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Thesis (MSc(R)) - University of Glasgow, 2008. / Submitted to the University of Glasgow for the degree of Master of Science (Medical Science) in the Faculty of Medicine, 2008. Includes bibliographical references. Print version also available.

published_or_final_version / abstract / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Research dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science. 28th May 2017. / The focal adhesion kinase or FAK plays an important role in detecting and transducing signals that are generated by cell-substrate attachment (Focal adhesions). When these pathways are activated under atypical conditions they may promote metastasis, uncontrolled proliferation and a chemo-resistant phenotype. However the mechanisms by which this protein is activated ectopically in human oesophageal squamous cell carcinomas cell lines (HOSCC) is unknown. In the current study it was hypothesised that the p90 ribosomal S6 kinase, a key member of multiple pro-survival pathways (activator of the Y-box binding protein-1), activates FAK. RSK may promote FAK activation directly, from its location at the plasma membrane, or it may modulate FAK activation indirectly via the regulation of one of its substrates. RSK inhibits the activation of the glycogen synthase kinase 3β (GSK3β) by phosphorylation at Ser9. GSK3β also localises at focal adhesions and may therefore play a role in mediating FAK activity. To ascertain the role RSK plays in FAK activation, 3 inhibition studies were performed. In the first assay, RSK was specifically inhibited within HOSCC and the levels of active FAK monitored (two different environmental conditions). FAK activation was monitored by detecting the auto-phosphorylation of FAK at Tyr397. A GSK3β inhibition assay was then performed in which GSK3β was specifically inhibited and the levels of active FAK monitored. Lastly, a dual inhibition assay was performed where both RSK and GSK3β were inhibited simultaneously and the levels of active FAK monitored. The overall net changes in the phospho-protein profile indicated that all of the HOSCC cells had distinct cellular responses to the three inhibitor combinations. However RSK did not appear to activate/inhibit FAK activity directly, in most of the HOSCC cells, but rather modulated FAK activation through the inhibition of GSK3β. The effects the RSK/GSK3β pathway had on FAK activation was partially dependent on the HOSCC cells containing active levels of PTEN. Interestingly, the inhibition of both GSK3β and RSK reduced the levels of active FAK in 3 of the 5 HOSCC cell lines, indicating that this might be a good anti-cancer therapeutic. RSK appeared to play a more context specific role in FAK activation within the HOSCC cells suggesting that the grading system for moderately differentiated carcinomas needs to be improved. This paper also highlights the importance of studying the effects the microenvironment has on neoplasmic transformation as varied environmental conditions, during the RSK inhibition studies, drastically impacted the effects the RSK inhibitor had on FAK activation. / MT 2017

Oesophageal Squamous cell Carcinoma (OSCC) has a poor survival rate and is highly prevalent in southern Africa. Cisplatin is the standard therapeutic drug for OSCC, but has poor efficacy due to drug resistance and toxicity. Development of therapies that can be used to reduce the dose of cisplatin or offer a more effective tumour response is of great importance. Metformin is an anti-diabetic drug that has demonstrated anti-proliferative effects in various cancer types. Metformin’s potential as a chemotherapeutic drug is highlighted by its low toxicity profile, ability to reduce growth factor signalling, and toxic effects against cancer stem cells. In this study we combined metformin and cisplatin to find that whilst metformin reduced the proliferation of OSCC cell lines, it antagonised the effects of cisplatin. This was attributed to increased levels of reduced thiols as a consequence of enhanced glycolysis, which leads to the formation of reducing equivalents such as NADPH. Since metformin enhances the intracellular reducing potential, we combined metformin with drugs that are activated in reducing environments. Two copper bis(thiosemicarbazones), Cu-ATSM and Cu-GTSM, both retained their toxicity in the presence of metformin. Disulfiram (DSF), an established anti-alcoholism drug, has previously demonstrated chemotherapeutic potential when conjugated to copper (Cu-DSF). DSF and Cu-DSF both exerted potent cytotoxic effects against OSCC cell lines which were enhanced by metformin. Metformin increased intracellular copper accumulation when combined with DSF and we found that DSF perturbed proteasome function, as observed in other studies. Furthermore, we identified a novel target of DSF, the lysosome, and found that DSF reduces lysosomal pH, which led to increased accumulation of lysosomal protein aggregates, thereby inhibiting autophagy in OSCC cell lines. Therefore, the co-prescription of metformin and cisplatin is not advised for OSCC treatment. However metformin can be effectively combined with DSF, which inhibits multiple protein degradation pathways, to offer a novel treatment option for OSCC.

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. / A member of the epidermal growth factor (EGF) family; transforming growth factor alpha (TGF-a) shares significant homology with EGF and binds to the EGF receptor (EGF-R). Like EGF TGF-a plays important roles in normal physiological processes; but, as its name signifies, it has potent transforming ability; often associated with autocrine stimulatory mechanisms. The purpose of this study is to investigate a possible role for TGF-a and its receptor in certam human oesophageal squamous cell carcinoma (SCC) cell lines - namely, WHCO-I, -3 and -5. The wellstudied A431 epidermoid. carcinoma cell line was used throughout for control purposes. (Abbreviation abstract) / Andrew Chakane 2018

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science Johannesburg, 2017 / Cancer is characterised by abnormal cell proliferation and is one of the leading causes of death in first world countries and the second leading cause in developing countries. In 2012 alone, over 14 million cases were reported and over 8 million deaths were attributed to cancer worldwide, with sub-Saharan Africa, especially South Africa having one of the highest oesophageal cancer rates in the world. An important aspect of cancer is the telomeres, which are 10-15kbp of TTAGGG DNA repeats in humans at the ends of chromosomes. These repeats are maintained by the enzyme telomerase. Up to 90% of all cancers show increased telomerase activity to overcome the "end-replication" problem in which the telomeres shorten after each cell division. This eventually leads to cellular senescence. Due to the high number of cancers relying on increased telomerase activity to bypass senescence, telomerase could be a viable target for anti-cancer therapies. The limiting factor of the multi-subunit telomerase enzyme is its telomerase reverse transcriptase component (hTERT). hTERT has also been shown to migrate to the mitochondria during times of high oxidative stress caused by reactive oxygen species (ROS). Here it confers protection to the mitochondria against ROS, potentially preventing the cell form undergoing apoptosis and reaching senescence. This can potentially be detrimental, as cells become damaged by the ROS and continue dividing. This could lead to further genetic damage. Metformin, a drug used for the treatment of type-2 diabetes, has been linked to lower incidences of cancer. The mode of action of metformin is not yet fully understood, however it is known that it affects the mitochondria. Since hTERT and metformin could co-localise, the drug may influence hTERT and potentially telomerase activity. This makes metformin an anticancer candidate to be used in conjunction with traditional anticancer therapies. To determine telomerase activity in metformin treated oesophageal carcinoma cells, qPCR based telomerase activity assays must be used. These assays can be very expensive and time consuming, so a faster and cheaper alternative would be beneficial. Therefore, the aim of this project was to alter and improve a nanoparticle based detection method for telomerase activity, by decreasing the time required to prepare the DNA functionalised nanoparticles as well as determining a more rapid method of data measurement, and compare it to conventional qPCR based techniques (TRAPeze RT Telomerase Activity Kit – Merck). Thereafter the effects of the metformin treatment on telomere dynamics, such as telomere length, telomerase activity and hTERT mRNA expression, in oesophageal squamous carcinoma cells were determined. Gold nanoparticles were synthesised and functionalised with thiolated-DNA (telomerase substrate). These functionalised particles were characterised using transmission electron microscopy. To assess telomerase activity the extracted protein was added to the functionalised nanoparticle solution and allowed to elongate the coupled DNA. A characteristic of gold nanoparticles is that the size of the particles as well as their proximity to one another determines the colour of the nanoparticle solution. Due to the steric hindrance caused by the now elongated DNA, a distinct colour change was observable. The change in absorption spectra of the nanoparticle solution was recorded after the enzyme elongated the substrate. This nanoparticle based assay was then compared to TRAPeze RT Telomerase detection kit (Merck-Millipore) as a positive control. Using the conventional qPCR based telomerase activity assay, it was found that metformin significantly decreased telomerase activity in oesophageal cancer cell lines, however this was not seen using the nanoparticle assay. A colour change was observed with the nanoparticle assay compared to the negative control reflecting detection of telomerase activity. However, no significant decrease in telomerase activity could be detected due to metformin treatment. More optimisation is required, however this technique has great potential, as nanoparticle based assays are also known for their high sensitivity. This technique is also far more rapid and significantly cheaper that the qPCR based method. The gold nanoparticle based telomerase activity assay could become an alternative to conventional qPCR based techniques. / MT2018