The Cancer Stem Cell (CSC) theory suggests that cancers arise from and are maintained by a subpopulation of cancer cells with stem cell properties. Molecular chaperones are key components of cellular regulation. The overexpression of chaperones has become synonymous with cancer cells with chaperones being recognized as bona fide anti-cancer drug targets. Although chaperone activity has been characterized in cancer cells, very little is known about the cellular functions of chaperones in cancer stem cells. We set out to compare the expression of selected molecular chaperones in non-stem cancer cell and cancer stem cell enriched populations isolated from breast cancer lines, in order to identify chaperones differentially expressed between the two populations for further biological characterization. In order to isolate breast cancer stem cells from the MCF-7 and MDA-MB-231 breast cancer cell lines, three cancer stem cell isolation and identification techniques were utilized based on (1) cell surface marker expression (CD44+/CD24- and CD44+/CD24-/EpCAM+ phenotypes), (2) aldehyde dehydrogenase enzyme activity (ALDHHi) and (3) ability to grow in anchorage-independent conditions. The MDA-MB-231 and MCF-7 breast cancer cell lines displayed CD44+/CD24- cell populations with the MCF-7 cell line additionally displaying a large CD44+/CD24-/EpCAM+ population. Although both cell lines showed similar ALDHHi populations, they differed substantially with respect to anchorage-independent growth. MCF-7 cells were able to form anchorage-independent colonies while the MDA-MB-231 cell line was not. Anchorage-independent MCF-7 cells showed enrichment in CD44+/CD24- and CD44+/CD24-/EpCAM+ cells compared to adherent MCF-7 cells, and were selected for gene expression studies. Gene expression studies identified 22 genes as being down-regulated at the mRNA level in the anchorage-independent MCF-7 cells, while only 2 genes (BAG1 and DNAJC12) were up-regulated. The down-regulation of selected chaperones in anchorage independent MCF-7 cells was confirmed at the protein level for selected chaperones, including DNAJB6, a type II DNAJ protein shown to be involved in the regulation of Wnt signaling. In order to characterize the effect of DNAJB6 expression on BCSCs we developed a pCMV mammalian expression plasmid for both DNAJB6 isoforms (DNAJB6L and DNAJB6S). We successfully constructed mutants of the conserved histidine-proline-aspartic acid (HPD) motif of the J domain of DNAJB6S and DNAJB6L. These constructs will allow the analysis of the role of DNAJB6 in cancer stem cell function. To the best of our knowledge, this is the first report to focus on the comparative expression of molecular chaperones in normal and cancer stem cell enriched breast cancer populations.

Breast cancer is one of the leading causes of death, with mortality rate estimates of 465 000 deaths per annum. It is estimated that 1.3 million women are diagnosed with the disease each year especially in the developing countries. Current chemotherapy relies on the use of high doses of non-specific toxic agents that possess adverse side effects and compromise patient’s compliance and adherence to treatment. Paclitaxel, one of the common drugs used in breast cancer chemotherapy results in sensory and motor neuropathy, whilst hormonal therapy e.g. Herceptin causes severe cardiovascular, gastrointestinal and cutaneous side effects. There has been a demand in developing newer cancer agents that demonstrate selective cytoxicity with minimal effect on normal body tissue. Numerous studies have shown that marine organisms produce a wide range of halogenated compounds that possess cytotoxic properties, and hence can be a source of new drug hits or leads for cancer therapy. Halomon, a polyhalogenated monoterpene from Portieria hornemannii, displayed interesting activity against brain, renal and lung cancer tumours with selective/differential cytotoxicity. This inspired us to focus our project on halogenated monoterpenes isolated from the same Rhodophyta class as P. hornemannii but with particular attention to Plocamium species. Several metabolites have been isolated from P. cornutum, P. corallorhiza and P. suhrii that possess interesting cytotoxicities against a breast cancer cell line (MCF7) and an oesophageal cancer line (WHCO1). The aim of the project was therefore centred at isolating target compounds for preliminary structure-activity studies against a breast cancer cell line, and use this information to synthesize a series of analogues that are more stable than the natural products and yet as active using a fragment-based type approach to map out pharmacophoric elements. Five metabolites were isolated from P. cornutum and five from P. corallorhiza. Cell-based assays were conducted using an MTT assay kit against MCF7 and MDA-MB-231 breast cancer cell lines and (1E,3E,5S,6R)-1,5,6-trichloro-2-(dichloromethyl)-6-methylocta-1,3,7-triene, isolated from P. cornutum was the most active with IC50 values of 3.0 μM and 6.15 μM respectively. Introduction of a terminal aromatic ring to enhance stability, together with varying substituents (H, CH3, CF3, Br, CN, CHO, CHCl2) on position 7 of the molecule, gave rise to a series of cinnamate ester derivatives inspired by (1E,3E,5S,6R)-1,5,6-trichloro-2-(dichloromethyl)-6-methylocta-1,3,7-triene. The analogues were synthesized from their benzaldehyde precursors via Aldol condensation, esterification and Wittig reactions. Their carboxylic acid counterparts were synthesized by hydrolysis of the parent esters in an attempt to promote water solubilities of the analogues. Biological activity assays were then conducted with the cinnamate analogues against the MDA-MB-231 breast cancer cell line using an MTT assay kit. Ester derivatives with -CHO and -CHCl2 functionalities had IC50 values of 43.45 μM and 100.01 μM respectively whilst the other ester derivatives were inactive. It was concluded that either an aldehyde (-CHO) or gem-dichlorides (-CHCl2) is specifically required for cytotoxic activity to be observed. None of the carboxylic acids were active which could have been due to failure of the compounds to enter the breast cancer cells and reach the target site because of their polar nature. Compounds with -CHO and -CHCl2 functionalities were therefore selected for future SARs studies.

Germ-line mutations within the breast cancer susceptibility genes, BRCA1 and BRCA2 are responsible for inherited susceptibility to breast and ovarian cancer. A wide spectrum of pathogenic mutations has been identified within both genes, but alterations within these genes occur far less frequently than originally believed. A large number of breast cancer families that showed linkage to BRCA1 were not found to carry a pathogenic BRCA1 mutation following the use of “classical” PCR based assays. In 1997, a large genomic rearrangement was reported within BRCA1, using Southern blotting. Numerous groups then employed semi-/quantitative methods to determine the presence and/or frequency of such alterations. This search extended the mutation spectrum of this gene, and to date at least 69 unique rearrangements have been reported. The contribution of these alterations to the burden of breast/ovarian cancer differs greatly between populations ranging from 0% to 36% of all BRCA1 mutations in the Finnish and Dutch populations respectively. Mutation screening has previously indicated that small mutations within the two BRCA genes are responsible for 59% of breast/ovarian cancer susceptibility in South Africa. To determine whether large rearrangements contribute to breast cancer susceptibility in South Africa, 74 BRCA1/2 small mutation negative patients from 58 breast / ovarian cancer families were screened for large intragenic BRCA1 rearrangements using Multiplex Ligation-dependent Probe Amplification (MLPA). In this first study of large genomic rearrangements within BRCA1 in South Africa, three genomic aberrations were detected. A deletion of exon 22 (IVS21-36del510) was identified in a Dutch immigrant. This deletion represents one of the Dutch founder mutations. Both exons 23 and 24 were found deleted in a South African family of Greek ancestry. The breakpoints of this deletion were not characterized. Simultaneous deletions of these two exons (where the breakpoints could not be characterized) have been reported in the Italian and Spanish populations. One of the genomic aberrations detected by MLPA in the present study erroneously appeared as a deletion of exon 18. Sequence analysis of this variant identified it as a single base pair substitution (c.5215G_A). This variant (R1699Q) has been reported previously, but its pathological significance is unconfirmed. In total, two large genomic rearrangements were detected in two families, of which only one is a South African, of Greek ancestry. This indicates that such mutations play a small role (1.75%; 1/57) in familial breast / ovarian cancer in South Africa (Dutch immigrant excluded). No rearrangements were identified in the Afrikaner population, indicating that such mutations do not contribute to the burden of familial breast/ovarian cancer in this population (0/40). The remaining South African breast/ovarian cancer risk may to some extent be explained by large rearrangements within BRCA2, or by mutations in other low penetrance breast cancer susceptibility gene(s). BRCA2 will now be screened by MLPA, followed by mutation screening of genes such as p53 and CHEK2 in high-risk families. / Dissertation (MSc)--University of Pretoria, 2011. / Genetics / unrestricted

Introduction: Positron Emission Tomography (PET) has revolutionized the diagnostic and imaging fields in cancer research. PET has opened new avenues in the pre-clinical study of radiotracers and radio-therapeutic compounds of which the full potentials are yet to be explored. To date 18F-Fluoro-Deoxy-Glucose1 (18F-FDG) is the most widely used radiotracer for PET imaging. The success of 18F-FDG is due to the existence of several trans-membrane proteins responsible for the facilitated transport of glucose. A related protein is a specific fructose selective trans-membrane transporter (GLUT 5) that has been observed to be over-expressed by some types of cancer cells suggesting that D-fructose is utilized by these cancer cells for energy production. Thus labeled D-fructose derivatives are potential candidates for selective PET imaging for cancer cells similar to 18F-FDG in active cells. Aim: The aim of this study was to investigate the effect of D-fructose on GLUT 5 positive and negative cell cultures and to evaluate the feasibility of GLUT 5 as a target for PET imaging of breast cancer. Objectives: The following were investigated: <ul> i. The extent of expression of GLUT 5 in three cancer cell lines: breast cancer cells (MCF-7), Baby Hamster Kidney cells (BHK) and cervical epithelial carcinoma cells (Hela). ii. The colony formation potential of D-fructose enriched medium (glucose-free) and its effect on proliferation of the investigated cell lines. iii. The effect of anti-GLUT 5 antibodies on the proliferation of breast cancer cells in vitro. iv. The synthesis and characterization of a non-radioactive fluorinated D-fructose derivative (1-deoxy-fluoro–D-fructose) </ul> Results: D-fructose was observed to mediate cell growth in MCF-7 cell lines but not in Hela and BHK cell lines. Glucose stimulated significantly greater cell proliferation than D-fructose for all 3 cell lines but more noticeably for the Hela (p<0.001) and BHK (p=0.0110) cell lines at all tested concentrations. Cell growth of MCF-7 cell lines where only D-fructose was present suggests a role for the highly expressed fructose specific transporter (GLUT 5) in the use of D-fructose for energy production and cell growth by these breast cancer cells. No significant differences were observed in the ability of D-fructose enriched medium to induce 3D colony formation among the three cell lines studied (p>0.05) suggesting that D-fructose is not linked directly with aggressive carcinogenesis in these cell lines despite the observed evidence of D-fructose involvement in cell proliferation and energy consumption. Anti-GLUT 5 antibodies did not show an inhibitory effect on MCF-7 cell proliferation at concentration up to 1 μg/ml (1:1000) despite these cells high expression of GLUT 5. GLUT 5 is highly expressed by MCF-7 but not by Hela and BHK cell lines making it an important selective target for imaging of this type of breast cancer and a possible therapeutic target for antibody targeted therapy of breast cancer. A chemical reaction sequence for the synthesis of 1-deoxy-fluoro–D-fructose (1-FDF) was carried out and an acceptable yield for an isotope labeling friendly reaction sequence was obtained and the product chemically characterized. Conclusion: The D-fructose transporter GLUT 5 shows potential for possible application with PET imaging of breast cancer. Isotope labeled 1-FDF can be synthesized in good yield and should be the object of further studies such as development of an automated synthesis module for its radio-labeled derivative as well as pre-clinical animal and human studies. Copyright / Dissertation (MSc)--University of Pretoria, 2013. / Pharmacology / unrestricted

The human epidermal growth factor receptor (HER-2) is over-expressed in 20-30% of breast carcinomas and is a prognostic marker for poor patient outcome. We previously identified the transcription/translation factor Y-box binding protein-1 (YB-1) to be a novel substrate of AKT which binds to epidermal growth factor receptor (EGFR) and HER-2 promoters once phosphorylated (Wu J et al. 2006). YB-1 is over-expressed in approximately 40% of breast cancers; its expression is strongly correlated with HER-2 and is associated with poor patient survival. In order to gain a deeper understanding of the functional role of YB-1 in HER-2 over-expressing breast cancer, we silenced the expression of this factor in BT474-m1 and MDA-MB-453 cells. The loss of YB-1 inhibited the growth of BT474-m1 and MDA-MB-453 cells in monolayer and/or in soft agar. Consistent with this, we found a decrease in the expression of YB-1 responsive gene egfr and/or her-2 in BT474-m1 and MDA-MB-453 cells, which could begin to explain how growth is promoted by this factor. Furthermore, loss of YB-1 expression induced apoptosis in BT474-m1 cells. Beyond its role in tumor growth, YB-1 is also strongly linked to drug resistance. We therefore addressed whether it could play a part in Herceptin sensitivity. Herceptin is currently being used to treat patients with HER-2 positive breast cancer; however, only 30% of the patients respond to the therapy and many of them develop resistance within the first year of treatment. Therefore, it is of utmost importance to understand the biology of HER-2 over-expressing breast cancer to develop novel therapies that can benefit more patients. First we established that Herceptin inhibited BT474-m1 cell growth in anchorage-independent conditions whereas MDA-MB-453 cells were resistant to this treatment. We subsequently demonstrated that knock-down of YB-1 increased sensitivity of BT474-m1 cells to Herceptin while MDA-MB-453 cells failed to respond to the combination treatment. The mechanism for Herceptin resistance in MDA-MB-453 cells still remains elusive and requires further investigation. Thus far, we conclude that YB-1 is needed for the growth and survival of HER-2 positive BT474-m1 and MDA-MB-453 breast cancer cells by inducing members of the HER family. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Early detection is one of the most important factors for successful treatment of cancer. Currently, scientists are searching for molecular markers that can help identify and predict outcome and chance of recurrence in patients. In this study, we demonstratet he potential impact of Y-Box binding protein-1 (YB-1) as a marker of aggressiveness and cancer recurrence in breast malignancies by screening one of the largest tissue microarrays in North America. YB-1 is an oncogenic transcription/translation factor, which is over-expressed in the majority of malignancies, including breast cancer. In the cohort of 4049 primary breast tumours, we show that YB-1 is a strong marker of aggressiveness, poor survival and cancer recurrence in all subtypes of human breast cancer with a particularly high frequency of expression in the ER negative basal-like and HER-2 breast cancer subtypes. This suggests that targeting YB-1 may provide a new avenue for therapeutic intervention in these breast cancers that are currently challenging to treat. Cox regression multivariate analysis indicates that YB-1 is second only to nodal status as a strong independent prognostic marker for poor outcome and relapse compared to established clinico-pathological biomarkers, including tumour size, age, grade, ER and HER-2 status. This finding suggests that YB-1 has great potential to be in a priority list of biomarkers for identifying the patients with a higher risk of relapse and poor outcome. Subsequently, we find an association between YB-1 and urokinase Plasminogen Activator (uPA) expression in the basal-like subtype. We then show that YB-1 is involved in the regulation of uPA expression. More importantly, silencing YB-1 or uPA results in a significant reduction in cancer cell invasion. As there are no commercially available YB-linibitors we examine the efficacy of BMS-536924, a small molecule inhibitor for activated IGF-1R/IR on SUM149 cells. We demonstrate that activated IGF-1R is associated with poor survival in primary breast tumours and, that BMS-536924 reduces uPA expression through inhibition YB-1 in SUM149 cells. We therefore conclude that YB-1 is a bio-marker for poor survival and relapse. We also indicate that YB-1 has potential use as a molecular marker in a clinical setting. Inhibiting YB-1 may provide an ideal opportunity for targeted therapy in breast cancer. / Medicine, Faculty of / Graduate

It is commonly believed that breast cancer dragon boating benefits survivors in a range of psychosocial areas, but there have been few empirical studies to investigate such relationships. An interpretive description design and a critical health promotion approach were used to explore the psychosocial experiences of women who breast cancer dragon boat. In-depth interviews with six participants were analyzed. Themes that arose from the data are: (1) moving past isolation — networks of like-minded support, (2) taking control,(3) journey into adventure, (4) affirmative outlook, (5) confronting painful experience, (6) rebuilding identity, (7) and spiritual engagement. The findings illustrate that dragon boating provides breast cancer survivors with a significant venue for change and the opportunity to move beyond traumatic elements of cancer. / Arts, Faculty of / Social Work, School of / Graduate

Chromosome changes in the haematological malignancies, lymphomas and sarcomas are known to be important events in the evolution of these tumours as they can, for example, form fusion oncogenes or disrupt tumour suppressor genes. The recently described recurrent fusion genes in prostate and lung cancer proved to be iconic examples as they indicated that important gene fusions are found in the common epithelial cancers also. Breast cancers often display extensive structural and numerical chromosome aberration and have among the most complex karyotyes of all cancers. Genome rearrangements are potentially an important source of mutation in breast cancer but little is known about how they might contribute to this disease. My first aim was to carry out a structural survey of breast cancer cell line genomes in order to find genes that were disrupted by chromosome aberrations in 'typical' breast cancers. I investigated three breast cancer cell lines, HCC1187, VP229 and VP267 using data from array painting, SNP6 array CGH, molecular cytogenetics and massively parallel paired end sequencing. I then used these structural genomic maps to predict fusion transcripts and demonstrated expression of five fusion transcripts in HCC1187, three in VP229 and four inVP267. Even though chromosome aberrations disrupt and fuse many genes in individual breast cancers, a major unknown is the relative importance and timing of genome rearrangements compared to sequence-level mutation. For example, chromosome instability might arise early and be essential to tumour suppressor loss and fusion gene formation or be a late event contributing little to cancer development. To address this question, I considered the evolution of these highly rearranged breast cancer karyotypes. The VP229 and VP267 cell lines were derived from the same patient before and after therapy-resistant relapse, so any chromosome aberration found in both cell lines was probably found in the common in vivo ancestor of the two cell lines. A large majority of structural variants detected by massively parallel paired end sequencing, including three fusion transcripts, were found in both cell lines, and therefore, in the common ancestor. This probably means that the bulk of genome rearrangement pre-dated the relapse. For HCC1187, I classified most of its mutations as earlier or later according to whether they occurred before or after a landmark event in the evolution of the genome-endoreduplication (duplication of its entire genome). Genome rearrangements and sequence-level mutations were fairly evenly divided between earlier and later, implying that genetic instability was relatively constant throughout the evolution of the tumour. Surprisingly, the great majority of inactivating mutations and expressed gene fusions happened earlier. The non-random timing of these events suggests many were selected.

Complaints of reduced cognitive abilities are frequent following chemotherapy. Research in the breast cancer population has revealed some patients may experience treatment-related decline in cognitive domains such as executive function, information processing speed, memory and learning, attention and concentration, and working memory. The extent and mechanism of action of this phenomenon remain poorly understood. Neuroimaging research can characterize the neural underpinnings of chemotherapy-related cognitive impairment; however, with few longitudinal studies, more prospective studies are needed to elucidate this important topic. The aim of this thesis was to use magnetic resonance imaging and contemporary analysis techniques to better understand the influence chemotherapy exerts on both the brain and cognition. This was achieved in two studies that measured cognitive function and brain structure and function at three time points: pre-treatment, one month post-chemotherapy, and at one-year follow-up. In the first study, the association between regions of brain structural changes and cognitive function was examined. The second study took a narrower approach and investigated the functional profile of brain activity during a working memory task. Patients had more pronounced structural and functional disruptions shortly after treatment, relative to both pre-treatment and one-year post-chemotherapy intervals. Regions of structural compromise were largely associated with information processing speed. Functional disruptions occurred in a frontoparietal network. Overall, this thesis provides more evidence of the injurious role chemotherapy plays on cognition, particularly in the short term. This thesis also provides the first longitudinal neuroimaging study to illustrate a complete resolution of working memory related brain disruption one year post-treatment.

Background: Arm morbidity continues to impact the lives of many breast cancer survivors long after acute treatments are completed. The most debilitating symptoms of arm morbidity are pain, lymphedema and limitation with range of motion (ROM). As a chronic condition, management of arm morbidity symptoms requires survivors to engage in self-management practices that alleviate symptoms. Objective: To explore self-management practices performed by breast cancer survivors, and the treatments women receive from healthcare practitioners in managing symptoms of arm morbidity. Methods: A secondary analysis of quantitative and qualitative data was undertaken. A descriptive correlational design was used to analyze data from breast cancer survivors (N = 740). Logistic regression identified variables related to self-management that were associated with pain, lymphedema and ROM limitations. A descriptive qualitative design was used to analyze data from a subset of participants (n = 40). Inductive content analysis approach was applied to develop codes, categories and themes related to how women self-manage arm morbidity symptoms and the treatments they received from healthcare providers to manage their arm morbidity. Results: Participants reported ongoing symptoms of pain (24%), lymphedema (21%), and range of motion limitation (34%) 30 to 36 months post-surgery. Pain was associated with experiencing swelling, taking pain medications, and discussing treatments for pain. Lymphedema was associated with swelling and receiving treatment for pain. ROM limitations were associated with swelling, receiving treatment for pain and taking pain medication. Two overarching qualitative themes emerged: 1) physical symptoms self-management, and 2) psychosocial self-management of uncertainty. Themes for treatments options included: rehabilitation and taking medications. Conclusion: Findings highlight that women living with symptoms of arm morbidity require ongoing monitoring and support for self-management, and there is a need for multidisciplinary approaches. Self-management practices reported are in line with the current lymphedema guidelines, however, the complexities associated with self-management practices can be burdensome to women. Chronic pain and ROM limitation necessitates further investigation to understand their cause and develop management strategies. Oncology nurses are well positioned to implement survivorship care plans that address survivorship needs with multidisciplinary teams and primary healthcare practitioners when women with breast cancer transition from acute cancer care to home. Future research is needed to provide an in depth understanding of self-management of arm morbidity in breast cancer survivors.