Estrogens in the causation of breast, endometrial and ovarian cancers - evidence and hypotheses from epidemiological findings

Estrogens along with progesterone/progestins, and other hormones, are important determinants of cancer in the breast, endometrium and ovary. Estrogens may increase the risk of breast cancer through various mechanisms and at various phases of life, with a possible synergistic effect of progesterone/progestins. Exposure to high doses of placental hormones, such as estrogens and/or progesterone, during pregnancy may play a pivotal role in reducing subsequent breast cancer susceptibility. Estrogens cause endometrial cancer, an effect that can be reduced, prevented or reversed by progesterone/progestin — if allowed to act for a sufficiently long period of each cycle. The role of sex hormones seems important for ovarian carcinogenesis. Intake of combined oral contraceptives has a substantial and well-documented protective effect on endometrial and ovarian cancer risks. Epidemiological observations and experimental data from an animal model indicate that estrogens may have an adverse effect, while progesterone/progestins have a risk reducing effect directly on the ovarian epithelium. Thus, estrogens and other sex hormones have potential effects on the three most important female cancers. Research has yet to define how some of the risk factors can be modified or treatment regimens can be improved to reduce these cancer risks.     

Hormonal carcinogenesis in women: from mechanisms to prevention

In spite of the explosion of basic knowledge, breast cancer remains a major problem of public health. Basic endocrinology was at the origin of the first targeted therapy in cancer with the anti-estrogens. Continuous breast cancer cell lines helped to specify the mechanism of the mitogenic activity of estrogens, the basis of their tumour promoter activity. They could not help to explain the deleterious effect of progestins after the menopause and to study the effect of ovarian hormones in the early steps of carcinogenesis. The variations of expression of ovarian hormone receptors in pre-malignant mammary lesions strongly suggest an increased sensitivity to these hormones. Moreover breast carcinogenesis is heterogeneous and the oestrogen receptor negative pathways require other targeted therapies. A better prevention of hormone-dependent cancers will need both increased basic researches translatable to human and good information of women on the avoidable risk factors.     

17beta-estradiol inhibits forskolin-induced vascular endothelial growth factor promoter in MCF-7 breast adenocarcinoma cells.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor whose expression is induced by the cAMP-dependent signalling pathway in several cell types, and by estrogens in some human breast cancer cells. Here, we investigated the cross-talk between estrogens and cAMP/PKA-dependent signalling pathway in human breast cancer MCF-7 cells. The results show that, in the absence of any CRE and ERE, forskolin induces whereas estrogens have no effect on VEGF promoter. Moreover, estrogens, through estrogen receptors, partly inhibit the forskolin-induced VEGF promoter in MCF-7 human breast cancer cells. Therefore, in breast cancers, estrogens could partly inhibit the effect of ligand-activated G protein-coupled receptors on VEGF expression.     

Endogenous estrogen, testosterone and progesterone levels in relation to breast cancer risk

Multiple lines of evidence support a central role of hormones in the etiology of breast cancer. In epidemiologic studies, considerable effort has focused on delineating the role of endogenous hormones in risk of breast cancer among postmenopausal women. Recently, substantial additional data has accrued from prospective studies where endogenous hormones are measured in study subjects prior to disease diagnosis. In this review, the epidemiologic evidence linking sex steroids—estrogens, testosterone, and progesterone, specifically—with subsequent risk of breast cancer in both premenopausal and postmenopausal women is summarized. Overall, a strong positive association between breast cancer risk and circulating levels of both estrogens and testosterone has now been well confirmed among postmenopausal women; women with hormone levels in the top 20% of the distribution (versus bottom 20%) have a two- to three-fold higher risk of breast cancer. Evidence among premenopausal women is more limited, though increased risk associated with higher levels of testosterone is consistent. However, both positive and null associations have been observed with estrogens and progesterone and clearly more evaluation is needed.     

Molecular epidemiology of breast cancer: genetic variation in steroid hormone metabolism

The age-specific incidence rate of breast cancer in women rises until menopause, levels off and then rises again at a much lower rate indicating a possible hormonal influence on the disease risk. A large amount of evidence has implicated hormones and other compounds with oestrogen activity in the pathogenesis of certain endocrine cancers, particularly breast cancer. Widely dispersed hormone-like chemicals, capable of disrupting the endocrine system and interfering with proliferation, have been described. Compounds such as dioxins, some polychlorinated biphenyls and the plastic ingredient bisphenol-A have been shown to interfere with human reproduction and hormonal regulation. The levels of these foreign compounds as well as the levels of endogenous oestradiol may influence the risk of breast cancer. Endogenous oestradiol is synthesised in the ovarian theca cells of premenopausal women or in the stromal adipose cells of the breast of postmenopausal women and minor quantities in peripheral tissue. These cells, as well as breast cancer tissue, express all the necessary enzymes for this synthesis: CYP17, CYP11a, CYP19, hydroxysteroid hydrogenase, steroid sulphatase as well as enzymes further hydroxylating oestradiol such as CYP1A1, CYP3A4, CYP1B1. Polymorphisms in these enzymes may have a possible role in the link between environmental estrogens and hormone-like substances and the interindividual risk of breast cancer.     

YTHDF1 promotes breast cancer cell growth,DNA damage repair and chemoresistance

Chemoresistance represents a major obstacle to the treatment of human cancers. Increased DNA repair capacity is one of the important mechanisms underlying chemoresistance. In silico analysis indicated that YTHDF1, an m6A binding protein, is a putative tumor promoter in breast cancer. Loss of function studies further showed that YTHDF1 promotes breast cancer cell growth in vitro and in vivo. YTHDF1 facilitates S-phase entry, DNA replication and DNA damage repair, and accordingly YTHDF1 knockdown sensitizes breast cancer cells to Adriamycin and Cisplatin as well as Olaparib, a PARP inhibitor. E2F8 is a target molecule by YTHDF1 which modulates E2F8 mRNA stability and DNA damage repair in a METTL14-dependent manner. These data demonstrate that YTHDF1 has a tumor-promoting role in breast cancer, and is a novel target to overcome chemoresistance.Subject terms: Breast cancer, Breast cancer     

Aromatase in the normal breast and breast cancer

Adipose tissue and muscle constitute the larger proportion of body mass, and therefore aromatization in these tissues is the major source of circulating estrogens in postmenopausal women. Although plasma estrogen concentrations are very low, levels in breast cancers from postmenopausal patients are reported to be 10-fold higher than in plasma and normal tissue. Whereas studies on aromatase activity in the tumor suggest that estrogen may be produced locally, the significance of this contribution has been questioned. Using immunocytochemistry (ICC) to an anti-aromatase antibody, a relatively strong immunoreaction was detected in tumor epithelial cells as well as in the terminal ductal lobular units (TDLUs) of the normal breast. Aromatase expression was detected in the cytoplasm of tumor epithelial cells and the surrounding stromal cells of over 50% of tumors in a series of 19 breast cancers. In situ hybridization (ISH) to aromatase mRNA confirmed the immunocytochemical result that the epithelial cells are the primary site of estrogen synthesis in the breast and breast cancers. In the 10 tumors which showed immunoreaction to aromatase, the average aromatase activity measured in cryosections was 286.5 ± 18.6 fmol estrogen/mg protein/h (SE), whereas in nine tumors with weak aromatase immunoreaction, the enzyme activity was 154.7 ± 19.3 fmol estrogen/mg protein/h (P < 0.05) (SE). The functional significance of tumor aromatase and locally produced estrogens on the growth of tumors was suggested by the correlation between aromatase activity and proliferating cell nuclear antigen (PCNA), a marker of cell proliferation (P < 0.005). Although intratumoral aromatase activity did not correlate with steroid receptors significantly, there was a trend for estrogen receptor (ER)-positive tumors to express aromatase. In addition, proliferation ([³H]-thymidine incorporation into DNA) during histoculture, was increased by both estradiol and testosterone in tumors with high aromatase activity. Our results suggest that some tumors synthesize sufficient estrogen to stimulate their proliferation. It may thus be important to inhibit tumor aromatase as well as to reduce circulating levels of estrogen for effective breast cancer treatment.     

BRCA1-directed,enhanced and aberrant homologous recombination

Despite intense studies, questions still remain regarding the molecular mechanisms leading to the development of hereditary breast and ovarian cancers. Research focused on elucidating the role of the breast cancer susceptibility gene 1 (BRCA1) in the DNA damage response may be of the most critical importance to understanding these processes. The BRCA1 protein has an N-terminal RING domain possessing E3 ubiquitinligase activity and a C-terminal BRCT domain involved in binding specific phosphoproteins. These domains are involved directly or indirectly in DNA double-strand break (DSB) repair. As the two terminal domains of BRCA1 represent two separate entities, understanding how these domains communicate and are functionally altered in regards to DSB repair is critical for understanding the development of BRCA1-related breast and ovarian cancers and for developing novel therapeutics. Herein, we review recent findings of how altered functions of these domains might lead to cancer through a mechanism of increased aberrant homologous recombination and possible implications for the development of BRCA1 inhibitors.     

Contributions of estrogen to ER-negative breast tumor growth

Breast cancer is a hormone-based disease with numerous factors contributing to the lifetime risk of developing the disease. While breast cancer risk is reduced by nearly 50% after one full term pregnancy, women over the age of 25 have a significantly greater risk of developing breast cancer immediately following parturition compared to their nulliparous counterparts. It is widely presumed that the increased risk of developing breast cancer following pregnancy is due to the ability of pregnancy-associated hormones to promote the further proliferation of an initiated target cell population. It is surprising however, that the majority of breast cancers that develop following pregnancy lack appreciable expression of either the estrogen or progesterone receptors. This important observation suggests that if hormones play a part in promoting breast cancer following pregnancy, they may not be doing so through direct binding to hormone receptor molecules expressed by breast cancer cells.

To reconcile this conceptual conflict we investigated the hypothesis that steroid hormones promote the outgrowth of ER-negative cancers by influencing host cell types distinct from the breast epithelium itself. We demonstrated that increasing the levels of circulating estrogens is sufficient to promote the formation and progression of ER-negative cancers while, pharmacologically inhibiting estrogen synthesis following pregnancy prevents ER-negative tumor formation. Moreover, we demonstrate that the effects of estrogen act via a systemic increase in host angiogenesis, in part through increased mobilization and recruitment of bone marrow stromal derived cells into sites of angiogenesis and to a growing tumor mass. Taken together, these data suggest that estrogen may promote the growth of ER-negative cancers by acting on cells distinct from the cancer cells to stimulate angiogenesis.     

Role of steroid sulfatase in local formation of estrogen in post-menopausal breast cancer patients

More than two-thirds of breast cancers occur in post-menopausal women, and depend on the estrogens for their proliferation and survival. For the treatment of estrogen-dependent breast cancers, two major treatment options are now available. One is selective estrogen receptor modulator (SERM) such as Tamoxifen and another is aromatase inhibitor such as Anastrozole, Letrozole and Exemestane, which reduce local in situ formation of estrogens. Although these therapies are clinically active for advanced and early breast cancers, de novo and/or acquired resistance to SERM and/or aromatase inhibitors are also clinical problem. Recent studies suggest that local formation of estrogens in the breast tumors is more important than circulating estrogen in plasma for the growth and survival of estrogen-dependent breast cancer in post-menopausal women. The rationale for the importance of local formation of estrogens is based on the following evidences. Estradiol (E2) levels in breast tumors are equivalent to those of pre-menopausal patients, although plasma E2 levels are 50-fold lower after menopause. E2 concentrations in breast tumors of post-menopausal women are 10–40 times higher than serum level. Biosynthesis of estrogens in breast tumors tissues occurs via two major different routes, one is aromatase pathway and another is steroid-sulfatase (STS) pathway. Whereas many studies has been reported about aromatase inhibitor and its clinical trial results in breast cancer patients, limited information are available regarding to other estrogen regulating enzymes including STS, its role in breast tumors and STS inhibitors. STS is the enzyme that hydrolyses estrone 3-sulfate (E₁S) and dehydroepiandrosterone-sulfate (DHEA-S) to their active un-sulfoconjugated forms, thereby stimulating the growth and survival of estrogen-dependent breast tumors. It has been well known that E₁S level are much higher than E2 level both in plasma and tumor of post-menopausal patients. Recent reports show that more than 80% of breast tumors are stained with anti-STS antibody and the expression of STS is an independent prognostic factor in breast cancer. Taking these findings into consideration, local formation of estrogens could be partially synthesized from large amount of E₁S by STS, which exist in breast cancer. On the other hand, aromatase localizes in stroma and adipocyte surrounding breast cancer. Furthermore, since estrogen formation from E₁S and DHEA-S (STS pathway) cannot be blocked by aromatase inhibitors, STS is thought to be a new molecular target for the treatment of estrogen-dependent tumor post-SERM and/or aromatase inhibitors. In this symposium, these recent rationale for the importance of STS in post-menopausal breast cancer patients is reviewed as well as STS inhibitor.     

Estrogen receptor mediated inhibition of cancer cell invasion and motility: An overview

In this overview of results from our laboratory, we address the question of the role of estrogens during early steps of metastasis, involving cell invasion through the basement membrane and cell motility. The motility of several estrogen receptor (ER) positive breast (MCF7, T47D) and ovarian (BG-1, SKOV3, PEO4) cancer cell lines was studied using a modified Boyden chamber assay. We observed, in all cases, estradiol induced inhibition of cancer cell invasion and motility. A similar inhibitory effect of estradiol was found when the wild-type ER was stably transfected in the ER-negative MDA-MB231 cells and 3Y1-Ad12 cancer cells. The mechanism of this inhibitory effect is unknown. In ovarian cancer, however, it may involve intermediary proteins such as fibulin-1, an extracellular matrix protein that strongly interacts with fibronectin and which is induced by estrogen and secreted by ovarian cancer cells. We conclude that estrogens in ER-positive breast and ovarian cancers have a dual effect, since they stimulate tumor growth but inhibit invasion and motility. This may be consistent with the good initial prognostic value of ER-positive breast cancers compared to ER negative breast cancers noted in several clinical studies.     

Hormones and cancer in humans

Hormones play a major role in the aetiology of several of the commonest cancers worldwide, including cancers of the endometrium, breast and ovary in women and cancer of the prostate in men. It is likely that the main mechanisms by which hormones affect cancer risk are by controlling the rate of cell division, the differentiation of cells and the number of susceptible cells. Hormones have very marked effects on cell division in the endometrium; oestrogens stimulate mitosis whereas progestins oppose this effect. The risk for endometrial cancer increases with late menopause, oestrogen replacement therapy and obesity, and decreases with parity and oral contraceptive use; thus risk increases in proportion to the duration of exposure to oestrogens unopposed by progestins, probably because unopposed oestrogens stimulate endometrial cell division. The effects of hormones on breast epithelial cell division in non-pregnant women are much less clear-cut than their effects on the endometrium, but both oestrogens and progestins appear to stimulate mitosis. Breast cancer risk increases with early menarche, late menopause and oestrogen replacement therapy, probably due to increased exposure of the breasts to oestrogen and/or progesterone. Early first pregnancy and multiparity reduce the risk for breast cancer, probably due to the hormonally-induced differentiation of breast cells and the corresponding reduction in the number of susceptible cells. Hormones do not have marked direct effects on the epithelial cells covering the ovaries, but hormones stimulate ovulation which is followed by cell division during repair of the epithelium. Risk for ovarian cancer increases with late menopause and decreases with parity and oral contraceptive use, suggesting that the lifetime number of ovulations may be a determinant of risk. For all three of these cancers risk changes within a few years of changes in exposure to sex hormones and some of the changes in risk persist for many years, indicating that hormones can affect both early and late stages of carcinogenesis. Understanding of the role of sex hormones in the aetiology of prostate cancer and of some rarer cancers is less complete.     

The selective estrogen enzyme modulators in breast cancer: a review

It is well established that increased exposure to estradiol (E(2)) is an important risk factor for the genesis and evolution of breast tumors, most of which (approximately 95-97%) in their early stage are estrogen-sensitive. However, two thirds of breast cancers occur during the postmenopausal period when the ovaries have ceased to be functional. Despite the low levels of circulating estrogens, the tissular concentrations of these hormones are significantly higher than those found in the plasma or in the area of the breast considered as normal tissue, suggesting a specific tumoral biosynthesis and accumulation of these hormones. Several factors could be implicated in this process, including higher uptake of steroids from plasma and local formation of the potent E(2) by the breast cancer tissue itself. This information extends the concept of 'intracrinology' where a hormone can have its biological response in the same organ where it is produced. There is substantial information that mammary cancer tissue contains all the enzymes responsible for the local biosynthesis of E(2) from circulating precursors. Two principal pathways are implicated in the last steps of E(2) formation in breast cancer tissues: the 'aromatase pathway' which transforms androgens into estrogens, and the 'sulfatase pathway' which converts estrone sulfate (E(1)S) into E(1) by the estrone-sulfatase. The final step of steroidogenesis is the conversion of the weak E(1) to the potent biologically active E(2) by the action of a reductive 17beta-hydroxysteroid dehydrogenase type 1 activity (17beta-HSD-1). Quantitative evaluation indicates that in human breast tumor E(1)S 'via sulfatase' is a much more likely precursor for E(2) than is androgens 'via aromatase'. Human breast cancer tissue contains all the enzymes (estrone sulfatase, 17beta-hydroxysteroid dehydrogenase, aromatase) involved in the last steps of E(2) biosynthesis. This tissue also contains sulfotransferase for the formation of the biologically inactive estrogen sulfates. In recent years, it was demonstrated that various progestins (promegestone, nomegestrol acetate, medrogestone, dydrogesterone, norelgestromin), tibolone and its metabolites, as well as other steroidal (e.g. sulfamates) and non-steroidal compounds, are potent sulfatase inhibitors. Various progestins can also block 17beta-hydroxysteroid dehydrogenase activities. In other studies, it was shown that medrogestone, nomegestrol acetate, promegestone or tibolone can stimulate the sulfotransferase activity for the local production of estrogen sulfates. All these data, in addition to numerous agents which can block the aromatase action, lead to the new concept of 'Selective Estrogen Enzyme Modulators' (SEEM) which can largely apply to breast cancer tissue. The exploration of various progestins and other active agents in trials with breast cancer patients, showing an inhibitory effect on sulfatase and 17beta-hydroxysteroid dehydrogenase, or a stimulatory effect on sulfotransferase and consequently on the levels of tissular levels of E(2), will provide a new possibility in the treatment of this disease.     

Proliferative effects of estradiol- or ethinylestradiol-progestogen combinations on human breast cancer cells in an intermitted and a long-term regimen

Currently the use of natural estradiol as estrogenic component in oral contraceptives is more and more extended. It is unknown whether the application of this estrogen is associated with a different breast cancer risk as compared to the common use of the synthetic ethinylestradiol. In addition with the intention to reduce menstruation associated symptoms and bleeding periods an extended-cycle regimen is currently considered. In the present in vitro work, we have compared the effect of these different estrogenic compounds and the different treatment regimens on breast cancer risk. Human breast cancer cells (ZR75-1 and HCC1500) were incubated with equimolar concentrations of estradiol or ethinylestradiol combined with various progestogens, dienogest, drospirenone, keto-desogestrel, levonorgestrel, and nomegestrel. Usual and extended cycle was mimicked by incubation periods of 3 days with 1 day hormones off and 4 days, respectively. Molecular markers for proliferation and apoptosis were investigated by Western blot. In both cell lines estradiol and ethinylestradiol elicited a significant increase in the proliferation rate without difference between the 2 estrogens. The effect in the long-term cycle tended to be more pronounced than in the intermitted cycle. Progestogen addition most significantly reduced the estrogen-induced proliferation rate. The molecular markers were influenced by the progestogens mostly in the same manner, reducing the proliferation/apoptosis rate. Our results indicate that both estrogenic based combinations with progestogens may not increase breast cancer risk independent from the regimen, intermitted or long-term cycle. However clinical studies are necessary to prove these in vitro results.     

Effects of thyroid hormones on human breast cancer cell proliferation

The involvement of estrogens in breast cancer development and growth has been well established. However, the effects of thyroid hormones and their combined effects with estrogens are not well studied. We investigated the response of human breast cancer cells to thyroid hormone, particularly the role of T3 in mediating cell proliferation and gene expression. We demonstrated that 17β-estradiol (E2) or triiodothyronine (T3) promoted cell proliferation in a dose-dependent manner in both MCF-7 and T47-D cell lines. The E2- or T3-dependent cell proliferation was suppressed by co-administration of the ER antagonist ICI. We also demonstrated that T3 could enhance the effect of E2 on cell proliferation in T47-D cells. Using an estrogen response element (ERE)-mediated luciferase assay, we determined that T3 was able to induce the activation of ERE-mediated gene expression in MCF-7 cells, although the effects were much weaker than that induced by E2. These results suggest that T3 can promote breast cancer cell proliferation and increase the effect of E2 on cell proliferation in some breast cancer cell lines and thus that T3 may play a role in breast cancer development and progression.     

Endogenous aromatization of testosterone results in growth stimulation of the human MCF-7 breast cancer cell line

Estrogens produced within breast tumors may play a pivotal role in growth stimulation of the breast cancer cells. However, it is elusive whether the epithelial breast cancer cells themselves synthesize estrogens, or whether the surrounding tumor stromal cells synthesize and supply the cancer cells with estrogen. The aromatase enzyme catalyzes the estrogen production, aromatizing circulating androgens into estrogens. The aim of this study was to investigate aromatase expression and function in a model system of human breast cancer, using the estrogen responsive human MCF-7 breast cancer cell line. Cells were cultured in a low estrogen milieu and treated with estrogens, aromatizable androgens or non-aromatizable androgens. Cell proliferation, expression of estrogen-regulated proteins and aromatase activity were investigated. The MCF-7 cell line was observed to express sufficient aromatase enzyme activity in order to aromatize the androgen testosterone, resulting in a significant cell growth stimulation. The testosterone-mediated growth effect was completely inhibited by the aromatase inhibitors letrozole and 4-hydroxy-androstenedione. Expression studies of estrogen-regulated proteins confirmed that testosterone was aromatized to estrogen in the MCF-7 cells. Thus, the results indicate that epithelial breast cancer cells possess the ability to aromatize circulating androgens to estrogens.     

Steroid hormone receptor signaling in tumorigenesis

Excessive activation of the hormone signaling pathways is implicated in several disorders of the target tissues, with cancer being one of the most serious fallouts. Steroid hormone receptors are key proteins through which steroid hormones convey their signals to the cells. Deregulated activity of the hormone receptors due to their altered activation; stability or sub-cellular localization is heavily implicated in the onset and progress of cancers. The role played by estrogen and its receptors in breast cancer remains the most thoroughly investigated steroid-dependent cancer system till date. Choosing it as an example, we have summarized the molecular mechanisms underlying the action of the estrogen receptors (ERs) in manifesting the effects of the estrogens in the cells. A special emphasis is placed on the molecular mechanism of their functionality, role of the coactivator proteins, and the reasons for the deregulated signaling. The therapeutic approaches resulting from the mechanistic study of the ER action and their efficacies are also discussed.