Review
Estrogen and its metabolites are carcinogenic agents in human breast epithelial cells

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Abstract

Estrogens play a crucial role in the development and evolution of human breast cancer. However, it is still unclear whether estrogens are carcinogenic to the human breast. There are three mechanisms that have been considered to be responsible for the carcinogenicity of estrogens: receptor-mediated hormonal activity, a cytochrome P450 (CYP)-mediated metabolic activation, which elicits direct genotoxic effects by increasing mutation rates, and the induction of aneuploidy by estrogen. To fully demonstrate that estrogens are carcinogenic in the human breast through one or more of the mechanisms explained above it will require an experimental system in which, estrogens by itself or one of the metabolites would induce transformation phenotypes indicative of neoplasia in HBEC in vitro and also induce genomic alterations similar to those observed in spontaneous malignancies. In order to mimic the intermittent exposure of HBEC to endogenous estrogens, MCF-10F cells that are ERα negative and ERβ positive were first treated with 0, 0.007, 70 nM and 1 μM of 17β-estradiol (E2), diethylstilbestrol (DES), benz(a)pyrene (BP), progesterone (P), 2-OH-E2, 4-hydoxy estradiol (4-OH-E2) and 16-α-OH-E2 at 72 h and 120 h post-plating. Treatment of HBEC with physiological doses of E2, 2-OH-E2, 4-OH-E2 induce anchorage independent growth, colony formation in agar methocel, and reduced ductulogenic capacity in collagen gel, all phenotypes whose expression are indicative of neoplastic transformation, and that are induced by BP under the same culture conditions. The presence of ERβ is the pathway used by E2 to induce colony formation in agar methocel and loss of ductulogenic in collagen gel. This is supported by the fact that either tamoxifen or the pure antiestrogen ICI-182,780 (ICI) abrogated these phenotypes. However, the invasion phenotype, an important marker of tumorigenesis is not modified when the cells are treated in presence of tamoxifen or ICI, suggesting that other pathways may be involved. Although we cannot rule out the possibility, that 4-OH-E2 may interact with other receptors still not identified, with the data presently available the direct effect of 4-OH-E2 support the concept that metabolic activation of estrogens mediated by various cytochrome P450 complexes, generating through this pathway reactive intermediates that elicit direct genotoxic effects leading to transformation. This assumption was confirmed when we found that all the transformation phenotypes induced by 4-OH-E2 were not abrogated when this compound was used in presence of the pure antiestrogen ICI. The novelty of these observations lies in the role of ERβ in transformation and that this pathway can successfully bypassed by the estrogen metabolite 4-OH-E2. Genomic DNA was analyzed for the detection of micro-satellite DNA polymorphism using 64 markers covering chromosomes (chr) 3, 11, 13 and 17. We have detected loss of heterozygosity (LOH) in ch13q12.2–12.3 (D13S893) and in ch17q21.1 (D17S800) in E2, 2-OH-E2, 4-OH-E2, E2 + ICI, E2 + tamoxifen and BP-treated cells. LOH in ch17q21.1–21.2 (D17S806) was also observed in E2, 4-OH-E2, E2+ICI, E2+tamoxifen and BP-treated cells. MCF-10F cells treated with P or P+E2 did not show LOH in the any of the markers studied. LOH was strongly associated with the invasion phenotype. Altogether our data indicate that E2 and its metabolites induce in HBEC LOH in loci of chromosomes 13 and 17, that has been reported in primary breast cancer, that the changes are similar to those induced by the chemical carcinogen (BP) and that the genomic changes were not abrogated by antiestrogens.

Introduction

Intensive epidemiological studies have identified a number of genetic risk factors associated with breast cancer [1]. An increased risk has also been associated with early onset of menstruation, nulliparity or delayed first childbirth, short duration of breast feeding, late menopause, use of hormone replacement therapy and increased bone density [2], [3], [4]. A principal culprit common for all these endocrine-related risk factors is the prolonged exposure to female sex hormones [5], [6], [7], [8]. The hormonal influences have been mainly attributed to unopposed exposure to elevated levels of estrogens [5], as has been indicated for a variety of female cancers, namely, vaginal, hepatic and cervical carcinomas [9], [10], [11]. Exposure to estrogens, particularly during the critical developmental periods (e.g. in utero, puberty, pregnancy, menopause), also affects affective behaviors (e.g. depression, aggression, alcohol intake) and increases breast cancer risk [12]. In addition, both environmental and genetic factors are believed to exert their influence by a hormonal mechanism [13], [14], [15], [16], [17], [18].

It is generally accepted that the biological activities of estrogens are mediated by nuclear estrogen receptors (ER) which, upon activation by cognate ligands, form homodimers with another ER–ligand complex and activate transcription of specific genes containing the estrogen response elements [19]. According to this classical model, the biological responses to estrogens are mediated by the ER universally identified until recently, which has been termed as ERα after the discovery of a second type of ER (ERβ). The presence of ERα in target tissues or cells is essential to their responsiveness to estrogen action. In fact, the expression levels of ERα in a particular tissue have been used as an index of the degree of estrogen responsiveness [20]. ERβ and ERα share high sequence homology, especially in the regions or domains responsible for specific binding to DNA and the ligands. ERβ can be activated by estrogen stimulation, and blocked with antiestrogens [21], [22]. Upon activation, ERβ can form homodimers as well as heterodimers with ERα [22], [23]. The existence of two ER subtypes and their ability to form DNA-binding heterodimers suggests three potential pathways of estrogen signaling: via the ERα or ERβ subtype in tissues exclusively expressing each subtype and via the formation of heterodimers in tissues expressing both ERα and ERβ. The pathways of the ER-mediated signal transduction have become even more complicated by the recent discovery of other types of ER [24], [25]. In addition, estrogens and antiestrogens can induce differential activation of ERα and ERβ to control transcription of genes that are under the control of an AP-1 element [23].

The most biologically active estrogen in breast tissue is 17β-estradiol (E2). Circulating estrogens are mainly originated from ovarian steroidogenesis in premenopausal women and peripheral aromatization of ovarian and adrenal androgens in postmenopausal women [26]. The importance of ovarian steroidogenesis in the genesis of breast cancer is highlighted by the fact that occurring naturally or induced early menopause prior to age 40 significantly reduces the risk of developing breast cancer [26]. However, the uptake of 17β-estradiol from the circulation does not appear to contribute significantly to the total content of estrogen in breast tumors, since the majority of estrogen present in the tumor tissues is derived from de novo biosynthesis [26]. In fact, the concentrations of 17β-estradiol in breast cancer tissues do not differ between premenopausal and postmenopausal women, even though plasma levels of 17β-estradiol decrease by 90% following menopause [27]. This phenomenon might be explained by the observation that enzymatic transformations of circulating precursors in peripheral tissues contribute 75% of estrogens in premenopausal women and almost 100% in postmenopausal women [28], [29], the data that highlight the importance of in situ metabolism of estrogens [26], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48].

Even though the breast is influenced by a myriad of hormones and growth factors [49], [50], [51], [52], estrogens are considered to play a major role in promoting the proliferation of both the normal and the neoplastic breast epithelium [49], [50]. Estradiol acts locally in the mammary gland, stimulating DNA synthesis and promoting bud formation, probably through an ER-mediated mechanism [49]. It is also known that the prevailing metabolic condition of an individual animal or human may significantly influence mammary gland responses to hormones. In addition, the mammary gland responds selectively to given hormonal stimuli for either cell proliferation or differentiation, depending upon specific topographic differences in gland development. In either case, the response of the mammary gland to these complex hormonal and metabolic interactions results in developmental changes that permanently modify both the architecture and the biological characteristics of the gland [49], [51].

The fact that the normal epithelium contains receptors for both estrogen and progesterone lends support to the receptor-mediated mechanism as a major player in the hormonal regulation of breast development. The role of these hormones on the proliferative activity of the breast, which is indispensable for its normal growth and development, has been for a long time, and still is, the subject of heated controversies [26]. There is little doubt, however, that the proliferative activity of the mammary epithelium in both rodents and humans varies with the degree of differentiation of the mammary parenchyma [49], [50], [51], [52], [53], [54], [55]. In humans, the highest level of cell proliferation is observed in the undifferentiated lobules type 1 (Lob 1) present in the breast of young nulliparous females [49], [50], [51], [52]. The progressive differentiation of Lob 1 into lobules types 2 (Lob 2) and 3 (Lob 3), occurring under the hormonal influences of the menstrual cycle, and the full differentiation into lobules type 4 (Lob 4), as a result of pregnancy, leads to a concomitant reduction in the proliferative activity of the mammary epithelium [49], [50], [51], [52], [53], [54], [55]. The content of ERα and progesterone receptor (PgR) in the lobular structures of the breast is directly proportional to the rate of cell proliferation, being also maximal in the undifferentiated Lob 1, and decreasing progressively in Lob 2, Lob 3, and Lob 4 [51], [56]. The findings that proliferating cells are different from those that are ERα- and PgR-positive support data that indicate that estrogen controls cell proliferation by an indirect mechanism. This phenomenon has been demonstrated using supernatant of estrogen-treated ERα-positive cells that stimulates the growth of ERα-negative cell lines in culture. The same phenomenon has been shown in vivo in nude mice bearing ER-negative breast tumor xenografts [57]. ERα-positive cells treated with antiestrogens secrete transforming growth factor-β that inhibits the proliferation of ERα-negative cells [58]. The findings that proliferating cells in the human breast are different from those that contain steroid hormone receptors explain many of the in vitro data [59], [60]. Of interest are the observations that while the ERα-positive MCF-7 cells respond to estrogen treatment with increased cell proliferation, and that the enhanced expression of the ERα by transfection also increases the proliferative response to estrogen [59], [60], [61], ERα-negative cells, such as MDA-MB-468 and others, when transfected with ERα, exhibit inhibition of cell growth under the same type of treatment [60]. Although the negative effect of estrogen on those ERα-negative cells transfected with the ERα has been interpreted as an interference of the transcription factor used to maintain estrogen independent growth [61], there is no definitive explanation for their lack of survival. However, it can be explained by the finding that proliferating and ERα-positive cells are two separate populations. Further support is the finding that when Lob 1 of normal breast tissue are placed in culture, they lose the ERα-positive cells, indicating that only proliferating cells that are also ERα-negative can survive and constitute the stem cells [62], [63].

Although 67% of breast cancers are manifested during the postmenopausal period, a vast majority, 95%, is initially hormone-dependent [26]. This indicates that estrogens play a crucial role in their development and evolution. It has been established that in situ metabolism of estrogens through aromatase-mediated pathway is correlated with the risk of developing breast cancer [37], [38]. A recent finding that expression of estrone sulfatase is inversely correlated with relapse-free survival of human breast cancer patients [42] reiterates the importance of estrone sulfatase-mediated local production of estrogen in the development and progression of human breast cancer. However, it is still unclear whether estrogens are carcinogenic to the human breast. Most of the current understanding of carcinogenicity of estrogens is based on studies in experimental animal systems and clinical observations of a greater risk of endometrial hyperplasia and neoplasia associated with estrogen supplementation or polycystic ovarian syndrome [26].

There are three mechanisms [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], [79], [80], [81], [82], [83], [84], [85], [86], [87], [88], [89], [90], [91], [92], [93], [94], [95], [96], [97], [98], [99], [100], [101], [102], [103], [104], [105], [106], [107], [108], [109], [110], [111], [112], [113], [114], [115], [116], [117], [118], [119], [120], [121], [122], [123], [124], [125], [126], [127], [128], [129], [130], [131], [132], [133], [134], [135], [136], [137], [138], [139], [140], [141], [142], [143], [144], [145], [146], [147], [148] that have been considered to be responsible for the carcinogenicity of estrogens: receptor-mediated hormonal activity, which has generally been related to stimulation of cellular proliferation, resulting in more opportunities for accumulation of genetic damages leading to carcinogenesis [56], [63], [75], [76], [77], [78], [79], [80], [81], [82], [83], [84], [85], [86], a cytochrome P450 (CYP)-mediated metabolic activation, which elicits direct genotoxic effects by increasing mutation rates [26], [64], [65], [87], [88], [89], [90], [91], [92], [93], [94], [95], [96], [97], [98], [99], [100], [101], [102], [103], [104], [105], [106], [107], [108], [109], [110], [111], [112], [113], [114], [115], [116], [117], [118], [119], [120], [121], [122], [123], [124], [125], [126], [127], [128], [129], [130], [131], [132], [133], and the induction of aneuploidy by estrogen [65], [66], [67], [68], [69], [70], [71], [72], [73], [134], [135], [136], [137], [138], [139], [140], [141], [142], [143], [144], [145], [146], [147], [148]. There is also evidence that estrogen compromises the DNA repair system and allows accumulation of lesions in the genome essential to estrogen-induced tumorigenesis [74].

To fully demonstrate that estrogens are carcinogenic in the human breast through one or more of the mechanisms explained above it will require an experimental system in which, estrogens by itself or one of the metabolites would induce transformation phenotypes indicative of neoplasia in HBEC in vitro and also induce genomic alterations similar to those observed in spontaneous malignancies, such as DNA amplification and loss of genetic material that may represent tumor suppressor genes [149], [150], [151], [152], [153], [154], [155], [156], [157], [158], [159], [160], [161], [162], [163], [164]. For this purpose, we have develop an in vitro system in which we have demonstrated that estrogens are transforming agents on human breast epithelial cells (HBEC), by utilizing the spontaneously immortalized HBEC MCF-10F [165], [166]. In order to mimic the intermittent exposure of HBEC to endogenous estrogens, all cells were treated repetitively with different concentrations of 17β-estradiol inducing phenotypic and genotypic changes indicative of cell transformation [167], [168]. In the present work, we further demonstrate that metabolites of estrogens are also able to induce phenotypic and genotypic changes in human breast epithelial cells furthering our understanding of the complex role of estrogen in breast carcinogenesis.

Section snippets

The in vitro model of cell transformation

The transforming potential of estrogens on human breast epithelial cells in vitro, have being evaluated by utilizing the spontaneously immortalized HBEC MCF-10F cells [167], [168]. The spontaneously immortalized MCF-10F cells, treated cells and derived clones were maintained in DMEM:F-12 (1:1) medium with a 1.05 mM Ca2+ concentration. All cell lines were regularly tested for correct identity using a fingerprint cocktail of three minisatellite plasmid probes (ATCC, Rockville, MD). Culture media

Transformation effect of estrogens and its metabolites in MCF-10F cells

We have determined the optimal doses for the expression of the cell transformation phenotype by treating the immortalized human breast epithelial cells MCF-10F with 17β-estradiol (E2) with 0.0, 0.07, 70 nM, or 1 μM of E2 twice a week for 2 weeks. The survival efficiency was increased with 0.007 and 70 nM of 17β-estradiol and decrease with 1 μM. The cells treated with either doses of E2 formed colonies in agar methocel (Fig. 2) and the size was not different among them, however, the CE increased

Discussion

We have demonstrated that 17β-estradiol induces cell transformation of the human breast epithelial cells MCF-10F. The cells treated with either doses of E2 formed colonies in agar methocel a phenotype indicative of neoplastic transformation [61], [75], [76], [167]. Non-transformed cells produce ductules like structure and transformed cells produce spherical or solid masses of cells [169], [176]. Cells treated with DMSO, cholesterol or progesterone at different concentrations was unable to alter

Acknowledgements

This work was supported by Grant DAMD 17-00-1-0247 and DAMD-17-00-1-0249.

Glossary

Definition of key terms

ATCC
American Tissue Culture Collection
BP
Benz(a)pyrene
BSA
Bovine serum albumin
CE-Q
Catechol estrogen-quinone
CENP-E
Centromere–kinetochore complex
CE
Colony efficiency
CGH
Comparative genomic hybridization
CS
Colony size
CYP
Cytochrome P450
DES
Diethylstilbestrol
DTT
Dithiothreitol
EDTA
Ethylene-diamino-tetraacetic-acid
E1
Estrone
E2
Estradiol
ER
Estrogen receptors
ERα
Estrogen receptor α
ERβ
Estrogen receptor β
4-OH-E2
4-Hydoxy estradiol
HBEC
Human breast epithelial cells
HCl
Hydrochloric acid
Lob 1
Lobule type 1
Lob 2
Lobule type 2

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      LOH was strongly associated with the invasion phenotype. Interestingly, MCF-10F cells treated with P4 or P4+E2 did not show LOH in the any of the markers studied (Russo et al., 2003). Similar results were obtained using ER-negative MCF-10A cells; treatment with 4-hydroxylated catechols (4-OHE2, 4,17β-OHEqn, 4OH-E1, 3-OHEqn) induced significantly more colony formation compared to parent phenols (E2, 17β-OHEqn, E1 and Eqn), with 4-OHEqn showing a concentration-dependent increase in colony formation and anchorage independent growth (Cuendet et al., 2004).

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