Implications of Estrogens in Human Breast Carcinogenesis

Although 67% of breast cancers are manifested during the postmenopausal period, a vast majority, 95%, are initially hormone-dependent [14]. 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 [66-69]. A recent finding that expression of estrone sulfatase is inversely correlated with re lapse-free survival of human breast cancer patients [78] 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 endome-trial hyperplasia and neoplasia associated with estrogen supplementation or polycystic ovarian syndrome [19-21].

There are two mechanisms 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 [139,140], and cytochrome P450-mediated metabolic activation, which elicits direct genotoxic effects by increasing mutation rates (Fig. 7) [140-142]. There is also evidence that estrogen compromises the DNA repair system and allows accumulation of lesions in the genome essential to estrogen-induced tu-morigenesis [143].

The receptor-mediated activity of estrogen is generally related to induction of expression of the genes involved in the control of cell cycle progression and growth of human breast epithelium [144]. The biological response to estrogen depends upon the local concentrations of the active hormone and its receptors. The level of ER expression is higher in breast cancer patients than in control subjects and is related to breast cancer risk in postmenopausal women [145]. It has been suggested that overexpression of ER in normal human breast epithelium may augment estrogen responsiveness and hence the risk of breast cancer [145]. The proliferative activity and the percentage of ERa-positive cells are

Fig. 7. Potential mechanisms of estrogen-induced carcinogenesis in human breast tissues

highest in Lob 1 in comparison with the various lobular structures composing the normal breast. These findings provide a mechanistic explanation for the higher susceptibility of these structures to be transformed by chemical carcinogens in vitro [146,147], supporting as well the observations that Lob 1 are the site of origin of ductal carcinomas [148].

The presence of ERa-positive and ERa-negative cells with different prolifer-ative activity in the normal human breast may help to elucidate the genesis of ERa-positive and ERa-negative breast cancers [149,150]. It has been suggested that either ERa-negative breast cancers result from the loss of the ability of the cells to synthesize ERa during clinical evolution of ERa-positive cancers, or that ERa-positive and ERa-negative cancers are different entities [150, 151]. Based on our observations, it is postulated that Lob 1 contain at least three cell types, ERa-positive cells that do not proliferate, ERa-negative cells that are capable of proliferating, and a small proportion of ERa-positive cells that can proliferate as well (Fig. 8) [129]. Therefore, estrogen might stimulate ERa-positive cells to produce a growth factor that in turn stimulates neighboring ERa-

er~ Non-Proliferating Ceils Estrogens or Estrogen V Agonists er~ Non-Proliferating Ceils Estrogens or Estrogen V Agonists

ER- Proliferating Cetls

Fig. 8. Schematic representation of the postulated pathways of estrogen actions on breast epithelial cells

ER- Proliferating Cetls

Fig. 8. Schematic representation of the postulated pathways of estrogen actions on breast epithelial cells

Fig. 9A, B. Expression of ERa or ER^ in human breast epithelial cells. Signal intensities of the ERa or ER^ products for each cell line were normalized using GAPDH products to produce arbitrary units of relative abundance. The average value for each cell line from three independent RT-PCR reactions is plotted in Fig. 9B, while a representative picture of the gel for ERa or ER^ reactions is shown in Fig. 9A (Reproduced with permission from ref. [154])

Fig. 9A, B. Expression of ERa or ER^ in human breast epithelial cells. Signal intensities of the ERa or ER^ products for each cell line were normalized using GAPDH products to produce arbitrary units of relative abundance. The average value for each cell line from three independent RT-PCR reactions is plotted in Fig. 9B, while a representative picture of the gel for ERa or ER^ reactions is shown in Fig. 9A (Reproduced with permission from ref. [154])

negative cells capable of proliferating (Fig. 8) [129]. In the same fashion, the small proportion of cells that are ERa-positive and can proliferate could be the source of ERa-positive tumors. The possibility exists, as well, that the ERa-negative cells convert to ERa-positive cells (Fig. 8) [129].

The newly discovered ER^ opens another possibility that those cells traditionally considered negative for ERa might be positive for ER^ [33-35, 152-158]. It has recently been found that ER^is expressed during the immortalization and transformation of ER-negative human breast epithelial cells (Fig. 9) [154], supporting the hypothesis of conversion from a negative to a positive receptor cell. The functional role of ER^-mediated estrogen signaling pathways in the pathogenesis of malignant diseases is essentially unknown. In the rats, ER^-

mediated mechanisms have been implicated in the up-regulation of PgR expression in the dysplastic acini of the dorsolateral prostate in response to treatment of testosterone and 17f-estradiol [159]. In the human, ERf has been detected in both normal and cancerous breast tissues or cell lines [153], and is the predominant ER type in normal breast tissue [158]. Expression of ERf in breast tumors is inversely correlated with the PgR status [157] and variant transcripts of ERf have been observed in some breast tumors [155,156]. ERf and ERa are co-expressed in some breast tumors and a few breast cell lines [153-155], suggesting an interesting possibility that ERa and ERf proteins may interact with each other and discriminate between target sequences leading to differential responsiveness to estrogens (Fig. 2). In addition, estrogen responses mediated by ERa and ERf may vary with different composition of their co-activators that transmit the effect of ER-ligand complex to the transcription complex at the promotor of target genes [160]. Recently, it has been shown that an increase in the expression of ERa with a concomitant reduction in ERf expression occurs during tumorigenesis of the breast [161] and ovary [162], but breast tumors expressing both ERa and ERf are lymph node-positive and tend to be of higher histopathological grade [158]. These data suggest a change in the interplay of ERa- and ERf-mediated signal transduction pathways during breast tumorigenesis.

Even though it is now generally believed that alterations in the ER-mediated signal transduction pathways contribute to breast cancer progression toward hormonal independence and more aggressive phenotypes, there is also mounting evidence that a membrane receptor coupled to alternative second messenger signaling mechanisms [163,164] is operational, and may stimulate the cascade of events leading to cell proliferation. This knowledge suggests that ERa-negative cells found in the human breast may respond to estrogens through this or other pathways. The biological responses elicited by estrogens are mediated, at least in part, by the production of autocrine and paracrine growth factors from the epithelium and the stroma in the breast [165, 166]. In addition, evidence has accumulated over the last decade supporting the existence of ER variants,mainly a truncated ER and an exon-deleted ER [167-171]. It has been suggested that expression of ER variants may contribute to breast cancer progression toward hormone independence [171]. Although more studies need to be done in this direction, it is clear that the findings that in the normal breast the proliferating and steroid hormone receptor-positive cells are different open new possibilities for clarifying the mechanisms through which estrogens might act on the proliferating cells to initiate the cascade of events leading to cancer.

More importantly, estrogen may not need to activate its nuclear receptors to initiate or promote breast carcinogenesis. There is evidence that oxidative ca-tabolism of estrogens mediated by various cytochrome P450 (CYP) complexes constitutes a pathway of their metabolic activation and generates reactive free radicals and intermediate metabolites, reactive intermediates that can cause oxidative stress and genomic damage directly [141,142].

17f-Estradiol and estrone, which are continuously interconverted by 17ff-estradiol hydroxysteroid dehydrogenase (or 17f-oxidoreductase), are the two major endogenous estrogens (Fig. 10). They are generally metabolized via two major pathways: hydroxylation at C16a position and at the C2 or C4 positions

16a-hydroxyestrone

4-hydroxyestrone: R1=H; R2=OH 4-hydroxyestradiol: R1=H; R2=OH 2-hydroxyestrone: R1 =OH; R2=H 2-hydroxyestradiol: R1 =OH; R2=H

16a-hydroxyestrone

4-hydroxyestrone: R1=H; R2=OH 4-hydroxyestradiol: R1=H; R2=OH 2-hydroxyestrone: R1 =OH; R2=H 2-hydroxyestradiol: R1 =OH; R2=H

estriol

4-methoxyestrone: R1=H; R2=OCH3 4-methoxyestradiol: R1=H; R2=OCH3 2-methoxyestrone: Rl=OCH3; R2=H 2-methoxyestradiol: Rl=OCH ; R2=H

Fig. 10. Biosynthesis and steady-state control of catechol estrogens in human breast tissues estriol

4-methoxyestrone: R1=H; R2=OCH3 4-methoxyestradiol: R1=H; R2=OCH3 2-methoxyestrone: Rl=OCH3; R2=H 2-methoxyestradiol: Rl=OCH ; R2=H

Fig. 10. Biosynthesis and steady-state control of catechol estrogens in human breast tissues

(Fig. 10) [172-174]. The carbon position of the estrogen molecules to be hy-droxylated differs among various tissues and each reaction is probably catalyzed by various CYP isoforms [175]. For example, in MCF-7 human breast cancer cells, which produce catechol estrogens in culture [176, 177], CYP1A1 catalyzes hy-droxylation of 17^-estradiol at C2, C15a and C16a, CYP1A2 predominantly at C2 [178], and a member of the CYP1B1 subfamily is responsible for the C4 hydrox-ylation of 17^-estradiol [179-181]. CYP3A4 and CYP3A5 have also been shown to play a role in the 16a-hydroxylation of estrogens in human [182, 183].

The hydroxylated estrogens are catechol estrogens that will easily be autooxi-dated to semiquinones and subsequently quinones, both of which are elec-trophiles capable of covalently binding to nucleophilic groups on DNA via a Michael addition and, thus, serve as the ultimate carcinogenic reactive intermediates in the peroxidative activation of catechol estrogens (Fig. 11) [184]. In addition, a redox cycle consisting of the reversible formation of the semiquinones and quinones of catechol estrogens catalyzed by microsomal P450 and cytochrome P450-reductase can locally generate superoxide and hydroxyl radicals to produce additional DNA damage (Fig. 11) [141, 142]. Furthermore, catechol estrogens have been shown to interact synergistically with nitric oxide present in human breast generating a potent oxidant that induces DNA strand breakage [185].

Steady-state concentrations of catechol estrogens are determined by the cytochrome P450-mediated hydroxylations of estrogens and monomethylation of

Fig. 11. Carcinogenic effects associated with the metabolisms of catechol estrogens in human breast tissues

catechols catalyzed by blood-borne catechol O-methyltransferase (Fig. 10) [173, 178,186,187]. Increased formation of catechol estrogens as a result of elevated hydroxylations of 17^-estradiol at C4 [181] and C16a [188-190] positions occurs in human breast cancer patients and in women at a higher risk of developing this disease. There is also evidence that lactoperoxidase, present in milk, saliva, tears and mammary glands, catalyzes the metabolism of 17^-estradiol to its phenoxyl radical intermediates, with subsequent formation of superoxide and hydrogen peroxide that might be involved in estrogen-mediated oxidative stress [191]. A substantial increase in base lesions observed in the DNA of invasive ductal carcinoma of the breast [192,193] has been postulated to result from the oxidative stress associated with metabolism of 17^-estradiol [191].

Clearly, the ability of the mammary gland to metabolize 17^-estradiol and/or to accumulate "genotoxic" metabolites could profoundly influence the neoplastic transformation of the epithelium [194]. Oxidative biotransformation of estradiol occurs in human mammary explant cultures [194,195). Treatment of normal mouse mammary epithelial cells with the mutagenic polycyclic hydrocarbon 7,12-dimethylbenzo[a]anthracene results in production of 16a-hydroxy-estrone as the predominant metabolite of estrogens, which increases unscheduled DNA synthesis, cellular proliferation and anchorage-independent growth, indicative of preneoplastic transformation [196]. In experimental animals, cat-echols have been implicated as mediators of estrogen-induced carcinogenesis [197]. Elevated metabolic conversion of 17^-estradiol to catechol estrogens has been documented in a number of organs susceptible to estrogen-induced carcinogenesis, including hamster kidney [174,178,198], mouse uterus [199,200] and rat pituitary [177, 198]. Modulation of catechol estrogen concentrations influences susceptibility to estrogen-inducible carcinogenesis. For instance, catecholamines, which are substrates and competitive inhibitors of the catechol O-methyltransferase, are present at much higher levels in the organs susceptible to estrogen-induced carcinogenesis [201]. Inhibition of the catechol O-methyl-transferase-catalyzed O-methylation of 2- and 4-hydroxy-17^-estradiol by quercetin, a flavonoid, increases accumulation of catechol estrogens [202] and augments the induction of estradiol-induced carcinogenesis [203]. In addition, a genetic polymorphism of catechol O-methyltransferase [i.e., low-activity allele] has been positively associated with an increased breast cancer risk in premenopausal women, especially in those overweight [204]. The carcinogenic potential of estrogens in normal human breast epithelial cells is currently under active investigation.

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