Methyl-CpG Binding Proteins (MCBPs) are thought to function as the interpreters of epigenetic information encoded in cytosine methylation. Their ability to translate DNA methylation into local transcriptional repression has sparked interest in the role of Methyl-Binding Domain Proteins (MBDs) in cancer, where repatterning of CpG methylation is common. In this dissertation I summarize and discuss observations made in the Ginder Lab linking MCBPs to the progression of neoplastic disease. It is clear from our work that the Methyl Binding Domain Protein 2 (MBD2) is necessary for the persistent repression of critical tumor suppressor genes in breast cancer. We show that stable knockdown of MBD2 also leads to growth suppression in cultured human mammary epithelial cancer lines (MCF-7, 49% suppression; MDA-MB-231, 77%; MDA-MB-435, 94%; SK-BR-3, 92%) with the peak cytotoxicity and anti-proliferative effect occurring as late as 2-3 weeks after knockdown. MBD2 knockdown also led to a decrease in viable tumor cells at equivalent doses of the histone deacetylase inhibitor, SAHA (Vorinostat™), and chemotherapeutic agents Doxorubicin, and Paclitaxel. Stable MBD2 knockdown in MCF7 cells led to an increased proportion of normal epithelial structures in 3D culture (70%, [CI=0.55-0.83]) when compared to untransfected (46%, [CI=0.39-0.53], p≤0.038) or scrambled shRNA transfected (37%, [CI=0.29-0.45], p≤0.012) controls. In vivo xenograft studies show tumor growth in BALB/C nu/nu mice was significantly impaired when mice were implanted with human breast cancer cells harboring MBD2 targeted shRNA. Following MBD2 knockdown, tumor suppressor promoter methylation remained unchanged despite sustained increases in gene expression, arguing against the convention that passive demethylation occurs with increased transcription. Our data suggest that uncoupling CpG methylation from histone modifications or other repressor functions by removing MBD2 is sufficient to initiate and maintain anti-tumor gene transcription in the absence of secondary changes in DNA methylation. In this dissertation I present evidence for the pathologic role of MBD2 in breast cancer and provide mechanistic support for the prospect of targeting MBDs in neoplastic disease..

Epigenetic misregulation of gene expression is known to be an important feature in cancer. This has mainly been studied at the level of changes in DNA methylation and histone modifications at individual genes. In this thesis I have set out to investigate whether there are long-range changes in chromatin structure linked to altered gene expression in breast cancer. From large published datasets, I used a computational approach to identify large genomic regions which are coordinately misregulated in breast cancer independent of copy number aberrations (genomic effects). I found 26 regions of co-ordinate regulation of neighbouring genes that are consistent between breast tumours and breast cancer cell lines. These regions had different expression phenotypes (activation, repression, no change) compared to normal breast and also with tumour subtype (luminal vs basal and ER status). The regions of epigenetic regulation (RER) identified in breast cancer were mostly cancer type specific. I investigated the mechanism of long-range misregulation at one such region on chromosome 16p11.2 which is aberrantly activated in breast cancer. Interestingly, in estrogen-receptor positive (ER+ve) cells, genes in this region are upregulated relative to estrogen receptor negative (ER-ve) cells. Using fluorescence in situ hybridisation (FISH) I found that in ER+ve breast cancer cell lines and tumour tissue this region is in a more decondensed chromatin architecture than in ER-ve cell lines and tumour tissue. Furthermore this region was very compact in a normal breast epithelial cell line and breast tissue corresponding to what would be expected from the expression data. Estrogen was found to play a key role in maintaining the aberrant decondensation of chromatin at this locus on chr16p11.2, as shown by compaction of the region by starving ER+ve cells of estrogen and decompaction upon subsequent estrogen treatment. Interestingly there was also an estrogen mediated repositioning of the 16p11.2 RER domain away from the nuclear centre in hormone starved conditions and towards the centre upon estrogen stimulation. Together these results show that estrogen is key to regulating the changes in nuclear organisation and chromatin decompaction at this locus, which are associated with aberrant patterns of gene expression in ER+ve breast cancer.

Introduction Aromatase inhibitors (AIs) have an established role in the treatment of estrogen receptor alpha positive (ER+) post-menopausal breast cancer. However, response rates are only 50-70% in the neoadjuvant setting and lower in advanced disease. There is a need to identify pre- or early on-treatment biomarkers to predict sensitivity which outperform those currently used, in a move towards stratified treatments and improved patient care. Given the heterogeneity known to exist in the breast cancer population, and the limited availability of matched pre- and on-treatment clinical material, this study also sought to develop novel data integration approaches allowing for the inclusion of similar previously published datasets, thus maximising the power of this study. Experimental Design Pre- and on-treatment (at 14 days and 3-months) biopsies were obtained from 34 postmenopausal women with ER+ breast cancer receiving 3 months of neoadjuvant letrozole. Illumina Beadarray gene expression data from these samples were combined with Affymetrix GeneChip data from a similar published study (n=55) and crossplatform integration approaches were evaluated. Dynamic clinical response was assessed for each patient from periodic 3D ultrasound measurements during treatment. Results Despite intrinsic differences between different microarray technologies, suitably similar studies can be directly integrated for robust and meaningful meta-analysis with improved statistical power. After mapping probe sequences to Ensembl genes it was demonstrated that, ComBat and cross platform normalisation (XPN), significantly outperform mean-centering and distance-weighted discrimination (DWD) in terms of minimising inter-platform variance. In particular it was observed that DWD, a popular method used in a number of previous studies, removed systematic bias at the expense of genuine biological variability, potentially reducing legitimate biological differences from integrated datasets. A pipeline for the successful integration of microarray datasets from different platforms was developed. Using this approach a classifier of clinical response to endocrine therapy in the neoadjuvant setting based on the expression of 4 genes was developed which predicted response with 96% and 91% accuracy in training (n=73) and independent validation (n=44) datasets respectively. An early on-treatment biopsy was found to improve predictive power in addition to pre-treatment alone. Conclusions Using a novel data integration approach developed as part of this study, a model comprising 4 novel biomarkers for accurate and robust prediction of clinical response to AIs by two weeks of treatment has been generated and validated. On-going work will investigate the applicability to other anti-estrogens, and the adjuvant setting and will assess the potential for a new therapy response test.

Philosophiae Doctor - PhD / Breast cancer is the second most common cancer amongst South African women. Despite ongoing efforts to combat breast cancer, current prognostic and/or therapeutic monitoring methods are limited since very little improvement, in the rate of long term recurrence of breast cancer, has been observed. Considering this, developing novel strategies to detect breast cancer recurrence – at an early onset – is crucial for monitoring the disease and potentially preventing disease progression. Methods currently used for the detection of BC are costly and can also be very uncomfortable for the patient. These methods are also too costly to use as a routine test, following surgery or treatment to assess disease progression. Thus, developing a cost-effective detection method appears to be an appealing alternative. Serum/blood-based biomarkers are ideal targets for the development of low cost detection assays. Two candidate biomarkers, unique ligand binding protein 2 (ULBP2) and glial cell line-derived neurotrophic factor family receptor alpha 1 (GFR1) were identified using bioinformatics and proteomics, respectively. These biomarkers have demonstrated to be useful prognostic biomarkers for breast cancer. The selection of aptamers against these biomarkers can facilitate the development of cost-effective detection methods. Aptamers are short DNA or RNA oligonucleotides that have very high affinity and specificity for its targets and can potentially replace antibodies as tools for molecular recognition in detection systems, such as the enzyme-linked immunosorbent assay (ELISA), lateral flow assays and electrochemical biosensors.

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

dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements of the degree of Master of Science. Johannesburg, 2014 / Cancer is a global burden due to high incidence and mortality rates and is ranked the second most diagnosed disease amongst non-communicable diseases in South Africa. A high expression level of the 37kDa/67kDa laminin receptor (LRP/LR) is one characteristic of cancer cells. This receptor is implicated in the pathogenesis of cancer cells by supporting tumor angiogenesis, metastasis and especially for this study, the evasion of apoptosis. In the current study, the role of LRP/LR on cellular viability of breast MCF-7, MDA-MB 231 and WHCO1 oesophageal cancer cells was investigated. Western blot analysis revealed that total LRP expression levels of MCF-7, MDA-MB 231 and WHCO1 were significantly downregulated by targeting the mRNA of LRP using siRNA-LAMR1 by 100, 44% and 73%, respectively. This knockdown of LRP expression resulted in a significant decrease of viability in the breast and oesophageal cancer cells as determined by an MTT assay by 72%, 52% and 45% in WHCO, MCF-7 and MDA-MB 231 cells, respectively. Moreover, the reduction in cellular viability was accompanied by a significant decrease in cell proliferation by 26%, 43% and 59% in MCF-7, WHCO1 and MDA-MB 231 cells, respectively. The exposure of the phosphatidylserine protein on the surface of breast MCF-7, MDA-MB 231 and oesophageal WHCO1 cancer cells as detected by the Annexin-V/7-AAD assay indicates that the reduction in cellular proliferation and viability is due to the induction of apoptosis which is elaborated by the loss of membrane symmetry as well as observations of nuclear morphological changes in all cell lines post transfection with siRNA-LAMR1. This data indicates that LRP/LR is crucial for the maintenance of cellular viability of cancerous cells and our findings recommend siRNA technology as a novel alternative therapeutic approach for the treatment of metastatic cancer.

This research project explores the use of virtual support groups by women diagnosed with breast cancer in South Africa. Through a content analysis of the online forums and eight in-depth interviews with women of various backgrounds (age, race and socio-economic status) it becomes evident that women use these virtual spaces for information exchange, sense of belonging, search for meaning and most significantly support. Various factors within the illness experience of breast cancer influence the initial use, continuation, breaks and withdrawal from of these online forums. The analysis of online forums is framed around concepts of lay consultation, gender, adaptation theory and social capital to conceptualise and make sense of these virtual interactions. The virtual groups allow women to read and write on breast cancer according to their perceptions and experiences, thereby lessening the dominant medical power to create space for their personal voices. The interactions on the forums foster a sense of empowerment, social support and social engagement critical to their wellbeing and adaptation to the condition. This study also brings attention to the lack of studies about virtual groups within the South African context which is increasingly becoming significant as more and more individuals use virtual groups as part of health lay interaction and consultation.

Thesis advisor: Pamela J. Grace / Purpose/Specific Aims: Van Manen’s hermeneutic phenomenological method was used to design this study aimed at better understanding the meaning of day-to-day living with advanced breast cancer in young women. Rationale/Significance of Study: There is a gap in knowledge about the particular needs and daily life experiences of this cohort. Very little data specifically addresses this population. The limited literature that exists suggests that, due to the particular stage of life, their needs differ from those at other life stages as well as those coping with earlier stages of breast cancer. These women have described themselves as being invisible and having to live with the knowledge that their future is uncertain. Thus, this qualitative study is an important initial step in expanding our understanding of what daily life is like for this population. Sample and Recruitment: Women aged 25–39 with Stage III or IV breast cancer were purposively recruited via private FacebookTM groups specifically for women with breast cancer. The final sample consisted of 12 participants from across the U.S. Incidentally, all were parents. Data Analysis: Data were collected through two or more semistructured interviews and written journals. Analysis followed van Manen’s method of immersion, reading, and rereading, and using manual coding and NVivo software to develop themes to capture the participants’ lifeworlds. Findings: The meaning of their experiences is captured by the overarching theme: Wearing the mask of wellness in the presence of life-threatening illness. Five major themes were identified: Wanting to be known as the person I am, I’m still Mom, Living is more than surviving, Getting through it, and Being connected to others. Conclusions: Findings highlight that these young women are managing multiple roles and responsibilities despite the ongoing challenges of treatment and symptom management. They feel that their needs and struggles are not well understood because to outsiders they do not look ill. This study provides a base for further research and eventually interventions. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Connell School of Nursing. / Discipline: Nursing.

The Matrix Metalloproteinase (MMP) family are conventionally considered as key enzymes contributing to cancer-cell invasion through remodelling of the extracellular matrix (ECM). In contrast, MMP-8 has been shown to exert an anti-cancer role. In normal breast, MMP-8 expression is restricted to tumour suppressor myoepithelial cells (MECs), which form the interface between glandular epithelium and the ECM. In ductal carcinoma in situ (DCIS), MECs are altered; a consistent change is up-regulation of αvβ6-integrin, which is associated with loss of suppressor activity. Preliminary observations indicated that there is also loss of MMP-8 expression in DCIS-MEC. The aim of this study is to investigate the impact of loss of MEC derived MMP-8 on MEC function and how this might modulate tumour progression. To generate a model of DCIS MEC, an αvβ6 over-expressing cell line (Myo-β6) was generated from normal MECs (Myo-Puro). These cells were found to lose MMP-8 expression. To dissect gain-of-function effect, MMP-8 was re-introduced into Myo-β6 (MMP-8 WT). A proteolytic inactive form of MMP-8 was used to dissect the dependence of function on proteolysis. In-vitro analysis demonstrated that MMP-8 WT but not inactive MMP-8 significantly up-regulates MEC adhesion (p=0.0001) and spread (p=0.0003) on ECM, but reduces migration towards ECM proteins including Collagen-I (p=0.006) and fibronectin (p=0.01). Furthermore MMP-8 WT results in reduced numbers of filopodia/retraction fibres (p=0.01) and reduced protrusion length (p=0.0001) on MEC cell surface. MMP-8 promotes the localisation of α6β4-integrin to hemidesmosomal adhesive structures (p=0.003), and inhibits MEC gelatinase (p=0.002) and TGF-β activity. Conversely, knock-down of endogenous MMP-8 in Myo-Puro MECs promotes migration and filopodia/retraction fibre formation (p=0.05), increases gelatinase activity (p=0.007) and TGF-β signalling. To analyse the paracrine effect of MEC-derived MMP-8 on breast cancer cell invasion, MDA MB 231 or SUM159 cells were co-cultured with modified Myo-β6 cells in Boyden chamber invasion assays. A significant reduction in breast cancer cell invasion was observed only in the presence of MMP-8 WT (p=0.004) but not with inactive MMP-8. In contrast, MMP-8 knock-down in Myo-Puro MECs significantly enhanced breast cancer cell invasion (p=0.001). In order to recapitulate the DCIS stromal micro-ecology, 3D-organotypic cultures were constructed. In these systems there was a significant reduction in invasion only in the presence of MMP-8 WT MECs (p<0.001). Conversely, in the absence of Myo-Puro-derived MMP-8 breast cancer cell invasion was significantly up-regulated (p=0.007). These results suggest that MMP-8 does contribute to MEC tumour suppressor function via mechanisms dependent upon its proteolytic activity. These data support the hypothesis that loss of MMP-8 may contribute to the progression of DCIS to invasive disease.

Tumour necrosis factor receptor associated factors (TRAFs) play a key role in signal transduction in mammalian cells. Several members of the TRAF family have been identified but only TRAF2 and TRAF6 are implicated in the regulation of both osteoclastic bone resorption and breast cancer. Here I studied the role of TRAF2 and TRAF6 in breast cancer induced osteoclastogenesis and osteolysis. I observed that TRAF2, but not TRAF6, is highly expressed in a highly metastatic bone-tropic clone of the human MDA-MB-231-BT (MDA-231-BT) breast cancer cells when compared to parental MDA-MB-231 (MDA-231) cells. Targeted knockdown of TRAF2, but not TRAF6, in both parental MDA-231 and bone-tropic MDA-231-BT breast cancer cells by siRNAs markedly reduced cell migration and significantly reduced the ability of these cells and their conditioned medium to induce osteoclast formation in RANKL stimulated bone marrow cultures. Encouraged by these data, I generated stable parental MDA-231 and bone-tropic MDA-231-BT breast cancer cell lines overexpressing TRAF2 using a retroviral approach. Then, I went on to show that overexpression of TRAF2 in parental MDA-231 cell line significantly stimulated directed cell migration and 3D invasion in vitro. Bone-tropic MDA-231-BT breast cancer cells over expressing TRAF2 or their conditioned medium were significantly effective in enhancing RANKL induced osteoclast formation in vitro. Mechanistic studies in parental MDA-231 and bone-tropic MDA-231-BT breast cancer cells revealed that over-expression of TRAF2 enhanced cell migration and osteoclastogenesis via a mechanism that involves the activation of the breast cancer oncogene IKKepsilon (IKKε) coupled with significant increase in levels of Vascular endothelial growth factor (VEGF). Ex vivo studies in human MDA-231-mouse calvaria organ co-cultures showed that conditioned medium obtained from MDA- 231 cells enhanced calvarial osteolysis. In vivo studies showed that overexpression of TRAF2 in the human breast cancer cells MDA-231 enhanced tumour incidence and tumour volume after orthotopic injection and exacerbated osteolysis after supracalvarial injection of conditioned medium from these cells. In conclusion, our studies showed that the TRAF2/IKK/VEGF axis in breast cancer cells regulates breast cancer cell motility in vitro, osteoclastogenesis in vitro and osteolysis ex vivo and in vivo. However, the role of TRAF2 in bone metastasis associated with breast cancer will require further in vivo investigation.