Endocrine disrupting chemicals and breast cancer cells.

Many hundreds of endocrine disrupting chemicals (EDCs) have been measured as entering human breast tissue from a range of environmental sources, and this review focuses on discussion of mechanisms by which such EDCs may be contributing to the globally rising incidence of breast cancer. Many of the distinguishing features of breast cancer may be accounted for by EDC exposure, including, but not limited to, the fact that many EDCs possess estrogenic activity and exposure to estrogen is a main risk factor for breast cancer. Studies of the actions of EDCs in human breast cancer cells are aided by use of the conceptual framework of the hallmarks of cancer, and, acting by a variety of genomic and nongenomic mechanisms, EDCs have now been shown to enable all the hallmarks of cancer to develop in human breast cancer cells. Many studies report that hallmarks can develop at concentrations which are within the range of those measured in human breast tissues, especially when added as mixtures. The varied levels of different EDCs measured in individual breast tissue samples together with the overlapping and complementary mechanisms of action of the EDCs imply that thematic mechanisms will be driven inevitably by different chemical mixtures. Despite the complexity, EDCs do need to now be acknowledged as a risk factor for breast cancer in order for preventative strategies to include reduction in EDC exposure.

Long-term exposure to triclosan increases migration and invasion of human breast epithelial cells in vitro.

Extensive use of triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) as an antimicrobial agent in household and personal care products has resulted in global exposure of the human population. Its presence in human tissues, including milk, and its oestrogen-disrupting properties raise concerns for an involvement in breast cancer. Because metastatic tumour spread is the main cause of breast cancer mortality, we have investigated the effects of triclosan on cell migration and invasion using three human breast epithelial cell lines and using concentrations comparable with those in human tissues. Long-term exposure to 10-7 M of triclosan resulted in increased migration and invasion as measured by xCELLigence technology for all three cell lines, for the immortalized but nontransformed MCF-10F breast epithelial cells (after 28 weeks), the oestrogen-responsive MCF-7 breast cancer cells (after 17 weeks) and the oestrogen-unresponsive MDA-MB-231 breast cancer cells (after 20 weeks). The effects were therefore not limited to cancerous cells or to oestrogen-responsive cells. This was paralleled in the MCF-10F and MCF-7 (but not MDA-MB-231) cells by a reduction in levels of E-cadherin mRNA as measured by reverse transcription-polymerase chain reaction (RT-PCR) and of E-cadherin protein as measured by western immunoblotting, suggesting a mechanism involving epithelial-to-mesenchymal transition. This adds triclosan to the increasing list of ingredients of personal care products that can not only enter human breast tissue and increase cell proliferation but also influence cell motility. If mixtures of components in household and personal care products contribute to increasing cell migration and invasion, then reduction in exposure could offer a strategy for reducing breast cancer spread.

Chemical components of plastics as endocrine disruptors: Overview and commentary.

Bisphenol A and phthalate esters are used as additives in the manufacture of plastic materials, but their ability to leach out with age and heat has resulted in their becoming ubiquitous contaminants of the ecosystem including within human body tissues. Over recent years, these compounds have been shown to possess endocrine disrupting properties with an ability to interfere in the actions of many hormones and to contribute to human health problems. Much of the reported disruptive activity has been in relation to the action of estrogens, androgens, and thyroid hormones, and concerns have been raised for adverse consequences on female and male reproductive health, thyroid function, metabolic alterations, brain development/function, immune responses, and development of cancers in hormone-sensitive tissues. A recurring theme throughout seems to be that there are windows of susceptibility to exposure in utero and in early postnatal life, which may then result in disease in later life without any need for further exposure. This commentary highlights key issues in a historical context and raises questions regarding the many data gaps.

Low-dose environmental endocrine disruptors, increase aromatase activity, estradiol biosynthesis and cell proliferation in human breast cells.

BACKGROUND: Phenolic endocrine-disrupting compounds (EDCs) have long been suspected of increasing human breast cancer risk, via aromatase up-regulation; however, the metabolic effects upon aromatase in human breast cells exposed to environmentally relevant concentrations of phenolic compounds, have not been addressed. OBJECTIVES: To examine the mechanistic responses of aromatase CYP19A1 mRNA, aromatase activity, estradiol biosynthesis and cellular proliferation, in three human breast cell lines, exposed to seven phenolic compounds, at environmentally relevant concentrations. METHODS: MCF-7 and ZR-75-1 breast cancer cells, and HMF3A breast fibroblasts were treated with specific concentrations of p,p'-DDT, methoxychlor, benzophenone-2, bisphenol A, bisphenol S, 4-phenylphenol and n-butylparaben, with and without the presence of aromatase inhibitors and estrogen receptor inhibitors. RESULTS: All test EDCs up-regulated aromatase mRNA, increased aromatase activity, significantly increased the aromatase-induced biosynthesis of the breast carcinogen 17ß-estradiol, and increased ERα-positive breast cell proliferation. CONCLUSION: Inadvertent exposures to 'phenolic' EDCs, increase estradiol biosynthesis, and estrogen-sensitive breast cancer proliferation.

Overview of air pollution and endocrine disorders.

Over recent years, many environmental pollutant chemicals have been shown to possess the ability to interfere in the functioning of the endocrine system and have been termed endocrine disrupting chemicals (EDCs). These compounds exist in air as volatile or semi-volatile compounds in the gas phase or attached to particulate matter. They include components of plastics (phthalates, bisphenol A), components of consumer goods (parabens, triclosan, alkylphenols, fragrance compounds, organobromine flame retardants, fluorosurfactants), industrial chemicals (polychlorinated biphenyls), products of combustion (polychlorinated dibenzodioxins/furans, polyaromatic hydrocarbons), pesticides, herbicides, and some metals. This review summarizes current knowledge concerning the sources of EDCs in air, measurements of levels of EDCs in air, and the potential for adverse effects of EDCs in air on human endocrine health.

Effects of exposure to six chemical ultraviolet filters commonly used in personal care products on motility of MCF-7 and MDA-MB-231 human breast cancer cells in vitro.

Benzophenone (BP)-1, BP-2, BP-3, octylmethoxycinnamate (OMC), 4-methylbenzilidenecamphor and homosalate are added to personal care products to absorb ultraviolet light. Their presence in human milk and their oestrogenic activity suggests a potential to influence breast cancer development. As metastatic tumour spread is the main cause of breast cancer mortality, we have investigated the effects of these compounds on migration and invasion of human breast cancer cell lines. Increased motility of oestrogen-responsive MCF-7 human breast cancer cells was observed after long-term exposure (>20 weeks) to each of the six compounds at ≥10-7  m concentrations using three independent assay systems (scratch assay, live cell imaging, xCELLigence technology) and increased invasive activity was observed through matrigel using the xCELLigence system. Increased motility of oestrogen-unresponsive MDA-MB-231 human breast cancer cells was observed after 15 weeks of exposure to each of the six compounds by live cell imaging and xCELLigence technology, implying the increased migratory activity was not confined to oestrogen-responsive cells. Molecular mechanisms varied between compounds and cell lines. Using MCF-7 cells, reduction in E-cadherin was observed following 24 weeks' exposure to 10-5  m BP-1 and 10-5  m homosalate, and reduction in ß-catenin was noted following 24 weeks' exposure to 10-5  m OMC. Using MDA-MB-231 cells, increased levels of matrix metalloproteinase 2 were observed after 15 weeks exposure to 10-7  m OMC and 10-7  m 4-methylbenzilidenecamphor. Although molecular mechanisms differ, these results demonstrate that exposure to any of these six compounds can increase migration and invasion of human breast cancer cells.

Endocrine Disruptors and Obesity.

PURPOSE OF REVIEW: The purpose of this review was to summarise current evidence that some environmental chemicals may be able to interfere in the endocrine regulation of energy metabolism and adipose tissue structure. RECENT FINDINGS: Recent findings demonstrate that such endocrine-disrupting chemicals, termed "obesogens", can promote adipogenesis and cause weight gain. This includes compounds to which the human population is exposed in daily life through their use in pesticides/herbicides, industrial and household products, plastics, detergents, flame retardants and as ingredients in personal care products. Animal models and epidemiological studies have shown that an especially sensitive time for exposure is in utero or the neonatal period. In summarising the actions of obesogens, it is noteworthy that as their structures are mainly lipophilic, their ability to increase fat deposition has the added consequence of increasing the capacity for their own retention. This has the potential for a vicious spiral not only of increasing obesity but also increasing the retention of other lipophilic pollutant chemicals with an even broader range of adverse actions. This might offer an explanation as to why obesity is an underlying risk factor for so many diseases including cancer.

Exposure to cyclic volatile methylsiloxanes (cVMS) causes anchorage-independent growth and reduction of BRCA1 in non-transformed human breast epithelial cells.

Dermal absorption of components of personal care products (PCPs) may contribute to breast cancer development. Cyclic volatile methylsiloxanes (cVMS) are used widely in the formulation of PCPs, and their presence has been recently detected in human blood. The objectives of this study were to investigate any genotoxic effects after short- (1 week) or longer-term (30 weeks) exposure to hexamethylcyclotrisiloxane (D3), octamethylcyclotetrasiloxane (D4) or decamethylcyclopentasiloxane (D5) in MCF-10 A and MCF-10F immortalized non-transformed human breast epithelial cells. Genotoxic effects were assessed by an ability of cells to grow in suspension culture, from DNA damage measured by comet assays, and from a reduction in levels of DNA repair proteins measured by RT-PCR and western immunoblotting. Dose-dependent anchorage-independent growth in methocel culture was observed after exposure to D3 (10-13 M-10-5 M) and D4/D5 (10-9 M-10-5 M). DNA damage was measured by the comet assay after 1-h exposure to D3 (10-6 M-10-5 M) and D4 (10-5 M). BRCA1 mRNA and BRCA1 protein levels were reduced after 30-week exposure to 10-5 M D4 and D5 in both cell lines. Reduced levels of mRNAs for other DNA repair proteins (BRCA2, ATM, ATR, CHK1 and CHK2) were also observed after exposure to 10-5 M D5 in both cell lines, and some reductions after exposure to D3 and D4. If cVMS can not only enable anchorage-independent growth of non-transformed breast epithelial cells and damage DNA, but also compromise DNA repair systems, then there is the potential for them to impact on breast carcinogenesis. Further risk assessment now requires information concerning the extent to which cVMS may be present in human breast tissues. Copyright © 2016 John Wiley & Sons, Ltd.

Effect of aluminium on migration of oestrogen unresponsive MDA-MB-231 human breast cancer cells in culture.

Aluminium (Al) has been measured in human breast tissue, and may be a contributory factor in breast cancer development. At the 10th Keele meeting, we reported that long-term exposure to Al could increase migratory properties of oestrogen-responsive MCF-7 human breast cancer cells suggesting a role for Al in the metastatic process. We now report that long-term exposure (20-25 weeks) to Al chloride or Al chlorohydrate at 10(-4) M or 10(-5) M concentrations can also increase the migration of oestrogen unresponsive MDA-MB-231 human breast cancer cells as measured using time-lapse microscopy and xCELLigence technology. In parallel, Al exposure was found to give rise to increased secretion of active matrix metalloproteinase MMP9 as measured by zymography, and increased intracellular levels of activated MMP14 as measured by western immunoblotting. These results demonstrate that Al can increase migration of human breast cancer cells irrespective of their oestrogen responsiveness, and implicate alterations to MMPs as a potential mechanism worthy of further study.

Parabens can enable hallmarks and characteristics of cancer in human breast epithelial cells: a review of the literature with reference to new exposure data and regulatory status.

A framework for understanding the complexity of cancer development was established by Hanahan and Weinberg in their definition of the hallmarks of cancer. In this review, we consider the evidence that parabens can enable development in human breast epithelial cells of four of six of the basic hallmarks, one of two of the emerging hallmarks and one of two of the enabling characteristics. In Hallmark 1, parabens have been measured as present in 99% of human breast tissue samples, possess oestrogenic activity and can stimulate sustained proliferation of human breast cancer cells at concentrations measurable in the breast. In Hallmark 2, parabens can inhibit the suppression of breast cancer cell growth by hydroxytamoxifen, and through binding to the oestrogen-related receptor gamma may prevent its deactivation by growth inhibitors. In Hallmark 3, in the 10 nm-1 µm range, parabens give a dose-dependent evasion of apoptosis in high-risk donor breast epithelial cells. In Hallmark 4, long-term exposure (>20 weeks) to parabens leads to increased migratory and invasive activity in human breast cancer cells, properties that are linked to the metastatic process. As an emerging hallmark methylparaben has been shown in human breast epithelial cells to increase mTOR, a key regulator of energy metabolism. As an enabling characteristic parabens can cause DNA damage at high concentrations in the short term but more work is needed to investigate long-term, low-dose mixtures. The ability of parabens to enable multiple cancer hallmarks in human breast epithelial cells provides grounds for regulatory review of the implications of the presence of parabens in human breast tissue.

Exposure to parabens at the concentration of maximal proliferative response increases migratory and invasive activity of human breast cancer cells in vitro.

Alkyl esters of p-hydroxybenzoic acid (parabens) are widely used as preservatives in personal care products, foods and pharmaceuticals. Their oestrogenic activity, their measurement in human breast tissue and their ability to drive proliferation of oestrogen-responsive human breast cancer cells has opened a debate on their potential to influence breast cancer development. As proliferation is not the only hallmark of cancer cells, we have investigated the effects of exposure to parabens at concentrations of maximal proliferative response on migratory and invasive properties using three oestrogen-responsive human breast cancer cell lines (MCF-7, T-47-D, ZR-75-1). Cells were maintained short-term (1 week) or long-term (20 ± 2 weeks) in phenol-red-free medium containing 5% charcoal-stripped serum with no addition, 10(-8) M 17ß-oestradiol, 1-5 × 10(-4) M methylparaben, 10(-5) M n-propylparaben or 10(-5) M n-butylparaben. Long-term exposure (20 ± 2 weeks) of MCF-7 cells to methylparaben, n-propylparaben or n-butylparaben increased migration as measured using a scratch assay, time-lapse microscopy and xCELLigence technology: invasive properties were found to increase in matrix degradation assays and migration through matrigel on xCELLigence. Western immunoblotting showed an associated downregulation of E-cadherin and ß-catenin in the long-term paraben-exposed cells which could be consistent with a mechanism involving epithelial to mesenchymal transition. Increased migratory activity was demonstrated also in long-term paraben-exposed T-47-D and ZR-75-1 cells using a scratch assay and time-lapse microscopy. This is the first report that in vitro, parabens can influence not only proliferation but also migratory and invasive properties of human breast cancer cells.

Hypersensitivity and growth adaptation of oestrogen-deprived MCF-7 human breast cancer cells.

BACKGROUND: Efficacy of endocrine therapy is compromised when human breast cancer cells circumvent imposed growth inhibition. The model of long-term oestrogen-deprived MCF-7 human breast cancer cells has suggested the mechanism results from hypersensitivity to low levels of residual oestrogen. MATERIALS AND METHODS: MCF-7 cells were maintained for up to 30 weeks in phenol-red-free medium and charcoal-stripped serum with 10(-8) M 17ß-oestradiol and 10 µg/ml insulin (stock 1), 10(-8) M 17ß-oestradiol (stock 2), 10 µg/ml insulin (stock 3) or no addition (stock 4). RESULTS: Loss of growth response to oestrogen was observed only in stock 4 cells. Long-term maintenance with insulin in the absence of oestradiol (stock 3) resulted in raised oestrogen receptor-alpha (ERα) levels (measured by western immunoblotting) and development of hypersensitivity (assayed by oestrogen-responsive reporter gene induction and dose response to oestradiol for proliferation under serum-free conditions), but with no loss of growth response to oestrogen. CONCLUSION: Hypersensitivity can develop without any growth adaptation and therefore is not a prerequisite for loss of growth response in MCF-7 cells.

Aluminium and breast cancer: Sources of exposure, tissue measurements and mechanisms of toxicological actions on breast biology.

This review examines recent evidence linking exposure to aluminium with the aetiology of breast cancer. The human population is exposed to aluminium throughout daily life including through diet, application of antiperspirants, use of antacids and vaccination. Aluminium has now been measured in a range of human breast structures at higher levels than in blood serum and experimental evidence suggests that the tissue concentrations measured have the potential to adversely influence breast epithelial cells including generation of genomic instability, induction of anchorage-independent proliferation and interference in oestrogen action. The presence of aluminium in the human breast may also alter the breast microenvironment causing disruption to iron metabolism, oxidative damage to cellular components, inflammatory responses and alterations to the motility of cells. The main research need is now to investigate whether the concentrations of aluminium measured in the human breast can lead in vivo to any of the effects observed in cells in vitro and this would be aided by the identification of biomarkers specific for aluminium action.

Effect of aluminium on migratory and invasive properties of MCF-7 human breast cancer cells in culture.

Aluminium (Al) has been measured in human breast tissue, nipple aspirate fluid and breast cyst fluid, and recent studies have shown that at tissue concentrations, aluminium can induce DNA damage and suspension growth in human breast epithelial cells. This paper demonstrates for the first time that exposure to aluminium can also increase migratory and invasive properties of MCF-7 human breast cancer cells. Long-term (32 weeks) but not short-term (1 week) exposure of MCF-7 cells to 10(-4) M aluminium chloride or 10(-4) M aluminium chlorohydrate increased motility of the cells as measured by live cell imaging (cumulative length moved by individual cells), by a wound healing assay and by migration in real time through 8 µm pores of a membrane using xCELLigence technology. Long-term exposure (37 weeks) to 10(-4) M aluminium chloride or 10(-4) M aluminium chlorohydrate also increased the ability of MCF-7 cells to invade through a matrigel layer as measured in real time using the xCELLigence system. Although molecular mechanisms remain to be characterized, the ability of aluminium salts to increase migratory and invasive properties of MCF-7 cells suggests that the presence of aluminium in the human breast could influence metastatic processes. This is important because mortality from breast cancer arises mainly from tumour spread rather than from the presence of a primary tumour in the breast.

Combinations of parabens at concentrations measured in human breast tissue can increase proliferation of MCF-7 human breast cancer cells.

The alkyl esters of p-hydroxybenzoic acid (parabens), which are used as preservatives in consumer products, possess oestrogenic activity and have been measured in human breast tissue. This has raised concerns for a potential involvement in the development of human breast cancer. In this paper, we have investigated the extent to which proliferation of MCF-7 human breast cancer cells can be increased by exposure to the five parabens either alone or in combination at concentrations as recently measured in 160 human breast tissue samples. Determination of no-observed-effect concentrations (NOEC), lowest-observed-effect concentrations (LOEC), EC50 and EC100 values for stimulation of proliferation of MCF-7 cells by five parabens revealed that 43/160 (27%) of the human breast tissue samples contained at least one paraben at a concentration ≥ LOEC and 64/160 (40%) > NOEC. Proliferation of MCF-7 cells could be increased by combining all five parabens at concentrations down to the 50(th) percentile (median) values measured in the tissues. For the 22 tissue samples taken at the site of ER + PR + primary cancers, 12 contained a sufficient concentration of one or more paraben to stimulate proliferation of MCF-7 cells. This demonstrates that parabens, either alone or in combination, are present in human breast tissue at concentrations sufficient to stimulate the proliferation of MCF-7 cells in vitro, and that functional consequences of the presence of paraben in human breast tissue should be assessed on the basis of all five parabens and not single parabens individually.

Parabens enable suspension growth of MCF-10A immortalized, non-transformed human breast epithelial cells.

Parabens (alkyl esters of p-hydroxybenzoic acid) are used extensively as preservatives in consumer products, and intact esters have been measured in several human tissues. Concerns of a potential link between parabens and breast cancer have been raised, but mechanistic studies have centred on their oestrogenic activity and little attention has been paid to any carcinogenic properties. In the present study, we report that parabens can induce anchorage-independent growth of MCF-10A immortalized but non-transformed human breast epithelial cells, a property closely related to transformation and a predictor of tumour growth in vivo. In semi-solid methocel suspension culture, MCF-10A cells produced very few colonies and only of a small size but the addition of 5 × 10(-4) M methylparaben, 10(-5) M n-propylparaben or 10(-5) M n-butylparaben resulted in a greater number of colonies per dish (P < 0.05 in each case) and an increased average colony size (P < 0.001 in each case). Dose-responses showed that concentrations as low as 10(-6) M methylparaben, 10(-7) M n-propylparaben and 10(-7) M n-butylparaben could increase colony numbers (P = 0.016, P = 0.010, P = 0.008, respectively): comparison with a recent measurement of paraben concentrations in human breast tissue samples from 40 mastectomies (Barr et al., 2012) showed that 22/40 of the patients had at least one of the parabens at the site of the primary tumour at or above these concentrations. To our knowledge, this is the first study to report that parabens can induce a transformed phenotype in human breast epithelial cells in vitro, and further investigation is now justified into a potential link between parabens and breast carcinogenesis.

Molecular mechanisms of oestrogen action on growth of human breast cancer cells in culture.

Growth responses to oestrogen can be reproducibly obtained using a selection of oestrogen-receptor-containing human breast cancer cell lines, and molecular mechanisms have been shown to include modulation to growth factor/receptor/signalling pathways, cell-cycle proteins, apoptosis, differentiation, adhesion, motility and migration. Considerable progress has been made in understanding the molecular basis of oestrogen action on gene expression through the ligand-activated transcription factors human oestrogen receptor α (ERα) and ERß and the resulting effects on global gene expression patterns, but the full profile of coordination of the alterations, which brings about changes in cell growth through genomic and non-genomic mechanisms remain to be fully elucidated. Oestrogen regulation of cell growth involves a complex cross-talk between oestrogen receptor and growth factor signalling pathways such that inhibition of one pathway may lead to stimulation of another, which may explain the remarkable ability of human breast cancer cells to escape from any mode of imposed growth inhibition be it oestrogen deprivation or administration of antioestrogen. Although studies on cell growth have focused to date on the effects of physiological oestrogens, many hundreds of environmental chemicals with oestrogenic properties have now been measured in the human breast. Whether or not the weight of evidence eventually establishes any causal link of complex mixtures of environmental oestrogenic chemicals with breast cancer, the presence of so many oestrogenic chemicals in the breast must influence resulting oestrogenic responses, and the impact of this additional oestrogenic burden needs to be taken into account in future studies on growth regulation of human breast cancer cells.

Analysis of aluminium content and iron homeostasis in nipple aspirate fluids from healthy women and breast cancer-affected patients.

Aluminium is not a physiological component of the breast but has been measured recently in human breast tissues and breast cyst fluids at levels above those found in blood serum or milk. Since the presence of aluminium can lead to iron dyshomeostasis, levels of aluminium and iron-binding proteins (ferritin, transferrin) were measured in nipple aspirate fluid (NAF), a fluid present in the breast duct tree and mirroring the breast microenvironment. NAFs were collected noninvasively from healthy women (NoCancer; n = 16) and breast cancer-affected women (Cancer; n = 19), and compared with levels in serum (n = 15) and milk (n = 45) from healthy subjects. The mean level of aluminium, measured by ICP-mass spectrometry, was significantly higher in Cancer NAF (268.4 ± 28.1 µg l(-1) ; n = 19) than in NoCancer NAF (131.3 ± 9.6 µg l(-1) ; n = 16; P < 0.0001). The mean level of ferritin, measured through immunoassay, was also found to be higher in Cancer NAF (280.0 ± 32.3 µg l(-1) ) than in NoCancer NAF (55.5 ± 7.2 µg l(-1) ), and furthermore, a positive correlation was found between levels of aluminium and ferritin in the Cancer NAF (correlation coefficient R = 0.94, P < 0.001). These results may suggest a role for raised levels of aluminium and modulation of proteins that regulate iron homeostasis as biomarkers for identification of women at higher risk of developing breast cancer. The reasons for the high levels of aluminium in NAF remain unknown but possibilities include either exposure to aluminium-based antiperspirant salts in the adjacent underarm area and/or preferential accumulation of aluminium by breast tissues.

Enhanced sensitivity to rapamycin following long-term oestrogen deprivation in MCF-7, T-47-D and ZR-75-1 human breast cancer cells.

Human breast cancer cells (MCF-7, T-47-D and ZR-75-1) can adapt to circumvent any reduced growth rate during long-term oestrogen deprivation, and this provides three model systems to investigate mechanisms of endocrine resistance in breast cancer. In this paper we report consistent differences in the effects of three growth inhibitors following long-term oestrogen deprivation in all three cell models. Long-term oestrogen deprivation of MCF-7, T-47-D and ZR-75-1 cells resulted in reduced growth inhibition by PD98059 (2-10 µg/ml), implying a loss of dependence on mitogen-activated protein kinase pathways for growth. The growth inhibitor LY294002 (2-10 µM) inhibited growth of both oestrogen-maintained and oestrogen-deprived cells with similar dose-responses, implying continued similar dependence on phosphoinositide 3-kinase (PI3K) pathways with no alteration after adaptation to oestrogen independent growth. However, by contrast, long-term oestrogen deprivation resulted in an increased sensitivity to growth inhibition by rapamycin, which was not reduced by readdition of oestradiol. The enhanced inhibition of long-term oestrogen-deprived MCF-7-ED, T-47-D-ED and ZR-75-1-ED cell growth by combining rapamycin with LY294002 at concentrations where each alone had little effect, offers preclinical support to the development of therapeutic combinations of rapamycin analogues with other PI3K inhibitors in endocrine-resistant breast cancer.