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Steroid receptors in human breast cancer

Robert B. Clarke1, Elizabeth Anderson2 and Anthony Howell1
1 2

CR-UK Department of Medical Oncology, Christie Hospital, Manchester, UK M20 4BX Tumour Biochemistry Laboratory, Christie Hospital, Manchester, UK M20 4BX

Ovarian steroids, acting through nuclear receptors, are crucial players in normal breast development and cancer. Estrogen, in particular, is the focus of breast cancer therapies because tumours are often dependent on this steroid for growth. Recently, novel genes and/or protein isoforms of receptors for both estrogen and progesterone have been discovered, leading us to reappraise their roles in breast development and cancer. Recognition of changes in estrogen receptor biology that occur in the transition from normal development to cancer has emphasized its contribution to tumorigenesis. In addition, complex interactions with other signalling pathways, particularly growth factor pathways, have recently come to the forefront. These interactions might explain resistance to endocrine treatments and offer solutions in terms of novel therapeutic targets. The ovarian steroids estrogen (E) and progesterone (P) are crucial for the normal development of the human mammary gland [1]. These hormones exert many of their effects via steroid receptors found largely, but not exclusively, in female reproductive tissues, which are

targets for E and P. In addition to their effects during normal reproductive development, it is clear that ovarian hormones can alter the risk of breast cancer (Box 1). For example, an early menarche and a late menopause increase breast cancer risk, whereas an early menopause is protective. These data suggest that breast cancer risk is related to cumulative exposure to ovarian hormones [24]. The greater risk from an increased reproductive lifespan might be related to the total number of times that the breast epithelium undergoes cyclical proliferation in response to variation in E and P levels during the menstrual cycle, which increases the chances of cancer initiation and promotion [5]. It is also clear from epidemiological studies that both a full-term pregnancy at a young age and breast-feeding are protective in terms of breast cancer risk [6]. This protective effect was initially thought to be related to the full lactational differentiation that the breast epithelium undergoes in response to hormones during pregnancy [7]. However, recent experiments in rodents suggest that the protective effect of pregnancy can be mimicked by a short exposure to pregnancy levels of E but not by induction of lactational differentiation [8]. There is emerging evidence

Box 1. Breast development and tumorigenesis: contribution of ovarian steroids and epithelial cell types
The adult breast consists of a branching, tree-like network of ducts lined by a double layer of epithelial cells that is surrounded by delimiting broblasts and embedded in an extracellular matrix [68]. Terminal duct lobuloalveolar units (TDLU) or lobules are the functional milk-producing units of the breast. Lobules exist initially as alveolar buds that mature after menarche into a variable number of blindending, secretory sacs, known as alveoli, which open into the intralobular terminal duct. The full development of the TDLUs is accelerated during pregnancy as the breast lobules expand in terms of the number of epithelial cells and alveoli that they contain in preparation for lactation. After lactation, the lobules involute to resemble those present in the non-pregnant gland, although they might retain a larger number of individual alveoli per lobule than before [68]. The alveoli within the lobules are lined by an inner layer of luminal epithelial cells surrounded by an outer layer of basal, myoepithelial cells. The luminal cells account for more than 90% of the epithelial cell proliferation that is seen in the non-pregnant gland [69]. Signicantly, more than 90% of breast tumours synthesize cytokeratins distinctive of the luminal phenotype, and greater than 70% synthesize steroid receptors, indicating that the luminal cell type is the major target for breast tumorigenesis [70]. The TDLU is the site from which all epithelial hyperplasias and carcinomas of the breast arise because this is where they are observed histopathologically [71]. Breast tumorigenesis is thought to result from a benign to malignant progression, in which the accumulation of genetic changes allows evolution from normal breast epithelium through benign and atypical proliferative lesions to carcinoma in situ and frankly invasive tumours [72]. The lesions associated with the greatest risk of invasive breast cancer are, in order of increasing risk: hyperplasia of usual type, atypical ductal hyperplasia, lobular carcinoma in situ and ductal carcinoma in situ (DCIS). These premalignant lesions, with the exception of high grade DCIS, are frequently dependent on estrogen (E) for their growth, as judged by the presence of steroid receptors for E and progesterone (P) [27]. E receptor a (ERa)-negative tumours often overexpress the genes encoding growth factor receptors, such as epidermal growth factor (EGFR or erbB1) and erbB2/HER2, and these are often overexpressed in DCIS of high nuclear grade [73]. Premalignant lesions synthesizing ERa and the P receptor might account for the success of the antiestrogens tamoxifen and raloxifene in breast cancer prevention, because it is their progression to invasive breast cancer that might be inhibited [74].

Corresponding author: Robert B. Clarke (rclarke@picr.man.ac.uk). Available online 30 July 2004

www.sciencedirect.com 1043-2760/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tem.2004.07.004

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that exposure to high pregnancy-like levels of E induces both a systemic and local resetting of hormonal sensitivity that affects lifetime breast cancer risk. There is evidence that steroid receptor biology is profoundly altered both during breast tumorigenesis and during tumour treatment with endocrine therapy. Most breast tumours express receptors for E and P, and a subset of these tumours is dependent on E for growth. The postmenopausal fall in E and P levels causes the normal breast epithelium to undergo a process of involution, whereas tumours can continue to grow. This suggests that there are changes in steroid receptor signalling or sensitivity in tumour cells compared with normal breast cells. The purpose of this review is to summarize the current state of research in the biology of the receptors for E and P (ER and PR, respectively). Second, we aim to describe how steroid receptor signalling is dysregulated during the breast tumorigenic process and subsequently during cancer progression. An understanding of the ways that steroid receptors contribute to these processes might provide novel strategies for the targeting of E and other steroid signalling pathways in breast cancer prevention and therapy. Ovarian steroid hormones: systemic control of epithelial proliferation In the adult, non-pregnant, non-lactating breast, epithelial proliferation is maximal approximately one week after ovulation, during the luteal phase of the menstrual cycle. This is when levels of both E and P are high [1] and is in contrast to the endometrium, where E-driven proliferation is highest during the follicular phase, leading to the assumption that P is the major breast mitogen, possibly after E priming. To study this assumption experimentally, our group developed a model in which small pieces of intact normal human breast tissue were implanted subcutaneously into adult female athymic nude mice. Subcutaneously implanted silastic pellets containing steroid hormone were tailored to give serum concentrations of E and/or P equivalent to those seen in the follicular or luteal phases of the menstrual cycle [9]. In tissue removed from untreated control mice or those treated with luteal phase levels of P, breast epithelial proliferation rates were very low. Follicular phase E levels had a small effect on proliferation, whereas luteal phase levels of E signicantly increased proliferation. The addition of P to E had no additional effect in this model. Therefore, E alone appeared to explain the effect of the menstrual cycle on breast proliferation because no proliferative effects of P were seen, either alone or in combination with E. The conclusion from this study was that a low dose of E equivalent to follicular phase levels induced some proliferation, but higher levels were necessary to induce maximal cell division [1,9]. However, recent studies of hormone replacement therapy (HRT) indicate that it is the combination of E and P in longterm HRT that correlates most strongly with an increased risk of breast cancer [1012]. This has been investigated further by looking at the proliferation rates of normal breast epithelium removed from postmenopausal women on either E-only,
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or ECP HRT, and comparing them with the rates seen in untreated postmenopausal women of a similar age [13]. Combined ECP HRT signicantly increased breast epithelial proliferation rates compared with those seen in untreated women or those taking E alone. The increased proliferation seen in normal breast epithelium taken from postmenopausal women being treated with combination ECP HRT correlates with increased breast cancer risk. Because the risk of breast cancer might be linked to the proliferation of normal epithelium, these data suggest that longterm P treatment has an effect on the breast that was not seen in our model of short-term treatment. We conclude from this that P has different effects in postmenopausal breast, especially when given as a longterm treatment. In breast cancer, there is much accumulated evidence that E stimulates the growth of both premalignant and invasive tumours. Beatson rst recorded the successful treatment of breast cancer by removal of the ovaries more than 100 years ago [14]. Irradiation of the ovaries was used as an effective breast cancer treatment in addition to surgery for much of the early 20th century. Later in the mid-20th century, pharmacological doses of the rst synthetic E, diethylstilbestrol (DES), were shown to be an effective treatment for breast cancer, particularly in postmenopausal women [14]. This is probably because at such high doses DES shuts down cellular responses to estrogens. Later, in the early 1970s, a failed contraceptive drug that behaved as an antiestrogen in E-stimulated rat uterine growth was used successfully to treat advanced breast cancer. The drug was tamoxifen, and this drug went on to become the most widely prescribed endocrine treatment in both advanced and adjuvant settings [14]. Its early use closely followed the discovery of the ER as the cellular mediator of the effects of E. This led to the examination of responses to tamoxifen in relation to breast tumour ER positivity and conrmation of a strong correlation. ER-containing breast cancer cell lines derived from patients were also shown to be E dependent. Presurgical studies where a tumour sample is taken before and during treatment with antiestrogens, such as tamoxifen and fulvestrant, have shown that proliferative rates fall while cell death increases in vivo, but only in ER-positive cases [14]. Antiproliferative and proapoptotic effects of antiestrogens have also been demonstrated in ER-positive ductal carcinoma in situ (DCIS) [15]. Treatment with tamoxifen has been so successful that much of the fall in breast cancer mortality since the mid-1980s has been attributed to its use [16]. Other antiestrogens have also been used successfully in breast cancer prevention and its treatment [17]. More recently, drugs that inhibit the synthesis of estrogens, in particular, new specic aromatase inhibitors such as letrozole and anastrozole, have been shown to be superior to tamoxifen in both adjuvant and advanced settings [18,19]. Steroid receptors: ligand-activated transcription factors that function as cellular sensors of systemic hormonal cues Classically, E and P exert their effects by binding to receptors in the nuclei of target cells and altering their

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structure such that they efciently bind specic DNA sequences within gene promoter regions, termed steroidresponse elements. Thus, the ER and the PR are essentially ligand-activated transcription factors that regulate expression of a specic subset of genes in the presence of their ligands [20]. The cells that express the genes encoding classic ER and PR (ESR and PGR, respectively) are found within the luminal epithelial, but not the myoepithelial or stromal, cells of the human breast [21]. In the past decade, a second ER-like gene has been discovered; the classic ER protein has been renamed the ERa, and the novel form is ERb [22]. ERa and PR are colocalized in w20% of luminal epithelial cells [23]. ERb appears to be much more widely synthesized than ERa in normal breast tissue, being detected in most luminal epithelial and myoepithelial cells, together with stromal broblasts and endothelial cells [24]. The PR also has two different isoforms, PRA and PRB, both of which are transcribed from the same gene using different promoters. PRB is the longer version containing an additional 164 amino acids at the N-terminal of the protein [25]. In vitro data support the view that PRB is the active PR, whereas PRA is either inactive or acts as an inhibitor of PRB [25]. However, in the normal physiology of human breast epithelium, both PRA and PRB appear to be synthesized in the same cells and at similar levels [26]. In normal human adult breast tissue, luminal cells expressing both ESRA and PGR are distributed evenly throughout the intralobular ducts and peripheral alveoli [21,23]. ERa synthesis levels in normal epithelium are inversely correlated with age, such that the proportion of ERa-positive cells is lower in premenopausal than in postmenopausal tissue [27]. In the premenopausal breast, w5% of epithelial cells are proliferating in response to steroid hormones although, surprisingly, these cells do not express ESRA, but are often adjacent or in close proximity to those that do [23]. This dissociation between steroid receptor synthesis and cell proliferation in the mammary epithelium has been conrmed by many investigators [2831]. Because it has been shown that receptors are not simply being downregulated during cell division, these data suggest a model where ovarian steroids stimulate proliferation via paracrine signals secreted by ERa/PR-positive cells (Figure 1a). For example, mouse mammary epithelium in which the PGR gene has been deleted [PGR-knockout (PGR-KO) mice] fail to undergo alveolar development. However, this growth decit in response to P can be overcome by mixing PGR-KO cells together with wild-type cells before transplantation into the cleared fat pad of syngeneic hosts [32]. This suggests that the wild-type cells communicate with the PGR-KO cells via paracrine growth signals (Figure 1b). One of these signals has been identied as the WNT4 gene product [33]. In contrast to ERa, ERb synthesis is much more widespread in the epithelium, it does not segregate with cells synthesizing estrogen-induced genes such as PGR and pS2, and deletion of the Esrb gene in the mouse mammary gland does not affect growth in response to E [24,34]. The dissociation between ERa synthesis and cell proliferation that is seen in vivo might help to explain longstanding
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data showing that normal breast epithelial cells in vitro do not synthesize ERa or PR [1]. Furthermore, there has been recent speculation that a quiescent stem cell population in normal epithelium might be a source of breast tumour formation [35]. There is some preliminary evidence that the quiescent ERa/PR-positive cells found in normal breast epithelium might have stem cell-like features that make them a candidate population for the production of ERa/PR-positive tumours, which are the most common type of breast cancer [36]. Although the number of proliferating cells decreases with increasing age and reduced circulating ovarian steroid hormone levels, the percentage of ERa-positive cells increases [27]. ERa synthesis is higher in normal breast tissue adjacent to cancer than in age-matched controls, and is greater in Caucasian women, who have a high breast cancer incidence, than in Japanese women, who are at low risk [37]. Taken together, these ndings indicate that increased ERa synthesis per se is a risk factor for breast cancer. Consistent with this, the separation between steroid receptor synthesis and cell proliferation seen in the normal mammary epithelium is dysregulated early in human breast tumorigenesis, such that proliferating ERa-positive cells are often seen [38]. This suggests that E can directly drive cell proliferation in ER a-positive cancers (Figure 1c). This autonomous response to E might be the consequence of an autocrine growth factor loop, in which the cell secretes growth factors and responds to them by dividing, or it might be that the biology of the cell is altered, such that E acting via the ER directly induces entry into the cell cycle (Figure 1d). This alteration in E action might increase the sensitivity of breast tumour cells to E and explain why E-dependent breast tumours arise postmenopausally, at a time when the normal epithelium undergoes a process of involution as ovarian steroid levels decline. Whereas ERa-positivity increases in the transition from normal to malignant tissue, the levels of ERb decrease [39,40]. ERb synthesis is correlated with lower proliferation and pathological grade, and the ratio of ERa to ERb synthesis increases with increasing proliferation and risk of breast cancer in premalignant lesions [39]. These data indicate that ERb might negatively regulate ERa signalling and contribute to the increased sensitivity of tumours to E. Elucidation of the role of ERb in predicting prognosis and response to antiestrogen treatment is awaited. The ratios of PRA to PRB have also been reported to change during progression from normal breast epithelium to malignant tissue. In normal breast, PRA and PRB are synthesized in equal amounts and there is little variation during the menstrual cycle [26]. The early premalignant lesion hyperplasia of usual type also synthesizes equal amounts of both PRA and PRB, but in atypical ductal hyperplasia, DCIS and invasive tumours the ratio is altered such that PRA predominates [26]. In general, PR synthesis is a good prognostic marker and an indicator of probable response to endocrine treatment in invasive breast cancer [14]. Thus, the predominance of PRA synthesis correlates with tumour formation, but loss of

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(a)

(b) Normal: paracrine

Growth factors

Proliferation

ER +ve

ER -ve

Epithelial cell

Epithelial cell

? Stromal cell (c) (d) Cancer: autocrine or cell autonomous

Growth factors

Proliferation

ER +ve

Epithelial cell
Figure 1. Steroid receptor expression and cell proliferation in human breast tissues. (a) A typical photomicrograph of an area of lobular epithelium showing double antibody uorescent staining for ERa (green) and the cell proliferation marker Ki67 (red). The proliferating cells are ERa negative because where the red and green are coincident cells appear yellow [e.g. the autouorescent red blood cell near to the epithelial cells (arrow)]. Scale barZ50 mM. (b) Because the ERa-positive cells (green nucleus) are often found to be adjacent to the proliferating cells (red nucleus), it is proposed that their proliferation is stimulated by the paracrine secretion of growth factors secreted by the ERa-positive cells in response to systemic estrogen. (c) The dissociation between steroid receptor synthesis and cell proliferation seen in normal breast epithelium is disrupted in breast cancer. The photomicrograph shows double antibody uorescent staining of a breast cancer sample where many of the tumour cells are positive for ERa (orange/red). In contrast to the normal tissue, the Ki67-positive proliferating cells appear yellow (arrows), showing that they are positive for the ERa protein. Scale barZ 50 mM. (d) This suggests a model where proliferating tumour cells in ERa-positive breast cancer respond to estrogen in a cell autonomous manner, where ERa directly induces proliferation or, alternatively, the cell responds to autocrine growth factors secreted in response to hormone. Abbreviation: ERa, estrogen receptor.

PR synthesis in a tumour indicates a more aggressive tumour with a greater likelihood of metastasis. Other steroid receptors that might play a role in breast cancer include those for androgens (AR), glucocorticoids (GR), retinoids (RAR and RXR) and vitamin D (VDR). ARs in particular might play a signicant role because they are synthesized in normal and malignant breast, where they might antagonize the effects of E during breast development [41]. In this setting, the presence of AR alleles that alter androgen sensitivity relative to the number of polyglutamine repeats in their N-terminal might affect the risk of breast cancer [42]. However, the contribution of AR to breast tumour formation and progression will need further study to assess its signicance. Even less is known about the relevance of other steroid receptors including GR, VDR and RAR/RXR, although retinoid signalling pathways might be important in tumorigenesis because
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RXR-specic retinoids can prevent mammary cancer in animal models [43]. Clearly, there is great potential in studying other steroid receptor signalling pathways in the breast, because they probably impinge on E and P signalling during breast development and affect breast cancer risk. Other important modulators of steroid receptor function in normal and malignant breast tissue are receptor coregulators. These are nuclear factors, termed coactivators and corepressors, which enhance or repress, respectively, the transcriptional activity of receptors [44]. The increases in coactivator synthesis seen during tumorigenesis might enhance or dysregulate E signalling through the ERa, or explain the observed changes in signalling in malignant compared with normal tissues. Several coactivators are increased in breast cancer, including SRA, AIB1, TIF2 and CBP [4547]. Corepressors such as N-CoR have

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been reported to be downregulated in invasive breast tumours. Experimentally, a change in coregulator expression can determine the response to endocrine treatments, and there are emerging in vivo data to support this mechanism [48]. Crosstalk with other signalling pathways There is accumulating evidence that ligand- and DNA-independent activation of steroid receptors, in particular ERa, inuences the response of breast cancer cells to estrogens and antiestrogens. This activation occurs through crosstalk with cell surface growth factor
(a)

receptors and the intracellular signalling cascades that they activate [49]. For example, activation of the phosphoinositol-3 (PI3) and mitogen-activated protein kinase (MAPK) signalling cascades by receptors, such as epidermal growth factor receptor, erbB2 and insulin-like growth factor receptor 1 (IGFR1) leads to phosphorylation of the ER and receptor coactivators (Figure 2a) [50,51]. It might be signicant that, in cultured breast cancer cells resistant to tamoxifen, these growth factor receptor pathways are usually activated [5254]. Activation of these intracellular pathways downstream of growth factor receptors could potentially increase the agonist properties

IGFR

EGFR, HER2 Ras Cell membrane

Cbl PI3 kinase

GRB2

SOS Raf

MEK1/2

ERK 1/2 AKT p90rsk

ER Tam

ER p160 Tam

CBP

Basal transcription machinery Target gene

Proliferation PR, etc.

ERE Nucleus

(b)
Ras SOS GRB2 Raf E ER SHC PI3 kinase MEK1/2 Cell membrane

ERK 1/2 AKT

Survival

ELK1

AP1

Basal transcription machinery Target gene

Proliferation

Nucleus
Figure 2. Models showing crosstalk mechanisms between ERa, growth factor receptors and their intracellular kinase cascade signalling pathways. The mechanisms proposed for crosstalk between growth factors, intracellular signalling and ERa are interpretations based on recent studies of these pathways [4967]. (a) Nuclear ERa can be activated by growth factor signalling pathways such as those from cell surface receptors for IGFR1, EGFR and the HER2 receptor, which is from the same protein family as EGFR. Multiple protein phosphorylation events involving PI3 kinase and MAP kinase intracellular signalling pathways sensitize nuclear ERa and coactivators to the agonist effects of tamoxifen or to low levels of estrogen, permitting the growth of breast cancer cells during treatment and inducing estrogen-regulated genes such as PGR. (b) In contrast to the nuclear ERa action shown in (a), there is evidence that ERa, either at the cell membrane or in the cytoplasm, can interact directly with and activate intracellular signalling pathways such as PI3 kinase and MAP kinase, leading to promotion of breast cell proliferation and survival. Abbreviations: EGFR, epidermal growth factor receptor; ERa, estrogen receptor; HER2, human EGFR-related; IGFR1, type 1 insulin-like growth factor receptor; MAP kinase, mitogen-activated protein kinase; PI3 kinase, phosphoinositol-3 kinase; PR, progesterone receptor.
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of tamoxifen because many of the ERa phosphorylation sites are in the AF-1 domain, which, signicantly, retains its transcriptional activity in the presence of tamoxifen. If coactivators are also being synthesized, this effect could be further potentiated. For example, MAPK directly phosphorylates the Ser118 residue in the ERa AF-1 domain, leading to recruitment of the p68/p72 coactivator, which activates target gene transcription in a ligand-independent manner [55]. In addition, MAPK also targets ERa coactivators such as p160 and CBP for phosphorylation, leading to an increase in their activity [5658]. There is good evidence that ERa phosphorylation by growth factor signal transduction pathways contributes to cancer growth in vivo. For example, there is a lower rate of recurrence of E-dependent, erbB2-positive breast tumours after treatment with aromatase inhibitors (AIs) compared with tamoxifen. Because AIs deplete E, ERa will bind with reduced afnity to an E-response element (ERE) in the gene promoters of treated tumours. By contrast, tamoxifen bound to ERa might bind an ERE in the gene promoters of tumours and become activated in the presence of MAPK activity induced by ERBB2 overexpression, leading to agonist effects of tamoxifen [18,19,48,5962]. Recently, it has been shown that, at least in vitro, the converse also occurs; ligand-activated ERa can regulate the PI3 and MAPK signal transduction pathways (Figure 2b) [63,64]. In one study, ERa localized at the membrane was extracted from MCF-7 breast cancer cells in membrane preparations. E-stimulated growth of the cells was inhibited by an anti-ERa antibody that competed with E for binding to the receptor. An antibody would not be expected to enter a live cell in sufcient amounts to bind to the ERa in the nucleus or cytoplasm [65]. It is proposed that this rapid and non-genomic mechanism for activation of intracellular pathways might be increasingly important in acquired E sensitivity of breast cancer cells when they have been grown in very low concentrations of estradiol for several months [66]. In a second study that examined longterm E deprivation of MCF-7 breast cancer cells, E hypersensitivity was also reported. However, the E sensitivity of breast cancer cells they derived was proposed to be caused by increased production of both IGFR and erbB2, and increased PI3 kinase, MAPK and ERa phosphorylation, rather than membrane ERa signalling [67]. In this scenario, the adaptation of breast cancer cells to low E levels equates closer to Figure 2a than to 2b, and it is therefore unclear whether both of these mechanisms will be operational in breast tumours in vivo. Conclusions Steroid hormones and their receptors, in particular those for E, are intrinsically involved in normal breast development, tumorigenesis and cancer progression. Their effects are modulated by other steroid receptors, particularly PRA and PRB which are synthesized in the same subset of cells as ERa in both normal and malignant tissues. Less is known about other steroid receptors, although there is accumulating evidence that ERb is more widely synthesized in normal tissue than ERa or PR, and its synthesis decreases during the tumorigenic process. The involvement of other steroid receptors merits further
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study, as does the contribution of receptor coregulators, ERa/coactivator phosphorylation mediated via PI3 and MAPKs, and the signicance of non-genomic signalling via a putative cell membrane ER [66]. The dysregulation of ERa signalling in breast cancer compared with normal tissue can be exploited to target drugs that prevent and treat human breast cancer. For this to happen, efforts must be redoubled to understand the molecular mechanisms that underlie these changes and to identify their importance in breast cancer formation and progression in vivo. Hopefully, in the future, these novel pathways and mechanisms of steroid receptor action will provide multiple opportunities for intervention in this disease.

Acknowledgements
RBC and AH are supported by Cancer Research UK; EA is supported by the Christie Hospital Research Endowment Fund.

References
1 Anderson, E. et al. (1998) Estrogen responsiveness and control of normal human breast proliferation. J. Mammary Gland Biol. Neoplasia 3, 2335 2 Lambe, M. et al. (1996) Parity, age at rst and last birth, and risk of breast cancer: a population-based study in Sweden. Breast Cancer Res. Treat. 38, 305311 3 Staszewski, J. (1971) Age at menarche and breast cancer. J. Natl. Cancer Inst. 47, 935940 4 Trichopoulos, D. et al. (1972) Menopause and breast cancer risk. J. Natl. Cancer Inst. 48, 605613 5 Cohen, S.M. and Ellwein, L.B. (1991) Genetic errors, cell proliferation, and carcinogenesis. Cancer Res. 51, 64936505 6 Collaborative Group on Hormonal Factors in Breast Cancer (2002) Breast cancer and breastfeeding: collaborative reanalysis of individual data from 47 epidemiological studies in 30 countries, including 50302 women with breast cancer and 96973 women without the disease. Lancet 360, 187195 7 Russo, J. and Russo, I.H. (1978) DNA labeling index and structure of the rat mammary gland as determinants of its susceptibility to carcinogenesis. J. Natl. Cancer Inst. 61, 14511459 8 Rajkumar, L. et al. (2001) Short-term exposure to pregnancy levels of estrogen prevents mammary carcinogenesis. Proc. Natl. Acad. Sci. U. S. A. 98, 1175511759 9 Laidlaw, I.J. et al. (1995) The proliferation of normal human breast tissue implanted into athymic nude mice is stimulated by estrogen but not progesterone. Endocrinology 136, 164171 10 Magnusson, C. et al. (1999) Breast-cancer risk following long-term oestrogen- and oestrogenprogestin-replacement therapy. Int. J. Cancer 81, 339344 11 Russouw, J.E. (2002) Risks and benets of estrogen plus progestin in healthy postmenopausal women: principal results from the Womens Health Initiative randomized controlled trial. J. Am. Med. Assoc. 288, 321333 12 Ross, R.K. et al. (2000) Effect of hormone replacement therapy on breast cancer risk: estrogen versus estrogen plus progestin. J. Natl. Cancer Inst. 92, 328332 13 Hofseth, L.J. et al. (1999) Hormone replacement therapy with estrogen or estrogen plus medroxyprogesterone acetate is associated with increased epithelial proliferation in the normal postmenopausal breast. J. Clin. Endocrinol. Metab. 84, 45594565 14 Howell, A. et al. (1997) Oestrogens, Beatson and endocrine therapy. Endocr. Relat. Cancer 4, 371380 15 Gandhi, A. et al. (2000) Effects of a pure antiestrogen on apoptosis and proliferation within human breast ductal carcinoma in situ. Cancer Res. 60, 42844288 16 Peto, R. et al. (2000) UK and U. S. A. breast cancer deaths down 25% in year 2000 at ages 2069 years. Lancet 355, 1822 17 Cummings, S.R. et al. (1999) The effect of raloxifene on risk of breast

322

Review

TRENDS in Endocrinology and Metabolism

Vol.15 No.7 September 2004

18

19

20

21

22 23

24 25

26

27 28

29

30

31

32

33

34

35 36 37

38 39

40

41

cancer in postmenopausal women: results from the MORE randomized trial. Multiple Outcomes of Raloxifene Evaluation. J. Am. Med. Assoc. 281, 21892197 Smith, I.E. (2003) Letrozole versus tamoxifen in the treatment of advanced breast cancer and as neoadjuvant therapy. J. Steroid Biochem. Mol. Biol. 86, 289293 Baum, M. et al. (2003) Anastrozole alone or in combination with tamoxifen versus tamoxifen alone for adjuvant treatment of postmenopausal women with early-stage breast cancer: results of the ATAC (arimidex, tamoxifen alone or in combination) trial efcacy and safety update analyses. Cancer 98, 18021810 Tsai, M.J. and OMalley, B.W. (1994) Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu. Rev. Biochem. 63, 451486 Petersen, O.W. et al. (1987) Frequency and distribution of estrogen receptor-positive cells in normal, nonlactating human breast tissue. Cancer Res. 47, 57485751 Kuiper, G.G. et al. (1996) Cloning of a novel receptor expressed in rat prostate and ovary. Proc. Natl. Acad. Sci. U. S. A. 93, 59255930 Clarke, R.B. et al. (1997) Dissociation between steroid receptor expression and cell proliferation in the human breast. Cancer Res. 57, 49874991 Speirs, V. et al. (2002) Distinct expression patterns of ERa and ERb in normal human mammary gland. J. Clin. Pathol. 55, 371374 Graham, J.D. and Clarke, C.L. (2002) Expression and transcriptional activity of progesterone receptor A and progesterone receptor B in mammalian cells. Breast Cancer Res. 4, 187190 Mote, P.A. et al. (2002) Loss of co-ordinate expression of progesterone receptors A and B is an early event in breast carcinogenesis. Breast Cancer Res. Treat. 72, 163172 Shoker, B.S. et al. (1999) Oestrogen receptor expression in the normal and pre-cancerous breast. J. Pathol. 188, 237244 Seagroves, T.N. et al. (2000) C/EBPb (CCAAT/enhancer binding protein) controls cell fate determination during mammary gland development. Mol. Endocrinol. 14, 359368 Russo, J. et al. (1999) Pattern of distribution of cells positive for estrogen receptor a and progesterone receptor in relation to proliferating cells in the mammary gland. Breast Cancer Res. Treat. 53, 217227 Zeps, N. et al. (1998) Estrogen receptor-negative epithelial cells in mouse mammary gland development and growth. Differentiation 62, 221226 Capuco, A.V. et al. (2002) Postnatal mammary ductal growth: threedimensional imaging of cell proliferation, effects of estrogen treatment, and expression of steroid receptors in prepubertal calves. Tissue Cell 34, 143154 Brisken, C. et al . (1998) A paracrine role for the epithelial progesterone receptor in mammary gland development. Proc. Natl. Acad. Sci. U. S. A. 95, 50765081 Brisken, C. et al. (2000) Essential function of Wnt-4 in mammary gland development downstream of progesterone signaling. Genes Dev. 14, 650654 Anderson, E. and Clarke, R.B. (2004) Steroid receptors and cell cycle in normal mammary epithelium. J. Mammary Gland Biol. Neoplasia 9, 313 Dontu, G. et al. (2003) Stem cells in normal breast development and breast cancer. Cell Prolif. 36(Suppl. 1), 5972 Clarke, R.B. et al. (2003) Regulation of human breast epithelial stem cells. Cell Prolif. 36(Suppl. 1), 4558 Lawson, J.S. et al. (1999) Low oestrogen receptor a expression in normal breast tissue underlies low breast cancer incidence in Japan. Lancet 354, 17871788 Shoker, B.S. et al. (1999) Estrogen receptor-positive proliferating cells in the normal and precancerous breast. Am. J. Pathol. 155, 18111815 Roger, P. et al. (2001) Decreased expression of estrogen receptor b protein in proliferative preinvasive mammary tumors. Cancer Res. 61, 25372541 Jensen, E.V. et al. (2001) Estrogen receptors and proliferation markers in primary and recurrent breast cancer. Proc. Natl. Acad. Sci. U. S. A. 98, 1519715202 Moinfar, F. et al. (2003) Androgen receptors frequently are expressed in breast carcinomas: potential relevance to new therapeutic strategies. Cancer 98, 703711

42 Chamberlain, N.L. et al. (1994) The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function. Nucleic Acids Res. 22, 31813186 43 Wu, K. et al. (2002) The retinoid X receptor-selective retinoid, LGD1069, prevents the development of estrogen receptor-negative mammary tumors in transgenic mice. Cancer Res. 62, 63766380 44 McKenna, N.J. et al. (1999) Nuclear receptor coregulators: cellular and molecular biology. Endocr. Rev. 20, 321344 45 Murphy, L.C. et al. (2000) Altered expression of estrogen receptor coregulators during human breast tumorigenesis. Cancer Res. 60, 62666271 46 List, H.J. et al. (2001) Expression of the nuclear coactivator AIB1 in normal and malignant breast tissue. Breast Cancer Res. Treat. 68, 2128 47 Kurebayashi, J. et al. (2000) Expression levels of estrogen receptor-a, estrogen receptor-b, coactivators, and corepressors in breast cancer. Clin. Cancer Res. 6, 512518 48 Osborne, C.K. et al. (2003) Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer. J. Natl. Cancer Inst. 95, 353361 49 Hall, J.M. et al. (2001) The multifaceted mechanisms of estradiol and estrogen receptor signaling. J. Biol. Chem. 276, 3686936872 50 Lee, H. et al. (2000) MEKK1 activation of human estrogen receptor a and stimulation of the agonistic activity of 4-hydroxytamoxifen in endometrial and ovarian cancer cells. Mol. Endocrinol. 14, 18821896 51 Campbell, R.A. et al. (2001) Phosphatidylinositol 3-kinase/AKTmediated activation of estrogen receptor a: a new model for antiestrogen resistance. J. Biol. Chem. 276, 98179824 52 Knowlden, J.M. et al. (2003) Elevated levels of epidermal growth factor receptor/c-erbB2 heterodimers mediate an autocrine growth regulatory pathway in tamoxifen-resistant MCF-7 cells. Endocrinology 144, 10321044 53 McClelland, R.A. et al. (2001) Enhanced epidermal growth factor receptor signaling in MCF7 breast cancer cells after long-term culture in the presence of the pure antiestrogen ICI 182,780 (Faslodex). Endocrinology 142, 27762788 54 Gee, J.M. et al. (2003) The antiepidermal growth factor receptor agent getinib (ZD1839/Iressa) improves antihormone response and prevents development of resistance in breast cancer in vitro. Endocrinology 144, 51055117 55 Kato, S. et al. (1995) Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270, 14911494 56 Shao, W. and Brown, M. (2004) Advances in estrogen receptor biology: prospects for improvements in targeted breast cancer therapy. Breast Cancer Res. 6, 3952 57 Rowan, B.G. et al. (2000) Phosphorylation of steroid receptor coactivator-1. Identication of the phosphorylation sites and phosphorylation through the mitogen-activated protein kinase pathway. J. Biol. Chem. 275, 44754483 58 Font de Mora, J. and Brown, M. (2000) AIB1 is a conduit for kinasemediated growth factor signaling to the estrogen receptor. Mol. Cell. Biol. 20, 50415047 59 Ellis, M.J. et al. (2001) Letrozole is more effective neoadjuvant endocrine therapy than tamoxifen for ErbB-1- and/or ErbB-2-positive, estrogen receptor-positive primary breast cancer: evidence from a phase III randomized trial. J. Clin. Oncol. 19, 38083816 60 Mouridsen, H. et al. (2001) Superior efcacy of letrozole versus tamoxifen as rst-line therapy for postmenopausal women with advanced breast cancer: results of a phase III study of the International Letrozole Breast Cancer Group. J. Clin. Oncol. 19, 25962606 61 Ellis, M.J. et al. (2003) Letrozole inhibits tumor proliferation more effectively than tamoxifen independent of HER1/2 expression status. Cancer Res. 63, 65236531 62 Adeyinka, A. et al. (2002) Activated mitogen-activated protein kinase expression during human breast tumorigenesis and breast cancer progression. Clin. Cancer Res. 8, 17471753 63 Kousteni, S. et al. (2001) Nongenotropic, sex-nonspecic signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 104, 719730

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64 Simoncini, T. et al. (2000) Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 407, 538541 65 Marquez, D.C. and Pietras, R.J. (2001) Membrane-associated binding sites for estrogen contribute to growth regulation of human breast cancer cells. Oncogene 20, 54205430 66 Song, R.X. et al. (2002) Linkage of rapid estrogen action to MAPK activation by ERaShc association and Shc pathway activation. Mol. Endocrinol. 16, 116127 67 Martin, L.A. et al. (2003) Enhanced estrogen receptor (ER) a, ERBB2, and MAPK signal transduction pathways operate during the adaptation of MCF-7 cells to long term estrogen deprivation. J. Biol. Chem. 278, 3045830468 68 Russo, I.H. and Russo, J. (1998) Role of hormones in mammary cancer initiation and progression. J. Mammary Gland Biol. Neoplasia 3, 4961

69 Perusinghe, N.P. et al. (1992) Effects of growth factors on proliferation of basal and luminal cells in human breast epithelial explants in serum-free culture. In Vitro Cell. Dev. Biol. 28A, 9096 70 Santini, D. et al. (1996) Differentiation pathways in primary invasive breast carcinoma as suggested by intermediate lament and biopathological marker expression. J. Pathol. 179, 386391 71 Wellings, S.R. et al. (1975) An atlas of subgross pathology of the human breast with special reference to possible precancerous lesions. J. Natl. Cancer Inst. 55, 231273 72 Allred, D.C. et al. (2001) Histological and biological evolution of human premalignant breast disease. Endocr. Relat. Cancer 8, 4761 73 Holland, P.A. et al. (1997) Assessment of hormone dependence of comedo ductal carcinoma in situ of the breast. J. Natl. Cancer Inst. 89, 10591065 74 Cuzick, J. et al. (2003) Overview of the main outcomes in breast-cancer prevention trials. Lancet 361, 296300

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