Managing vasomotor symptoms in women after cancer.

Women with estrogen-sensitive cancer or survivors of these neoplasms are generally not candidates for systemic menopausal hormone therapy or tibolone for the treatment of bothersome vasomotor symptoms (hot flashes or night sweats). However, menopausal symptoms negatively affect quality of life and need to be addressed by clinicians. For mild vasomotor symptoms, optimizing lifestyle changes or mind-brain behavior may be sufficient. For women with moderate to severe vasomotor symptoms unresponsive to these measures, non-hormone pharmacologic therapy may be needed. Randomized controlled trials have shown efficacy for vasomotor symptoms with selective serotonin reuptake inhibitors (paroxetine, citalopram, and escitalopram) and serotonin-norepinephrine reuptake inhibitors (venlafaxine and desvenlafaxine), as well as gabapentin, pregabalin, and clonidine. Therapies in development include neurokinin B inhibitors (neurokinin 3 receptor), stellate ganglion blockade, and a natural estrogen, estetrol. Individualizing therapy is important. As the physiology of menopausal hot flashes becomes better understood, it will drive development of future non-hormone pharmacotherapies.

Cause or prevention of breast cancer with estrogens: analysis from tumor biologic data, growth kinetic model and Women's Health Initiative study.

The existing medical literature suggests that estrogens may cause breast cancer but, paradoxically, can also prevent this neoplasm under specific circumstances. Appropriate interpretation of this complex data requires an understanding of emerging concepts of tumor biology. A substantial body of data, including animal models and epidemiologic studies, suggests that estrogens contribute to the development of breast cancer. Additionally, pre-clinical experiments indicate that the responsible mechanisms include both estrogen receptor α-dependent and -independent effects (ERα-dependent and ERα-independent effects). We recently developed two models to describe the growth kinetics of occult breast tumors, one based on autopsy studies and tumor doubling time and the other, computer-based. Validation of the models involved comparison of the predicted incidence of breast cancer with the actual incidence in population-based studies. Utilization of these models allowed us to determine that 16 years on average are required for tumors to undergo the 30 doubling times necessary for the occult tumors to reach the threshold for clinical detection. These models suggest that menopausal hormone therapy with estrogen plus a progestogen in the Women's Health Initiative (WHI) study accelerated the doubling time of occult, pre-existing tumors from 200 to 150 days and thus, increased the rate of tumor diagnosis. Based on estrogen-induced apoptosis data, the model accurately predicted the prevention of diagnosed breast cancer in the estrogen-alone arm of the WHI. Notably, pre-clinical studies demonstrated that conjugated equine estrogen, as used in the WHI, has unique, pro-apoptotic properties compared to the anti-apoptotic effects of estradiol, a finding providing an explanation for the reduction in breast cancer with conjugated equine estrogen.

Aromatase expression in atypical ductal hyperplasia in women.

PURPOSE: To determine the levels of aromatase in atypical ductal hyperplasia (ADH) lesions, tissue surrounding the ADH, and in dense and non-dense normal breast tissue. We postulated that excess aromatase in breast tissue might, through production of increased estrogen, drive the carcinogenic process. Estrogens and their metabolites are thought to contribute to the development of breast cancer through estrogen receptor-mediated mechanisms and genotoxic effects of estrogen metabolites. ADH is a benign lesion of the breast which is associated with substantially increased risk for subsequent development of breast cancer. After 25 years, approximately 30% of women with ADH develop breast cancer. In women with three or more separate ADH lesions at the same time, 47% will develop breast cancer over that time period. Another important risk factor for breast cancer is the presence of mammographically dense breast tissue. METHODS: We utilized quantitative immunochemical analysis of aromatase in biopsy tissue to test this possibility. Previously published results comparing dense with non-dense breast tissue in normal women (Vachon et al. Breast Cancer Res Treat 125:243-252, 2011) were used for comparisons with ADH. A well-characterized histochemical H-score was employed for quantitative assessment of aromatase in the various tissue studied. RESULTS: The H-score of aromatase staining was statistically significantly higher (p = 0.003) in the ADH epithelium than surrounding epithelial tissue. In order of H-score from highest to lowest were ADH, issue surrounding ADH, dense normal and non-dense normal breast tissues. The levels of aromatase in a subset of women with ADH who went on to develop breast cancer were not higher than in women who did not. CONCLUSIONS: We suggest from these studies that overexpression of aromatase in breast tissue and its resultant increase in estradiol levels may contribute to the later development of breast cancer in women with ADH.

Treatment of symptoms of the menopause: an Endocrine Society Clinical Practice Guideline

OBJECTIVE: The objective of this document is to generate a practice guideline for the management and treatment of symptoms of the menopause. PARTICIPANTS: The Treatment of Symptoms of the Menopause Task Force included six experts, a methodologist, and a medical writer, all appointed by The Endocrine Society. EVIDENCE: The Task Force developed this evidenced-based guideline using the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) system to describe the strength of recommendations and the quality of evidence. The Task Force commissioned three systematic reviews of published data and considered several other existing meta-analyses and trials. CONSENSUS PROCESS: Multiple e-mail communications, conference calls, and one face-to-face meeting determined consensus. Committees of The Endocrine Society, representatives from endorsing societies, and members of The Endocrine Society reviewed and commented on the drafts of the guidelines. The Australasian Menopause Society, the British Menopause Society, European Menopause and Andropause Society, the European Society of Endocrinology, and the International Menopause Society (co-sponsors of the guideline) reviewed and commented on the draft. CONCLUSIONS: Menopausal hormone therapy (MHT) is the most effective treatment for vasomotor symptoms and other symptoms of the climacteric. Benefits may exceed risks for the majority of symptomatic postmenopausal women who are under age 60 or under 10 years since the onset of menopause. Health care professionals should individualize therapy based on clinical factors and patient preference. They should screen women before initiating MHT for cardiovascular and breast cancer risk and recommend the most appropriate therapy depending on risk/benefit considerations. Current evidence does not justify the use of MHT to prevent coronary heart disease, breast cancer, or dementia. Other options are available for those with vasomotor symptoms who prefer not to use MHT or who have contraindications because these patients should not use MHT. Low-dose vaginal estrogen and ospemifene provide effective therapy for the genitourinary syndrome of menopause, and vaginal moisturizers and lubricants are available for those not choosing hormonal therapy. All postmenopausal women should embrace appropriate lifestyle measures.

Vaginal administration of estradiol: effects of dose, preparation and timing on plasma estradiol levels.

CONTEXT: The use of systemic estrogens for the treatment of menopausal symptoms has declined by approximately 80% following the initial publication of the Women's Health Initiative in 2002. Current attention focuses on vaginal estrogen as a local therapeutic means to achieve control of symptoms due to vulvovaginal atrophy without increasing plasma estradiol levels. A key issue is whether or not vaginally administered estrogens are absorbed and produce systemic effects. EVIDENCE ACQUISITION: Medline and PubMed were searched for relevant English-language articles using pertinent key words. The bibliographies of the pertinent articles were then read to identify further relevant articles. EVIDENCE SYNTHESIS: Several confounding factors influenced the data analysis including: (1) estradiol assay sensitivity and specificity; (2) acute versus chronic absorption; (3) delivery systems, doses, timing, and formulation; and (4) effect of atrophic versus mature vaginal mucosa on absorption. Each preparation was associated with acute estradiol absorption with peaks at approximately 8 h and return to baseline at 12 h. Low-dose vaginal estrogen, arbitrarily defined as the 7.5-µg vaginal ring and 10-µg tablet, increased plasma estradiol levels during chronic administration but not above the normal range of ≤ 20 pg/ml. Surprisingly, these increments were associated with systemic effects to lower plasma levels of low density lipoprotein cholesterol and bone resorption rates. Intermediate doses (i.e. 25 µg estradiol or 0.3 mg conjugated equine estrogen) resulted in plasma estradiol levels approaching or exceeding 20 pg/ml. The higher doses (50-2000 µg estradiol or 0.625-2.5 mg conjugated equine estrogen) resulted in premenopausal levels of estrogen. CONCLUSIONS: Low-dose vaginal estrogen appears to be an effective strategy for managing women whose symptoms result from vulvovaginal atrophy. These regimens limit but do not completely eliminate systemic absorption. Low-doses regimens should be preferred clinically to intermediate- or high-dose methods.

Oncology in midlife and beyond.

The onset of the menopause is often a time when women's concerns can act as a powerful trigger to encourage healthy modifications in lifestyle which will maintain, or improve, their general health. This document aims to help women to understand their potential risks, to encourage them to find proactive preventive strategies by modifying some of their attitudes, and to use health resources (when available) to be screened. Cancer is an important cause of death but not the primary cause of mortality. Cardio/circulatory diseases represent 35-40% of causes of death in most developed countries and 20-25% of women will die from cancers in Western Europe, Australasia, high-income North America, high-income Asia Pacific, East Asia and Southern Latin America. Breast cancer, lung cancer and colorectal cancer are prevalent in most regions of the world. Cervical cancer remains a hallmark of low access to health care. Preventive strategies (decreasing smoking and alcohol consumption, losing weight, eating a healthy diet and undertaking physical activity) and implementation of screening could help to significantly decrease the incidence of and mortality from cancer. The mortality/incidence ratio is higher in developing countries compared to high-income regions as well as in subgroups of populations in developed countries with lower socioeconomic levels. Implementation of better diagnostic methods and management of cancer according to the local resources will help to decrease the mortality rate in developing countries, and effort has to be made to decrease social inequities and improve access to health care for low-income groups. In conclusion, cancer incidence is increasing as a consequence of longer life expectancy all over the world. National health programs are mandatory to implement screening and to improve individual management. Finally, educating women so that they are aware of ways to improve their general health, to minimize their own risk factors and to identify signs of change in their own health which may be markers of impending cancer will help to reduce the burden of disease and improve the prognosis for tumors detected at an earlier stage.

Metabolic effects of oral versus transdermal 17ß-estradiol (E2): a randomized clinical trial in girls with Turner syndrome.

CONTEXT: The long-term effects of pure 17ß-estradiol (E2) depending on route of administration have not been well characterized. OBJECTIVE: Our objective was to assess metabolic effects of oral vs transdermal (TD) 17ß-E2 replacement using estrogen concentration-based dosing in girls with Turner syndrome (TS). PATIENTS: Forty girls with TS, mean age 16.7 ± 1.7 years, were recruited. DESIGN: Subjects were randomized to 17ß-E2 orally or TD. Doses were titrated using mean E2 concentrations of normally menstruating girls as therapeutic target. E2, estrone (E1), and E1 sulfate (E1S) were measured by liquid chromatography tandem mass spectrometry and a recombinant cell bioassay; metabolites were measured, and dual-energy x-ray absorptiometry scan and indirect calorimetry were performed. MAIN OUTCOME: Changes in body composition and lipid oxidation were evaluated. RESULTS: E2 concentrations were titrated to normal range in both groups; mean oral dose was 2 mg, and TD dose was 0.1 mg. After 6 and 12 months, fat-free mass and percent fat mass, bone mineral density accrual, lipid oxidation, and resting energy expenditure rates were similar between groups. IGF-1 concentrations were lower on oral 17ß-E2, but suppression of gonadotropins was comparable with no significant changes in lipids, glucose, osteocalcin, or highly sensitive C-reactive protein between groups. However, E1, E1S, SHBG, and bioestrogen concentrations were significantly higher in the oral group. CONCLUSIONS: When E2 concentrations are titrated to the normal range, the route of delivery of 17ß-E2 does not affect differentially body composition, lipid oxidation, and lipid concentrations in hypogonadal girls with TS. However, total estrogen exposure (E1, E1S, and total bioestrogen) is significantly higher after oral 17ß-E2. TD 17ß-E2 results in a more physiological estrogen milieu than oral 17ß-E2 administration in girls with TS.

Hormone therapy and breast cancer risk 10 years after the WHI.

Ten years after the publication of the first Women's Health Initiative (WHI) report, a substantial decrease in hormone replacement therapy (HRT) has been observed world-wide. Fear of developing breast cancer represents one of the reasons for an important shift toward alternatives for treatment of menopause symptoms or abstention from therapy altogether. Many publications in the scientific and lay press have emphasized the magnitude of the relative risk of breast cancer but have not focused on excess or attributable risk. Since the original report of the WHI study, new information has been published on risk factors for breast cancer related to hormone therapy use. Accordingly, we believe it important to review current data and examine excess rather than relative or absolute risk. A balanced perspective on excess risk determined from existing data suggests that the benefits of HRT for quality of life can outweigh the risks in management of a large number of postmenopausal women. In addition, alternative strategies for relief of menopausal symptoms are not as effective as HRT in treating the climacteric symptoms.

Clinical review: Effect of endocrine therapies on bone in breast cancer patients.

CONTEXT: Two common strategies are used to treat estrogen receptor-positive breast cancer in women: tamoxifen to inhibit estrogen action, and aromatase inhibitors (AIs) to block estrogen biosynthesis. Recent data suggest that AIs are more effective than tamoxifen in the adjuvant and advanced disease settings and are now being more commonly used. Tamoxifen, as a selective estrogen receptor modulator, exerts estrogenic effects to preserve bone, whereas the AIs profoundly lower estrogen levels and cause bone loss. Recent comparative studies of these agents provide extensive data on fracture rates, bone mineral density, and markers of bone formation and resorption. OBJECTIVE: The aim of the study was to review the mechanistic effects of estrogen on bone and clinical data regarding bone density, bone turnover markers, and fracture rates in women with breast cancer taking tamoxifen or AIs. EVIDENCE ACQUISITION AND SYNTHESIS: Data presented reflect a review of the literature and data integration from the perspective of the author's knowledge of the field. RESULTS: Tamoxifen increases bone density and reduces fractures in postmenopausal women with breast cancer, whereas AIs increase rate of fracture, accelerate loss of bone mineral density, and enhance levels of markers of bone formation and resorption. Bisphosphonates and denosumab counteract the effects of the AIs on bone. Guidelines for management of AI-induced bone loss are available from several sources, but a simple algorithm guides decision making most effectively. CONCLUSIONS: Endocrine therapy for postmenopausal women with breast cancer exerts substantial effects on bone, and guidelines are available to assist in the management of bone-related problems.

History of aromatase: saga of an important biological mediator and therapeutic target.

Aromatase is the enzyme that catalyzes the conversion of androgens to estrogens. Initial studies of its enzymatic activity and function took place in an environment focused on estrogen as a component of the birth control pill. At an early stage, investigators recognized that inhibition of this enzyme could have major practical applications for treatment of hormone-dependent breast cancer, alterations of ovarian and endometrial function, and treatment of benign disorders such as gynecomastia. Two general approaches ultimately led to the development of potent and selective aromatase inhibitors. One targeted the enzyme using analogs of natural steroidal substrates to work out the relationships between structure and function. The other approach initially sought to block adrenal function as a treatment for breast cancer but led to the serendipitous finding that a nonsteroidal P450 steroidogenesis inhibitor, aminoglutethimide, served as a potent but nonselective aromatase inhibitor. Proof of the therapeutic concept of aromatase inhibition involved a variety of studies with aminoglutethimide and the selective steroidal inhibitor, formestane. The requirement for even more potent and selective inhibitors led to intensive molecular studies to identify the structure of aromatase, to development of high-sensitivity estrogen assays, and to "mega" clinical trials of the third-generation aromatase inhibitors, letrozole, anastrozole, and exemestane, which are now in clinical use in breast cancer. During these studies, unexpected findings led investigators to appreciate the important role of estrogens in males as well as in females and in multiple organs, particularly the bone and brain. These studies identified the important regulatory properties of aromatase acting in an autocrine, paracrine, intracrine, neurocrine, and juxtacrine fashion and the organ-specific enhancers and promoters controlling its transcription. The saga of these studies of aromatase and the ultimate utilization of inhibitors as highly effective treatments of breast cancer and for use in reproductive disorders serves as the basis for this first Endocrine Reviews history manuscript.

Adaptation of estrogen-regulated genes in long-term estradiol deprived MCF-7 breast cancer cells.

First line treatment of hormone dependent breast cancer initially causes tumor regression but later results in adaptive changes and tumor re-growth. Responses to second line treatments occur but tumors again begin to progress after a period of 12???18??months. In depth understanding of the adaptive process would allow the identification of targets to abrogate the development of hormonal resistance and prolong the efficacy of endocrine therapy. We have developed a model system to examine adaptive changes in human MCF-7 breast cancer cells. Upon deprivation of estradiol for a prolonged period of time, a maneuver analogous to surgical oophorectomy in pre-menopausal women and use of aromatase inhibitors in post-menopausal patients, tumor cells adapt and become hypersensitive to estradiol. We reasoned that the expression pattern of multiple genes would change in response to estradiol deprivation and that cDNA microarrays would provide an efficient means of assessing these changes. Accordingly, we examined the transcriptional responses to estradiol in long-term estradiol deprived (LTED) MCF-7 cells with a cDNA microarray containing 1901 known genes and ESTs. To assess the changes induced by long-term estradiol deprivation, we compared the effects of estradiol administration in LTED cells with those in MCF-7 cells, which we had previously reported, and confirmed with real time PCR using the parental and LTED cells. Seven genes and one EST were induced by estradiol in LTED but not in wild type MCF-7 cells, whereas ten genes were down-regulated by estradiol only in LTED cells. The expression of seven genes increased concurrently and five decreased in response to estradiol in both cell types. From these observations, we generated testable hypotheses regarding several genes including DKFZP, RAP-1, ribosomal protein S6, and TM4SF1. Based upon the known functions of these genes and the patterns of observed changes, we postulate that divergent regulation of these genes may contribute to the different biologic responses to estrogen in these cell lines. These results provide targets for further mechanistic studies in our experimental system. Our findings indicate that long-term estradiol deprivation causes expression changes in multiple genes and emphasizes the complexity of the process of cellular adaptation.

Consensus statement. Workshop on therapeutic resistance in breast cancer: impact of growth factor signalling pathways and implications for future treatment.

Anti-hormones (notably tamoxifen), chemotherapy and modern radiotherapeutic approaches are invaluable in the management of breast cancer, and collectively have contributed substantially to the improved survival in this disease. Moreover, there is promise that these successes will continue with the emergence of other endocrine agents (for example, aromatase inhibitors and pure anti-oestrogens). However, de novo and acquired resistance comprises a significant problem with all treatment approaches examined to date. This Workshop aimed to evaluate the contribution made by growth factor signalling pathways in the various resistant states, primarily focusing on resistance to anti-hormonal strategies and spanning experimental models and, where possible, clinical breast cancer data. The successes and limitations of therapeutic targeting of these pathways with various signal transduction inhibitors (STIs) were evaluated in model systems and from emerging clinical trials (including epidermal growth factor receptor inhibitors such as gefitinib). It was concluded that growth factor signalling is an important contributor in the development of endocrine resistance in breast cancer and that use of STIs provides a promising therapeutic strategy for this disease. However, the cancer cell is clearly able to harness alternative growth factor signalling pathways for growth and cell survival in the presence of STI monotherapy and, as a consequence, the efficacy of STIs is likely to be limited by the acquisition of resistance. A number of strategies were proposed from studies in model systems that appeared to enhance anti-tumour actions of existing STI monotherapy, notably including combination therapies targeting multiple pathways. With the increased availability of diverse STIs and improved drug delivery, there is much hope that the more complex therapeutic strategies proposed may ultimately be achievable in clinical practice.

Long-term estradiol deprivation in breast cancer cells up-regulates growth factor signaling and enhances estrogen sensitivity.

Deprivation of estrogen causes breast tumors in women to adapt and develop enhanced sensitivity to this steroid. Accordingly, women relapsing after treatment with oophorectomy, which substantially lowers estradiol for a prolonged period, respond secondarily to aromatase inhibitors with tumor regression. We have utilized in vitro and in vivo model systems to examine the biologic processes whereby long-term estradiol deprivation (LTED) causes cells to adapt and develop hypersensitivity to estradiol. Several mechanisms are associated with this response, including up-regulation of estrogen receptor-alpha (ERalpha) and the MAP kinase, phosphoinositol 3 kinase (PI3-K) and mammalian target of rapamycin (mTOR) growth factor pathways. ERalpha is four- to tenfold up-regulated and co-opts a classical growth factor pathway using Shc, Grb-2 and Sos. This induces rapid non-genomic effects which are enhanced in LTED cells. The molecules involved in the non-genomic signaling process have been identified. Estradiol binds to cell membrane-associated ERalpha, which physically associates with the adaptor protein Shc, and induces its phosphorylation. In turn, Shc binds Grb-2 and Sos, which result in the rapid activation of MAP kinase. These non-genomic effects of estradiol produce biologic effects as evidenced by Elk-1 activation and by morphologic changes in cell membranes. Additional effects include activation of the PI3-K and mTOR pathways through estradiol-induced binding of ERalpha to the IGF-I and epidermal growth factor receptors. A major question is how ERalpha locates in the plasma membrane since it does not contain an inherent membrane localization signal. We have provided evidence that the IGF-I receptor serves as an anchor for ERalpha in the plasma membrane. Estradiol causes phosphorylation of the adaptor protein, Shc and the IGF-I receptor itself. Shc, after binding to ERalpha, serves as the 'bus' which carries ERalpha to Shc-binding sites on the activated IGF-I receptors. Use of small inhibitor (si) RNA methodology to knockdown Shc allows the conclusion that Shc is needed for ERalpha to localize in the plasma membrane. In order to abrogate growth factor-induced hypersensitivity, we have utilized a drug, farnesylthiosalicylic acid, which blocks the binding of GTP-Ras to its membrane acceptor protein, galectin 1, and reduces the activation of MAP kinase. We have also shown that this drug is a potent inhibitor of mTOR as an additional mechanism of inhibition of cell proliferation. The concept of 'adaptive hypersensitivity' and the mechanisms responsible for this phenomenon have important clinical implications. The efficacy of aromatase inhibitors in patients relapsing on tamoxifen could be explained by this mechanism and inhibitors of growth factor pathways should reverse the hypersensitivity phenomenon and result in prolongation of the efficacy of hormonal therapy for breast cancer.

Adaptive hypersensitivity to estrogen: mechanisms and clinical relevance to aromatase inhibitor therapy in breast cancer treatment.

Breast tumors in women can adapt to endocrine deprivation therapy by developing hypersensitivity to estradiol. For this reason, aromatase inhibitors can be effective in women relapsing after treatment with tamoxifen or following oophorectomy. To understand the mechanisms responsible, we examined estrogenic stimulation of cell proliferation in a model system and provided in vitro and in vivo evidence that long-term estradiol deprivation (LTED) causes "adaptive hypersensitivity". The primary mechanisms responsible involve up-regulation of ER alpha as well as the MAP kinase, PI-3 kinase, and mTOR growth factor pathways. ER alpha is 4-10-fold up-regulated and co-opts a classical growth factor pathway using Shc, Grb2, and Sos. This induces rapid non-genomic effects which are enhanced in LTED cells. Estradiol binds to cell membrane associated ER alpha, physically associates with the adaptor protein Shc, and induces its phosphorylation. In turn, Shc binds Grb2 and Sos which result in the rapid activation of MAP kinase. These non-genomic effects of estradiol produce biologic effects as evidenced by Elk activation and by morphologic changes in cell membranes. Additional effects include activation of PI-3 kinase and mTOR pathways through estradiol induced binding of ER alpha to the IGF-1 and EGF receptors. Further proof of the non-genomic effects of estradiol involved use of "designer" cells which selectively express ER alpha in nucleus, cytosol, and cell membrane. We have used a new downstream inhibitor of these pathways, farnesyl-thio-salicylic acid (FTS), to block proliferation in hypersensitive cells as a model for a potentially effective strategy for treatment of patients.

The validation of new aromatase monoclonal antibodies for immunohistochemistry--a correlation with biochemical activities in 46 cases of breast cancer.

Intratumoral aromatase is a therapeutic target for the treatment of post-menopausal estrogen-dependent breast cancers. Therefore, reliable methods should be developed for routine application for the detection of intratumoral aromatase. Immunohistochemistry (IHC) is considered one of the most suitable methods in this regard. A multi-centre collaborative group has been established to generate and validate new aromatase monoclonal antibodies. We have selected two monoclonal antibodies, #677 against native aromatase protein and F2 against formalin-fixed protein for this purpose. With these two monoclonal antibodies 43 cases of invasive ductal carcinoma, which had been previously assayed for aromatase activity by product isolation methodology, were immunostained in three laboratories in UK, USA and Japan and independently evaluated by three pathologists (H.S., T.A. and S.G.S.). Staining of malignant epithelium, adipose tissue, normal/benign and stromal compartments of the tumors were assessed by estimating the proportion of positive staining cells and the relative intensity of staining in this fashion. Immunoreactivity could be detected in each component of the tissue specimens but a significant positive correlation with biochemical activity was detected only in malignant epithelium stained with 677 not in other components with #677 and not in any of the components. Staining using F2 as a primary antibody did not produce a positive correlation in any components with aromatase activity. These results suggest that we now have a monoclonal antibody against aromatase (#677) which may be used to stain archival materials. A methodology and scoring system is recommended whereby staining significantly correlates with aromatase activity of the resected tissue specimens of breast cancer.

Down-regulation of Bcl-2 enhances estrogen apoptotic action in long-term estradiol-depleted ER(+) breast cancer cells.

Postmenopausal women with estrogen receptor positive (ER(+)) breast cancer frequently respond paradoxically to estrogen administration with tumor regression. Using both LTED and E8CASS cells derived from MCF-7 breast cancer cells by long-term estrogen-deprivation, we previously reported that 17beta -estradiol (estradiol) is a powerful, pro-apoptotic hormone which kills the cancer cells through activation of the Fas/FasL death receptor pathway. We postulated that the mitochondrial interactive protein Bcl-2 might play a role in the regulation of estradiol-induced apoptosis in both LTED and E8CASS cells. In this study, we assessed estradiol effects on cell growth, proliferation and apoptosis. Additionally we investigated the effect of estradiol on caspase activation, NF-KB and Bcl-2 expression. The functional role of Bcl-2 in estradiol-induced apoptosis was further studied by knockdown or decrease of Bcl-2 with siRNA. Our results show that estradiol significantly inhibited cell growth primarily through a pro-apoptotic action involving caspase-7 and 9 activations (p < 0.01). Basal Bcl-2 and NF-KB levels were greatly elevated and estradiol decreased NF-KB, but not Bcl-2 expression. Knockdown of Bcl-2 expression with siRNA decreased the levels of this protein by 9 fold (p < 0.01). This reduction markedly sensitized both LTED and E8CASS cells to the pro-apoptotic action of estradiol, leading to a synergistic induction of apoptosis and a concomitant reduction in cell number (p < 0.01). Therefore, down-regulation of Bcl-2 synergistically enhanced estradiol-induced apoptosis in ER(+) postmenopausal breast cancer cells.

Validation of new aromatase monoclonal antibodies for immunohistochemistry: progress report.

Intratumoral aromatase is a potential therapeutic target for the treatment of postmenopausal estrogen-dependent breast cancers. Therefore, reliable methods should be developed for routine application for the detection of intratumoral aromatase. A multi-center collaborative group has been established to generate and validate new aromatase monoclonal antibodies (MAbs). A recombinant GST-aromatase fusion protein was expressed in baculovirus and the purified protein was used for immunization of mice either as a native or formalin-fixed antigen. Hybridomas were generated using standard techniques and screened biochemically prior to immunohistochemistry (IHC) evaluation in human placenta, ovary and breast cancer tissues. Twenty-three MAbs selected by biochemical assays were further evaluated by IHC of paraffin-embedded tissue sections including normal ovary, and placenta, and a small series of 10 breast carcinomas. Of the 23 MAbs, 2 (clones 677 and F2) were determined to specifically stain cell types known to express aromatase in normal tissues. In breast carcinomas staining of malignant epithelium, adipose tissue, normal/benign and stromal compartments was detected. IHC was performed and independently evaluated by three pathologists (HS, TJA and SGS), each using the same evaluation criteria for staining intensity and proportion of immunopositive cells. With these two MAbs, interpathologist and intralaboratory variations were minimal in comparison with differences which could be detected between tissue specimens and antibodies.

Genotoxic metabolites of estradiol in breast: potential mechanism of estradiol induced carcinogenesis.

Long term exposure to estradiol increases the risk of breast cancer in a variety of animal species, as well as in women. The mechanisms responsible for this effect have not been firmly established. The prevailing theory proposes that estrogens increase the rate of cell proliferation by stimulating estrogen receptor-mediated transcription and thereby the number of errors occurring during DNA replication. An alternative hypothesis proposes that estradiol can be metabolized to quinone derivatives which can react with DNA and then remove bases from DNA through a process called depurination. Error prone DNA repair then results in point mutations. We postulate that these two processes, increased cell proliferation and genotoxic metabolite formation, act in an additive or synergistic fashion to induce cancer. If correct, aromatase inhibitors would block both processes whereas anti-estrogens would only inhibit receptor-mediated effects. Accordingly, aromatase inhibitors would be more effective in preventing breast cancer than use of anti-estrogens. Our studies initially demonstrated that catechol estrogen (CE) quinone metabolites are formed in MCF-7 human breast cancer cells in culture. Measurement of estrogen metabolites and conjugates involved utilization of an HPLC separation coupled with an electrochemical detector. We then utilized an animal model that allows dissociation of estrogen receptor-mediated function from that of the effects of estradiol metabolites. Wnt-1 transgenic mice harboring a knock-out of ERalpha provides a means of examining the effect of estrogen deprivation in the absence of the ER in animals with a high incidence of breast tumors. ERbeta was shown to be absent in the breast tissue of these animals by RNase protection assay. In the breast tissue of these estrogen receptor alpha knock-out (ERKO)/Wnt-1 transgenic mice, we demonstrated formation of genotoxic estradiol metabolites. The ERKO/Wnt-1 breast extracts contained picomole amounts of the 4-catechol estrogens, but not their methoxy conjugates nor the 2-CE or their methoxy conjugates. The 4-CE conjugates with glutathione or its hydrolytic products (cysteine and N-acetylcysteine) were detected in picomole amounts in both tumors and hyperplastic mammary tissue, demonstrating the formation of CE-3,4-quinones. These results are consistent with the hypothesis that mammary tumor development is primarily initiated by metabolism of estrogens to 4-CE and, then, to CE-3,4-quinones, which may react with DNA to induce oncogenic mutations. The next set of experiments examined the incidence of tumors formed in Wnt-1 transgenic mice bearing wild type ERalpha (ER+/+), the heterozygous combination of genes (ER+/ER-) or ERalpha knock-out (ER-/-). To assess the effect of estrogens in the absence of ER, half of the animals were oophorectomized on day 15 and the other half were sham operated. Castration reduced the incidence of breast tumors in all animal groups and demonstrated the dependence of tumor formation upon estrogens. A trend toward reduction in tumor number (not statistically significant at this interim analysis) occurred in the absence of functional ER since the number of tumors was markedly reduced in ERKO animals which were castrated early in life. In aggregate, our results support the concept that metabolites of estradiol may act in concert with ER mediated mechanisms to induce breast cancer.

Adaptive hypersensitivity to estrogen: mechanism for superiority of aromatase inhibitors over selective estrogen receptor modulators for breast cancer treatment and prevention.

Clinical observations suggest that human breast tumors can adapt to endocrine therapy by developing hypersensitivity to estradiol (E(2)). To understand the mechanisms responsible, we examined estrogenic stimulation of cell proliferation in a model system and provided in vitro and in vivo evidence that long-term E(2) deprivation (LTED) causes "adaptive hypersensitivity". The enhanced responses to E(2) do not involve mechanisms acting at the level of transcription of estrogen-regulated genes. We found no evidence of hypersensitivity when examining the effects of E(2) on regulation of c-myc, pS2, progesterone receptor, several estrogen receptor (ER) reporter genes, or c-myb in hypersensitive cells. Estrogen deprivation of breast cells long-term does up-regulate both the MAP kinase and phosphatidyl-inositol 3-kinase pathways. As a potential explanation for up-regulation of these signaling pathways, we found that ERalpha is 4- to 10-fold up-regulated and co-opts a classic growth factor pathway using Shc, Grb-2 and Sos. This induces rapid non-genomic effects which are enhanced in LTED cells. E(2) binds to cell membrane-associated ERalpha, physically associates with the adapter protein SHC, and induces its phosphorylation. In turn, Shc binds Grb-2 and Sos, which results in the rapid activation of MAP kinase. These non-genomic effects of E(2) produce biological effects as evidenced by Elk activation and by morphological changes in cell membranes. Further proof of the non-genomic effects of E(2) involved use of cells which selectively expressed ERalpha in the nucleus, cytosol and cell membrane. We created these COS-1 "designer cells" by transfecting ERalpha lacking a nuclear localization signal and containing a membrane localizing signal. The concept of "adaptive hypersensitivity" and the mechanisms responsible for this phenomenon have important clinical implications. Adaptive hypersensitivity would explain the superiority of aromatase inhibitors over the selective ER modulators (SERMs) for treatment of breast cancer. The development of highly potent third-generation aromatase inhibitors allows reduction of breast tissue E2 to very low levels and circumvents the enhanced sensitivity of these cells to the proliferative effects of E(2). Clinical trials in the adjuvant, neoadjuvant and advanced disease settings demonstrate the greater clinical efficacy of the aromatase inhibitors over the SERMs. More recent observations indicate that the aromatase inhibitors are superior for the prevention of breast cancer as well. These observations may be explained by the hypothesis that estrogens induce breast cancer both by stimulating cell proliferation and by their metabolism to genotoxic products. The SERMs block ER-mediated proliferation only, whereas the aromatase inhibitors exert dual effects on proliferation and genotoxic metabolite formation.