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The transition from normal mammary development to the formation of aberrant cancerous tissues requires numerous mutations to occur. These alterations allow cancer cells to evade the usually strictly controlled pathways of survival, proliferation and self-renewal. The cell of origin for most tumours remains unknown but evidence that a sub-population of stem-like cells, termed cancer initiating cells, exists within solid cancers is strong.

I have investigated epigenetic mechanisms of acquired endocrine-resistance in breast cancer using an in vitro model system based on estrogen-dependent MCF7 cells and their derivatives, LCC1 and LCC9. LCC1 cells, derived from MCF7 after passage in ovariectomised mice and routinely cultured in vitro in the absence of estrogen, exhibit estrogen-independent growth. They retain sensitivity to tamoxifen and fulvestrant. LCC9 cells, derived from LCC1 cells by growing them in increasing concentrations of fulvestrant, are completely estrogen-independent and are resistant to fulvestrant and cross-resistant to tamoxifen. When compared to MCF7 cells, LCC1 cells have marked up-regulation of the estrogen receptor α (ERα) protein that is not concomitant with increased estrogen receptor 1 (ESR1) transcription, suggesting a role for estrogen in controlling the proteasomal degradation of ERα. However, despite being grown in the same estrogen-deprived conditions, LCC9 cells do not have up-regulated ERα levels. As LCC1 cells retain sensitivity to tamoxifen and fulvestrant, these data suggest that LCC1 have developed estrogen-independence through ERα uncoupled from its ligand. However, LCC9 cells appear to have developed an alternative mechanism which is not dependent on ERα, presumably explaining their resistance to fulvestrant. I have studied global gene expression changes in the presence and absence of estrogen in these cell lines, using oligonucleotide microarrays, and correlated these data with global DNA methylation data derived from methylation arrays, which interrogate the methylation status of approximately 27,000 CpG dinucleotides in the genome. The analysis led to the discovery of more than 5,000 genes that were potentially either up-regulated or down-regulated by estrogen in MCF7 cells, either directly or indirectly. The transcriptional response to estrogen was generally muted in LCC1 and LCC9 compared with MCF7, but was not completely absent. I used various methods based on differential gene expression to parse the data, including gene ontology analysis, aiming to select genes for further mechanistic study. However, none of these methods led to the conclusive identification of a specific gene (or set of genes) that might have accounted for the physiological differences between the cell lines. In one strategy, I reasoned that, as the endocrine-resistant cells had lost their estrogen-dependence, genes involved might be regulated in an estrogen-dependent manner in MCF7 cells, without exhibiting misregulation in LCC9. This led to the identification of DUSP1 as a candidate gene, which was taken forward for mechanistic study because of its potential role in regulating ERα expression. However, when over-expressing DUSP1 in LCC9 cells, I could not demonstrate any effect on ERα levels. The final approach taken was to identify genes that might have been epigenetically deregulated, being both estrogen-regulated and deregulated in association with aberrant DNA methylation in the estrogen-independent cell lines. Surprisingly, given the phenotypic differences between the cell lines, only a very few genes were significantly methylated between cell lines. Of those that were differentially methylated between MCF7 cells and LCC1/9, only three exhibited the expected inverse correlation between methylation and expression. Of these, the gene CYBA was selected for further investigation. CYBA is a critical component of the NAPDH oxidase complex which is involved in generating oxygen free-radicals. My work suggests CYBA expression is estrogen-dependent, and that chronic estrogen deprivation leads to the epigenetic inactivation of CYBA in breast cancer cells. I speculate that the epigenetic suppression of CYBA may protect cells from the oxidant damage that results from estrogen deprivation and may be part of the mechanism that leads to acquired endocrine-resistance in previously sensitive cells.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. / Objective: This study was undertaken to evaluate the association between components defining insulin resistance and breast cancer in women. Study Design: We conducted a systematic review of four databases (PubMed-Medline, EMBASE, Web of Science, and Scopus) for observational studies evaluating components defining insulin resistance in women with and without breast cancer. A meta-analysis of the association between insulin resistance components and breast cancer was performed using random effects models. Results: Twenty-two studies (n = 33,405) were selected. Fasting insulin levels were not different between women with and without breast cancer (standardized mean difference, SMD 20.03, 95%CI 20.32 to 0.27; p = 0.9). Similarly, non-fasting/ fasting C-peptide levels were not different between the two groups (mean difference, MD 0.07, 20.21 to 0.34; p = 0.6). Using individual odds ratios (ORs) adjusted at least for age, there was no higher risk of breast cancer when upper quartiles were compared with the lowest quartile (Q1) of fasting insulin levels (OR Q2 vs. Q1 0.96, 0.71 to 1.28; OR Q3 vs. Q1 1.22, 0.91 to 1.64; OR Q4 vs. Q1 0.98, 0.70 to 1.38). Likewise, there were no differences for quartiles of non-fasting/fasting C-peptide levels (OR Q2 vs. Q1 1.12, 0.91 to 1.37; OR Q3 vs. Q1 1.20, 0.91 to 1.59; OR Q4 vs. Q1 1.40, 1.03 to 1.92). Homeostatic model assessment (HOMAIR) levels in breast cancer patients were significantly higher than in people without breast cancer (MD 0.22, 0.13 to 0.31, p, 0.00001). Conclusions: Higher levels of fasting insulin or non-fasting/fasting C-peptide are not associated with breast cancer in women. HOMA-IR levels are slightly higher in women with breast cancer.

<p>The ABL family of non-receptor tyrosine kinases, ABL1 (also known as c-ABL) and ABL2 (also known as Arg), links diverse extracellular stimuli to signaling pathways that control cell growth, survival, adhesion, migration and invasion. ABL tyrosine kinases play an oncogenic role in human leukemias. However, the role of ABL kinases in solid tumors including breast cancer progression and metastasis is just emerging. </p><p>To evaluate whether ABL family kinases are involved in breast cancer development and metastasis, we first analyzed genomic data from large-scale screen of breast cancer patients. We found that ABL kinases are up-regulated in invasive breast cancer patients and high expression of ABL kinases correlates with poor prognosis and early metastasis. Using xenograft mouse models combined with genetic and pharmacological approaches, we demonstrated that ABL kinases are required for regulating breast cancer progression and metastasis to the bone. Using next generation sequencing and bioinformatics analysis, we uncovered a critical role for ABL kinases in promoting multiple oncogenic pathways including TAZ and STAT5 signaling networks and the epithelial to mesenchymal transition (EMT). These findings revealed a role for ABL kinases in regulating breast cancer tumorigenesis and bone metastasis and provide a rationale for targeting breast tumors with ABL-specific inhibitors.</p> / Dissertation

A dissertation submitted to the Faculty of Science, University of Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Science. Gauteng, Johannesburg, 2013. / Retinoblastoma Binding Protein 6 (RBBP6) is a multi-domain protein that uses its ring finger domain to interact with p53 and pRb tumour suppressor genes. The mechanism by which RBBP6 uses to degrade p53 is still unknown. Nonetheless, it is well known that RBBP6 promotes cell proliferation in several cancers by negatively regulating p53 via its E3 ubiquitin ligase activity (Ntwasa, 2008). Degradation of p53 by RBBP6 may compromise p53-mediated apoptosis in breast cancer. This study is intended to investigate the potential applications of RNA interference (RNAi) to block RBBP6 expression as well as its subsequent effect on cell growth and apoptosis. To achieve these methodologies, the following techniques were used: RT-PCR, western blotting, xCELLigence system and flow cytometry. Our studies indicate that the knockdown of RBBP6 expression by siRNA modulates p53 gene involved in cell death pathways and apoptosis, showing statistically significant gene expression differences. RBBP6siRNA significantly reduced cell index (CI) compared to the control samples and we observed an inhibition of cellular proliferation in the interval of between 24 and 48 h, as shown in the data obtained by dynamic evaluation using the xCELLigence System. These results were further confirmed by flow cytometry which showed some apoptotic activity. About 20.7% increase in apoptosis was observed in cells co-treated with RBBP6 siRNA and camptothecin when compared to camptothecin-only whereas in siRBBP6 and Staurosporine treated there was only 8.8% increase in apoptosis. These findings suggest that silencing RBBP6 may be a novel strategy to promote staurosporine- and camptothecin-induced apoptosis in breast cancer cells. Keywords: Retinoblastoma Binding Protein 6, staurosporine, camptothecin

MSc (Med), Faculty of Health Sciences, University of the Witwatersrand / The HER2 gene is amplified in 20-30% of breast cancers, a common cancer amongst South African women. HER2 amplification is associated with a poor prognosis and predicts response to treatments such as Herceptin. The gold standard for HER2 testing is Fluorescent in situ Hybridisation (FISH) with dual colour probes for the HER2 gene and chromosome 17 centromere (CEP17) internal control. According to international guidelines, a HER2/CEP17 ratio >2.2 is considered positive. The HER2 FISH test is complicated by the emergence of ambiguous cases with increased CEP17 signals that cannot be accounted for by chromosome 17 polysomy (> 6 copies of CEP17) and that may hide true HER2 gene amplification. The aims of this study were to characterise the HER2 amplicon, in particular the copy number of genes in the vicinity of the HER2 gene, and to design an alternative control probe that could clarify the HER2 gene status in ambiguous cases. In addition, results on 1558 breast cancer specimens sent for routine testing were analysed to determine the trends of HER2 amplification amongst South African women. The rate of HER2 gene amplification was significantly higher (p < 0.05) in African patients (52%) than in Caucasian patients (43%). In Caucasian women, the rate of HER2 amplification in the younger group (68%) was significantly higher (p < 0.05) than in the general Caucasian group (43%), while the same was not seen in the African cohort. Nineteen ambiguous cases with more than 9 copies of CEP17 were further investigated. FISH assays with four different probe kits (PathVysion HER-2: Poseidon Repeat free TOP2A, HER2, CEP17: and Vysis PML-RARA respectively) were performed to determine the copy number of the HER2, TOP2A, RARA genes and CEP17. An in-house dual colour probe kit was designed using the ACTG1 gene as a control for HER2. Of the 19 ambiguous cases, 16 had centromeric amplification, showing that CEP17 is no longer an adequate internal control in FISH HER2 testing. The TOP2A gene was only amplified in HER2 positive cases and the RARA gene was only amplified when the TOP2A gene was also amplified. FISH with ACTG1 as v a control clearly revealed HER2 amplification in ambiguous cases on image analysis and gave HER2/ACTG1 ratios significantly higher than HER2/CEP17 ratios. However, screening of an additional 40 unambiguous cases showed an increased copy number, although limited ( 8), of the ACTG1 gene in four patients; this warrants further testing to assess the value of this gene as a control. Interestingly, a trend was observed for ACTG1 increased copy number in HER2 negative cases, this may point to the presence of a driver gene whose amplification tends to be mutually exclusive from HER2 amplification.

Matrix Metallopeptidase 1 (MMP-1) expression has repeatedly been correlated to tumorigenesis and metastasis. Yet, MMP-1 regulation in a metastatic context remains largely unknown. Here we confirm differential MMP-1 expression in mammary carcinoma cells with varied metastatic potentials and identify a mechanism differentially regulating MMP-1. We show that MMP-1 expression is regulated by an AP-1 element in its promoter in highly metastatic MDA-MB-231 mammary carcinoma cell derivatives. Fra-1, an AP-1 family transcription factor, differentially binds this element in highly metastatic derivatives compared to low-metastatic cells and is required for MMP1 expression. Fra-1 mRNA levels are unchanged in the cell variants, however its protein levels are higher in the metastatic cells. There was no change in protein degradation rates, while protein synthesis rates of Fra-1 increased. These results suggest that protein translation of Fra-1 is differentially regulated in these cells. Consistent with the importance of Fra-1 for tumor growth, we found that Fra-1 overexpression is sufficient to increase cell motility and anchorage independent growth. These results suggest that Fra-1 regulation is critical for regulation of MMP-1 and metastasis.

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