There is no abstract available for this dissertation. / Department of Educational Studies

Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer, with a particularly poor prognosis. Identification of epidemiologic risk factors for IBC might shed light on causes of the disease, and guide screening and perhaps treatment. Previous studies have suggested that race, geographic location, body mass index (BMI), menopausal status, age at menarche, parity, duration of lactation, and exposure to mouse mammary tumor virus may be key risk factors in the development of IBC. This retrospective epidemiologic study examines the risk factors for IBC in predominantly Caucasian patients treated at the Dana Farber Cancer Institute (DFCI) in Boston, MA. The risk factors that were examined in this study include the following: BMI, family history of having breast cancer, comorbidities, duration of symptoms associated with IBC prior to diagnosis, season of diagnosis, and molecular subtypes of breast cancer. Additionally, the descriptive statistics for the mean age of diagnosis, race, menopausal status, genetic predisposition, and the presence of metastases in distant organs were also determined. This study showed that there is some evidence of a hereditary component and seasonal variation to the disease. Furthermore, this study reiterates the association of high body mass index (BMI) and IBC. The data collected from the DFCI IBC patient population suggest that modifiable lifestyle factors, perhaps due to a lack of awareness of the disease, might be crucial in the development of IBC. Further research is needed to explore the unique risk factors in developing IBC elucidated in this study in order to better understand and prevent such an aggressive disease.

O câncer de mama é a neoplasia mais comum entre as mulheres. Sua etiologia é complexa, onde tanto fatores ambientais como genéticos podem contribuir para o desenvolvimento tumoral. Estima-se que 5 a 10% dos casos de carcinomas de mama sejam representados pelos carcinomas de mama familial e 90 a 95% sejam representados pelos carcinomas de mama esporádicos. Independente da etiologia, um dos principais agravantes é consequência da habilidade das células tumorais metastizar. Mutações podem levar a mudança ou perda de expressão de diferentes genes e isto possibilita que as células adquiram particularidades genéticas e fenotípicas que contribuem para a progressão do tumor através da aquisição de vantagens que medeiam a sua sobrevivência. Dentre estas vantagens adquiridas está a capacidade das células tumorais de escaparem da destruição pelas células imunológicas ou, até mesmo, utilizarem estas células a seu favor, na promoção de um microambiente tumoral inflamatório que pode auxiliar o desenvolvimento da angiogênese que posteriormente facilitará a metástase. As características dos carcinomas de mama são as principais ferramentas para avaliação do diagnóstico e prognóstico da doença. Portanto, o objetivo de nosso trabalho foi a análise de quatro variantes polimórficas de genes que codificam importantes moléculas do sistema imunológico, duas relacionadas aos genes que codificam os receptores de quimiocinas, CCR2 e CCR5, e duas relacionadas ao gene HLA-G em 188 mulheres com carcinoma de mama (105 com câncer de mama familial e 83 com câncer de mama esporádico) e em 151 mulheres sem carcinoma e sem história familiar de câncer (grupo controle), como possíveis marcadores de diagnóstico e prognóstico do carcinoma de mama. Para a análise da variante do CCR2, denominada CCR264I, e para a análise de uma das variantes que codifica a molécula de HLA-G, denominada +3142, utilizou-se a técnica PCR-RFLP. Para a análise da variante do gene CCR5, denominada CCR5delta32 e para a análise da outra variante do HLA-G, caracterizada pela inserção de 14pb na região 3’UTR do gene, utilizou-se a técnica PCR. As frequências alélicas, genotípicas e haplotípicas foram estimadas e comparadas entre os grupos de mulheres, usando o Teste Qui-Quadrado ou o Teste Exato de Fisher e, posteriormente, foram relacionadas a fatores de diagnóstico e prognóstico. Observou-se maiores frequências dos alelos selvagens do CCR2, Val (p=0,040, OR 0,61, IC 95% = 0,38 – 0,98) e do CCR5, Wt (p=0,032, OR 0,46, IC 95% = 0,23 – 0,94) e maior frequência do haplótipo duplo selvagem Wt/Val destas mesmas variantes gênicas dos genes CCR2 e CCR5, nas mulheres do grupo controle (p=0,030) em relação às mulheres com câncer de mama familial. Quando as variantes foram avaliadas em conjunto com os parâmetros clínicos, observou-se que as mulheres com carcinoma de mama esporádico apresentavam a doença em idade mais elevada (57,29 ± 8,457 anos e 44,23 ± 12,092 anos para mulheres com câncer de mama esporádico e familial, respectivamente, p < 0.001) e de forma mais agressiva, com maior frequência dos carcinomas invasores (p = 0,001) que as mulheres com carcinoma de mama familial. Além disso, as variantes de inserção/deleção de 14 pb do HLA-G e CCR264I, mostraram relação positiva com a agressividade tumoral nestas mulheres (p = 0,039 e p = 0,005). Nossos dados sugerem que os carcinomas invasores possam estar relacionados a uma maior infiltração de células imunológicas e com o aumento da inflamação no microambiente tumoral, mediados pelo receptor CCR2 e pela molécula HLA-G, respectivamente. / Breast cancer is the most common cancer among women. Its etiology is complex, where genetic, environmental and endocrine factors contribute to tumor development. It is estimated that 5 to 10% of the breast cancers are represented by familial breast cancers and 90 to 95% are represented by sporadic breast cancers. Independent of the etiology, the major aggravating consequence is the ability of tumor cells to metastasize. Mutations can lead to a change or loss of expression a different genes and this allows the appearance of genetic and phenotypic features which contribute to tumor progression. Among these features is the ability of tumor cells to evade from the immune cells or even use immune cells in the promotion of a inflammatory microenvironment promotion which may help angiogenesis and, later, metastasis. The aim of our study was to evaluate four important polymorphic variants of genes which encode important immune system molecules, two related genes encoding chemokine receptors, CCR2 and CCR5, and two related to HLA-G gene in 188 women with breast cancer (105 women with familial breast cancer and 83 with sporadic breast cancer) and 151 women without cancer and family history of cancer (control group), such as potential markers for diagnosis and prognosis of breast cancer. CCR2 polymorphism, CCR264I, and one HLA-G polymorphism, +3142, were genotyped by PCR-RFLP. CCR5delta32 and 14pb HLA-G polymorphism were genotyped by PCR. Allelic, genotypic and haplotypic frequencies were estimated and compared between the groups using the Chi-square test or Fisher's exact test and subsequently were associated to diagnostic and prognostic factors. We observed a higher allelic frequency of the CCR2 wild type allele, Val (p = 0.040, OR 0.61, 95% CI = 0.38 - 0.98) e CCR5 wild type allele, Wt (p = 0.032, OR 0.46, CI 95% = 0.23 - 0.94) and higher haplotype frequency of the double wild type variants (Wt/Val) of these same genes (CCR2 and CCR5) in women in the control group (p = 0.030) compared to women with familial breast cancer. All polymorphisms were evaluated together with the clinical parameters and it was observed that women with breast cancer showed sporadic cancer latter (57.29 ± 8.457 years and 44.23 ± 12.092 years for women with sporadic breast cancer and familial breast cancer, respectively, p < 0.001) and more invasiveness (p = 0.001) as compared to women with familial breast cancer. Moreover, the HLA-G 14pb and CCR264I polymorphism, showed a positive association with tumor aggressiveness in women with sporadic breast cancer (p = 0.039 and p = 0.005, respectively). Our data suggest that invasive cancers may be associated with increased immune cells infiltration and inflammation in the tumor microenvironment mediated by both CCR2 receptor and HLA-G molecule.

O câncer de mama é a neoplasia mais comum entre as mulheres. Sua etiologia é complexa, onde tanto fatores ambientais como genéticos podem contribuir para o desenvolvimento tumoral. Estima-se que 5 a 10% dos casos de carcinomas de mama sejam representados pelos carcinomas de mama familial e 90 a 95% sejam representados pelos carcinomas de mama esporádicos. Independente da etiologia, um dos principais agravantes é consequência da habilidade das células tumorais metastizar. Mutações podem levar a mudança ou perda de expressão de diferentes genes e isto possibilita que as células adquiram particularidades genéticas e fenotípicas que contribuem para a progressão do tumor através da aquisição de vantagens que medeiam a sua sobrevivência. Dentre estas vantagens adquiridas está a capacidade das células tumorais de escaparem da destruição pelas células imunológicas ou, até mesmo, utilizarem estas células a seu favor, na promoção de um microambiente tumoral inflamatório que pode auxiliar o desenvolvimento da angiogênese que posteriormente facilitará a metástase. As características dos carcinomas de mama são as principais ferramentas para avaliação do diagnóstico e prognóstico da doença. Portanto, o objetivo de nosso trabalho foi a análise de quatro variantes polimórficas de genes que codificam importantes moléculas do sistema imunológico, duas relacionadas aos genes que codificam os receptores de quimiocinas, CCR2 e CCR5, e duas relacionadas ao gene HLA-G em 188 mulheres com carcinoma de mama (105 com câncer de mama familial e 83 com câncer de mama esporádico) e em 151 mulheres sem carcinoma e sem história familiar de câncer (grupo controle), como possíveis marcadores de diagnóstico e prognóstico do carcinoma de mama. Para a análise da variante do CCR2, denominada CCR264I, e para a análise de uma das variantes que codifica a molécula de HLA-G, denominada +3142, utilizou-se a técnica PCR-RFLP. Para a análise da variante do gene CCR5, denominada CCR5delta32 e para a análise da outra variante do HLA-G, caracterizada pela inserção de 14pb na região 3’UTR do gene, utilizou-se a técnica PCR. As frequências alélicas, genotípicas e haplotípicas foram estimadas e comparadas entre os grupos de mulheres, usando o Teste Qui-Quadrado ou o Teste Exato de Fisher e, posteriormente, foram relacionadas a fatores de diagnóstico e prognóstico. Observou-se maiores frequências dos alelos selvagens do CCR2, Val (p=0,040, OR 0,61, IC 95% = 0,38 – 0,98) e do CCR5, Wt (p=0,032, OR 0,46, IC 95% = 0,23 – 0,94) e maior frequência do haplótipo duplo selvagem Wt/Val destas mesmas variantes gênicas dos genes CCR2 e CCR5, nas mulheres do grupo controle (p=0,030) em relação às mulheres com câncer de mama familial. Quando as variantes foram avaliadas em conjunto com os parâmetros clínicos, observou-se que as mulheres com carcinoma de mama esporádico apresentavam a doença em idade mais elevada (57,29 ± 8,457 anos e 44,23 ± 12,092 anos para mulheres com câncer de mama esporádico e familial, respectivamente, p < 0.001) e de forma mais agressiva, com maior frequência dos carcinomas invasores (p = 0,001) que as mulheres com carcinoma de mama familial. Além disso, as variantes de inserção/deleção de 14 pb do HLA-G e CCR264I, mostraram relação positiva com a agressividade tumoral nestas mulheres (p = 0,039 e p = 0,005). Nossos dados sugerem que os carcinomas invasores possam estar relacionados a uma maior infiltração de células imunológicas e com o aumento da inflamação no microambiente tumoral, mediados pelo receptor CCR2 e pela molécula HLA-G, respectivamente. / Breast cancer is the most common cancer among women. Its etiology is complex, where genetic, environmental and endocrine factors contribute to tumor development. It is estimated that 5 to 10% of the breast cancers are represented by familial breast cancers and 90 to 95% are represented by sporadic breast cancers. Independent of the etiology, the major aggravating consequence is the ability of tumor cells to metastasize. Mutations can lead to a change or loss of expression a different genes and this allows the appearance of genetic and phenotypic features which contribute to tumor progression. Among these features is the ability of tumor cells to evade from the immune cells or even use immune cells in the promotion of a inflammatory microenvironment promotion which may help angiogenesis and, later, metastasis. The aim of our study was to evaluate four important polymorphic variants of genes which encode important immune system molecules, two related genes encoding chemokine receptors, CCR2 and CCR5, and two related to HLA-G gene in 188 women with breast cancer (105 women with familial breast cancer and 83 with sporadic breast cancer) and 151 women without cancer and family history of cancer (control group), such as potential markers for diagnosis and prognosis of breast cancer. CCR2 polymorphism, CCR264I, and one HLA-G polymorphism, +3142, were genotyped by PCR-RFLP. CCR5delta32 and 14pb HLA-G polymorphism were genotyped by PCR. Allelic, genotypic and haplotypic frequencies were estimated and compared between the groups using the Chi-square test or Fisher's exact test and subsequently were associated to diagnostic and prognostic factors. We observed a higher allelic frequency of the CCR2 wild type allele, Val (p = 0.040, OR 0.61, 95% CI = 0.38 - 0.98) e CCR5 wild type allele, Wt (p = 0.032, OR 0.46, CI 95% = 0.23 - 0.94) and higher haplotype frequency of the double wild type variants (Wt/Val) of these same genes (CCR2 and CCR5) in women in the control group (p = 0.030) compared to women with familial breast cancer. All polymorphisms were evaluated together with the clinical parameters and it was observed that women with breast cancer showed sporadic cancer latter (57.29 ± 8.457 years and 44.23 ± 12.092 years for women with sporadic breast cancer and familial breast cancer, respectively, p < 0.001) and more invasiveness (p = 0.001) as compared to women with familial breast cancer. Moreover, the HLA-G 14pb and CCR264I polymorphism, showed a positive association with tumor aggressiveness in women with sporadic breast cancer (p = 0.039 and p = 0.005, respectively). Our data suggest that invasive cancers may be associated with increased immune cells infiltration and inflammation in the tumor microenvironment mediated by both CCR2 receptor and HLA-G molecule.

O câncer de mama é a neoplasia mais comum entre as mulheres. Sua etiologia é complexa, onde tanto fatores ambientais como genéticos podem contribuir para o desenvolvimento tumoral. Estima-se que 5 a 10% dos casos de carcinomas de mama sejam representados pelos carcinomas de mama familial e 90 a 95% sejam representados pelos carcinomas de mama esporádicos. Independente da etiologia, um dos principais agravantes é consequência da habilidade das células tumorais metastizar. Mutações podem levar a mudança ou perda de expressão de diferentes genes e isto possibilita que as células adquiram particularidades genéticas e fenotípicas que contribuem para a progressão do tumor através da aquisição de vantagens que medeiam a sua sobrevivência. Dentre estas vantagens adquiridas está a capacidade das células tumorais de escaparem da destruição pelas células imunológicas ou, até mesmo, utilizarem estas células a seu favor, na promoção de um microambiente tumoral inflamatório que pode auxiliar o desenvolvimento da angiogênese que posteriormente facilitará a metástase. As características dos carcinomas de mama são as principais ferramentas para avaliação do diagnóstico e prognóstico da doença. Portanto, o objetivo de nosso trabalho foi a análise de quatro variantes polimórficas de genes que codificam importantes moléculas do sistema imunológico, duas relacionadas aos genes que codificam os receptores de quimiocinas, CCR2 e CCR5, e duas relacionadas ao gene HLA-G em 188 mulheres com carcinoma de mama (105 com câncer de mama familial e 83 com câncer de mama esporádico) e em 151 mulheres sem carcinoma e sem história familiar de câncer (grupo controle), como possíveis marcadores de diagnóstico e prognóstico do carcinoma de mama. Para a análise da variante do CCR2, denominada CCR264I, e para a análise de uma das variantes que codifica a molécula de HLA-G, denominada +3142, utilizou-se a técnica PCR-RFLP. Para a análise da variante do gene CCR5, denominada CCR5delta32 e para a análise da outra variante do HLA-G, caracterizada pela inserção de 14pb na região 3’UTR do gene, utilizou-se a técnica PCR. As frequências alélicas, genotípicas e haplotípicas foram estimadas e comparadas entre os grupos de mulheres, usando o Teste Qui-Quadrado ou o Teste Exato de Fisher e, posteriormente, foram relacionadas a fatores de diagnóstico e prognóstico. Observou-se maiores frequências dos alelos selvagens do CCR2, Val (p=0,040, OR 0,61, IC 95% = 0,38 – 0,98) e do CCR5, Wt (p=0,032, OR 0,46, IC 95% = 0,23 – 0,94) e maior frequência do haplótipo duplo selvagem Wt/Val destas mesmas variantes gênicas dos genes CCR2 e CCR5, nas mulheres do grupo controle (p=0,030) em relação às mulheres com câncer de mama familial. Quando as variantes foram avaliadas em conjunto com os parâmetros clínicos, observou-se que as mulheres com carcinoma de mama esporádico apresentavam a doença em idade mais elevada (57,29 ± 8,457 anos e 44,23 ± 12,092 anos para mulheres com câncer de mama esporádico e familial, respectivamente, p < 0.001) e de forma mais agressiva, com maior frequência dos carcinomas invasores (p = 0,001) que as mulheres com carcinoma de mama familial. Além disso, as variantes de inserção/deleção de 14 pb do HLA-G e CCR264I, mostraram relação positiva com a agressividade tumoral nestas mulheres (p = 0,039 e p = 0,005). Nossos dados sugerem que os carcinomas invasores possam estar relacionados a uma maior infiltração de células imunológicas e com o aumento da inflamação no microambiente tumoral, mediados pelo receptor CCR2 e pela molécula HLA-G, respectivamente. / Breast cancer is the most common cancer among women. Its etiology is complex, where genetic, environmental and endocrine factors contribute to tumor development. It is estimated that 5 to 10% of the breast cancers are represented by familial breast cancers and 90 to 95% are represented by sporadic breast cancers. Independent of the etiology, the major aggravating consequence is the ability of tumor cells to metastasize. Mutations can lead to a change or loss of expression a different genes and this allows the appearance of genetic and phenotypic features which contribute to tumor progression. Among these features is the ability of tumor cells to evade from the immune cells or even use immune cells in the promotion of a inflammatory microenvironment promotion which may help angiogenesis and, later, metastasis. The aim of our study was to evaluate four important polymorphic variants of genes which encode important immune system molecules, two related genes encoding chemokine receptors, CCR2 and CCR5, and two related to HLA-G gene in 188 women with breast cancer (105 women with familial breast cancer and 83 with sporadic breast cancer) and 151 women without cancer and family history of cancer (control group), such as potential markers for diagnosis and prognosis of breast cancer. CCR2 polymorphism, CCR264I, and one HLA-G polymorphism, +3142, were genotyped by PCR-RFLP. CCR5delta32 and 14pb HLA-G polymorphism were genotyped by PCR. Allelic, genotypic and haplotypic frequencies were estimated and compared between the groups using the Chi-square test or Fisher's exact test and subsequently were associated to diagnostic and prognostic factors. We observed a higher allelic frequency of the CCR2 wild type allele, Val (p = 0.040, OR 0.61, 95% CI = 0.38 - 0.98) e CCR5 wild type allele, Wt (p = 0.032, OR 0.46, CI 95% = 0.23 - 0.94) and higher haplotype frequency of the double wild type variants (Wt/Val) of these same genes (CCR2 and CCR5) in women in the control group (p = 0.030) compared to women with familial breast cancer. All polymorphisms were evaluated together with the clinical parameters and it was observed that women with breast cancer showed sporadic cancer latter (57.29 ± 8.457 years and 44.23 ± 12.092 years for women with sporadic breast cancer and familial breast cancer, respectively, p < 0.001) and more invasiveness (p = 0.001) as compared to women with familial breast cancer. Moreover, the HLA-G 14pb and CCR264I polymorphism, showed a positive association with tumor aggressiveness in women with sporadic breast cancer (p = 0.039 and p = 0.005, respectively). Our data suggest that invasive cancers may be associated with increased immune cells infiltration and inflammation in the tumor microenvironment mediated by both CCR2 receptor and HLA-G molecule.

Magister Artium - MA / Breast cancer is a common cause of death among women worldwide. It has long been recognized as a major public health burden in high-income countries, however, the majority of cases are said to occur in low and middle-income countries, such as in South Africa. A breast cancer diagnosis and treatment heralds a series of frightening events and can be a traumatic experience. The manner in which women perceive and cope with their illness is predictive of emotional and physical health outcomes. It is thus imperative to explore the experiences of South African women, whose voices may have been silenced in the past. The purpose of my study was to explore the subjective lived experiences of women with early stage breast cancer undergoing treatment. The objectives of the study were to; explore the emotional experiences of women with early stage breast cancer undergoing treatment and secondly to explore how women perceive their bodies through their experience of early stage breast cancer while undergoing treatment. Phenomenology was used as the theoretical position conceptualising the study as well as the research design. This research study adopted a qualitative approach utilising in-depth face to face semi-structures interviews for collecting data. The participants were selected through purposive sampling and comprised six women aged between 30 and 40 who are undergoing treatment for early stage breast cancer. The data was analysed using interpretative phenomenological analysis. Emotions experienced were characterised by the shock of the diagnosis due to factors such as lack of family history and age. Participants reported positive changes and viewpoints which they gained through their breast cancer journey. Emotions were heightened during treatment due to the physical change experienced and the effects this had on family members and the general public. Furthermore, results indicated that participants, even though they discovered a new found love for life and for their wellbeing, neglected their emotional needs in order to protect family members. An additional reason for this neglect centered on the lack of understanding other individuals may have regarding the experiences of participants. Recommendations involves the encouragement of accessing counselling services and that interventions tailored to the needs of each patient especially according to age. All ethical considerations as stipulated by the University of the Western Cape were adhered to.

One group of chemotherapeutics that are used successfully to treat breast cancer, alone or in combination with other agents, are the taxanes; paclitaxel and docetaxel. They act by interfering with the spindle microtubule dynamics of the cell causing cell cycle arrest. However, the complexities underlying the mechanism of action are yet to be fully elucidated. Arguably, one of the most significant problems with taxanes is chemoresistance. Unfortunately, some patients are intrinsically resistant to taxanes and others acquire resistance to taxanes as treatment advances. This problem is exacerbated by a lack of understanding of the mechanisms underlying taxane resistance. Isogenic breast cancer cell lines that were taxane resistant were generated to use as an experimental model. Paclitaxel resistant (PACR) MDA-MB-231, paclitaxel resistant ZR75-1 and docetaxel resistant (DOCR) ZR75-1 cell lines were successfully generated by incrementally increasing taxane dose in respective native cell lines in vitro. An extensive characterisation of each of the resistant cell lines was conducted, focussing primarily on the 25nM resistant cells which were determined to be the most clinically relevant dose of taxane. A suboptimal dose of 5nM, a “superoptimal” dose of 50nM and the native, taxane sensitive cells was included. Dose response cell count experiments were performed that confirmed taxane resistant cells had been generated. It was shown that MDA-MB-231 native cells were more sensitive to paclitaxel than the ZR75-1 native cells, suggesting that ZR75-1 cells may already have low level inherent resistance. The MDA-MB-231 25nM PACR cells were tested to determine whether they retained PACR when maintained in media containing no paclitaxel. MDA-MB-231 25nM PACR cells were maintained in a taxane free environment for six months and then rechallenged with taxane. When rechallenged, the PACR cells previously maintained in the absence of paclitaxel mirrored the pattern of growth of corresponding PACR cells that had been maintained in the presence of paclitaxel. This proved that in the absence of paclitaxel, PACR cells did not revert to parent phenotype. This meant that experiments could be designed to grow cell lines as xenografts in mice, (in the absence of paclitaxel) & bring in vitro experiments into an in vivo setting. Effects of taxane treatment on both native and resistant cells were analysed using flow cytometry. Paclitaxel treatment exerted G2/M block in native MDA-MB-231 cells but when PACR cells were treated with the same dose of paclitaxel no G2/M block was observed, suggesting that PACR cells had developed a mechanism for escaping G2/M block. ZR75-1 native lines were also investigated and we established that treatment with paclitaxel also exerted a G2/M block in these lines. In future studies this process will be repeated to investigate the effect of taxane treatment on the ZR75-1 PACR and DOCR lines. CD 1 nude mice were injected with cells from all five cell lines to grow xenografts, unfortunately MDA-MB-231 PACR cells failed to grow so they could not be used for further xenograft experiments. PACR, DOCR and Native ZR75-1 cells did successfully grow as xenografts in mice and confirmed that all 3 groups showed very similar growth patterns. A cross resistance experiment was conducted and it was determined that the DOCR xenografts maintained a taxane resistant phenotype to docetaxel, and not paclitaxel and the PACR xenografts may be perpetuate the paclitaxel resistant phenotype in xenografts and that there may be cross resistance to docetaxel in the paclitaxel resistant xenografts. This is the first time that taxane resistant cell lines grown in this way have been established as xenografts in mice. These cross resistance experiments represent novel findings and merit further investigation. Extensive genomic and transcriptomic analyses were carried out on the cell lines to help identify potential taxane resistance markers. aCGH experiments were carried out to compliment the illumina experiments. The first set of experiments used DNA from pooled whole female blood as ref sample and DNA from each of the native and taxane resistant cell lines as test samples. The second set of experiments used DNA from native cells as a ref sample and DNA from their respective taxane resistant cells as a test, which allowed areas of loss or gain to be tracked in the genome as resistance increased. In the MDA-MB-231 cell lines the following areas of loss extended with increasing resistance: 1p36.13-q44, 6p25.3-q12, 8p, 10p, 19q, X Chr and the following areas of gain 2p25.3-23.3, 3p24.3-q13.3, 4p16.1-q12, 5q14.3-q31.1, 8q21.13-24.3, 11q15.1-q25, centromeric 12, and centromeric 14. In the ZR75-1 PACR and DOCR cell lines the areas of loss extended with increasing resistance in the following regions: 7q, 12p and 16q. For gene expression analysis RNA was extracted from the MDA-MB-231 cell lines, labelled and hybridised them to illumina human ref 8 vs. 2 chips. Data showed a progressive increase in mRNA dysregulation as paclitaxel resistance increased. Eleven genes were dysregulated across all resistance levels in the PACR MDA-MB-231 cells when compared to the relative cell lines; RGS16, CLDN1, IL7R, P&PP1R14C, COBL, TRPV4, TSPAN8, CD33, NLRP2, P13, and PAGE5. The experiment was repeated using MDA-MB-231 PACR, ZR75-1 PACR and DOCR cells and resulting data was analysed to determine genes commonly dysregulated across resistance levels, between MDA-MB-231 PACR and ZR75-1 PACR and between ZR75-1 PACR and DOCR cell lines. An extensive literature search was conducted and established four genes of interest in the context of our genomic and transcriptomic experiments including AURKA, Mdr-1, Stathmin and YY1. The novel biomarkers identified in the illumina experiments were validated with complimentary qPCR gene expression experiments looking at expression levels of the eleven commonly dysregulated genes identified and a panel of 19 other genes with significantly increased or decreased expression as resistance increased including AURKA, Mdr-1, Stathmin and YY1. Western blots were performed with lysates from the cell lines using a standard panel of predictive breast cancer markers and AURKA, Mdr-1, Stathmin and YY1. Combining the data from the genomic study, the gene expression profile, qPCR and Western blotting it was established that Mdr-1 had increased expression in the taxane resistant ZR75-1 lines and YY1 had increased expression in the MDA-MB-231 PACR line. Material from the LAPATAX trial was used to observe any transcriptomic changes occurring in tumours following treatment with docetaxel and to compare them to changes identified in our in vitro and xenograft models, this allowed the final step to be taken into a translational environment. LAPATAX (EORTC 10054) is a phase I-II study of Lapatanib and Docetaxel as neoadjuvant treatment for HER-2 +ve locally advanced/inflammatory or large operable breast cancer. Tumour material from eighteen core biopsies pre and post treatment was obtained, the mRNA was extracted, labelled and hybridised to the illumina array. This allowed the changes in gene expression pre and post docetaxel treatment to be tracked. The gene expression data from the LAPATAX trial was combined with gene expression data from our cell line panel and identified two novel putative markers of taxane resistance DUSP1 and FOS. Although sample size is small this has provided extremely valuable evidence directly from the clinic. These two novel putative biomarkers are extremely intriguing and certainly merit further investigation, ideally using additional taxane treated breast tumour tissue. Ultimately, an isogenic in vitro model of taxane resistance was developed in two different cell lines and with two different taxanes within one cell line. The cell lines were characterised and the effect of the taxanes on the cell cycle was determined in the native and taxane resistant lines. Selected cell lines were grown as xenografts in mice and performed successful cross resistance studies upon them. A large transcriptomic and genomic analysis was conducted and has identified a panel of potential taxane resistance markers and areas of loss and gain in the genome perpetuated by increasing taxane resistance. This analysis was validated using qPCR and Western blotting. This allowed a panel of novel taxane resistance markers to be identified. In future studies it is hoped that these targets will be knocked down with shRNA to observe if the taxane resistant cell lines revert to the parental phenotype. In vitro studies will be conducted to find agents that may be used to reduce expression of these markers and restore sensitivity to taxanes and consequently restore the efficacy of these drugs in a clinical setting. As far as the author is aware this is the first time that isogenic taxane resistant cell lines have been generated and investigated in this way.

No description available.

No description available.

In the Western world, breast cancer is the most frequent malignancy in women and still the leading cause of cancer related deaths, therefore, a better understanding of the disease is needed. Adequate therapeutic targets for all breast cancer types have not been identified yet, and patients with the same type of cancer have often different outcomes. Polycomb proteins are emerging as important factors involved in breast cancer formation. Polycomb proteins play a crucial role in embryogenesis, early development, stem cell renewal and establishing and maintaining cell identity. Their alteration leads to mis-regulation of several important cellular factors including tumour suppressors, DNA repair factors, cell cycle regulation factors and cell-cell interaction factors. In this thesis the importance of several polycomb proteins in breast cancer has been investigated. The effect of EZH2 knockdown has been tested in breast cancer cell lines expressing different level of the protein and with different features. The results obtained are in line with other studies and suggest that the effect of EZH2 down-regulation in breast cancer cells is dependent on cellular context. In vitro experiments, using both established breast cell lines and primary epithelial cells have been used for investigating the importance of CBX8 in breast cancer. The results obtained showed that the polycomb proteins CBX8 does not play a central role in malignant transformation of the mammary epithelial cells tested.