"October 2003" / Bibliography: leaves 223-271. / xiii, 271 leaves : ill. (some col.), plates (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / 1. Hormonal control of human breast cancer -- 2. General materials and methods -- 3. Proliferative effects of androgens -- 4. Cross-talk between androgen and estrogen signalling pathways -- 5. Androgen receptor function in the MDA-MB-453 cell line -- 6. Gene expression profiles in breast cancer cells: identification of androgen regulated genes -- 7. Regulation of BRCA1 expression -- 8. Regulation of prostate specific antigen expression -- 9. General discussion. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 2004

Despite the medical advancements, the woman breast cancer incidence rates keep rising in the past few decades. Scientists have proposed the increased exposure to endocrine disrupting chemicals as a possible factor for the rises. Zearalenone (ZEA) and aflatoxin B1 (AFB1) are common mycotoxins that are present in cereal crops worldwide. ZEA has long been recognized as a xenoestrogen, while the endocrine disrupting effects of AFB1 on steroidogenesis have been identified recently. Due to the co-occurrence and the endocrine disrupting potentials of ZEA and AFB1, the hypothesis of this project was proposed as exposure to low doses of ZEA and AFB1 might affect the growth of hormonal dependent breast cancer. In order to address the hypothesis, the aim of the first study was to examine the ultimate effects on growth and cell cycle progression in breast cancer MCF-7 cell line, following low dose exposure to ZEA and AFB1 individually and in combination. The effects on viability, cell growth, DNA synthesis, cell cycle progression and cyclin gene expressions were determined. Significant interactions were detected for their effects on viability and DNA synthesis. While ZEA promoted growth, DNA synthesis and cell cycle progression in MCF-7 cells, AFB1 was cytotoxic and counteracted the effects of ZEA. This study confirmed the growth promoting properties of ZEA, and is the first to report the combined effects of ZEA and AFB1 on breast cancer cell growth, suggesting endocrine-disrupting mycotoxins that co-occur in human food can interact and modulate the effects of each other on human health. The second study aimed to reveal the modulation of breast cancer genes and the underlying pathways that were directly affected by ZEA and AFB1. By using a real time reverse transcription polymerase chain reaction array, it was shown that ZEA was capable of altering the expressions of a large number of breast cancer related genes, whereas AFB1 had minimal effects on the breast cancer gene expressions. With the use of specific inhibitors, estrogen receptor α, G protein-coupled estrogen receptor 1, and mitogen-activated protein kinases (MAPKs) were found to be responsible for ZEA’s effects on cell growth and myelocytomatosis oncogene activation; while MAPK pathways might be involved in the cytotoxic effects by AFB1. Further confirmation is necessary for linking the activations of estrogen receptors and MAPKs to breast cancer cell growth by ZEA and AFB1. The last study aimed at assessing the impacts of ZEA and AFB1 on steroidogenic and steroid metabolic enzymes in MCF-7 cells, which might result in hormonal imbalance and undesirable breast cancer cell growth. By evaluating the mRNA expressions, it was found that ZEA significantly modulated the expressions of all the steroidogenic and steroid metabolic enzymes tested while AFB1 altered the expression of cytochrome P450 (CYP) 1A1 and CYP 1B1. Coexposure of MCF-7 cells to the two mycotoxins revealed that AFB1 antagonized the effects of ZEA on expression of CYP 1A1, CYP 3A4 and CYP 1B1. Further research into the alternations of enzyme levels and activities by ZEA and AFB1 is necessary before a solid conclusion can be made. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Breast cancer is the most common malignant cancer in western countries. It can be classified into various types/stages according to patient age, tumor size, histological grade or hormone receptor status. Obesity is a well-known risk factor of breast tumor. Studies have shown that overweight or obese postmenopausal women have a threefold higher risk to develop breast cancer in comparison to their lean or normal counterparts. There are many mechanisms that can link obesity with breast cancer and one of the major contributors is adipokines. The main focus of this study is adiponectin. Many cellular and animal studies have illustrated the inhibitory action of adiponectin on breast cancer cell proliferation. In this study, the effect of complete loss of adiponectin expression on breast cancer development in Mouse Mammary Tumor Virus-polyomavirus middle T antigen(MMTV-PyVT)mice [PyVT(+/-)]will be investigated. Mice with [ADN(+/+)]or without [ADN(-/-)] adiponectin gene were used for comparison. It was found that PyVT(+/-)ADN(-/-)mice had earlier tumor onset time and larger tumor volume than PyVT(+/-)ADN(+/+) mice. Histological analysis has demonstrated that increased and more dispersed metastasis existed in lung tissue of PyVT(+/-)ADN(-/-)mice in comparing with PyVT(+/-)ADN(+/+)mice. The aggressiveness of adiponectin deficient tumor was preserved after implantation into immune-deficient mice. Gene expression and protein expression studies of PyVT tumor have indicated a different expression level and pattern of two important molecules: P63 and YY1. In conclusion, tumor developed under microenvironment of adiponectin deficient will give rise to a more aggressive tumor. This tumor consistsof modified genotypes and phenotypes that are permanent and can be preserved after re-implantation into immuno-compromised mice. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

"July, 2003" / "Amendments of the thesis" and "abbreviations (additional)" inside back cover. / Includes bibliographical references (leaves 195-231) / x, 231, [20] leaves : ill., plates ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2003

The growth and survival of normal breast epithelial cells and breast cancer cells is promoted by estrogens. In contrast, androgens inhibit the proliferation of normal and malignant breast epithelial cells. While this effect of androgens on breast cells appears to be androgen receptor (AR) dependent, the precise mechanism of inhibition and its functional significance are unknown. The aims of this thesis were to investigate the effect of androgen signalling on growth of normal and malignant breast epithelial cells, and to assess the interactions between androgen and estrogen signalling in the breast. To investigate the role of androgen signalling in the growth and development of the normal mammary gland, female mice were treated with either the native androgen 5α- dihydrotestosterone (DHT) or the antiandrogen, flutamide. Analysis of the mammary glands at the end of the treatment period demonstrated that DHT reduced ductal branching and mammary epithelial cell proliferation when treatment commenced mid-puberty. Conversely, flutamide treatment that commenced post-puberty significantly increased ductal branching and proliferation of mammary epithelial cells. This data demonstrates that androgen signalling inhibits proliferation in the normal mammary gland, and may therefore oppose to the growth stimulatory effects of estrogen signalling to regulate breast growth and development. The antiproliferative effects of androgens on breast epithelial cells may be due in part to direct AR-mediated activation of androgen regulated genes, or alternatively, androgens could act indirectly through AR to inhibit estrogen receptor alpha (ERα) activity. Expression of fulllength AR or a truncated, constitutively active AR (AR-T707) significantly inhibited the activity of ectopically expressed ERα in MDA-MB-231 breast cancer cells (ERα- and ARnegative), in a dose-dependent manner. The functional consequences of inhibition of estrogen signalling by overexpressing AR were investigated in the T-47D breast cancer cell line (ERα- and AR-positive). Expression of AR-T707 in T-47D cells resulted in inhibition of both basal and estradiol-induced cell proliferation and a marked reduction in the steady-state protein levels of the estrogen regulated gene, PR. The final chapter investigated the mechanism by which AR inhibits ERα activity. A coimmunoprecipitation assay demonstrated an interaction between ectopically expressed AR and ERα in COS-1 cells, but not endogenous AR and ERα in a breast cancer cell line. To delineate the regions of AR required for inhibition of ERα signalling, various functional domains of the AR were mutated or deleted. Reporter gene assays showed that the inhibitory effects of AR were abrogated by deletion or mutation of the DNA binding domain (DBD). Furthermore, overexpression of the AR-DBD alone was sufficient to inhibit ERα activity. Consistent with a requirement for the DBD of AR to inhibit ERα activity, mobility shift assays demonstrated binding of AR to the Xenopus vitellogenin A2 consensus estrogen response element (cERE); however AR/ERα heterodimers were not detected on a cERE. Consistent with these findings, molecular modelling demonstrated that it is feasible for the DBD of AR to bind to a cERE and that it is unlikely that AR/ERα heterodimers could bind. Chromatin immunoprecipitation demonstrated recruitment of AR to the promoters of endogenous estrogen regulated genes. The findings suggest that the inhibitory effect of AR on ERα activity may occur either via formation of non-functional AR/ERα heterodimers that are unable to bind to EREs, or AR homodimers competing effectively for binding to EREs, in ERα target genes. The results in this thesis demonstrate an inhibitory effect of androgen signalling on growth of normal and malignant breast epithelial cells. Additionally, the inhibition of breast epithelial cell proliferation by androgen signalling can be attributed, at least in part, to inhibition of ERα activity. These studies have provided insight into androgen action in the breast, and support a model whereby androgens balance the stimulatory effects of estrogen signalling in normal and malignant breast epithelial cells. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

[Truncated abstract] Breast cancer is the cause of significant suffering and death in our community. It is now estimated that the risk of developing breast cancer for an Australian woman before the age of 85 is 1 in 8, with this risk rising for unknown reasons. While mortality rates from breast cancer are falling due to increased awareness and early detection, few new treatments have been developed from an advanced understanding of the molecular basis of the disease. From decades of scientific research it is clear that estrogen (E2) has a large role to play in breast cancer. However, the basic mechanism behind E2 action in breast cancer remains unclear. E2 plays a fundamental role in breast cancer cell proliferation and is highly expressed in breast cancers, thus, it is important to understand both E2 and its receptor, the estrogen receptor (ER). The ER is a member of the nuclear receptor (NR) superfamily. The NR superfamily consists of a large group of proteins which regulate a large number of homeostatic proteins together with regulator proteins termed coregulators and corepressors. SRA (steroid receptor RNA activator) is the only known RNA coactivator and augments transactivation by NRs. SRA has been demonstrated to play an important role in mediating E2 action (Lanz et al., 1999; Lanz et al., 2003) and its expression is aberrant in many human breast tumors, suggesting a potential role in breast tumorigenesis (Murphy et al., 2000). Despite evidence that an alternative splice variant of SRA exists as a protein (Chooniedass-Kothari et al., 2004), it has been conclusively shown that SRA can function as an RNA transcript to coactivate NR transcription (Lanz et al., 1999; Lanz et al., 2002; Lanz et al., 2003). The precise mechanism by which SRA augments ER activity remains unknown. However, it is currently hypothesized that SRA acts as an RNA scaffold for other coregulators at the transcription initiation site. Several SRA stem loops have been identified as important for SRA function, including structure (STR) 1, 5 and 7 (Lanz et al., 2002; Zhao et al., 2007). Previously, I sought to identify SRA-binding proteins using a specific stem-loop structure of SRA (STR7) that was identified as both important for its coactivator function (Lanz et al., 2002) and also as a target for proteins from breast cancer cell extracts (Hatchell, 2002). From a yeast E. Hatchell Abstract iii III hybrid screen using STR7 as bait, I identified a novel protein which was named SLIRP (Patent Number: WO/2007/009194): SRA stem-Loop Interacting RNA-binding Protein (Hatchell, 2002; Hatchell et al., 2006). '...' This thesis demonstrates that SLIRP modulates NR transactivation, provides mechanistic insight into interactions between SRA, SRC-1, HSP-60 and NCoR and suggests that SLIRP may regulate mitochondrial function. These studies contribute significantly to the growing field of NR biology, and contribute more specifically to the elucidation of estrogen action in breast cancer. Furthermore, it lays a strong and exciting foundation for further studies to evaluate SLIRP as a biomarker and potential therapeutic target in hormone dependent cancers.

Indiana University-Purdue University Indianapolis (IUPUI) / The time course and biological mechanisms by which breast cancer (BC) and/or alterations in estrogen status lead to cognitive and brain changes remain unclear. The studies presented here use neuroimaging, cognitive testing, genetics, and biomarkers to investigate how post-chemotherapy interval (PCI), chemotherapy-induced amenorrhea (CIA), and genetic variation in the estrogen pathway affect the brain. Chapter 1 examines the association of post-chemotherapy interval (PCI) with gray matter density (GMD) and working memory-related brain activation in BC survivors (mean PCI 6.4, range 3-10 years). PCI was positively associated with GMD and activation in the right frontal lobe, and GMD in this region was correlated with global neuropsychological function. In regions where BC survivors showed decreased GMD compared to controls, this was inversely related to oxidative DNA damage and learning and memory scores. This is the first study to show neural effects of PCI and relate DNA damage to brain alterations in BC survivors. Chapter 2 demonstrates prospectively, in an independent cohort, decreased combined magnitudes of brain activation and deactivation from pre-to post-chemotherapy in patients undergoing CIA compared to both postmenopausal BC patients undergoing chemotherapy and healthy controls. CIA’s change in activity magnitude was strongly correlated with change in processing speed, suggesting this activity increase reflects effective cognitive compensation. These results demonstrate that the pattern of change in brain activity from pre- to post-chemotherapy varies according to pre-treatment menopausal status. Chapter 3 presents the effects of variation in ESR1, the gene that codes for estrogen receptor-α, on brain structure in healthy older adults. ESR1 variation was associated with hippocampus and amygdala volumes, particularly in females. Single nucleotide polymorphism (SNP) rs9340799 influenced cortical GMD and thickness differentially by gender. Apolipoprotein E (APOE)-ε4 carrier status modulated the effect of SNP rs2234693 on amygdala volumes in women. This study showed that genetic variation in estrogen relates to brain morphology in ways that differ by sex, brain region and APOE-ε4 carrier status. The three studies presented here explore the interplay of BC, estrogen, and cognition, showing that PCI, CIA, and ESR1 genotype influence brain phenotypes. Cognitive correlates of neuroimaging findings indicate potential clinical significance of these results.