Introduction: Estrogen and Progesterone receptor (ER and PR respectively) status are widely used to predict response to hormonal therapy in both the adjuvant and metastatic setting. However a proportion of patients who are ER and PR positive will not respond to hormonal therapy and some patients will also develop resistance to estrogen based therapies. The aim of this study is to identify the role of ERβ, its isoforms and other biological markers in female breast cancer. Methods: Tissue microarrays were created to evaluate the expression of ERβ and its isoforms as well other hormone receptors (Androgen receptor (AR), Progesterone receptor B (PRB), Estrogen a receptors at phosphorylated at serines 118 (ERαS 118) and 167 (ERαS 167) and their correlation with clinico-pathological variables, overall survival (OS) and disease free survival (DFS). Experiments to identify eo- localisation of ERβ and also the effect of estrogen on the phospho specific ERa expression were also carried out. Results : Most of the biological markers showed significant correlation with clinicopathological variables, OS and DFS on univariate analysis. However the nuclear expression of ERβ2 also showed significant correlation with OS on multivariate analysis. The same fmding was noted on multivariate analysis of DFS with AR and ERα S167expression Conclusion: ERβ, AR and ERα S167expression appear to have significant roles as prognostic indicators in breast cancer.

Altered glycosylation patterns have been reported in breast cancer cells but despite some understanding of these changes, the role of glycosidases remains an underresearched area of glycobiology. The aim of this research project was to establish an in vitro model for the biochemical study of glycosidases and their isoforms in normal and cancer breast cells grown in vitro. A glycosidase assay based on paranitrophenol sugar substrates was adapted, and for this purpose showed that there was a significant increase (p<0.05) in a-fucosidase, f3-mannosidase, f3-N-acetylgalactosaminidase and f3-N-acetylglucosaminidase in the breast cancer cell lines compared to the normal breast cell line but the optimum pH for maximum activity was similar in all the five cell lines. The kinetic analysis showed a statistically significant (p<0.05) two-fold increase in Vmax for f3-N-acetylglucosaminidase in the cancerous cells compared to the normal breast cell line but the Km remained unaltered. Two isoforms of f3-N-acetylgalactosaminidase and f3-N-acetylglucosaminidase were isolated from the cell lysate using ion exchange chromatography and no significant differences were found between their pH optima and the enzyme kinetics. The correlation between glycosidase activity and lysosome number in the normal and cancerous cell lines was studied using DND 99 Iysotracker dye and confocal microscopy. The results showed a two fold increase in lysosome number in the MDA MB 435 and MCF 7 compared with the HB4a cells. The activity of the glycosidases secreted into the media surrounding the cells, after 48h incubation, also showed a two-fold increase (p< 0.05) in f3-N-acetylgalactosaminidase and f3-N-acetylglucosaminidase activity in the MDA MB 435 and MCF 7 compared with the HB4a cells. Further treatment of MDA MB 435 cells seeded on Matrigel™ material, with imino sugar and protease inhibitors resulted in a significant 86 percent decrease in secreted f3-N-acetylglucosaminidase activity and a 70 percent decrease in cell adhesion and a decrease in cell invasion in this model system. This is the first time that intracellular and secreted glycosidase activity has been studied in a systematic manner in the above breast cell lines. The Iysotracker data suggests that there is a correlation between the number of Iysosomes and cell lysate glycosidase level. The decrease in MDA MB 435 cell migration through the extracellular Matrigel™ material suggests that glycosidases have a functional role in cancer cell migration.

WWP1 is a ubiquitin ligase of the Nedd4-protein like family. Previous analysis in our laboratory suggested a possible relationship between WWP1 expression and breast cancer. This work attempted to characterise WWP1 expression and potential function in breast oncology. WWP1 mRNA expression was found to be up regulated mostly in ER+ breast tumour cell lines compared with non tumourigenic cell lines. In our experiments, WWP1 expression was not activated by oestrogen.

No description available.

To date, germline mutations in known high-penetrance genes, mainly BRCAI and BRCA2, and in moderate- and low-penetrance genes are responsible for approximately 30- 35% of breast cancer familial clustering, leaving the majority of them unexplained. In addition, the variability of the risk conferred by BRCAI and BRCA2 mutations suggests the presence of genetic modifiers of this risk. Therefore, the identification and characterization of as many as possible of genetic factors is crucial for risk prediction in members of breast cancer families. In this context, the aim of this thesis was firstly to investigate the role of the two Fanconi Anemia (FA) genes PALB2 and SLX4 as breast cancer predisposing loci. In the PALB2 screening, I observed a frequency of deleterious mutation of 2.1 % in familial cases recruited in cancer centers in Milan. Interestingly, I also identified the recurrent mutation c. l 027C > T, detected with 10-fold increased frequency in cases from Bergamo with respect to those ascertained in Milan, suggesting a founder effect. On the contrary. the SLX 4 analysis failed to identify any clearly deleterious mutation, excluding a major role of this gene in breast cancer susceptibility in the Italian population. In addition, I genotyped the candidate low-risk rs895819 polymorphism, located in the gene coding for miR-27a, to evaluate its role in reducing breast cancer risk, previously reported in the German population. No such an association was observed in our sample set. Finally, I investigated the role of the CASPS rs3834129 ins/del polymorphism as a genetic modifier in Italian BRCA1 and BRCA2 mutation carries and I observed an association of this SNP with increased breast cancer risk only in individuals carrying BRC1 mutations. In conclusion, our investigation contributed to assess the role of candidate predisposing loci and genetic modifiers of breast cancer risk, providing further knowledge on the susceptibility to this disease.

Breast cancer is the most common cancer in the UK and approximately 1 in 8 women will be affected by the disease. Estrogen regulates breast cancer growth through the action of the estrogen receptors ERα and ERβ. Antiestrogens, in particular tamoxifen, have contributed greatly to the reduction in breast cancer mortality. Tamoxifen is a tissue selective antiestrogen; it is antiestrogenic in the breast but estrogenic in other tissues, thereby enabling it to promote the beneficial effects of estrogen, such as maintaining bone density. However, like estrogen, tamoxifen also promotes endometrial cancer, so there is an impetus for the development of novel tissue selective ERα ligands. Regulation of gene expression by the estrogen receptors requires the ligand-regulated recruitment of transcription coregulator proteins. In breast cancer ERα-coactivator interactions are associated with tumour progression while ERα-corepressor interactions are associated with receptor antagonism and a therapeutic block of ERα signalling. This thesis details the development of a luciferase fragment complementation assay to image the interaction of ERα with the coactivator AIB1 and corepressor SMRT. It is hoped that elucidation of these interactions will enable a greater appreciation of the tissue selective actions of ERα ligands and aid in the screening of novel ERα antagonists. By means of complimentary luciferase fragment fusion proteins, it is shown that ligand dependent ERα-coregulator interaction can be imaged in vitro and in vivo. ERα and AIB1 luciferase fusion proteins indicate an E2 induced increase in luciferase fragment complementation which is modulated by antiestrogens. The complementation observed correlates with ERα transcriptional activity and the specificity has been further validated by ERα fusion protein mutants. Consistent with the notion that the ERα-SMRT interaction is characteristic of ERα antagonism, ERα and SMRT fusion proteins show increased luciferase fragment complementation with antiestrogens compared with estrogen.

No description available.

No description available.

Cytochromes(CYP) P450 are class of heme-containing enzymes involved in Phase I metabolism of a large number of xenobiotics. The CYP family member CYP2E1 is involved in the metabolism of many xenobiotics and procarcinogens including ethanol, acetone, nitrosamine, pyridine, isoniazid and carbon tetrachloride (CCl4), which are also CYP2E1-inducing agents. CYP2E1 comprises approximately 7% of the liver CYP content and it is also expressed in kidney, lung, brain, gastrointestinal tract and breast tissue implying that this enzyme is implicated in other biological processes aside from its role in Phase I metabolism. Several studies converge to the conclusion that CYP2E1 is induced under many pathological conditions including cancer, obesity, and type 2 diabetes. Increased hepatic ketogenesis and insulin resistance are the possible mechanisms mediating CYP2E1 induction in these conditions. Elevated CYP2E1 expression has been reported in the presence of proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). CYP2E1 generates the highest level of reactive oxygen species (ROS) among the CYP450 superfamily and transient overexpression of this CYP family member in COS cells increases the production of mitochondrial ROS, even in the absence of substrate. Oxidative stress induced by CYP2E1 disturbs the folding capacity of the endoplasmic reticulum (ER), with concomitant alterations in the mRNA and protein expression of the ER stress proteins GRP78 and GRP94. Initially the unfolded protein response (UPR) restores ER homeostasis, and cell viability, but at later stages the accumulation of unfolded proteins stimulates pro-apoptotic signals. Cell death induced by ER stress has been reported in several conditions including hypoxia, and diseases such as diabetes and heart disease. To gain better understanding of the factors regulating CYP2E1 gene expression in breast cancer cells we studied CYP2E1 mRNA and protein levels in MCF7 (p53wt) and MDA-MB-231 (p53 mutated) breast cancer cells and identified that CYP2E1 was under p53 and HIF-1α transcriptional control in both of these cell lines treated with etoposide or desferrioxamine respectively. In addition, CYP2E1 was differentially expressed in the low metastatic potential MCF7 cell line compared to highly metastatic MDA-MB-231 cells in accord with clinical studies indicating that CYP2E1 isoenzyme is expressed in lower levels in patients at clinical stages II, III, and IV which exhibit higher metastatic potential than in patients at stage I of breast cancer. To further investigate the functional significance of the difference in CYP2E1 levels the generation of ROS in breast cancer cells ectopically overexpressing CYP2E1 or in cells in which CYP2E1 expression had been silenced was assessed. Our results indicated that CYP2E1 overexpression resulted in higher ROS generation, which coincided with increased autophagy biomarker expression, increased endoplasmic reticulum stress, detected by XBP1 mRNA splicing in fluorescent reporter assays, and inhibition of metastatic potential. These studies show that CYP2E1 exerts an important role in breast cancer cells by inducing generation of oxidants the levels of which differentially influence the unfolding protein response and apoptosis in high and low metastatic potential breast cancer cells and suggest that targeting CYP2E1 might be a powerful approach to modulate breast cancer initiation, progression, and metastasis.

Breast cancer remains one of the most common causes of death amongst women today. Worldwide approximately 1.3 million women are diagnosed as having breast cancer every year. Much research has been done in trying to unravel the cause and behaviour of breast cancer and hormonal influences are known to play a role. In particular, two hormones known as growth hormone (GH) and insulin-like growth factor (IGF-1) have been found to play an important role in breast cancer growth and development. It has been found that women with breast cancer have higher levels of IGF-1 in their blood compared with women without breast cancer. Further studies have shown that if IGF-1 is added to breast cancer cells in laboratory conditions they grow rapidly. This evidence points to a strong link between IGF-1 and breast cancer. The majority of circulating IGF-1 is made by the liver in response to growth hormone, but as IGF-1 is also found in most cells in the human body, it is uncertain whether its possible effects on breast cancer may be due to local production of IGF-1 by breast cancer cells or to IGF-1 circulating in the blood. This research aims to study the link between IGF-1, GH and breast cancer. In order to do this, small specimens of breast tissue will be taken from women who are undergoing surgery for treatment of their breast cancer. Most cells removed will be cancer cells, but a rim of normal cells will surround them. I will then look at the different levels of IGF-1, growth hormone and other related hormones produced by the cancer or normal cells. During further research, I will aim to keep these cells alive for a few hours and see how they respond when various hormones are added to them. This research will give us more information about the hormonal control of breast cancer growth and may lead to novel treatments for breast cancer in the future.