Thesis (Ph.D. (Biochemistry)) -- University of Limpopo, 2021 / Breast cancer is the leading cause of female deaths in the world. Varying types of therapy options are available, yet these conventional treatments for the malignancy are known to have numerous side effects. Similar to other diseases, herbal remedies are being explored as alternative treatment options as well as starting points for development of new drugs to treat breast cancer. Bidens pilosa is a weed distributed throughout the world with known medicinal properties. Its anti-cancer activity has been established in various cancers. This study aimed to investigate the epigenetic patterns affected by a bioactive extract of B. pilosa in breast cancer. A crude methanol extract of B. pilosa was fractionated with n-hexane, chloroform, ethyl acetate, n-butanol, 65% methanol and water. Healing properties of plants are often an attribute of the presence of phenolic compounds within the plant and the sub-fractions of the methanol extract of B. pilosa were, therefore, assayed for these compounds. The water sub-fraction showed the highest content of total phenolic compounds, however, when the sub-fractions were analysed for the presence of two classes of specific phenolic compounds, the butanol sub-fraction boasted the highest concentration of flavonoids and tannins, affording it superior antioxidant activity in a quantitative DPPH assay. Distribution of the antioxidant compounds in TLC-DPPH analysis also supported this finding. Despite its high antioxidant compound content, cytotoxicity of the butanol sub-fraction in MCF-7 breast cancer cells was not impressive in the MTT viability assay. Treatment with varying concentrations of the chloroform sub-fraction resulted in a better dose- and time-dependent decrease in cell viability of MCF-7 cells than all the other sub-fractions as well as the crude methanol extract. Analysis of breast cancer genes affected by the chloroform sub-fraction on the Human Breast Cancer RT2 Profiler PCR array showed repression in BRCA1 and BRCA2, genes classified as tumour suppressors. Bisulfite pyrosequencing showed no significant modification in methylation of selected CpG islands within the promoter regions of both genes. Results of the array also showed decreased expression of CDH1 which is associated with invasiveness and aggression of tumours. Its investigated CpG island was also shown not to be differentially methylated by treatment of the cells with the chloroform sub-fraction of the extract. As a well-appreciated biomarker for breast cancer risk, BRCA1 protein expression was further investigated. Western blot analysis showed parallel findings to those of the PCR array, with down-regulation of BRCA1 within 24 hours of treatment of MCF-7 cells with the sub-fraction. Repression of the BRCA genes is strongly linked to arrest of cells at the G2/M phase of the cell division cycle, and this was therefore also assessed. Treatment of MCF-7 cells with the chloroform sub-fraction effected a dose-dependent accumulation of cells at the G2/M phase of the cell cycle as determined by flow cytometry. Results of global DNA methylation analysis showed an increase in chromosomal instability by a significantly reduced level of methylation of the genome. This hypomethylation also supports arrest of the cells at the G2/M phase of the cell cycle, as cells accumulate at this checkpoint, awaiting repair to prevent segregation of broken chromosomes during mitosis. However, the lack of BRCA1 suggests that repair proteins were not recruited to the sites of repair and the cells were consequently directed to apoptosis. Analysis of the effect of the chloroform sub-fraction of the methanol extract of B. pilosa in the Mitopotential assay showed an increase in the number of dead cells with depolarised mitochondrial membranes, alluding to the intrinsic mode of apoptotic cell death in MCF-7 cells treated with the sub-fraction. Down-regulation of BRCA1 is further associated with telomerase inactivation in cancer cells. Treatment of MCF-7 cells with the chloroform sub-fraction reduced telomerase activity within 24 hours of treatment, with an absence of activity following treatment with 100 and 125 μg/ml of the sub-fraction. This lack of telomerase activity resulted in shortened telomeres which limit proliferative ability of the cells. Characterisation of the six sub-fractions of the methanol extract of B. pilosa with GC-MS showed an abundance of fatty acids in the chloroform sub-fraction, specifically α-linolenic acid, palmitic acid and linoleic acid. Palmitic acid is alleged to play a role in down-regulation of BRCA1 and its abundance in this sub-fraction leads to the conclusion that palmitic acid may be responsible for the decreased expression of BRCA1 in MCF-7 breast cancer cells. The down-regulation results in hypomethylation of the genome leading to cell cycle arrest at the G2/M checkpoint and subsequent apoptosis as a result of this repression of BRCA1. Repression of BRCA1 also leads to inactivation of telomerase, inhibiting cell proliferation. Taken together, the observed antioxidant activity and pro-apoptotic potential attributed to epigenetic modifications validate B. pilosa as an anticancer agent. Our findings merit the plant for use in development of potential breast cancer drugs. / SAMRC and University of Limpopo (UL)

Exosomes are small secreted vesicles of endocytic origin with a size range of 50-150 nm. They are secreted by many cell types and display multiple biological functions including immune-activation, immune-suppression, antigen presentation, and the shuttling of mRNA and miRNA, as well as other cargo. We have characterised the exosomes secreted from two breast cancer cell lines, MDA-MB-231 and MCF7. Exosomes secreted from both cell lines display typical markers including ALIX, Tsg101, CD9 and CD63, and were capable of inducing apoptosis of the Jurkat T cell line, indicating the potential immune-suppressive function of such tumour-derived exosomes. To further investigate the biological potential of exosomes, we loaded purified exosomes with gene specific siRNAs using electroporation, and observed the targeted inhibition of both a known component of the exosome pathway, Rab27a, and also the arthritis associated gene ERAP1, demonstrating the potential novel use of exosomes as therapeutic gene delivery vectors. We have also shown that exosomes derived from MDA-MB-231 cells and the parental cells have different lipid composition, as analysed by lipidomics study. Nanoparticle tracking analysis (NTA), which allows the rapid detection of size and concentration of nanoparticles within the size range 10 nm-1000 nm was tested for its ability to accurately measure size and concentration of exosomes and microvesicles under different conditions. NTA was capable of detecting apoptotic vesicles induced by Taxol and Curcumin treatment. Immunodepletion was used to determine the percentage of CD9 and CD63 positive vesicles. Our data suggest that NTA is a useful technique for measuring size and concentration of exosomes and microvesicles. We hypothesized that NTA could assist in the screening of agents that interfere or promote exosome release. NTA was therefore used to detect increases in exosomes secretion induced by Tamoxifen and Thimerosal treatment, and to monitor the inhibition of exosome secretion from MDA-MB-231 breast cancer cells expressing inhibitory RNA targeted for Rab27a, a component of the exosome pathway. Increases in exosome release induced by Tamoxifen and Thimerosal was detected by NTA and a significant reduction in the release of exosomes by inhibition of Rab27a expression was also observed. Treatment with the known exosomal pathway inhibitor DMA also reduced exosome release, establishing the principle of NTA as a screening technique. We further compared the siRNA targeted cells for their ability to migrate, invade and form anchorage-independent colonies, which were all significantly reduced. Supplementation with MDA-MB-231 derived exosomes restored the ability to form colonies, suggesting exosomes may contribute to metastatic lesion formation. These data suggest that the exosomal pathway is a valid target to disrupt the behaviour of tumour cells and NTA can be used to monitor its activity.

This dissertation examines the production and management of genetic risk for breast and ovarian cancer in the United States in the new era of multi-gene panel testing. Drawing on three years of ethnographic fieldwork and in-depth interviews with genetics health professionals and women with mutations, this project is the first social science study to examine how breast and ovarian cancer genetic risk is constructed and managed among women with variants of uncertain significance or moderate-risk mutations. Moving beyond an individual-level focus on women’s risk management decisions, this project instead explores how the structures, practices, and organization of genetic medicine constrain and enable those decisions. There are four key findings from this study. First, the adoption of panel testing has shifted the boundaries of risk, disease, and patienthood and contributed to a spectrum of medicalization of breast and ovarian cancer risk. Women with high-risk breast and ovarian cancer mutations are now typically viewed and treated like full patients with a "disease," while women with moderate-risk mutations occupy a liminal space of qualified patienthood. Second, the structures and organization of genetic medicine in the United States point women with breast and ovarian cancer mutations toward risk-reducing mastectomy and breast reconstruction and encourage choosing those surgical responses over breast surveillance or staying flat. Mastectomy has become the standard “treatment” for the “disease” of genetic risk for breast cancer, regardless of whether women have high- or moderate-risk mutations and despite more conservative recommendations in clinical guidelines. Third, the structures of genetic medicine and the contemporary gender order in the United States are mutually constituted and co-produced. Breast reconstruction and gynecologic surgery practices both emerge from and reinforce gendered social and cultural norms that prioritize women's appearance and their reproductive capacity over their embodied experiences and daily quality of life. Finally, the discourses and practices of genetic medicine leave many women un- or under-prepared for the duration and severity of the side effects and consequences associated with breast reconstruction and risk-reducing salpingo-oophorectomy. By closely examining the social and structural dimensions of how cancer genetic risk is produced and managed in the United States, this project illuminates how clinical practices that magnify and focus on reducing certain risks simultaneously obscure and generate exposure to others.

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[Truncated abstract] Introduction: Breast cancer is a heterogeneous disease, resulting in very different outcomes for women with apparently similar tumour characteristics. In order for patients to have optimal treatment, a better understanding of the molecular nature of their disease is required. Aims: The aims of this thesis were: 1) To determine whether methylation of RARβ2, ER, CDH1, BRCA1, CCND2, p16 and TWIST genes are associated with phenotypic features of breast cancer and the prognostic significance of methylation of these genes. 2) To investigate for possible associations between the frequency of methylation at RARβ2, CDH1, ER, BRCA1, CCND2, p16 and TWIST genes and the presence of germ-line variants in the TS, MTHFR, MS, CBS, MTHFD1 and DNMT3B genes, as well as for possible correlations between these polymorphisms and clincopathological features of breast cancer including patient outcome. 3) To determine whether PIK3CA mutations determined clinical phenotype and the prognostic significance of PIK3CA mutations in a large and well characterized cohort of breast cancer patients. Methods: A large and well characterized series of primary breast tumours were selected for methylation of RARβ2, ER, CDH1, BRCA1, CCND2, p16 and TWIST genes using MSP, and for polymorphisms in TS, MTHFR, MS, CBS, MTHFD1 and DNMT3B genes using PCR, PCR-RFLP and PCR-SSCP. Mutations to PIK3CA were detected using F-SSCP. Results and Conclusions: Methylation frequencies ranged from 11% for CCND2 to 84% for ER. More frequent hypermethylation was observed in tumours with poor histological differentiation compared to those with well/moderate differentiation, as well as trends for association with larger tumour size and mutant TP53. Tumours with ER and CDH1 methylation were associated with significantly lower hormone receptor levels, younger age at diagnosis and the presence of mutant p53. TWIST methylation is firstly reported to be associated with significantly older patient age at diagnosis and larger tumour size. Our data suggests that gene methylation may be linked to various pathological features of breast cancer. However, there appears to be little support for a distinctive CpG island methylator phenotype in breast cancer.

Background. While the prevalence of a pathogenic variant in the BRCA1 and BRCA2 genes occurs in about 1:400 (0.25%) in the general population, the prevalence is as high as 1:40 (2.5%) among the Ashkenazi Jewish population. Despite cost-effective preventive measures for mutation carriers, Orthodox Jews constitute a cultural and religious group that presents challenges to BRCA1 and BRCA2 genetic testing. This study analyzed a dialogue of key stakeholders and community members to explore factors that influence decision-making about BRCA1 and BRCA2 genetic testing in the New York Orthodox Jewish community. Methods. Qualitative research methods, based in Grounded Theory and Narrative Research, were utilized to analyze the narratives of key stakeholders and community members in an analysis of qualitative data collected from 49 stakeholders. A content analysis was conducted to identify themes; inter-rater reliability was 71%. Results. Facilitators to genetic testing were prevention and education, while barriers to genetic testing included negative emotions, impact on family/romantic relationships, cost, and stigma. The role of religious figures and healthcare professionals in medical decision-making were viewed as controversial. Education, health, and community were discussed as influential factors. There were issues around disclosure, implementation, and information needs. Conclusion. This study revealed the voices of the Orthodox Jewish women (decision-makers) and key stakeholders (influencers) who play a critical role in the medical decision-making process. The findings have broad implications for engaging community stakeholders within faith-based or culturally distinct groups to ensure better utilization of healthcare services for cancer screening and prevention designed to improve population health.

[Truncated abstract] Background: A review of the literature indicated there was a need for more long-term randomised controlled studies on the effects of BRCA counselling/testing on high risk women, including improved strategies for risk communication. Reviews have also shown women are confused about the significance of inconclusive or non informative results with a need for more research in this area. Aims: The general aim of this study was to evaluate the impact of breast cancer genetic counselling on psychological distress levels, perception of risk, genetic knowledge and understanding of BRCA testing/test results in a cohort of 207 women from high risk breast cancer families who were referred for genetic counselling in Perth during the period 1997 to 2001. Short- and long-term impact of BRCA genetic counselling/testing was determined in women with and without cancer in a randomised controlled trial as part of which women were randomised to either receive immediate versus delayed genetic counselling. This included family communication patterns before BRCA testing, anticipated outcomes of testing on oneself and family including intentions for result disclosure. Comprehension of index and predictive BRCA testing with possible results was assessed both in the short- and the long-term and understanding of individual or family BRCA test results was evaluated at long-term. The effect of genetic counselling on breast cancer risk perception in unaffected women was evaluated. This study considered a theoretical framework of educational learning theories to provide a basis for risk communication with possible relevance for future research. ... Only 25% of the original study population (52/207) reported BRCA results and women's understanding of results is concerning. Key findings were: 1. The majority of affected women received an inconclusive result. 2. Out of twelve unaffected women who reported results, seven were inconclusive which are not congruent with predictive testing. This implies that these women did not understand their test result. 3. A minority of untested relatives did not know whether a family mutation had or had not been found in their tested family member or what their actual test result was. This implies either a lack of disclosure or that woman did not understand the rationale for and significance of testing for a family mutation. 4. Three relatives did not understand a positive result was a mutation. Conclusion: The implication of this research for breast cancer counselling and testing services is that women who wait for counselling are no worse off in terms of short- or long-term general psychological distress than women who receive the intervention early. There is a suggestion that unaffected women without the disease found counselling more advantageous than affected women. The meaning of BRCA results as reported by women is concerning particularly women's understanding of negative and inconclusive results and further research is needed in this area. Too much information presented at counselling may affect women's comprehension of risk, BRCA testing and future test results and further research is required to evaluate the effects of information overload.

It is increasingly recognized by the scientific community that the field of epigenetics is a key step for a better understanding of human biology in both normal and pathological states. Its implication in cancer, and in particular in breast cancer, is now well accepted. Breast cancer, responsible for more than 450,000 deaths worldwide yearly, is a heterogenous disease at the histological and clinical levels as well as at the molecular level. Despite considerable efforts to develop new treatments and improve patient management, patients with a same “profile” of breast cancer can respond differently to therapies and have completely different clinical outcomes. There is therefore a critical need to improve our understanding of breast cancer biology and diversity, in order to find new markers that should provide a better management of patients and the development of new therapies. An increasing number of biologists, pathologists as well as clinicians are currently working towards these goals. During my PhD, we have conducted two studies in order to gain new insights into the contribution of epigenetics in breast cancer biology.<p>In the first study, by performing large genome-scale DNA methylation profiling of numerous breast tumors as well as of normal breast tissues, we first revealed the existence of six groups of breast tumors based on their DNA methylation profiles. Three of these groups showed a strong association with the basal-like, HER2 and luminal A breast cancer subtypes, previously identified by gene expression profiling. Interestingly, the three other groups were found to be a mixture of several gene expression-based subtypes, thus revealing the capacity of DNA methylation profiling to improve breast tumor taxonomy. Second, our study suggests that the establishment of DNA methylation patterns of breast tumors might help to determine their cell type of origin. Finally, we also showed that DNA methylation profiling can reflect the cell type composition of the tumor microenvironment and that a signature of T cell tumoral infiltration is associated with a good prognosis in particular categories of breast cancer patients. <p>In the second study, we revealed the clinical relevance of the KDM5 histone demethylases in breast cancer. The expression of these histone demethylases was deregulated in the analyzed breast tumors as well as in the pre-invasive samples as compared to normal breast samples. This suggests that KDM5 enzymes might be good markers for early diagnosis of breast cancer. Moreover, we showed a prognostic value of the KDM5C histone demethylase.<p>In conclusion, the above data should provide a better understanding of breast cancer biology and diversity, and this should bring new insights to improve breast cancer patient management.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

De nombreux travaux ont démontré la modulation, par les tumeurs, de certaines fonctions des cellules du système immunitaire. Dans le cadre de notre travail, nous avons étudié les lymphocytes T CD4+, cellules clefs de la réponse immune spécifique, chez des patientes atteintes d’un cancer du sein.<p>Sur base de l’établissement des profils d’expression génique des lymphocytes T infiltrant les tumeurs, nous avons dérivé la « tumor-infiltrating CD4+ signature » (TICD4S) composée de 61 gènes immuns et qui reflète l’état d’activation immunitaire. Cette signature présente une valeur prédictive chez les patientes porteuses de tumeurs ERBB2-positives et ER-négative/PR-négative/ERBB2-négative: une plus forte expression de ces gènes est associée à une meilleure survie.<p>Nous avons également étudié conjointement les profils géniques établis au départ des lymphocytes T CD4+ de la tumeur, du ganglion axillaire et du sang de dix patientes. Nous avons constaté que ces profils d’expression génique des TIL CD4+ diffèrent selon le statut ER de la tumeur qu’ils infiltrent. Les lymphocytes T ganglionnaires CD4+ subissent également les effets de la masse tumorale et, tout comme les TIL, sont moins activés chez les patientes porteuses de tumeurs ER-négatives. Par contre, les lymphocytes T sanguins semblent subir dans une moindre mesure les effets de la tumeur et peu de différences ont été notées par rapport à leurs homologues isolés chez des donneuses saines.\ / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / Telomere maintenance is a critical component of genomic stability. An increasing body of evidence suggests BRCA1, a tumor suppressor gene with a variety of functions including DNA repair and cell cycle regulation, plays a role in telomere maintenance. Mutations in BRCA1 account for approximately half of all hereditary breast and ovarian cancers, and the gene is silenced via promoter methylation and loss of heterozygosity in a proportion of sporadic breast and ovarian cancers. The objective of this study was to determine whether GRN163L, a telomerase inhibitor, currently in clinical trials for the treatment of cancer, has enhanced anti-cancer activity in BRCA1 mutant breast/ovarian cancer cell lines compared to wild-type cancer cells. BRCA1 mutant cancer cells were observed to have shorter telomeres and increased sensitivity to telomerase inhibition, compared to cell lines with wild-type BRCA1. Importantly, GRN163L treatment was synergistic with DNA-damaging drugs, suggesting potential synthetic lethality of the BRCA1 cancer subtype and telomerase inhibition In a related study to examine the roles of BRCA1/2 in telomere maintenance, DNA and RNA extracted from peripheral blood were used to investigate the age-adjusted telomere lengths and telomere-related gene expression profiles of BRCA1 and BRCA2 individuals compared to individuals who developed sporadic cancer and healthy controls. BRCA1 mutation carriers and breast cancer patients showed the shortest average telomere lengths compared to the other groups. In addition, distinct genomic profiles of BRCA mutation carriers were obtained regarding overexpression of telomere-related genes compared to individuals who developed sporadic or familial breast cancer. In summary, telomerase inhibition may be a viable treatment option in BRCA1 mutant breast or ovarian cancers. These data also provides insights into further investigations on the role of BRCA1 in the biology underlying telomere dysfunction in cancer development.