The Journal of Steroid Biochemistry and Molecular Biology
The role of estrogen in the initiation of breast cancer☆
Introduction
Breast cancer is a malignancy whose dependence on ovarian function was first recognized through the regression of both advanced cancer [1] and metastatic disease [2] induced by oophorectomy in premenopausal women. Ulterior correlation of ovarian function with estrogen production [3], and the isolation of the estrogen receptor protein [4], [5], combined with the observed greater incidence of estrogen receptor positive tumors in postmenopausal women [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], led to the identification of a strong association of estrogen dose and length of exposure with increased breast cancer risk [6], [10], [13], [14]. The importance of ovarian steroidogenesis in normal breast development and in the genesis of breast cancer is highlighted by the facts that early menarche and late menopause are associated with greater breast cancer risk, whereas late menarche and early menopause, that occurring before 40 years of age, result in a significant reduction of the same [17], [18], [19], [20]. Breast development at puberty and during sexual maturity is stimulated by 17β-estradiol (E2), which is the predominant circulating ovarian steroid and the most biologically active hormone in breast tissue [21], [22]. At menopause E2 plasma levels decrease by 90% [17], [18], [19]. In spite of the markedly different circulating levels of estrogens in pre- and post-menopausal women, the concentrations of E2 in breast cancer tissues do not differ between these two groups of women, an indication that its uptake from the circulation might not contribute significantly to the total content of this hormone in breast tumors, but rather that de novo biosynthesis, i.e., peripheral aromatization of ovarian and adrenal androgens, plays a more significant role [23], [24].
Considerable epidemiological and clinical evidence link cumulative and sustained exposure to estrogens with increased risk of developing breast cancer. However, there is no clear understanding of the mechanisms through which estrogens cause cancer. In experimental animal models it has been demonstrated that E2, 11β-methoxyethinylestradiol (Moxestrol), and diethylstilbestrol (DES), as well as their 4-hydroxycatechols, induce kidney cancer in castrated male Syrian golden hamsters [25], [26], [27]. In rats, continuous administration of supraphysiological doses of estrogens induces a high percentage of mammary adenocarcinomas, whereas low doses given over long periods induce fibroadenomas [28]. In both models, however, the tumorigenic effects of estrogens are associated with marked hyperprolactinemia and pituitary hyperplasia resulting from an increase in number of hyperplastic prolactin secreting cells. The dependence on a functional pituitary gland has been further confirmed in hypophysectomized rats in which estrogens are ineffective as carcinogens [29]. Nevertheless, the most widely acknowledged mechanism of estrogen carcinogenicity is its binding to its specific nuclear receptor alpha (ER-α) for exerting a potent stimulus on breast cell proliferation through its direct and/or indirect actions on the enhanced production of growth factors [21], [22]. However, the fact that ER-α knockout mice expressing the Wnt-1 oncogene (ERKO/Wnt-1) develop mammary tumors provides direct evidence that estrogens may cause breast cancer through a genotoxic, non-ER-α-mediated mechanism [30], [31]. This postulate is further supported by the observations that when ovariectomized mice are supplemented with E2 they develop a higher tumor incidence with shorter latency time than controls, even in the presence of the pure antiestrogen ICI-182,780. Experimental studies on estrogen metabolism [32], [33], formation of DNA adducts [34], carcinogenicity [35], [36], [37], mutagenicity [38], and cell transformation [39], [40], [41], [42] have supported the hypothesis that reaction of specific estrogen metabolites, namely, catechol estrogen-3,4-quinones (CE-3,4-Q) and to a much lesser extent, CE-2,3-Q, can generate critical DNA mutations that initiate breast, prostate and other cancers [43].
Section snippets
Rationale for an in vitro–in vivo model of cell transformation
In order to definitively outline the pathways through which estrogens act as carcinogens in the human breast and for assessing whether one or more of the mechanisms described above are responsible of carcinogenic initiation, it is needed an experimental system in which E2 by itself or its metabolites induce transformation of human breast epithelial cells (HBEC) in a well controlled environment, preferentially in vitro. Recently it has been reported an in vitro–in vivo system of cell
The experimental model of transformation of MCF-10F cells by 17-β estradiol treatment
Treatment of the spontaneously immortalized ER-α and progesterone receptor (PgR) negative human breast epithelial cell line MCF-10F (Fig. 1) with 70 nM E2 twice a week for 2 weeks formed colonies in agar methocel (Fig. 2) and the colony efficiency increased from 0 in controls to 12.0 ± 1 in the treated cells. The positive control cells BP1-Tras and MDA-MB231 cells had a moderately (p < 0.02) and significantly (p < 0.001) higher colony efficiency than E2-transformed cells, respectively [44]. This
Genomic pathway of 17 beta estradiol induced neoplastic transformation
Using comparative genomic hybridization (CGH) that is a molecular cytogenetic method for screening gains and losses at chromosomal and subchromosomal levels, it has been detected that MCF-10F cells transformed by E2 had lost 9p11-13, a loss that persisted in the invasive cell line E2-70nM-C5 (Fig. 7). This locus contains the serine protease family member PRSS3 (trypsinogen-IV), a putative tumor-suppressor gene [50] in which an allelic imbalance has been reported in hepatocellular carcinoma [51]
Genomic changes during the tumorigenic stage of malignant transformation
E2 induces, in addition to the expression of early phenotypes of neoplastic transformation, tumorigenesis in a heterologous host (Fig. 8). This phenomenon became possible only after the selection of invasive cells that exhibit specific changes, such as the deletion of chromosome 4p15.3-16, which was the first one, detected (Fig. 7, Fig. 8). Interestingly enough, injection of these cells to SCID mice resulted in the formation of tumors in which the entire chromosome 4 was deleted, a change that
Conclusions
17-β-Estradiol is able to induce complete neoplastic transformation of human breast epithelial cells, as proven by the formation of tumors in SCID mice. This model demonstrates a sequence of chromosomal changes that correlates with specific stages of neoplastic progression. The data also support the concept that 17-β-estradiol can act as a carcinogenic agent without the need of the ER-α, although we cannot rule out thus far the possibility that other receptors such as ER-β, or other mechanisms
Acknowledgement
This work was supported by the U.S. Army Medical and Research Materiel Command under grants DAMD17-00-1-0247 and DAMD17-03-1-0229.
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Lecture presentation at the 17th International Symposium of the Journal of Steroid Biochemistry & Molecular Biology, ‘Recent Advances in Steroid Biochemistry and Molecular Biology’, Seefeld, Tyrol, Austria, 31 May–3 June, 2006.