohort. The novel virus investigations however, proved to be inconclusive and there appeared to be no link between HERV insertions and somatic mutations present in the patients. Very significantly, it was determined that numerous somatic mutations were present in the MUC3A gene of the patient cohort, an interesting observation as no such previous association with OSCC has been recorded. MUC3A is a membrane-bound glycoprotein component of mucous gels, and its aberrant expression has been correlated with invasion and metastasis in a variety of other cancers. However, due to the complexity of the particular gene sequence and the known inconsistencies of variant calling performed on complex data sets, these mutations should be viewed with extreme caution as they are likely to be false positives. Analysis of RNA-seq data showed a 4.6 log2 fold increase in MUC3A expression in the tumour samples of these OSCC patients, with a P-adjusted value of 7.05e-06, suggesting highly significant differential gene expression. Functional enrichment analysis further showed that MUC3A was significantly associated with one of the top 5 gene ontologies (extracellular matrix structural constituent) for molecular function ontology class together with a number of collagen (COL) and MMP genes known to play a role in oncogenic progression and membrane stiffness. GSEA and KEGG analysis indicated predominantly chemokine/cytokine pro-inflammatory enriched pathways. Immunohistochemistry staining showed 10 out of 13 of the samples had no detectable levels of MUC3A protein, suggesting that the production of a non-functional truncated protein may lead to the upregulation of MUC3A expression that could possibly play a role in downstream pro-oncogenic signalling.

Yes / Background: High HOX gene expression has been described in many cancers, including oral squamous cell carcinoma and the functional roles of these genes are gradually being understood. The pattern of overexpression suggests that inhibition may be useful therapeutically. Inhibition of HOX protein binding to PBX cofactors by the use of synthetic peptides, such as HXR9, results in apoptosis in multiple cancers. Methods: Activity of the HOX-PBX inhibiting peptide HXR9 was tested in immortalised normal oral (NOK), potentially-malignant (PMOL) and squamous cell carcinoma (OSCC) cells, compared to the inactive peptide CXR9. Cytotoxicity was assessed by LDH assay. Expression of PBX1/2 and c-Fos was assessed by qPCR and western blotting. Apoptosis was assessed by Annexin-V assay. Results: PMOL and OSCC cells expressed PBX1/2. HOX-PBX inhibition by HXR9 caused death of PMOL and OSCC cells, but not NOKs. HXR9 treatment resulted in apoptosis and increased expression of c-Fos in some cells, whereas CXR9 did not. A correlation was observed between HOX expression and resistance to HXR9. Conclusion: Inhibition of HOX-PBX interactions causes selective apoptosis of OSCC/PMOL, indicating selective toxicity that may be useful clinically. / Intercalated Degree Scholarship from the Harry Bottom Trust; scholarship by Becas Chile, Comisión Nacional de Investigación Científica y Tecnológica de Chile (CONICYT), Grant 72160041

Oral squamous cell carcinoma (OSCC) accounts for majority of head and neck cancers and ranks as the sixth most common cancer in the world. OSCC belongs to the most understudied cancers and little is known about molecular mechanisms underlying its etiology and progression to metastasis. A hallmark of cancer is the enhanced posttranslational modification of cell surface proteins with complex N-glycans. Our studies have shown that induced protein N-glycosylation via activation of the core N-glycosylation-regulating gene, DPAGT1, is associated with reduced E-cadherin adhesion, as well as deregulation of several oncogenic signaling pathways, including Wnt/β-catenin and Hippo. Modest increases in DPAGT1 expression are associated with dramatic amplification of Wnt/β-catenin activity and increased expression and nuclear localization of the Hippo pathway effectors TAZ /YAP. The goal of this study was to align the expression and localization of DPAGT1, complex N-glycans, β-catenin, and TAZ/YAP with the progression of oral cancer in vivo from dysplasia to OSCC. Human oral tissues from different stages of OSCC pathogenesis were characterized for DPAGT1/β-catenin/α-catenin/YAP/TAZ expression and localization and correlated with cell surface expression of complex N-glycans by PHA lectin staining and with expression of primitive cell surface markers, CD44, CD24 and CD29. Results showed that high DPAGT1 expression and nuclear TAZ became increasingly associated with disorganized E-cadherin junctions as oral epithelium progressed from mild to severe dysplasia to OSCC. This correlated with increasing expression of cell surface complex N-glycans and CD44. These studies suggest that DPAGT1/β-catenin/TAZ and high PHA staining represent novel signatures for OSCC pathogenesis.

There is some evidence to suggest that human papilloma virus (HPV) may play a causal role in head and neck carcinoma (HNSCC). The aim of this study was to investigate the prevalence of HPV DNA in HNSCC and to determine whether any correlation exists with p16 or survival. An initial pilot study of sixty formalin-fixed HNSCC was carried out in order to optimise the methodology for the PCR and immunohistochemistry. A further 84 benign lesions, 12 dysplasias and additional 80 HNSCC were also included. In the pilot study the prevalence of all HPV types was 67% of which 18% were high risk-HPV (HR-HPV) and for the combined carcinoma sample it was 59% of which 25% were HR-HPV. The overall HPV prevalence was 51% and 42% for benign lesions and dysplasias with HR-HPV accounting for 14% and 8% respectively. A total of four alpha HPV types were identified and eleven beta HPV types. Multiple HPV types co-existed in the same tissue and in some cases both alpha and beta HPV. The results may suggest that HR-HPV may play a role in a small subset of HNSCC. An association was found between HPV status and gender, age group, survival, nodal metastasis and T3 tumour size and smoking. HPV16 was predominantly present in female patients and was associated with an improved overall survival and recurrence free survival. p16 positivity varied from 76-78% in carcinomas, 51% in benign lesions and 66% in dysplasias. p16 status was not associated with disease recurrence or nodal metastasis. Positive p16 staining and high staining intensity was associated with a poorer overall survival and the male gender, an older age group, anatomic site, and T2 tumour size. Overall HPV status was not correlated with p16 expression but a correlation found between p16 and HPV16 may suggest that p16 could potentially act as a surrogate marker of HPV16. However, the lack of concordance would suggest that in isolation p16 may not be a reliable marker for HR-HPV and should not be relied upon in isolation. Our findings could suggest that HPV16 and p16 status may be independent predictors for prognosis and disease recurrence.

AIM & OBJECTIVES: Human papilloma virus (HPV) has been related to cervical infection, however, its part in Head and Neck Squamous Cell Carcinoma (HNSCC) is still debatable and is easy to refute. Suspicion of HPV causation is heightened when carcinomas arise in patients that are young and have never smoked. The present UK based study undertaken at Northampton NHS Trust endeavoured to determine the extent to which HPV is an entity in HNSCC in the UK. Furthermore, the study investigated whether sexual behaviour (as measured by sexual health clinic (SHC) attendance) is linked the acquisition of HPV associated HNSCC in young age groups. HNSCC incidences and sexual trends in the UK were collected from publicly available databases to identify if there were any changes at a national level in sexual behaviours and their influence on HNSCC in young age groups. MATERIALS & METHODS: PCR was used to evaluate the presence of HPV in biopsy samples from of 99 patients diagnosed with HNSCC at Northampton Hospital from 2006 to 2014. Patient demographics on age, sex, smoking, alcohol use and SHC attendance were also collected. All HPV PCR positive biopsies were further genotyped using an ABI 3130xl genetic analyser. Databases in the UK; including GLOBOCAN, NATSAL and PHE were searched for data on HNSCC prevalence, sexual behaviour trends and vaccine uptake. Multinomial regression explored the relationship between HPV positivity and sex, age, smoking, drinking, race and SHC attendance. RESULTS: PCR showed that 25.2% (25/99) of biopsies tested were positive for HPV and were all obtained from white participants. Most specimens (23, 92%) were high-risk (HR) HPV 16 positive with a mean age of 56 for HPV positivity and 72% of the cases 50-60 years old. Smokers were 11% in total (11/99) with most 88.9% participants (88/99) being non-smokers. HPV positivity was strongly linked with non-smoking history (p < 0.001); no alcohol abuse (p < 0.001); male gender (p < 0.001); young age less than 60 years (p < 0.001) and SHC attendance (p < 0.001). A Kruskal-Wallis post hoc test affirmed the impact of age on HPV positivity (p= < 0.05). GLOBOCAN and Cancer Research demonstrated a rising UK HNSCC pattern of over 200% for both sexes from 1975 to 2011. The three NATSAL surveys undertaken in 1990-1991, 1999-2001 and 2010-2012 demonstrated an overall increase in opposite and same sex partners. The UK average of individuals engaging in oral sex was in the younger age groups of between 16 and 54 with at least 70% of males and 63% females of that age engaging in oral sex. Finally, NASTAL 1, 2 and 3 surveys reported 20 vs 15; 25 vs 55; 55 vs 65 of males and females respectively with more than 10 sexual partners to have attended the SHC. The UK immunization take-up was over 90% countrywide. CONCLUSION: Few research studies have been conducted to date on HPV as a cause of HNSCC in the UK. The present research showed 25.2% of HNSCC to be caused by HPV, with the high risk (HR) genotype 16 (the leading cause of cervical cancer) accounting for 92% (23/25) of the cases. These outcomes affirmed the high prevalence of HR-HPV in HNSCC, with a rate of 25.2% similar to those reported previously. Routine HPV testing in those aged below 60 is therefore warranted. Smoking and drinking showed negative correlation; the young age of below 60 and attendance of the SHC for both sexes showed a positive correlation with HPV positive HNSCC. NATSAL data showed increased sexually risky behaviour coupled with attending the SHC in younger ages for both sexes. Increased sexually risky behaviour as shown in NASTAL surveys may be the reason why young age and SHC attendance is positively correlated with HPV HNSCC. The study highlights a conceivable relationship between HPV positive HNSCC in those under 60 years with no smoking history who attended the SHC. Smoking and drinking are known risks for HNSCC in those past 65 years of age; the negative association with HPV HNSCC in the young in the present research revealed smoking and drinking to have reduced association with HPV HNSCC. The reported HR-HPV positive HNSCC in young age groups inform future vaccination strategies and consequently decrease the quantity of HPV HNSCC's.

No description available.