Catechol estrogen quinones as initiators of breast and other human cancers: implications for biomarkers of susceptibility and cancer prevention

Exposure to estrogens is associated with increased risk of breast and other types of human cancer. Estrogens are converted to metabolites, particularly the catechol estrogen-3,4-quinones (CE-3,4-Q), that can react with DNA to form depurinating adducts. These adducts are released from DNA to generate apurinic sites. Error-prone base excision repair of this damage may lead to the mutations that can initiate breast, prostate and other types of cancer. The reaction of CE-3,4-Q with DNA forms the depurinating adducts 4-hydroxyestrone(estradiol) [4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1-N7Gua. These two adducts constitute more than 99% of the total DNA adducts formed. Increased levels of these quinones and their reaction with DNA occur when estrogen metabolism is unbalanced. Such an imbalance is the result of overexpression of estrogen activating enzymes and/or deficient expression of the deactivating (protective) enzymes. This unbalanced metabolism has been observed in breast biopsy tissue from women with breast cancer, compared to control women. Recently, the depurinating adduct 4-OHE1(E2)-1-N3Ade has been detected in the urine of prostate cancer patients, but not in urine from healthy men. Mutagenesis by CE-3,4-Q has been approached from two different perspectives: one is mutagenic activity in the lacI reporter gene in Fisher 344 rats and the other is study of the reporter Harvey-ras gene in mouse skin and rat mammary gland. A-->G and G-->A mutations have been observed in the mammary tissue of rats implanted with the CE-3,4-Q precursor, 4-OHE2. Mutations have also been observed in the Harvey-ras gene in mouse skin and rat mammary gland within 6-12 h after treatment with E2-3,4-Q, suggesting that these mutations arise by error-prone base excision repair of the apurinic sites generated by the depurinating adducts. Treatment of MCF-10F cells, which are estrogen receptor-alpha-negative immortalized human breast epithelial cells, with E2, 4-OHE2 or 2-OHE2 induces their neoplastic transformation in vitro, even in the presence of the antiestrogen ICI-182,780. This suggests that transformation is independent of the estrogen receptor. The transformed cells exhibit specific mutations in several genes. Poorly differentiated adenocarcinomas develop when aggressively transformed MCF-10F cells are selected and injected into severe combined immune depressed (SCID) mice. These results represent the first in vitro/in vivo model of estrogen-induced carcinogenesis in human breast epithelial cells. In other studies, the development of mammary tumors in estrogen receptor-alpha knockout mice expressing the Wnt-1 oncogene (ERKO/Wnt-1) provides direct evidence that estrogens may cause breast cancer through a genotoxic, non-estrogen receptor-alpha-mediated mechanism. In summary, this evidence strongly indicates that estrogens can become endogenous tumor initiators when CE-3,4-Q react with DNA to form specific depurinating adducts. Initiated cells may be promoted by a number of processes, including hormone receptor stimulated proliferation. These results lay the groundwork for assessing risk and preventing disease.     

Contribution of estrogen receptors alpha and beta to the effects of estradiol in the brain

Clinical and experimental studies show a modulatory role of estrogens in the brain and suggest their beneficial action in mental and neurodegenerative diseases. The estrogen receptors ER and ERβ are present in the brain and their targeting could bring selectivity and reduced risk of cancer. Implication of ERs in the effect of estradiol on dopamine, opiate and glutamate neurotransmission is reviewed. The ER agonist, PPT, is shown as estradiol to modulate hippocampal NMDA receptors and AMPA receptors in cortex and striatum of ovariectomized rats whereas the ERβ agonist DPN is inactive. Striatal DPN activity suggests implication of ERβ in estradiol modulation of D2 receptors and transporters in ovariectomized rats and is supported by the lack of effect of estradiol in ERβ knockout (ERKOβ) mice. Both ER and ERβ agonists modulate striatal preproenkephalin (PPE) gene expression in ovariectomized rats. In male mice PPT protects against MPTP toxicity to striatal dopamine; this implicates Akt/GSK3β signaling and the apoptotic regulators Bcl2 and Bad. This suggests a role for ER in striatal dopamine neuroprotection. ERKO mice are more susceptible to MPTP toxicity and not protected by estradiol; differences in ERKOβ mice are subtler. These results suggest therapeutic potential for the brain of ER specific agonists.     

Estrogen receptor alpha is required for mammary development and the induction of mammary hyperplasia and epigenetic alterations in the aromatase transgenic mice

Aromatase transgenic mice exhibit hyperplastic and dysplastic changes, attesting to the importance of local estrogen in breast carcinogenesis. These mice also show increased levels of the estrogen receptor and β (ER, ERβ) suggesting that this receptor may play an important role in the initiation of estrogen-mediated mammary hyperplasia observed in these mice. To address the specific role of ER in the mammary development and in the induction of estrogen-mediated hyperplasia in aromatase transgenic mice, we have generated MMTV-aromatase × ER knockout cross (referred as aromatase/ERKO). Even though ERβ is expressed in aromatase/ERKO mice, lack of ER leads to impaired mammary growth in these mice. The data suggest that ER plays an important role in the mammary gland development as well as in the induction of mammary hyperplasia in aromatase transgenic mice. Lack of ER expression in the aromatase/ERKO mice resulted in a decrease in the expression of Cyclin D1, PCNA and TGFβ relative to the aromatase parental strain. The studies involving aromatase/ERKO mice show that lack of ER results in impaired mammary development even in the presence of continuous tissue estrogen, suggesting estrogen/ER-mediated actions are critical for mammary development and carcinogenesis.     

Recent data on intratumor estrogens in breast cancer

The contribution of local synthesis versus circulatory delivery of normal breast as well as breast cancer tissue estrogens has remained a controversial area for decades. Novel data on tissue estrogen levels confirm a positive normal breast tissue to plasma concentration gradient for estrone, and to a smaller extent estradiol. Remarkably, this gradient is similar for pre- and post-menopausal women. Together with pharmacokinetic data on estrogen disposition, these findings suggest plasma and breast tissue estrogens to rapidly equilibrate, with circulating estrogens being a major contributor to breast tissue estrone levels. A likely explanation to the concentration gradient could be the fact that non-polar estrogens easily dissolve into tissue fat compartments as compared to plasma. While intratumor estrone levels are low as compared to benign tissue concentrations, intratumor estradiol is elevated in ER+ tumors. The correlation between intratumor estradiol levels and expression levels of dehydrogenases reducing estrone into estradiol but also intratumor ER concentrations are consistent with intratumor estrogen activation but also a scavenger effect of the ER.     

Diaryl dimers of estradiol and of estrone may be formed as major metabolites by mouse mammary glands

The formation of a novel estrogen metabolite by mammary tissues was investigated. Polar and nonpolar metabolites of endogenous estrogens are formed in liver and other tissues. Polar products such as the catechol estrogens are implicated in tumorigenesis in breast tissue, whereas a nonpolar metabolite, 2-methoxyestradiol, may be protective. Diaryl ether dimers, as a novel form, have been reported as nonpolar products from liver microsomes. We have noted major amounts of nonpolar metabolites in other tissues that were neither 2-methoxyestrogens nor estrogen fatty acid esters. The possible formation of such novel metabolites by breast tissues from adult nulliparous mice with [³H]-labeled estrogens as substrates was considered. Steroids were recovered from media by solid-phase extraction and profiles were obtained from HPLC (acetonitrile:water). Saponification was done with an internal standard of estradiol stearate. Major amounts of nonpolar metabolites were formed in all instances, with one or two principal peaks. Alkaline hydrolysis had no effect on the nonpolar product(s) but released estradiol from its stearate. Strong acid treatment also had no effect as shown by HPLC. Thus, it is suggested that diaryl dimers of estrogens may be formed as major metabolites by mouse mammary glands.     

Deconstructing estradiol: Removal of B-ring generates compounds which are potent and subtype-selective estrogen receptor agonists

Estradiol and related estrogens have been widely used as supplements to relieve menopausal symptoms, but they lead to an increased risk of breast and endometrial cancer. Here we report the synthesis of a new family of compounds where we have removed the B-ring from the steroid ABCD structure, and functionalized the A-ring. These A-CD compounds show a preferential affinity for the estrogen receptor subtype ERβ. Some show binding affinities which are greater than estradiol. The presence of electron-withdrawing substituents on the A-ring should reduce the tendency of these compounds to form carcinogenic metabolites, so they might lead to a safer approach to hormone replacement therapy.     

Estrogen resistance and aromatase deficiency in humans

The primordial role of estrogens in female reproductive function is well known. The recent production of transgenic mice deficient in estrogen receptors (ERKO) or in aromatase (ArKO) and the discovery in man of inactivating mutations of the corresponding genes (ER) have contributed to the understanding of the role of estrogens in metabolic processes in female as well as male. To date 8 well documented cases (5 women and 3 men) of congenital deficiencies in estrogens have been reported. As mice deficient in ERa had been previously described, these cases definitely proved that estrogen absence was compatible with survival and disproved the "lethality concept" previously held because the role of estrogens in implantation and gestation maintenance. ERKO mice are phenotypically normal though sterile, but their bone density is lower (20-25%) than that of controls. Similarly, men with no aromatase or no ER display continuous growth, osteoporosis and also (but not necessarily) alterations in testicular functions. How much do primordial functions such as bone development, control of gonadotrophin secretion and lipid metabolism depend on estrogens? These interrogations, elegantly clarified following testosterone and estradiol treatment in an aromatase deficient man are considered in this present synthesis.     

Unbalanced metabolism of endogenous estrogens in the etiology and prevention of human cancer

Among the numerous small molecules in the body, the very few aromatic ones include the estrogens and dopamine. In relation to cancer initiation, the estrogens should be considered as chemicals, not as hormones. Metabolism of estrogens is characterized by two major pathways. One is hydroxylation to form the 2- and 4-catechol estrogens, and the second is hydroxylation at the 16α position. In the catechol pathway, the metabolism involves further oxidation to semiquinones and quinones, including formation of the catechol estrogen-3,4-quinones, the major carcinogenic metabolites of estrogens. These electrophilic compounds react with DNA to form the depurinating adducts 4-OHE(1)(E(2))-1-N3Ade and 4-OHE(1)(E(2))-1-N7Gua. The apurinic sites obtained by this reaction generate the mutations that may lead to the initiation of cancer. Oxidation of catechol estrogens to their quinones is normally in homeostasis, which minimizes formation of the quinones and their reaction with DNA. When the homeostasis is disrupted, excessive amounts of catechol estrogen quinones are formed and the resulting increase in depurinating DNA adducts can lead to initiation of cancer. Substantial evidence demonstrates the mutagenicity of the estrogen metabolites and their ability to induce transformation of mouse and human breast epithelial cells, and tumors in laboratory animals. Furthermore, women at high risk for breast cancer or diagnosed with the disease, men with prostate cancer, and men with non-Hodgkin lymphoma all have relatively high levels of estrogen-DNA adducts, compared to matched control subjects. Specific antioxidants, such as N-acetylcysteine and resveratrol, can block the oxidation of catechol estrogens to their quinones and their reaction with DNA. As a result, the initiation of cancer can be prevented.     

Relationship between estrogen receptors, 17 beta-hydroxysteroid dehydrogenase and estrogen content in human breast cancer.

Estrone and estradiol levels in tumor tissue cytosols were determined in 11 premenopausal and 20 postmenopausal women at the same time that 17 beta-hydroxysteroid dehydrogenase and estrogen receptors (ER) were carried out on their breast cancers. Estrogen receptor positive tumors showed significantly higher levels of estrone and estradiol. However, all ER negative tumors contained measurable amounts of both estradiol and estrone. Higher levels of estrone were observed in ER negative tumors which correlates well with high 17 beta-hydroxysteroid dehydrogenase activity. These results suggest that false negative receptor assays in the premenopausal women is not likely to be due to occupancy of receptors by endogenous estrogens. Furthermore, the higher estrone content in the ER negative group is probably due to high 17 beta-hydroxysteroid dehydrogenase activity inherent to these tumor cells.     

The complexity of anti-estrogen responses

The actions and biological responses of anti-estrogens are a function of: the experimental conditions, the parameters, the organ and the animal species considered. Target tissues for estrogens in the guinea-pig during the perinatal period are interesting models to explore the action of anti-estrogens. The summary of the data indicates: (1) In the fetal uterus of guinea-pig in in vivo experiments (after injection to the maternal compartment) tamoxifen acts as a real agonist concerning growth, as a partial agonist concerning the stimulation of the progesterone receptor. (2) In in vitro experiments (in organ culture of fetal uterus or in isolated cells) anti-estrogens (tamoxifen or 4-hydroxy-tamoxifen) act as antagonists and also inhibit the effects provoked by estrogens. (3) In the uterus and vagina of newborn guinea-pigs, tamoxifen and its derivatives: 4-hydroxytamoxifen and N-desmethyltamoxifen act as real agonists concerning the uterotrophic and vaginotrophic effects, and also stimulate the amount of DNA per organ, but concerning the progesterone receptor in the uterus, in the short treatment anti-estrogens act as partial agonists but they have no effect in the long treatment. In the vagina in the short treatment anti-estrogens provoke no significant effects, but in the long treatment they are full agonists. In neither of the two biological responses studied (growth and progesterone receptor) does tamoxifen and its derivatives block the action of estradiol. (4) The use of a monoclonal antibody to the estrogen receptor revealed quantitative differences in the activation of the estrogen receptor when bound to estradiol or tamoxifen. This observation was in agreement with the lesser extent of binding to DNA-cellulose of the tamoxifen-estrogen receptor complex as compared with the estradiol-estrogen receptor complex. This fact suggests an impaired activation of the estrogen receptor induced by tamoxifen which might be related to the different biological responses provoked by estrogens and anti-estrogens.     

HER-2/neu x aromatase double transgenic mice model: the effects of aromatase overexpression on mammary tumorigenesis

A majority of breast cancers are hormone-responsive, and require estrogen for growth, and respond to hormonal therapy that blocks estrogen receptor action. Breast tumors with low levels of or completely lacking estrogen receptor fail to respond to antiestrogen therapy yet require estrogen for tumor initiation. To address the importance of local estrogen in oncogene-mediated breast tumorigenesis, we have crossed MMTV-aromatase with MMTV-HER2/neu and examined the incidence of breast cancer in double transgenic mice in comparison with parental strains. Double transgenic mice show normal mammary development and express both transgenes at similar levels to that of parental strains. Tumor incidence in double transgenic mice (<5%) decreased compared to HER2/neu mice (>65%). In addition to a significant decrease in tumorigenesis, these mice expressed ER as well as high levels of ERβ along with decreased levels of cyclin D1 and phosphorylated pRb among other changes. Furthermore, experiments using THC (ER-agonist and ERβ-antagonist) clearly demonstrate the critical role of ERβ in HER2/neu-mediated tumorigenesis. These studies provide the first genetic evidence that estrogen receptor, mainly ERβ than ER and its dependent changes play an important role in regulating mammary tumorigenesis. These findings provide further evidence for development and testing of novel therapeutic approaches based on selective regulation of estrogen receptors (ER and β)-dependent actions for the treatment and prevention of breast cancers.     

Formation of the depurinating N3adenine and N7guanine adducts by reaction of DNA with hexestrol-3',4'-quinone or enzyme-activated 3'-hydroxyhexestrol. Implications for a unifying mechanism of tumor initiation by natural and synthetic estrogens

The nonsteroidal synthetic estrogen hexestrol (HES), which is diethylstilbestrol hydrogenated at the C-3-C-4 double bond, is carcinogenic. Its major metabolite is the catechol, 3'-OH-HES, which can be metabolically converted to the catechol quinone, HES-3',4'-Q. Study of HES was undertaken with the scope to substantiate evidence that natural catechol estrogen-3,4-quinones are endogenous carcinogenic metabolites. HES-3',4'-Q was previously shown to react with deoxyguanosine to form the depurinating adduct 3'-OH-HES-6'-N7Gua by 1,4-Michael addition [Jan S-T, Devanesan PD, Stack DE, Ramanathan R, Byun J, Gross ML, et al. Metabolic activation and formation of DNAadducts of hexestrol,a synthetic nonsteroidal carcinogenic estrogen. Chem Res Toxicol 1998;11:412-9.]. We report here formation of the depurinating adduct 3'-OH-HES-6'-N3Ade by reaction of HES-3',4'-Q with Ade by 1,4-Michael addition. The structure of the N3Ade adduct was established by NMR and MS. We also report here formation of the depurinating 3'-OH-HES-6'-N7Gua and 3'-OH-HES-6'-N3Ade adducts by reaction of HES-3',4'-Q with DNA or by activation of 3'-OH-HES by tyrosinase, lactoperoxidase, prostaglandin H synthase or 3-methylcholanthrene-induced rat liver microsomes in the presence of DNA. The N3Ade adduct was released instantaneously from DNA, whereas the N7Gua adduct was released with a half-life of approximately 3 h. Much lower (<1%) levels of unidentified stable adducts were detected in the DNA from these reactions. These results are similar to those obtained by reaction of endogenous catechol estrogen-3,4-quinones with DNA. The similarities extend to the instantaneously-depurinating N3Ade adducts and relatively slowly-depurinating N7Gua adducts. The endogenous estrogens, estrone and estradiol, their 4-catechol estrogens and HES are carcinogenic in the kidney of Syrian golden hamsters. These results suggest that estrone (estradiol)-3,4-quinones and HES-3',4'-Q are the ultimate carcinogenic metabolites of the natural and synthetic estrogens, respectively. Reaction of the electrophilic quinones by 1,4-Michael addition with DNA at the nucleophilic N-3 of Ade and N-7 of Gua is suggested to be the major critical step in tumor initiation by these compounds.     

Estrogen receptor 1 modulates circadian rhythms in adult female mice

Estradiol influences the level and distribution of daily activity, the duration of the free-running period, and the behavioral phase response to light pulses. However, the mechanisms by which estradiol regulates daily and circadian rhythms are not fully understood. We tested the hypothesis that estrogens modulate daily activity patterns via both classical and “non-classical” actions at the estrogen receptor subtype 1 (ESR1). We used female transgenic mice with mutations in their estrogen response pathways; ESR1 knock-out (ERKO) mice and “non-classical” estrogen receptor knock-in (NERKI) mice. NERKI mice have an ESR1 receptor with a mutation in the estrogen-response-element binding domain, allowing only actions via “non-classical” genomic and second messenger pathways. Ovariectomized female NERKI, ERKO, and wildtype (WT) mice were given a subcutaneous capsule with low- or high-dose estradiol and compared with counterparts with no hormone replacement. We measured wheel-running activity in a light:dark cycle and constant darkness, and the behavioral phase response to light pulses given at different points during the subjective day and night. Estradiol increased average daily wheel-running, consolidated activity to the dark phase, and shortened the endogenous period in WT, but not NERKI and ERKO mice. The timing of activity onset during entrainment was advanced in all estradiol-treated animals regardless of genotype suggesting an ESR1-independent mechanism. We propose that estradiol modifies period, activity level, and distribution of activity via classical actions of ESR1 whereas an ESR1 independent mechanism regulates the phase of rhythms.     

Estrogen, DNA damage and mutations

Estrogen administration to rodents results in various types of DNA damage and ultimately leads to tumors in estrogen-responsive tissues. Yet these hormones have been classified as nonmutagenic, because they did not induce mutations in classical bacterial and mammalian mutation assays. In this review, we have discussed the induction by estrogens of DNA and chromosomal damage and of gene mutations, because the classical assays were designed to uncover mutations only at one specific locus and could not have detected other types of mutations or changes in other genes. Various types of estrogen-induced DNA damage include: (a) direct covalent binding of estrogen quinone metabolites to DNA; (b) enhancement of endogenous DNA adducts by chronic estrogen exposure of rodents; (c) free radical generation by metabolic redox cycling between quinone and hydroquinone forms of estrogens and free radical damage to DNA such as strand breakage, 8-hydroxylation of purine bases of DNA and lipid hydroperoxide-mediated DNA modification. Two different types of chromosomal damage have also been induced by estrogen in vivo and in cells in culture such as numerical chromosomal changes and also structural chromosomal aberrations. Gene mutations have been induced in several cell types in culture either by the parent estrogen or by reactive estrogen quinone metabolites. Furthermore, in estrogen-induced kidney tumors in hamsters, several mutations have been observed in the DNA polymerase beta gene mRNA. Estradiol also induces microsatellite instability in these kidney tumors and in premalignant kidney exposed to estradiol. Although this work is still ongoing, it can be concluded that estrogens are complete carcinogens capable of tumor initiation by mutation potentially in critical genes. The hormonal effects of estrogens may complete the development of tumors.     

Tissue estradiol is selectively elevated in receptor positive breast cancers while tumour estrone is reduced independent of receptor status

Previous studies have suggested elevated estrogen production in tumour-bearing breast quadrants as well as in breast cancers versus benign tissue. Using highly sensitive assays, we determined breast cancer tissue estrogen concentrations together with plasma and benign tissue estrogen concentrations in each quadrant obtained from mastectomy specimens (34 postmenopausal and 13 premenopausal women). We detected similar concentrations of each of the three major estrogens estradiol (E₂), estrone (E₁) and E₁S in tumour-bearing versus non-tumour-bearing quadrants. Considering malignant tumours, intratumour E₁ levels were reduced in cancer tissue obtained from pre- as well as postmenopausal women independent of tumour ER status (average ratio E₁ cancer: benign tissue of 0.2 and 0.3, respectively; p < 0.001 for both groups), suggesting intratumour aromatization to be of minor importance. The most striking finding was a significant (4.1–8.6-fold) increased E₂ concentration in ER positive tumours versus normal tissue (p < 0.05 and <0.001 for pre- and postmenopausal patients, respectively), contrasting low E₂ concentrations in ER− tumours (p < 0.01 and <0.001 comparing E₂ levels between ER+ and ER− tumours in pre- and postmenopausals, respectively). A possible explanation to our finding is increased ligand receptor binding capacity for E₂ in receptor positive tumours but alternative factors influencing intratumour estrogen disposition cannot be excluded.     

Analysis of 3D models of octopus estrogen receptor with estradiol: evidence for steric clashes that prevent estrogen binding

Relatives of the vertebrate estrogen receptor (ER) are found in Aplysia californica, Octopus vulgaris, Thais clavigera, and Marisa cornuarietis. Unlike vertebrate ERs, invertebrate ERs are constitutively active and do not bind estradiol. To investigate the molecular basis of the absence of estrogen binding, we constructed a 3D model of the putative steroid-binding domain on octopus ER. Our 3D model indicates that binding of estradiol to octopus ER is prevented by steric clashes between estradiol and amino acids in the steroid-binding pocket. In this respect, octopus ER resembles vertebrate estrogen-related receptors (ERR), which have a ligand-binding pocket that cannot accommodate estradiol. Like ERR, octopus ER also may have the activation function 2 domain (AF2) in a configuration that can bind to coactivators in the absence of estrogens, which would explain constitutive activity of octopus ER.     

Regioselective deuterium labeling of estrone and catechol estrogen metabolites

Increased exposure to estrogens and estrogen metabolites is linked with increased rates of breast, ovarian and other human cancers. Metabolism of estrogen can led to formation of electrophilic o-quinones capable of binding to DNA. In order to gain insight into the mechanism of estrogen-induced DNA damage, estrone and catechol estrogens derived from estrone, have been regioselectively labeled with deuterium at the 1-position. Estrone-1-d, estrone-1,2,4-d₃, 4-hydroxyestrone-1-d and 2-hydroxyestrone-1-d have been synthesized with or without deuteriums at the 16-position. The key labeling step involves deuterated trifluoroacetic acid exchange catalyzed by t-butyl alcohol. This economical, straightforward labeling technique makes available a range of estrone compounds containing deuterium at the 1-position.