Changes in the plasma membrane phospholipid composition may alter the structure and/or fluidity and lead to a variety of changes in membrane functions. Dietary fats are known to influence the composition of lipids in the plasma membrane. The purpose of this investigation was to compare effects of dietary linoleic and stearic acid on the composition of the phospholipids in the plasma membranes of mammary tumors in A/St mice.Plasma membranes were isolated and lipids were extracted. Phospholipids were separated by thin-layer chromatography and identified by detection with molybdenum blue reagent. The Rf values and integration of optical densities were used to compare phospholipid composition in membranes of tumors from mice fed experimental diets. It appears that the amount of dietary fat, but not the type, affects the phospholipid distributions. The phosphatidylinositol was the phospholipid most affected, representing the smallest amount in membranes from tumors in mice fed the low fat diets. / Department of Biology

Prostaglandin E2 (PGE2), a byproduct of arachidonic acid metabolism, has been suspected to be involved in tumor promotion. It has been suggested that diet may modulate PGE2 level in organisms thus affecting the implantation and growth of the tumor tissue. PGE2 content was investigated in mice fed ad libitum four types of fatty acid diets: saturated fatty acid diets: a stearic acid and a palmitic acid, and polyunsaturated fatty acid diets: a low fat (safflower 1%) and a high fat diet (safflower 15%). Tumor cells were implanted subcutaneously in mice and harvested when tumors reached .05- 4g. The extracted PGE2 were derivatized and quantified by High Performance Liquid Chromatography (HPLC). The results showed that there is a negative correlation between the level of PGE2 and the size of the tumors. PGE2 level declined as the tumor grew. This suggests that during the early stage of growth the tumor requires higher level of PGE2 to boost its growth. As the tumor becomes more adapted to its environment, it no longer depends on PGE2 to survive. Diet was also seen to be important in tumor suppression. Saturated fatty acid diet (SA-1) showed a suppressive effect on tumor growth. A visual comparison showed that polyunsaturated high fat diet produced more PGE2 than saturated fatty acid. This high level of PGE2 correlate with the highest tumor weights obtained in the Polyunsaturated high fat diet group. / Department of Biology

The specific objective of this study was to determine if Fgf-3 gene expression is mediated by dietary fatty acids and to confirm mouse mammary tumor virus infection. It is well known that dietary linoleic acid enhances growth and dietary stearic acid inhibits growth of mammary tumors. Tumor RNA was extracted from female strain A/St mice fed one of four diets. A radioactively labeled anti-sense RNA probe was generated, invitro, from isolated and purified pFgf-3c (int-2c clone contained in the vector pSP65). The Fgf-3c probe was hybridized to extracted tumor RNA using the ribonuclease protection assay.Electron microscopy confirmed MMTV infection by visualization of type A and B particles in tumor tissue. Expression of Fgf-3c, qualified by RNase protection assay, ranged from 0.02 to 5.89 (relative band density) in all of the diet groups. A positive association between Fgf-3c expression and weight was observed among the tumors of the SA-1 diet (R = 0.947). The SF, SF-1, and PA experimental diets, individually, did not appear to show strong correlation with respect to tumor size. Fgf-3 expression was less in small tumors (<275 mg) and enhanced in large tumors (>275 mg) (p<0.05). / Department of Biology

Breast cancer is characterized in part by chronic inflammation and tissue remodelling in the mammary gland. Adipocyte enhancer binding protein 1 (AEBP1), a pro-inflammatory protein, is up-regulated in breast cancer and enhances cytokine secretion in the mammary tumour microenvironment. AEBP1 over-expression in cultured macrophages resulted in increased enzymatic activity of MMP-9, a matrix metalloproteinase implicated in processing cytokines and stimulating tumour cell growth and mobility. MMP-9 activates the cytokine tumour necrosis factor-alpha (TNF?), and is required for the transformation of epithelial cells by the cytokine interleukin 6 (IL6). Treatment of epithelial cells with TNF? and IL6, both of which promote tumourigenesis, induced AEBP1 expression. Chromatin immunoprecipitation results suggested AEBP1 induction is directly mediated by pro-inflammatory transcription factors NF-?B and STAT3, downstream effectors of TNF? and IL6, respectively. AEBP1 induction may enhance inflammation, thereby contributing to cell proliferation and survival.

In der vorliegenden prospektiven Studie wurde der postoperative Krankheitsverlauf von 93 Hündinnen mit Mammatumoren untersucht. Ziel der Studie war es, eine präoperative Einschätzung der Dignität der Tumoren und der Prognose für die Hündin anhand der Relaxin-Plasmakonzentration zu gewinnen. In einer humanmedizinischen Studie konnte gezeigt werden, dass an Brustkrebs erkrankte Frauen mit einer hohen Relaxin-Plasmakonzentration häufiger an einem malignen Tumor erkrankt sind, der Tumor häufiger bereits metastasiert hatte und die Frauen früher starben. Der Kastrationsstatus (p = 0,132), eine hormonelle Läufigkeitsunterdrückung (p = 0,960), vorausgegangene Graviditäten (p = 0,780) und das Auftreten von Pseudograviditäten (p = 0,138) bei den an Mammatumoren erkrankten Hündinnen hatten keinen Einfluss auf die präoperativ bestimmte Relaxin-Plasmakonzentration. An Mammatumoren erkrankte Hündinnen und gesunde Kontrolltiere hatten keine unterschiedlichen Relaxin-Plasmakonzentrationen (p = 0,813). Die Relaxin-Plasma-konzentrationen von Hündinnen mit einer Herzerkrankung aus der Patientengruppe waren identisch mit denen der herzgesunden Hündinnen aus der Kontrollgruppe (p = 0,328). Innerhalb der Patientengruppe war es hinsichtlich der gemessenen Relaxin-Plasmakonzentration unerheblich, ob die Hündinnen einerseits an einem solitären oder an multiplen Mammatumoren erkrankt waren (p = 0,470), oder ob andererseits bei ihnen einseitig oder beidseitig Mammatumoren feststellbar waren (p = 0,371). Weder die Tumorgröße (p = 0,518) noch eine Ulzeration (p = 0,746) wirkten sich auf die Relaxin-Plasmakonzentration aus. Das Vorliegen von Nahmetastasen (p = 0,131) oder eines malignen Mammatumors (p = 0,240) führte zu keiner erhöhten Relaxin-Plasmakonzentration. Entsprechend war auch das Stadium der Erkrankung ohne Einfluss auf das gemessene Relaxin (p = 0,829). Im Rahmen der Verlaufsuntersuchung gab es keinen Unterschied zwischen den präoperativ und den sechs Monate postoperativ bestimmten Relaxin-Plasmakonzentrationen (p = 0,983). Weder eine Rezidivierung des Mammatumors (p = 0,084) noch eine Metastasierung des Tumors in die Lunge sechs Monate postoperativ (p = 0,200) waren anhand der präoperativ bestimmten Relaxin-Plasmakonzentrationen vorhersehbar. Auch lieferte Relaxin keinen Hinweis auf einen Tod infolge der Mammatumoren (p = 0,205). In dieser Arbeit konnte nach Auswertung der vorliegenden Daten kein Hinweis auf die Verwendbarkeit der Relaxin-Plasmakonzentration als prognostischer Marker für an Mammatumoren erkrankte Hündinnen gefunden werden. Es konnte, wie in vorherigen Studien, bestätigt werden, dass Hündinnen mit Tumoren kleiner 3 cm (p = 0,001) und Hündinnen im Stadium I der Erkrankung (p = 0,009, p = 0,022) eine signifikant niedrigere Wahrscheinlichkeit haben innerhalb des ersten Jahres postoperativ an den Folgen des Mammatumors zu versterben als Hündinnen mit größeren Tumoren oder in einem höheren Stadium der Erkrankung. Hündinnen, die an einem ulzerierenden Mammatumor erkrankt waren (p = 0,002) oder bei denen histopathologisch nachweisbare Metastasen in den regionären Lymphknoten vorlagen (p = 0,001), hatten eine signifikant niedrigere Wahrscheinlichkeit das erste postoperative Jahr zu überleben. Die Tiere, bei denen sechs Monate postoperativ Metastasen in der Lunge festgestellt werden konnten (p = 0,001) oder bei denen es zu einer Rezidivierung des Mammatumors kam (p = 0,001), hatten eine sehr hohe Wahrscheinlichkeit innerhalb des ersten postoperativen Jahres zu versterben.

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

Abstract Peroxisome proliferator-activated receptors (PPARs) are transcription factors that belong to the family of nuclear hormone receptors and exist as three isoforms namely PPARα, PPARβ and PPARγ. PPARs function as key regulators of glucose and lipid metabolism and are potential targets for drugs used in the treatment of glucose and lipid metabolism dysregulation. PPARs also regulate the expression of genes involved in the process of cellular proliferation and differentiation. Since it was discovered that PPAR ligands cause liver tumourigenesis in rodents, PPARs and their modulators have been investigated widely in in vitro and in vivo studies of carcinogenesis of the liver, colon, prostate, lung and skin. PPARα and PPARγ are the most studied PPAR isoforms in relation to cancer, while the association of PPARβ with cancer is increasingly being investigated. Some studies suggest that PPARβ and its ligands may have anticancer activity, while other studies identify a role for PPARβ in tumour promotion and progression. Breast cancer is one of the most common types of cancer in women with the majority caused by non-hereditary mechanisms. The activation of PPARα in breast cancer cells is associated with an increase in proliferation, while PPARγ activation in breast cancer cells is related to differentiation and an inhibition of cell proliferation. The role of PPARβ and its modulators in breast cancer is uncertain, as there have been limited studies addressing the effects of PPARβ modulation in breast cancer cell lines. Environmental contaminants such as the phthalate plasticizers and alcohol are putative risk factors for breast cancer. The phthalates di(2-ethylhexyl)phthalate (DEHP) and di-n-butyl phthalate (DBP) are plasticizers that are used in a range of common household, medical and beauty products and as a consequence humans are exposed to significant levels of these compounds. DEHP and DBP are known teratogens in rodents and DEHP induces hepatocarcinogenesis in a process thought to be mediated via PPARα. DEHP and DBP are metabolized in vivo by esterases to the monoesters, mono-(2-ethylhexyl) phthalate (MEHP) and mono-n-butyl phthalate (MBP), and these compounds have been identified in human biological samples. MEHP and MBP modulate PPARs in various tissues and cell types, but their ability to modulate PPARs in human breast cancer cells is not known. Like phthalates, ethanol is another modulator of PPARs and alcohol consumption is associated positively with breast cancer development, but the molecular mechanisms involved are unknown and there are no studies that examine the effects of ethanol and its metabolite acetaldehyde on PPARs in breast cancer cell lines. This thesis describes studies establishing and validating a breast cancer cell line that conditionally expresses human PPARβ under the control of a tetracycline regulator. Using this model, the ability of PPARβ over-expression and/or activation by the PPARβ specific ligand GW0742 to promote breast cancer cell proliferation was studied. Furthermore, putative PPARβ regulated genes were examined for alterations in expression in the presence of the PPARβ ligand. This work determined that over-expression of PPARβ and/or its activation by GW0742 does not promote proliferation in MCF-7 breast cancer cells. This thesis also investigated the effects of the phthalate monoesters MEHP and MBP on PPARs in MCF-7 breast cancer cells. It was found that MEHP activated both PPARα and PPARγ but was unable to activate PPARβ, whereas MBP could not activate any of the PPAR isoforms. MBP was an antagonist for both PPARγ and PPARβ. Using breast cancer cell lines, studies were conducted addressing the effects of an increasing concentration of ethanol (0-300 mM) on the transcription and transactivation of PPARα and PPARβ isoforms. Estrogen receptor positive MCF-7 breast cancer cells were more sensitive to the effects of ethanol than estrogen receptor negative MDA-MB-231 cells, with changes in PPARα mRNA more pronounced than PPARβ mRNA. Studies in MCF-7 cells conditionally expressing either PPARα or PPARβ in the presence of their respective specific ligands, GW7647 and GW0742, revealed that ethanol concentrations of 20 mM and 100 mM suppressed the maximal response to ligand-mediated activation for PPARα. Studies using the ethanol metabolism enzyme inhibitors 4-methylpyrazole and cyanamide, suggested that while ethanol was responsible for the modulation of PPARβ transactivation, the primary metabolite acetaldehyde was responsible for the effects on PPARα transactivation. Lastly, it was determined that ethanol and/or GW0742 did not increase the proliferation of MCF-7 Tet-off cells. The findings in this thesis suggest that given the different consequences of MEHP, MBP and ethanol on PPARs, PPAR expression and activation by ligands may have tissue specific consequences and that PPARβ may have a complex role in mammary gland tumourigenesis.

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly occurring in association with gene silencing. However, understanding the dynamics of epigenetic changes is often hindered due to the absence of adequate in vitro model systems that accurately reflect events occurring in vivo. Human mammary epithelial cells (HMECs) grown under standard culture conditions enter a growth arrest termed selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection cells, have many of the hallmarks seen in the earliest lesions of breast cancer, including transcriptional silencing and hypermethylation of the p16INK4A tumour suppressor gene. The overall aim of my thesis was to use post-selection HMECs as model system to identify and dissect the mechanism involved in early epigenetic aberrations. Firstly, using a microarray approach, I found that multiple members of the TGF-β signalling pathway were concordantly suppressed in post-selection cells, and this was associated with functional disruption of the TGF-β pathway. Interestingly, concordant gene suppression was not associated with aberrant DNA methylation, but with repressive chromatin remodelling. Secondly, to further understand the mechanism underpinning epigenetic silencing, I demonstrated using laser capture technology, that p16INK4A silencing is a precursor to DNA methylation and histone remodelling. Thirdly, I found that individual post-selection HMEC strains during the early passages shared a common 'wave' pattern of regional-specific methylation within the p16INK4A CpG island. Interestingly, the 'wave' pattern of early de novo methylation correlated with the apparent footprint of nucleosomes within the p16INK4A CpG island. Lastly, to further characterise the properties of the HMEC culture system, I demonstrated that post-selection cells do not possess a natural tumour-inducing property when transplanted into the mammary fat pad of immunocompromised mice. However, post-selection HMECs were associated with high expression of a variety of stem/progenitor markers, as well as stem/progenitor associated polycomb genes, thus demonstrating that these cells share some common features of stem/progenitor cells. The research presented in this thesis demonstrate that epigenetic changes occur early in the growth of post-selection HMECs and many of these changes are common in breast cancer.

Content removed due to copyright restriction: Appendix 6 Cooper, C. V., Stelwagen, K., Singh, K., Farr, V. C., Prosser, C. G., and Davis, S. R. (2004): Expression of the tight junction protein zonula occludens-1 during mammary engorgement. Proceedings of the New Zealand Society of Animal Production 64,43-47. Singh, K., Dobson, J., Phyn, C. V. C., Davis, S. R., Farr, V. C., Molenaar, A. J., and Stelwagen, K. (2005): Milk accumulation decreases expression of genes involved in cell-extracellular matrix communication and is associated with induction of apoptosis in the bovine mammary gland. Livestock Production Science 98,67-78. Appendix 7 McMahon, C.D., Farr, V.C., Singh, K., Wheeler, T.T. and Davis, S.R. (2004). Decreased expression of ß1-integrin and focal adhesion kinase in epithelial cells may initiate involution of mammary glands. Journal of Cellular Physiology 200, 318-325 / Extended periods of milk accumulation result in loss of secretory activity, increased apoptosis and eventually, involution of mammary glands. This process is associated with increased permeability of the tight junction (TJ) complexes between adjacent mammary epithelial cells (MECs). The change in cell shape during mammary engorgement from a cuboidal to a flattened morphology may initiate changes in protein and gene expression (mechanotransduction) that trigger these processes. Therefore, this study examined the regulation of the major TJ protein components during mammary engorgement, and in particular the role of physical distension of the mammary epithelium in the regulatory process. Expression of the integral transmembrane TJ proteins, occludin and claudin-1, and the cytoplasmic TJ protein, ZO-1, were down-regulated in both bovine and rat mammary glands during the early stages of mammary apoptosis and involution following the abrupt cessation of milk removal. In the rat, these responses were locally regulated as they occurred only in teat-sealed glands in a hemi-suckled model. Furthermore, the down-regulation of TJ proteins is consistent with a loss of TJ integrity during mammary engorgement. Induced physical distension of rat mammary glands in vivo transiently up-regulated the expression levels of occludin protein and mRNA, and ZO-1 mRNA, followed by an accelerated decrease in expression compared with the effects of milk accumulation alone. This was associated with the initiation of apoptosis, the up-regulation of the pro-apoptotic factor pSTAT3, and the down-regulation of the cell-ECM survival factor βl-integrin. An in vitro model was also developed to stretch MECs, mimicking the flattening in cell shape during mammary engorgement in vivo. While stretching MECs in vitro did not conclusively alter TJ protein expression, the overall results of this project support further investigation into the role of the TJ complex in mechanotransduction pathways. In addition, the results point to crosstalk between cell-ECM survival signalling and STAT3 death signalling as a candidate for regulation by physical distension of the mammary epithelium. In conclusion, this study supports the hypothesis that physical distension during engorgement of the mammary glands with milk is a primary trigger initiating apoptosis of MECs through changes in the regulation of gene pathways controlling cell survival and death, and the disruption of TJ function.

Thesis (Ph.D.)--McMaster University, 2003. / Advisor: W. J. Muller. Includes bibliographical references (leaves 259-282). Also available via World Wide Web.