Effects of estradiol with oral or intravaginal progesterone on risk markers for breast cancer in a postmenopausal monkey model

OBJECTIVE: To evaluate the effects of oral estradiol given with either oral or intravaginal micronized progesterone (P4) on risk biomarkers for breast cancer in a postmenopausal monkey model. DESIGN: This experiment was a two-way crossover study in which 20 ovariectomized adult female cynomolgus macaques were treated (in equivalent doses for women) with oral estradiol (1 mg/d) + oral micronized P4 (200 mg/d) or intravaginal P4 delivered by Silastic rings (6- to 10-mg/d release rate). Hormone treatments lasted 2 months and were separated by a 1-month washout period. The primary outcome measure was breast epithelial proliferation. RESULTS: Serum P4 concentrations were significantly greater in subjects receiving oral P4 (10.9 ng/mL) compared with intravaginal P4 (3.8 ng/mL) at 2 to 3 hours after oral dosing (P<0.0001) but not at 24 to 28 hours after oral dosing (2.9 ng/mL for oral P4 vs 3.2 ng/mL for intravaginal P4 at 2 months, P=0.19). Serum estradiol concentrations were significantly lower after oral P4 than after intravaginal P4 (P<0.05 for all time points). Oral P4 resulted in significantly decreased body weight (-2.5%) compared with intravaginal P4 (+3.6%) (P=0.0001). Markers of breast proliferation, sex steroid receptor expression, and endometrial area did not differ significantly between oral P4 and intravaginal P4 treatments (P>0.1 for all). CONCLUSIONS: Despite different pharmacodynamic profiles, oral and intravaginal P4 had similar effects on biomarkers in the postmenopausal breast.     

Effects of mifepristone on proliferation and apoptosis of human endometrium in new users of medroxyprogesterone acetate

BACKGROUND: Mifepristone has been demonstrated to decrease breakthrough bleeding (BTB) in users of progestin-only contraceptives. METHODS: Endometrial biopsies were collected from 50 normal cycling women who were new users of depot medroxyprogesterone acetate (DMPA) randomized to receive either mifepristone or placebo before, during and after treatment. Proliferation, apoptosis and sex steroid receptors were evaluated by either immunohistochemistry or TUNEL assay. RESULTS: Administration of mifepristone to DMPA-exposed endometrium for 1 week significantly increased endometrial expression of Ki-67 (MKI67), estrogen receptor (ER)alpha and progesterone receptors A and B (PRAB) and decreased the number of TUNEL-positive and caspase-3 (CASP3)-active cells in the endometrial stroma. However, after 10 weeks of mifepristone treatment, no significant difference in proliferation, apoptosis and the expression of ERalpha or PRAB could be detected between the endometrium treated with DMPA alone and endometrium treated with mifepristone and DMPA. CONCLUSIONS: Administration of mifepristone to DMPA users significantly increases endometrial proliferation and decreases endometrial stromal apoptosis in the short term. Prolonged exposure to mifepristone does not counteract the inhibitory effects of progestin therapy on endometrial proliferation. Estrogen and progesterone receptors may play an important role in these effects.     

Apoptosis and apoptosis-associated parameters in relation to tamoxifen exposure in postmenopausal endometrium

Tamoxifen increases endometrial cell proliferation and the incidence of endometrial cancer in postmenopausal women. The purpose of this study was to evaluate apoptosis and apoptosis-related factors in endometrium in relation to tamoxifen exposure. We analyzed benign postmenopausal endometrium from breast cancer patients receiving tamoxifen (n = 35) and from controls (n = 24), and endometrial cancer tissue from tamoxifen-treated breast cancer patients (n = 15) and endometrial cancer from women without tamoxifen exposure (n = 51). Apoptosis was examined morphologically, and the percentage of apoptotic epithelial cells was defined as the apoptotic index. In the benign samples, the presence of apoptotic cells was also evaluated immunohistochemically by the expression of caspase-3 and the monoclonal antibody M30. The expression of Fas, FasL, and Bcl-2 was analyzed in all tissue samples. No differences were observed in the mean apoptotic index in benign endometrium in tamoxifen users (0.17%) versus controls (0.08%), or in tamoxifen-exposed (2.46%) versus nonexposed endometrial cancer (2.28%). However, the ratio of the apoptotic index with the previously reported proliferation index was lower in benign endometrium from tamoxifen users than in controls (0.02 +/- 0.026 vs. 0.05 +/- 0.03, Mann-Whitney U <0.005). In benign endometrium FasL was more frequently expressed in tamoxifen-users than in controls (chi(2) <0.05). We conclude that the apoptosis/proliferation ratio in benign endometrium from tamoxifen users is lower than in controls, indicating that the tamoxifen-induced higher proliferation is not compensated for by increased apoptosis. An imbalance between cell proliferation and apoptosis, and possibly suppression of the antitumor immune response by FasL overexpression in tamoxifen-exposed endometrium might play a role in the development of endometrial cancer in tamoxifen users.     

Hormonal regulation and localization of estrogen, progestin and androgen receptors in the endometrium of nonhuman primates: effects of progesterone receptor antagonists

This article reviews the effects of estradiol (E(2)), progesterone (P) and P receptor antagonists (PA) on the rhesus macaque endometrium. Ovariectomized macaques can be treated with implants of estradiol (E(2)) and P to induce precisely controlled, artificial menstrual cycles. During these cycles, treatment with E(2) alone induces an artificial proliferative phase marked by extensive endometrial epithelial cell proliferation and increased expression of stromal and epithelial estrogen receptor (ER) and P receptor (PR). Androgen receptor (AR) is also upregulated by E(2) but is expressed only by the endometrial stroma. Progesterone acts on the E(2) primed endometrium to induce secretory differentiation and causes suppression of epithelial and stromal ER, epithelial PR, and stromal AR in the functionalis zone. However, epithelial ER and PR are retained in the basalis zone during the secretory phase. When potent P antagonists (PA) are administered acutely at the end of an E(2) + P induced cycle, menses typically ensues similar to P withdrawal at the end of the menstrual cycle. When PAs are administered chronically there is significant blockage of all P- dependent effects including upregulation of ER, PR and AR and suppression of glandular secretory function. However, chronic PA administration also inhibits estrogen-dependent endometrial cell proliferation and growth. This endometrial antiproliferative effect is the basis of the clinical use of PA to control various diseases such as endometriosis.     

The Effects of Sex Protein Receptors and Sex Steroid Hormone Gene Polymorphisms on Breast Cancer Risk

Breast cancer is a common disease and a major cause of death among women throughout the world. Various genes are believed to be involved in the initiation and progression of the disease. Some polymorphisms of these genes increase susceptibility to breast cancer in particular ethnicities. This study used electronic literature search to review the effects of different sex steroid hormone gene polymorphisms on breast cancer risk. Our findings indicated that some polymorphisms in estrogen receptor alpha (ER-α), ER-β, progesterone receptor (PGR), pregnane X receptor (PXR), and cytochrome P450 enzymes (CYPs) affected breast cancer susceptibility, especially in African American women.     

Endometrial oestrogen and progesterone receptors and their relationship to sonographic appearance of the endometrium

The rapid development of ultrasonographic equipment now permits instantaneous assessment of follicles and endometrium. The sonographic appearance of the endometrium has been discussed in relation to in-vitro fertilization (IVF) cycles. However, a generally agreed view of the relationship of the sonographic appearance to fecundity in IVF cycles has not emerged. We have studied the relationship between steroid receptors and the sonographic appearance of the preovulatory endometrium in natural cycles and ovulation induction cycles. Preovulatory endometrial thickness was not found to be indicative of fecundity, although a preovulatory endometrial thickness of <9 mm related to an elevated miscarriage rate. The preovulatory endometrial echo pattern did not predict fecundity. No relationships were found among endometrial appearance, endometrial steroid receptors and steroid hormone concentrations in serum. Oestrogen or progesterone receptor concentrations were not related to endometrial thickness or to concentrations of serum oestradiol, the only significant correlation being found between the endometrial concentrations of oestrogen and progesterone receptors. The ratio of progesterone:oestrogen receptor concentration was somewhat less in echo pattern B (not triple line) endometrium compared with pattern A (triple line) endometrium. Oestrogen and progesterone receptor concentrations appeared stable on gonadotrophin induction, though fewer numbers were found during clomiphene cycles than in natural cycles. With regard to the distribution of receptor concentration between clomiphene and natural cycles, most women using clomiphene had very low oestrogen receptor populations. Pregnancy rates were low, in spite of high ovulatory rates during clomiphene treatment and were mainly related to low oestrogen receptor concentrations in preovulatory endometrium.     

Promoter hypermethylation and expression of sprouty 2 in endometrial carcinoma

Sprouty 2 is a key antagonist regulator of receptor tyrosine kinases, and downstream signaling pathways, like fibroblastic growth factor (FGF) and Ras-mitogen-activated protein kinase (RAS-MAPK). By controlling these pathways, sprouty 2 is involved in regulation of cell proliferation, differentiation, and angiogenesis. Alterations in fibroblastic growth factor receptor (FGFR) and members of the RAS-MAPK pathway are frequent in endometrial carcinoma. The expression of sprouty 2 has been found to be decreased in several types of human cancer, by mechanisms of promoter methylation. In the present study, we have assessed the expression of sprouty 2 in endometrial carcinoma, in correlation with sprouty 2 promoter methylation. Sprouty 2 immunohistochemical expression was assessed using 3 different tissue microarrays: one constructed from paraffin blocks of 80 samples of normal endometrium and 2 tissue microarrays containing samples of 157 endometrial carcinoma (1 tissue microarray constructed with 95 endometrial carcinomas previously studied for microsatellite instability and alterations in phosphatase and tensin homolog (PTEN), k-ras, and b-catenin, and 1 tissue microarray containing 62 endometrial carcinoma, which were also subjected to sprouty 2 promoter methylation analysis). The immunohistochemical expression of sprouty 2 was correlated with cellular proliferation (Ki67) and clinicopathologic data. Sprouty 2 promoter methylation was assessed by methylation-specific polymerase chain reaction, with DNA obtained from fresh-frozen samples of endometrial carcinoma and corresponding normal tissues, and correlated with promoter methylation of RAS association domain family-1A (RASSF1A). A highly significant decrease in sprouty 2 immunoexpression was seen in the proliferative phase of normal endometrium (P < .001). Differences were detected between types I and II endometrial carcinoma, but they were not statistically significant. Reduced immunoexpression of sprouty 2 was seen in 19.85% of endometrial carcinoma and was strongly and inversely associated with increased cell proliferation (Ki67; r = -0.367; P = .001). Sprouty 2 promoter methylation was detected in 31 (53.4%) of 58 endometrial carcinomas. Results from our study show that alterations in sprouty 2 may be involved in endometrial carcinogenesis by controlling cell proliferation.     

The effects of tamoxifen on proliferation and steroid receptor expression in postmenopausal endometrium

AIM: To study the effects of tamoxifen on the proliferation index and oestrogen receptor (ER) and progesterone receptor (PR) expression in postmenopausal endometrium. METHODS: A total of 125 endometrial specimens of postmenopausal women, comprising benign endometria from tamoxifen users (n = 35) and non-users (n = 24), and endometrial cancer from tamoxifen users (n = 15) and non-users (n = 51), were immunohistochemically examined using MIB-1, anti-ER, and anti-PR antibodies in endometrial epithelium and stroma. RESULTS: In benign endometrium the mean MIB-1 index in the epithelium was higher in tamoxifen users than in non-users (mean, 13% (SD, 13%) v mean, 2% (SD, 2%); p < 0.05), whereas in endometrial cancer the MIB-1 index was higher, but similar in tamoxifen users and non-users (mean, 32% (SD, 24%) and mean, 35% (SD, 18%)). The expression of ER was comparably high in benign epithelium from tamoxifen users and non-users (97% and 92%, respectively), but in endometrial cancer it was lower in tamoxifen users (60% and 88%; p < 0.05). The expression of PR in stromal cells was higher in tamoxifen users, both in benign (84% v 54%) and in malignant endometrium (33% v 10%; p < 0.05). CONCLUSION: The proliferation index (as measured by MIB-1) in benign endometrial epithelium is higher in tamoxifen users than in non-users, and this might play a role in the reported higher incidence of endometrial cancer in postmenopausal tamoxifen users. The increased expression of PR in stroma from tamoxifen users with both benign and malignant endometrium demonstrates an additional oestrogenic effect of tamoxifen on the endometrial stroma.     

Ovarian steroids and the human breast: regulation of stem cells and cell proliferation

Ovarian steroidal control of mammary gland proliferation and differentiation is not well defined in the human. We therefore developed the athymic nude mouse model in which intact normal human breast tissue is xenografted subcutaneously and treated with human physiological serum levels of oestrogen (E) and/or progesterone (P). We showed that: (i) E, and not P, is the major steroid hormone inducing proliferation of epithelial cells in the adult non-pregnant, non-lactating breast; (ii) E induces progesterone receptor (PR) expression; and (iii) PR expression is maximally induced at low E concentrations while a higher amount of E was required to induce proliferation. Using double label immuno-fluorescence, we demonstrated that cells expressing the oestrogen receptor- (ER) invariably contained the PR but that steroid receptor expression and cell proliferation (Ki67 antigen) were dissociated. Recently, we have demonstrated that some ER/PR-positive epithelial cells are quiescent breast stem cells suggesting that they act as “steroid hormone sensors” that secrete paracrine factors to regulate the proliferative activity of adjacent ER/PR-negative epithelial cells. The dissociation between steroid receptor expression and cell proliferation in normal epithelium was lost at an early stage in ER/PR-positive breast tumour formation perhaps indicating that they arise from deregulation of the normally quiescent breast stem cells.     

A new trend of breast cancer research in the genome era (Review)

The decipherment of the human genome, as accomplished recently in USA and in Europe, now enables us to search for the cause of a disease at the levels of the whole spectrum of gene expressions (mRNAs) of the human genome. A set of investigations came to the world in AD 2000 to show that: a) A total of 50 genes of the estrogen receptor positive (ER+) human breast cancer cell line MCF-7 in their gene expressions were found to undergo stimulation from a physiological concentration of 17beta-estradiol. b) Comparison of estrogen-responsiveness among a number of estrogen-responsive genes, among cancer cell lines of both the breast and endometrium with and without active ER, and among cell lines of normal tissues revealed that association between the presence of active ER and estrogen-responsive genes is quantitative rather than qualitative including some exceptions, and that none of the estrogen-responsive genes tested was classified as of breast cancer specific. For this review, we collected information from our and other laboratories to investigate problems that remain to be disputed. Five items of discussion are given as follows: a) The dose of a steroid used for the production of an experimental tumor was fixed not to a physiological concentration but to a pharmacological concentration. In the case of estradiol, the latter was higher than the former by over 3 orders. The mitotic activity of MCF-7 underwent stimulation from the former but distinct suppression from the latter. b) A massive dose of a single steroid, when given at a good time of the host age, could produce a tumor of any kind. The timing of treatment rather than the nature of a steroid was found critical. c) Experience with the morphological development of Drosophila suggests the possibility that deficiency rather than amplification of gene expression in the infant age of Drosophila is responsible for the induction of morphological changes in an adult fly. Likewise, deficiencies of some escort steroids rather than overflow of estradiol may have more chance of occurrence in the genesis of spontaneous breast cancer, as suggested by many researchers including us. The plasma concentration of estradiol was found to be normal in patients with cancers of both the breast and endometrium. Future studies of breast cancer as well as other cancers should be directed to the multisteroidal carcinogenesis hypothesis rather than the monosteroidal carcinogenesis hypothesis. d) The necessity of recruiting an appropriate case-control data set and the difficulty of data interpretation were emphasized in the search for good biomarkers of breast cancer. e) Case-control studies of tamoxifen use was found useful for the prevention and clinical control of breast cancer of non-hereditary type but not for the breast cancer of hereditary type. Both decreased risk of breast cancer and increased risk of endometrial cancer were detected in the same population of tamoxifen use. The observed dualism of both human breast cancer and tamoxifen action can be taken as evidence to support the multi-steroidal carcinogenesis hypothesis rather than the mono-steroidal carcinogenesis hypothesis.     

The expression of S100P increases and promotes cellular proliferation by increasing nuclear translocation of β-catenin in endometrial cancer

There is increasing evidence suggesting that S100P has a significant role in cancer, and is associated with poor clinical outcomes. The expression of S100P mRNA and protein in endometrial cancer and normal endometrium tissues was detected by real-time quantitative RT-PCR and immunohistochemistry. Moreover, we reduced the expression of S100P in HEC-1A and Ishikawa endometrial cancer cell lines by siRNA transfection. Based on the reduced S100P mRNA expression, we measured the effects of S100P on cellular proliferation by the cell-counting kit-8. Nuclear β-catenin protein level was detected by western blotting. Cyclin D1 and c-myc mRNA expression regulated by β-catenin was detected by real-time quantitative RT-PCR. We found that the expression of S100P mRNA and protein increased in endometrial cancer tissues compared with the normal endometrium. Local S100P expression progressively increased from pathologic differenciation grade 1 to 3. After reducing the S100P expression, the cellular proliferation ability, nuclear β-catenin protein level, cyclin D1 and c-myc mRNA levels reduced. It indicated that S100P could promote cell proliferation by increasing nuclear translocation of β-catenin. The expression of S100P mRNA and protein in endometrial cancer significantly increased and is associated with pathologic differenciation grade. S100P may promote endometrial cell proliferation by increasing nuclear translocation of β-catenin.     

Steroid receptors in blood vessels of the rhesus macaque endometrium: a review

Estradiol (E) and progesterone (P) act on the primate endometrium to induce dramatic changes in the vascular system during the menstrual cycle. These changes include vessel breakdown and bleeding during menses, heightened angiogenesis during the early proliferative phase, and extensive growth of the spiral arteries in the luteal phase of the cycle. Because steroid hormone action is dependent upon the presence of specific nuclear receptors in target tissues, we used immunocytochemistry with receptor-specific monoclonal antibodies to characterize the spatial and temporal expression of estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta), progesterone receptor PR and androgen (A) receptor (AR) in the endometrial vessels of rhesus macaques (Macaca mulatta). The only sex steroid receptor that was present in the endothelium and smooth muscle walls of endometrial vessels was ERbeta. ERalpha, PR, and AR were not detectable in either the endothelium or vascular smooth muscle cells of primate endometrial vessels. However, all of these receptors were strongly expressed by the perivascular stroma, and in these cells, all were modulated by the changes in levels of E and P during the cycle. We concluded that any direct effects of E on endometrial vessels would be mediated by ERbeta, and that the actions of P and A, and possibly some of E, were indirectly mediated through perivascular stromal cells.     

Effects of tibolone and hormone replacement therapy on the breast of cynomolgus monkeys

OBJECTIVE: To measure the effects of 2 years of treatment with tibolone on the breasts of cynomolgus macaques (Macaca fascicularis) in comparison with conventional hormone replacement therapy. DESIGN: Ovariectomized cynomolgus macaques were randomized into five groups and treated for 2 years. Groups included controls (n = 31) and four drug treatments, including tibolone at 0.05 mg/kg (LoTIB, n = 30) or 0.2 mg/kg (HiTIB, n = 31), conjugated equine estrogens at 0.042 mg/kg (CEE, n = 28), or CEE + medroxyprogesterone acetate (MPA) at 0.167 mg/kg (CEE + MPA, n = 29). Endpoints included histologic, histomorphometric, and immunohistochemical assessment of the mammary gland. RESULTS: Tibolone did not cause stimulation of the breast in contrast to distinct proliferative responses of the breast to CEE and CEE + MPA, as measured by increases in breast epithelial tissue area and expression of the proliferation marker Ki67 in breast epithelial cells. Tibolone at the higher dose increased progesterone receptor expression in the breast relative to controls, indicating partial estrogen-agonist activity, but without induction of proliferation. Progesterone receptor expression was also induced by CEE. CONCLUSIONS: Tibolone may have an advantage over conventional hormone replacement therapy because it does not stimulate proliferation in the breast. This lack of mammotrophic effect may reflect a lower risk for promotion of breast cancer.     

Breast cancer: hormones and other risk factors

In North America and Northern Europe, breast cancer incidence rates begin increasing in the early reproductive years and continue climbing into the late seventies, whereas rates plateau after menopause in japan and less developed countries. Female gender, age and country of birth are the strongest determinants of disease risk. Family history and mutations in the BRCA1 and BRCA2 genes are important correlates of lifetime risk. Genetic polymorphisms associated with estrogen synthesis and metabolism are currently under study. Atypical hyperplasia and molecular alterations in benign breast lesions appear to be involved in the pathogenesis of invasive carcinoma. In postmenopausal women, increased breast density on mammograms increases risk. Bone density and breast cancer are associated, presumably through the mechanism of endogenous estrogen levels. Serum estrogen levels are higher in breast cancer cases than controls. Many established risk factors for breast cancer may function through and endocrine mechanism. Current use of oral contraceptives and prolonged, current or recent use of hormone replacement therapy moderately increase risk. Tamoxifen and possibly other selective estrogen receptor modulators reduce breast cancer risk in high risk women. Relationships between various dietary micro and macronutrients and breast cancer have been suggested but require evaluation in clinical trials. Whereas alcohol consumption is associated with increased risk, most environmental factors, including polychlorinated compounds and electromagnetic fields, are not. Conclusion: Breast cancer etiology is becoming clearer through the study of molecular alterations in germline and somatic cell genes, and the interaction of these genes with steroid hormones and relevant growth factors. This knowledge should be useful for breast cancer prevention.     

Cancer and obesity

The prevalence of obesity is increasing worldwide. Obesity is also a major risk factor for developing chronic diseases, including malignancies thereby increasing the risk of several types of tumors such as breast, endometrium, colon, prostate and kidney cancer. The mechanisms associated with obesity and insulin resistance, hormonal regulation and other proinflammatory factors are also involved in neoplastic processes including: cell proliferation, carcinogenesis, and angiogenesis vascularization. In addition to contributing to cancer pathogenesis obesity is associated with comorbidities and poor prognosis in cancer patients. The aim of this review is to describe some of the mechanisms involved in the association of obesity and malignancies.