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published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2012 / Cell-extracellular matrix (ECM) detachment triggers a cell survival mechanism known as autophagy. A link between attachment and autophagy suggests a form of adhesion-based regulation, involving mechanotransduction of extracellular-originating signals to the cellular machinery controlling autophagy induction. This implies a role for integrin-linked kinase (ILK), which transmits mechanical stimuli to the mammalian target of rapamycin (mTOR) signalling pathway. Cells with a propensity for metastasis may negate these adhesive signals, inducing autophagy inappropriately. Metastasis is a hallmark of transformation frequently associated with human oesophageal squamous cell carcinoma (HOSCC). Additionally, hyperactive mTOR/mTORC1 signalling correlates increasingly with HOSCC. Therefore, the protein expression of significant signal transduction pathway intermediates was investigated in response to both soluble and ECM-originating stimuli. Measurements by SDS-PAGE and western-blotting coupled to semi-quantitative densitometry, during standard tissue culture conditions, revealed that HOSCC’s expressed moderate-to-high levels of mTOR, p-RPS6(Ser 235/236) and mATG-13; indicating elevated levels of autophagy induction despite aberrant signalling through mTOR/mTORC1. Additionally, an 80 kDa mTORβ isoform was identified in HOSCC cells with lower mTOR abundance, presumably to maintain aberrant mTORC1 signalling. A canonical role for the PI3K/PKB pathway was also identified; where autophagy induction accompanied diminished mTORC1 signalling in response to specific PI3K inhibition with LY294002 and serum withdrawal. However, autophagy induction varied in response to a dose-dependent decrease in mTORC1 signalling after exposure of HOSCC cells to rapamycin. Moreover, specific inhibition of p90RSK with BI-D1870, suggests that mTORC1 phosphorylates RPS6(Ser 235/236) in the absence of MAPK signals. Furthermore, ectopic ILK expression indicated an enhanced potential for adhesion-based signalling. Correspondingly, HOSCC cells commonly increased mTOR and p-RPS6(Ser 235/236) expression following growth on fibronectin or collagen. However, co-immunoprecipitation analysis revealed that signals transduction to mTOR precludes a direct interaction with ILK or FAK. Rather, ECM-modulation of mTOR occurs in a integrin-triggered, but PI3K-depedant manner; since specific inhibition of PI3K negated fibronectin-induced increases of mTOR concentration and RPS6(Ser 235/236) phosphorylation. Thus, these data strongly suggest mTOR is a target for adhesion-based signal transduction, where the ECM influences cell survival through mTORC1. Moreover, exploitation of autophagy induction post cell-ECM detachment in HOSCC may promote the survival of metastases during dissemination.

Previous data have unveiled a novel autoregulatory feedback loop between iASPP and p63 in the stratified epithelia; this involves two microRNAs, miR-574-3p and miR-720, and is critical for epidermal homeostasis. The iASPP oncoprotein, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is a key inhibitor of p53 and NF-κB and is highly expressed in many cancers. Non-melanoma skin cancer, comprising of cutaneous squamous carcinoma (cSCC) and basal cell carcinoma, is currently the most common malignancy in the UK. In view of this newly-identified iASPP-p63 axis, I hypothesised a potential role for dysregulation of this feedback loop in the pathogenesis of cSCC and aimed to assess the role of iASPP in human cSCC. Protein and mRNA expression patterns were assessed in a panel of 10 cSCC cell lines generated by our group. In addition, immunostaining of iASPP and p63 was performed in 107 cSCC clinical samples of variable differentiation status. The data reveal an overall increase in expression of iASPP and ΔNp63 in cSCC but also suggest a significant alteration of the cellular localisation of iASPP dependent on the differentiation status of the tumour. To further assess the effects mediated by the iASPP/p63 axis, iASPP and p63 have been silenced by RNAi technology in a subset of cSCC cell lines. Whilst data shows the direct effects of iASPP and p63 upon each other's expression are maintained in cSCC, epigenetic dysregulation of the feedback loop at the microRNA level may be occurring via a novel p63 regulator, miR-211-5p. Functionality of iASPP in cSCC (proliferation, apoptosis, cell motility/migration and invasiveness) provides evidence for a role of iASPP in preventing epithelial-mesenchymal transition in cSCC via a p63/miR-205-5. These findings provide potential future directions for development of clinical biomarkers and novel therapeutic targets for cSCC and may ultimately provide the tools for tackling the increasing morbidity and mortality associated with this malignancy.

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. It is a result of numerous aetiological factors such as genetic predisposition, smoking, excessive alcohol consumption and viruses such as the human papilloma virus. Due to late diagnosis it has a high mortality and morbidity rates which has remained unchanged over the last 5 decades. Currently no screening is available for high risk patients for better monitoring. Diagnosing OSCC relies on histopathology of biopsy tissue, reviewed for dysplasia and advancing lesions. Although the technique has been used for decades for successful diagnosis it fails to identify the molecular signature of OSCC which appears much before the visual signs. It also falls short in predicting the malignant transformation of pre-malignant oral lesions. Identifying the molecular and genetic changes leading to OSCC lesion will aid in more specific (quantitative) and early diagnosis of the disease reducing the financial burden of treating late-stage OSCC patients on the healthcare system. This study focuses on developing new adjuncts which can be used alongside histopathology for early diagnosis. There is a need to monitor high risk patients through non-invasive methods causing less patient discomfort. We therefore explored the potentials of exosomes which are extracellular vesicles secreted by normal and tumour cells. They can be isolated from body fluids such as blood and saliva. In cancer biology exosomes offer both diagnostic and therapeutic advantage. Their involvement in cell-cell communication indicates their influence in tumour development, progression, metastasis and therapeutic efficacy. Exosomes released by cancerous cells carry numerous biomarkers, which are passed on to healthy cells via microenvironment, causing stromal and angiogenic activation along with immune escape. In this study exosomes were successfully isolated from body fluids (blood, saliva and plasma) and cell line supernatant through ultracentrifugation and characterised by visual and particle size quantification techniques including Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), Zetasizer and Nanosight Tracking Analysis (NTA). Exosomal specific membrane proteins were identified through Western blotting. 5 We report the presence of a potential protein biomarker located exclusively on the outer membrane of cancer exosomes. Since body fluids consist of a heterogeneous population of exosomes derived from multiple cell types, such surface biomarker can potentially be used to isolate OSCC exosomes. Characterisation of exosomal mRNA cargo was done using Agilent Bioanalyzer (for RNA quantity and quality assurance) and reverse transcription-quantitative PCR (RT-qPCR; for gene specific quantitation). Functional significance of exosomes was studied by transfecting normal oral keratinocyte cells with self and cancer-derived exosomes. Through gene-expression microarray and subsequent RT-qPCR verification, we report a panel of differentially expressed genes involved in essential cellular functions being modulated by exosome transfection. A previously developed molecular diagnostic system by our research group called quantitative malignancy index diagnostic system (qMIDS) based on FOXM1 oncogene and its downstream targets was validated on archival formalin fixed paraffin embedded OSCC patient biopsy samples. We report that qMIDS index successfully correlates with the disease stages including dysplasia, tumour and lymph node metastasis. Furthermore, through meta-analysis of 8 OSCC microarray studies we identified a panel of six genes including PLAU, FN1, CDCA5, CRNN, CLEC3B and DUOX1 (q6) which are able to identify two clinically distinct sub-groups of OSCC patient population. Through RT-qPCR the expression of q6 biomarkers was established in 100 OSCC biopsy samples. This information can be of immense importance in developing personalized treatment strategies based on the molecular makeup of the presenting tumour.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

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.

Cisplatin is an effective chemotheraputic agent for head and neck squamous cell carcinoma particularly in conjunction with radiation therapy. Unfortunately, its cytotoxic profile and associated systemic side effects limit its clinical efficacy. A localized delivery system was developed for cisplatin by processing calcium polyphosphate (CPP) in a multistep gelling protocol, with the goal of limiting its systemic toxicity and enhancing its overall clinical applicability. In addition, a novel method for processing the material was examined utilizing cold isostatic pressure (CIP) to allow for miniaturization of the system into an implantable device. The integration of cisplatin into the matrix was examined for efficient and dose dependent loading via dissolution of the final product and measurement of platinum concentrations by inductively coupled plasma optical emission spectrometry (ICP-OES). Drug release was measured in vitro by placing the CPP-cisplatin matrix into TRIS buffer solution while measuring the platinum concentration at given intervals from 0.5 hours to 14 days. The cytotoxicity of the cisplatin against L1210 cells was examined using an MTT assay following a 12-hour elution. The material demonstrated a predictable and dose dependent loading of cisplatin, although the release of the drug showed variability exemplified by a more pronounced burst release with aging of the stock CPP glass particulate. The CPP/cisplatin matrix exhibited cytotoxic effects after processing. This work suggests that further evaluation of this material as a matrix for cisplatin delivery should be undertaken in an attempt to normalize release, maximize the concentration within the system and further optimize the bead format in order to improve the potential for clinical usage.