Hypoxia, a state of low oxygen, is a feature of most solid tumours as a consequence of poor tumour vascularisation. The mechanisms, which allow cancer cells to survive and continue to grow in hypoxia, are coordinated by the transcription factor HIF. The tumour suppressor gene van Hippel-Lindau (vHL) that targets HIF for degradation is mutated in the vast majority of renal cell carcinomas (RCCs), highlighting the importance of hypoxia to tumour biology. There is, therefore, an important need to understand the adaptive changes mediated by hypoxia and to target this clinically. One class of genes regulated by HIF are microRNAs (miRNAs). MiRNAs are short, single stranded RNA that primarily inhibit protein expression from target m RNA. The first aim of this project was to identify novel hypoxia regulated miRNAs in bladder cancer and assess their functional significance. It was found that a number of miRNAs were induced in hypoxic conditions. The hypoxic induction of miR-210 was conserved in all cell lines tested. MiR-145 was found to be highly induced by hypoxia in RT4, a cell line derived from a low- grade, non-muscle invasive tumour. We showed that miR-145 was a novel, HIF target gene with two hypoxia response elements identified within the promoter. Functionally we found that miR-145 induces apoptosis in RT4 cells. MiR-100 was downregulated in hypoxia, but this downregulation did not involve HIF. Regulation of miR-100 was of interest, as it is known to target FGFR3, a gene commonly overexpressed or mutated in bladder cancer. Concomitant with a decrease in miR-100, both the mRNA and protein level of FGFR3 were found to increase in hypoxia in RT4 and RT112 cells. Increased FGFR3 expression in hypoxia was involved in sustaining activation of the downstream signaling targets phospho-PKB and phospho-ERK. In addition, we demonstrate a role for FGFR3 in regulating both 2D and 3D growth and of miR-100 in regulating 3D growth of RT4 cells. We also showed that miR-100 decreased the protein levels of mammalian target of rapamycin (mTOR). However, transfection of miR-l00 into RT4 cells did not affect the sensitivity of this cell line to rapamycin. The genetic and biochemical changes that occur in (hypoxic) tumours may alter their responsiveness to chemotherapeutic agents such as rapamycin. The second aim of this project was to investigate the responsiveness of RCCs to clinically approved chemotherapeutic agents, with the goal of correlating any differences in response to alterations in expression of specific genes. Although hypoxia regulated miR-100 did not affect sensitivity to rapamycin, we extended these studies and investigated the role of vHL status on response of renal cancer cell lines to sorafenib, sunitinib, rapamycin and metformin. We found that the presence of vHL increased resistance to rapamycin. Sensitivity to these drugs was also tested in 10 primary cell lines. There was varying sensitivity to these drugs across the cell lines representing the heterogeneity of renal cancer. We analysed the expression of a number of genes in the m TOR and hypoxic pathways in these tumours, we found the expression of a known hypoxic gene REDDl correlated with sensitivity to rapamycin. REDDl expression levels were also higher in tumour tissue when compared to normal renal parenchymal tissue and was associated with other prognostic markers such as CA9, miR-210 and vascular invasion suggesting a role as a diagnostic or prognostic marker and in patient selection for treatment with rapamycin.

The cancer stem cell (CSC) theory proposes that a minor population in tumor cells with specific features, such as self-renewal and reproducible tumor phenotype could contribute to tumor relapse and chemotherapy resistance. Several studies have convincingly documented the existence of ovarian CSC, but questions related to the biologic behavior and specific biomarkers of ovarian CSC remain to be clarified. In the present study, we firstly established a tumor cell line with capability of regenerating tumors through serial transplantation of ovarian tumor tissue in non-obese/severe combined immunodeficient (SCID) mice. After separation of CD133+ cells with magnetic beads, we compared the phenotype and biologic behavior of CD133+ versus CD133-cells. It was found that the CD133+ cells were much more potent to produce colonies in semi-solid agar culture than CD133-cells. The proportion of the cells in G0/1 cell cycle is much higher in CD133+ cells than in CD133-cells. Furthermore, in vivo experiments demonstrated that the CD133+ cells were capable of repeatedly regenerate tumors in NOD/SCID mice, while the CD133-cells were not. Compared with CD133-cells, the CD133+ cells expressed much higher levels of the stem cell markers Oct4, Sox2, Nanog and Mcl-1. Clinically, among a total of 290 ovarian epithelial cancers, increased level of CD133 expression was positively correlated with a high cancer stage and had a worse 5-year survival rate. Taken together, the results suggest that the CD133+ cells from human ovarian cancer have the characteristics of CSC, which may contribute to ovarian cancer relapse and anti-apoptotic activity. The method of ovarian CSC enrichment we established provides a feasible and practical way of ovarian cancer research in a molecular level. In addition, CD133 may be used as a prognostic marker for ovarian epithelial cancer, which may have a role for future therapeutic effect.

Philosophiae Doctor - PhD / Introduction: Globally, there are over 190,000 new reported cases of ovarian cancers per annum. This comprises 3% to 4% of all cancers in women. Ovarian cancer is one of the leading causes of deaths in women. Ovarian cancer is the second most diagnosed gynaecological malignancy and over all the fifth cause leading to death among all types of cancer in the UK in 2004. More than 70% of epithelial ovarian cancers are diagnosed at an advanced stage. Consequently, the prognosis is poor and the mortality rate high. Thus, the survival rate is affected by how far the disease has progressed or spread. A dire need exists to identify ovarian cancer biomarkers, which could be used as good indicators of expression in ovarian cancer cells in vitro Aim: The aim of this study was to analyse selected cancer biomarkers, which are currently under intense investigation for their suitability to diagnose epithelial ovarian cancer at an early stage. These biomarkers were analysed in terms of their in vitro expression in normal epithelial cells and ovarian cancer cell lines, which allows for their genomic and proteomic classification. The expression analysis of each biomarker is related to the malignancy of a tumour and, therefore, advocates its use for potential future improvement of sensitive tumour markers. Methods: The primary human ovarian surface epithelial cell line (HOSEpiC), SKOV-3 cells and the OAW42 human epithelial ovarian tumour cell lines were used to evaluate the selected cancer biomarkers. Cells were cultured using appropriate media and supplements, and real-time quantitative polymerase chain reaction (RT-PCR) utilized to validate expression levels of the following genes: HDAC1, HDAC2, HDCA3, HDAC5, HDAC6, HDAC7, HDAC8, LPAR1, LPAR2, MUC16 and FOSL1, against normal housekeeping genes GAPDH and HPRT. In addition, immunocytochemistry was also used in the validation process of the aforementioned genes. Significance: ovarian cancer cells express gene signatures, which pose significant challenges for cancer drug development, therapeutics, prevention and management. The present study is an effort to explore ovarian cancer biomarkers to provide a better diagnostic method that may offer translational therapeutic possibilities to increase five- year survival rate. Results: HDAC5, HDAC6, LPAR1, LPAR2 and MUC16 expressed distinctively in ovarian cancers matched to other tissues or cancer types have already been identified by RT-QPCR and confirmed by immunocytochemistry and efforts to generate monoclonal antibodies to the other six genes (HDAC1, HDAC2, HDAC3, HDAC7, HDAC8 and FOSL1) encoded proteins are underway. Conclusions: here we provide strong evidence suggesting that HDAC5, HDAC6, LPAR1, LPAR2, except MUC16 are up regulated in ovarian cancer. These data were confirmed by examining Human Protein Atlas (HPA) databases, in addition to protein expression of HDAC5, HDAC6, LPAR1, LPAR2 and MUC16 in cells cytoplasm. For future prospective, using other techniques that assess the variant expression that could explain the release of these gene candidates into the circulation with serum tumour markers, and protein expression will be strengthened.

Breast cancer is a complex heterogenous disease that consists of several different subtypes displaying distinct behaviors and responses to different treatments. It is the second leading cause of cancer death among women, and is the most commonly diagnosed cancer in women. Although recent developments have helped shed light into this disease, there is still much to investigate. One particular subtype of breast cancer, known as triple-negative breast cancer, remains the most aggressive, as this tumor type is of high histological grade and preferentially affects women with BRCA1 mutations and women who are younger than 40 years of age. Unlike other subtypes with better prognoses, triple-negative breast cancer still has no targeted therapy, and chemotherapy remains the primary systemic treatment. Recently, there has been an increase of interest in the SOXC family of high mobility group transcription factors and their roles in tumor development. Studies have revealed some of the effects that SOXC genes may have on various tumor types. However, further studies are still needed to elucidate the roles, functions, regulations, and mechanisms of these transcription factors. This study aims to focus on one particular gene in the SOXC family known as sex determining region Y-box 11. Recent studies have shown that sex determining region Y-box 11, also known as SOX11, is one of the factors required for maintaining the basal-like breast cancer phenotype and is also critical in regulating growth, migration, invasion, and expression of signature basal-like breast cancer genes. Emerging evidence also reveals that this transcription factor may have an impact on homologous recombination repair when DNA damage occurs, in triple-negative breast cancer. Using SOX11 overexpression and knockout cell models combined with basic science laboratory techniques and omics, the next generation of laboratory tools, this study seeks to explore the role and function of SOX11 in DNA damage in triple-negative breast cancer. The results of this study have confirmed the recent findings of the role of SOX11 in cell proliferation and growth in triple-negative breast cancer. It has also revealed that overexpression of SOX11 in triple-negative breast cancer cell lines leads to an increase in DNA damage, loss of BRCA1 function, and dysregulation in the cell cycle. High expression of SOX11 is also associated with worse prognostic outcomes in triple-negative breast cancer patients. Because overexpression of SOX11 resulted in a loss of BRCA1 function, there may be a potential role for SOX11 in inducing the BRCAness phenotype commonly seen in basal-like breast cancers. The results of this study strongly suggest that SOX11 is involved in defective DNA damage repair pathways. Further studies need to be conducted in order to evaluate SOX11 as an inducer of the BRCAness phenotype, which occurs when there is a homologous recombination repair defect and no germline BRCA1 mutation present. Because of this, SOX11 may also have the potential to act as a functional biomarker for therapies targeting DNA damage, as recent developments in identifying therapies that could potentially target homologous recombination repair defects have been promising.

A thesis submitted to the Faculty of Science under the school of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Doctor of Philosophy. Gauteng, Johannesburg, 2016. / Overexpression of RBBP6 in cancers of the colon, lung and oesophagus makes it a potential target in anticancer therapy. This is especially important because it associates with the tumour suppressor gene p53, inactivation of which has been linked to over 50% of all cancer types. Cancer is an enormous burden of a disease globally. Today, more people die from cancer than HIV/AIDS, tuberculosis and malaria combined. And in females, breast and cervical malignancies remain the most common types. Currently, cervical cancer is the most diagnosed gynaecological cancer type, whose mortality rate is the highest in developing countries due to the asymptomatic nature of the disease coupled with inadequate cancer control tools and facilities. Breast cancer incidence rate has increased beyond that of lung cancer, making it the most common malignancy among women. / GR2016

MSc., Faculty of Science, University of the Witwatersrand, 2011 / Aim: This study aimed at evaluating the accuracy of the treatment setup margin in external beam radiotherapy in cervical cancer patients treated supine with or without the CIVCO “kneefix and feetfix”TM immobilizing devices. Methods and materials: 2 groups of 30 cervical cancer patients each, who were treated supine with two parallel opposed fields or a four-field “box” technique were selected randomly. The treatment fields were planned with a 2 cm setup margin defined radiographically. The first group was treated without any immobilization and the second group was treated with the “kneefix and feetfix”TM immobilization device. Both groups of patients were selected from the patients treated on one of two linear accelerators (linac), which had weekly mechanical quality control (QC). All patients had pre-treatment verifications on the treatment machine in which a megavoltage Xray film was taken to compare with the planning simulation film. Both films were approved by the radiation oncologist managing the patient. In this study the position of the treatment couch as at the approved machine film was taken as the intended or planned position for the immobilized patients. The digital readouts of the daily treatment position of the couch were recorded for each patient as the absolute X (lateral), Y (longitudinal), and Z (vertical) position of the couch from the record and verify system interfaced to the treatment machine. A total of 1241 (582 for the immobilized and 659 for the non-immobilized patient group) daily treatment setup positions were recorded in terms of the X, Y and Z coordinates of the couch corresponding to the Medio-lateral (ML), Supero-inferior (SI) and Antero-posterior (AP) directions of the patient, respectively. The daily translational setup deviation of the patient was calculated by taking the difference between the planned (approved) and daily treatment setup positions in each direction. Each patient’s systematic setup error (mi) and the population mean setup deviation (M), was calculated. Random ( ) and systematic ( ) setup errors were then calculated for each group in each direction. The translational setup variations found in the AP, iii ML, SI directions were compared with the 2 cm x 2 cm x 2 cm Planning Target Volume (PTV). Couch tolerance limits with the immobilization device were suggested based on the ± 2SD (standard deviation) obtained for each translational movement of the treatment couch. Result: The random and systematic errors for the immobilized patient group were less than those for the non-immobilized patient group. For the immobilized patient group, the systematic setup error was greater than the random error in the ML and SI direction as shown in Table I. Table I: The random and systematic errors in the setup in the Antero-posterior (AP), Medio-lateral (ML) and Supero-inferior (SI) directions and the suggested couch tolerance limits for both patient groups. Almost all treatment setup positions had less than 2 cm variation in the AP setup for both patient groups however; one third of the immobilized positions had more than 2 cm variation in the setup in the ML and SI directions. Conclusion: The “kneefix and feetfix”TM immobilizing device resulted in a minor improvement in both the random and systematic setup errors. The systematic setup errors need to be investigated further. There are measurable patient rotations of more than 2 cm in the setup margin with the immobilizing device and this should be confirmed with an imaging study. The 2 cm margin in the ML and SI directions Immobilized patient group Non-immobilized patient group AP (cm) ML (cm) SI (cm) AP (cm) ML (cm) SI (cm) Random error (!) 0.30 1.35 1.26 0.37 2.74 7.83 Systematic error (") 0.19 1.55 1.64 0.33 1.70 8.11 Suggested couch tolerance limits (±2SD) 0.70 4.04 4.08 0.88 4.76 N/A iv established at simulation should not be changed for these patients. A 1 cm tolerance in the AP setup margin could be introduced at this institution.

University of Macau / Institute of Chinese Medical Sciences

University of Macau / Institute of Chinese Medical Sciences

BACKGROUND: Radiation exposure to the abdominal region causes intestinal toxicity and is also capable of inducing colorectal cancers (CRC). Genotype-phenotype studies provide some evidence explaining the variation in familial adenomatous polyposis (FAP) patients caused by modifiers of adenomatous polyposis coli (APC). This study aims to extend our understanding of irradiation-induced modifiers of ApcMin/+ mice and CRC. METHODS: By using a pre-existing backcross between recombinant inbred line of ApcMin/+ mice to the irradiation sensitive inbred BALB/c mouse, we obtained panels of 2Gy-irradiated and sham-irradiated N2 ApcMin/+ mice for genotyping with a genome-wide panel of microsatellites markers. Using the number of adenomas in different intestinal segments to represent polyp multiplicity, we carried out a genome wide quantitative trait loci (QTL) scan followed by statistical epistasis modelling and bioinformatics analysis. RESULTS: We identified five significant QTLs responsible for radiation induced tumour multiplicity in the upper small intestine defined as Mom (Modifier of Min) radiation-induced polyposis (Mrip1-5) on chromosome 2 (LOD 2.8, p = 0.0003), two regions within chromosome 5 (LOD 5.2, p=0.00001, 6.2, p=0.00001) and two regions within chromosome 16 (LOD 4.1, p=4x10-5 and 4.8, p=0.00001). Suggestive QTLs were found for sham-irradiated mice on chromosomes 3, 6 and 13 (LOD 1.7, 1.5 and 2.0 respectively; p,0.005). Two significant QTLs were detected in the 2large intestine on chromosome 2 and 7 (LOD 2.7, p=1.2x10-3 and 2.2, p=1.2x10-3, 12 respectively). Using statistical epistasis modelling and logical selection of target genes though in silico sequence based on BALB/c specific non-synonymous polymorphisms which are predicted deleterious we selected target genes and further eliminated genes by sequencing and mRNA expression. CONCLUSIONS: Our study locates the QTL regions responsible for increased radiation-induced intestinal tumorigenesis in ApcMin/+ mice and identifies candidate genes with predicted functional polymorphisms that are involved in spindle checkpoint and chromosomal stability (Bub1b, Bub1r, and Casc5), Wnt pathway (Tiam1, Rac1), DNA repair (Recc1 and Prkdc) and inflammation (Duox2, Itgb2l and Cxcl5).

Background: Colorectal cancer is the result of dysregulation within classic regulatory pathways in epithelial stem-cell(s): the precursors giving rise to all other intestinal lineages. The resulting cancer stem-cell (CSC) generates tumours utilising its innate properties, e.g. self-renewal and lineage plasticity. CSCs appear to persist within a tumour as a distinct subtype responsible for local recurrence/metastasis. Therefore, therapies targeting colorectal CSCs may lead to improved cancer-specific outcome measures. The PROM-1/CD/AC133 cell surface marker has been associated with colorectal CSCs and its expression is reported as an independent negative prognostic marker. Therefore, this thesis sought to investigate the role of PROM-1/CD/AC133 in colorectal cancer. Methods: Tissue-culture, RT-qPCR, IHC, Western blotting, siRNA, PCR-array and FACS analyses were used to quantify and profile mRNA/protein expression patterns. Results: PROM-1/CD/AC133 was widely expressed in patient-matched colorectal tumour, adjacent normal epithelium, vascular invasion, lymph node metastases with significantly decreased expression in liver metastases. Furthermore, PROM- 1/CD/AC133 expression was not found to enrich colorectal cancer cell line populations for additional stem cell phenotypes (expression of ABCB1/ABCG2/BMIJ Murphy 1/CD44/LGR5/MSI-1). The data confirm the presence of alternative splice variants of PROM-1, and show that transcripts specifying PDZ binding predominate in colorectal cancer cell lines. Concomitantly, siPROM-1 was shown to modulate the expression of several key transcripts in colorectal tumourigenesis as well as regulate signal transduction pathways including the central cancer, colorectal cancer, NF-kB and p53 signalling cascades. Conclusions: PROM-1/CD/AC133 does not identify rare colorectal cancer cells responsible for tumourigenesis. However, it is associated with the regulation of signalling networks associated with cell growth, differentiation and apoptosis suggesting a potential role for this marker in colorectal tumourigenesis. The identification of specific target genes and signalling pathways in this thesis provides a springboard for further investigations into the functional role of this marker in colorectal cancer, with the potential for better treatments for this disease.