Progesterone from ovulatory menstrual cycles is an important cause of breast cancer.

Many factors, including reproductive hormones, have been linked to a woman's risk of developing breast cancer (BC). We reviewed the literature regarding the relationship between ovulatory menstrual cycles (MCs) and BC risk. Physiological variations in the frequency of MCs and interference with MCs through genetic variations, pathological conditions and or pharmaceutical interventions revealed a strong link between BC risk and the lifetime number of MCs. A substantial reduction in BC risk is observed in situations without MCs. In genetic or transgender situations with normal female breasts and estrogens, but no progesterone (P4), the incidence of BC is very low, suggesting an essential role of P4. During the MC, P4 has a strong proliferative effect on normal breast epithelium, whereas estradiol (E2) has only a minimal effect. The origin of BC has been strongly linked to proliferation associated DNA replication errors, and the repeated stimulation of the breast epithelium by P4 with each MC is likely to impact the epithelial mutational burden. Long-lived cells, such as stem cells, present in the breast epithelium, can carry mutations forward for an extended period of time, and studies show that breast tumors tend to take decades to develop before detection. We therefore postulate that P4 is an important factor in a woman's lifetime risk of developing BC, and that breast tumors arising during hormonal contraception or after menopause, with or without menopausal hormone therapy, are the consequence of the outgrowth of pre-existing neoplastic lesions, eventually stimulated by estrogens and some progestins.

Turning scientific serendipity into discoveries in breast cancer research and treatment: a tale of PhD students and a 50-year roaming tamoxifen team.

PURPOSE: This retrospective, about a single "mobile" laboratory in six locations on two continents, is intended as a case study in discovery for trainees and junior faculty in the medical sciences. Your knowledge of your topic is necessary to expect the unexpected. HISTORICAL METHOD: In 1972, there was no tamoxifen, only ICI 46, 474, a non-steroidal anti-estrogen with little chance of clinical development. No one would ever be foolish enough to predict that the medicine, 20 years later, would achieve legendary status as the first targeted treatment for breast cancer, and millions of women would benefit from long-term adjuvant tamoxifen therapy. The secret of tamoxifen's success was a translational research strategy proposed in the mid 1970's. This strategy was to treat only patients with estrogen receptor (ER)-positive breast cancer and deploy 5 or more years of adjuvant tamoxifen therapy to prevent recurrence. Additionally, tamoxifen prevented mammary cancer in animals. Could the medicine prevent breast cancer in women? RESULTS: Tamoxifen and the failed breast cancer drug raloxifene became the first selective estrogen receptor modulators (SERMs): a new drug group, discovered at the University of Wisconsin, Comprehensive Cancer Center. Serendipity can play a fundamental role in discovery, but there must be a rigorous preparation for the investigator to appreciate the possibility of a pending discovery. This article follows the unanticipated discoveries when PhD students "get the wrong answer." The secret of success of my six Tamoxifen Teams was their technical excellence to create models, to decipher mechanisms, that drove the development of new medicines. Discoveries are listed that either changed women's health or allowed an understanding of originally opaque mechanisms of action of potential therapies. These advances in women's health were supported entirely by government-sponsored peer-reviewed funding and major philanthropy from the Lynn Sage Breast Cancer Foundation, the Avon Foundation, and the Susan G. Komen Breast Cancer Foundation. The resulting lives saved or extended, families aided in a time of crisis and the injection of billions of dollars into national economies by drug development, is proof of the value of Federal or philanthropic investment into unencumbered research aimed at saving millions of lives.

Pharmacology and Molecular Mechanisms of Clinically Relevant Estrogen Estetrol and Estrogen Mimic BMI-135 for the Treatment of Endocrine-Resistant Breast Cancer.

Long-term estrogen deprivation (LTED) with tamoxifen (TAM) or aromatase inhibitors leads to endocrine-resistance, whereby physiologic levels of estrogen kill breast cancer (BC). Estrogen therapy is effective in treating patients with advanced BC after resistance to TAM and aromatase inhibitors develops. This therapeutic effect is attributed to estrogen-induced apoptosis via the estrogen receptor (ER). Estrogen therapy can have unpleasant gynecologic and nongynecologic adverse events. Here, we study estetrol (E4) and a model Selective Human ER Partial Agonist (ShERPA) BMI-135. Estetrol and ShERPA TTC-352 are being evaluated in clinical trials. These agents are proposed as safer estrogenic candidates compared with 17ß-estradiol (E2) for the treatment of endocrine-resistant BC. Cell viability assays, real-time polymerase chain reaction, luciferase reporter assays, chromatin immunoprecipitation, docking and molecular dynamics simulations, human unfolded protein response (UPR) RT2 PCR profiler arrays, live cell microscopic imaging and analysis, and annexin V staining assays were conducted. Our work was done in eight biologically different human BC cell lines and one human endometrial cancer cell line, and results were compared with full agonists estrone, E2, and estriol, a benchmark partial agonist triphenylethylene bisphenol (BPTPE), and antagonists 4-hydroxytamoxifen and endoxifen. Our study shows the pharmacology of E4 and BMI-135 as less-potent full-estrogen agonists as well as their molecular mechanisms of tumor regression in LTED BC through triggering a rapid UPR and apoptosis. Our work concludes that the use of a full agonist to treat BC is potentially superior to a partial agonist given BPTPE's delayed induction of UPR and apoptosis, with a higher probability of tumor clonal evolution and resistance. SIGNIFICANCE STATEMENT: Given the unpleasant gynecologic and nongynecologic adverse effects of estrogen treatment, the development of safer estrogens for endocrine-resistant breast cancer (BC) treatment and hormone replacement therapy remains a priority. The naturally occurring estrogen estetrol and Selective Human Estrogen-Receptor Partial Agonists are being evaluated in endocrine-resistant BC clinical trials. This work provides a comprehensive evaluation of their pharmacology in numerous endocrine-resistant BC models and an endometrial cancer model and their molecular mechanisms of tumor regression through the unfolded protein response and apoptosis.

The Structure-Function Relationship of Angular Estrogens and Estrogen Receptor Alpha to Initiate Estrogen-Induced Apoptosis in Breast Cancer Cells.

High-dose synthetic estrogen therapy was the standard treatment of advanced breast cancer for three decades until the discovery of tamoxifen. A range of substituted triphenylethylene synthetic estrogens and diethylstilbestrol were used. It is now known that low doses of estrogens can cause apoptosis in long-term estrogen deprived (LTED) breast cancer cells resistant to antiestrogens. This action of estrogen can explain the reduced breast cancer incidence in postmenopausal women over 60 who are taking conjugated equine estrogens and the beneficial effect of low-dose estrogen treatment of patients with acquired aromatase inhibitor resistance in clinical trials. To decipher the molecular mechanism of estrogens at the estrogen receptor (ER) complex by different types of estrogens-planar [17ß-estradiol (E2)] and angular triphenylethylene (TPE) derivatives-we have synthesized a small series of compounds with either no substitutions on the TPE phenyl ring containing the antiestrogenic side chain of endoxifen or a free hydroxyl. In the first week of treatment with E2 the LTED cells undergo apoptosis completely. By contrast, the test TPE derivatives act as antiestrogens with a free para-hydroxyl on the phenyl ring that contains an antiestrogenic side chain in endoxifen. This inhibits early E2-induced apoptosis if a free hydroxyl is present. No substitution at the site occupied by the antiestrogenic side chain of endoxifen results in early apoptosis similar to planar E2 The TPE compounds recruit coregulators to the ER differentially and predictably, leading to delayed apoptosis in these cells. SIGNIFICANCE STATEMENT: In this paper we investigate the role of the structure-function relationship of a panel of synthetic triphenylethylene (TPE) derivatives and a novel mechanism of estrogen-induced cell death in breast cancer, which is now clinically relevant. Our study indicates that these TPE derivatives, depending on the positioning of the hydroxyl groups, induce various conformations of the estrogen receptor's ligand-binding domain, which in turn produces differential recruitment of coregulators and subsequently different apoptotic effects on the antiestrogen-resistant breast cancer cells.

The SERM Saga, Something from Nothing: American Cancer Society/SSO Basic Science Lecture.

BACKGROUND: The discovery of nonsteroidal antiestrogens created a new group of medicines looking for an application; however, at the time, cytotoxic chemotherapy was the modality of choice to treat all cancers. Antiestrogens were orphan drugs until 1971, with the passing of the National Cancer Act. This enabled laboratory innovations to aid patient care. METHODS: This article traces the strategic application of tamoxifen to treat breast cancer by targeting the estrogen receptor (ER), deploying long-term adjuvant tamoxifen therapy, and becoming the first chemopreventive for any cancer. Laboratory discoveries from the University of Wisconsin Comprehensive Cancer Center (UWCCC) are described that address a broad range of biological issues with tamoxifen. These translated to improvements in clinical care. RESULTS: Tamoxifen was studied extensively at UWCCC in the 1980s for the development of acquired resistance to long-term therapy. Additionally, the long-term metabolism of tamoxifen and regulation of growth factors were also studied. A concern with tamoxifen use for chemoprevention was that an antiestrogen would increase bone loss and atherosclerosis. Laboratory studies with tamoxifen and keoxifene (subsequently named raloxifene) demonstrated that 'nonsteroidal antiestrogens' maintained bone density, and this translated into successful clinical trials with tamoxifen at UWCCC. However, tamoxifen also increased endometrial cancer growth; this discovery in the laboratory translated into changes in clinical care. Selective estrogen receptor modulators (SERMs) were born at UWCCC. CONCLUSIONS: There are now five US FDA-approved SERMs, all with discovery origins at UWCCC. Women's health was revolutionized as SERMs have the ability to treat multiple diseases by switching target sites around a woman's body on or off.

Endoxifen, 4-Hydroxytamoxifen and an Estrogenic Derivative Modulate Estrogen Receptor Complex Mediated Apoptosis in Breast Cancer.

Estrogen therapy was used to treat advanced breast cancer in postmenopausal women for decades until the introduction of tamoxifen. Resistance to long-term estrogen deprivation (LTED) with tamoxifen and aromatase inhibitors used as a treatment of breast cancer inevitably occurs, but unexpectedly low-dose estrogen can cause regression of breast cancer and increase disease-free survival in some patients. This therapeutic effect is attributed to estrogen-induced apoptosis in LTED breast cancer. Here, we describe modulation of the estrogen receptor (ER) liganded with antiestrogens (endoxifen and 4-hydroxytamoxifen) and an estrogenic triphenylethylene (TPE), ethoxytriphenylethylene (EtOXTPE), on estrogen-induced apoptosis in LTED breast cancer cells. Our results show that the angular TPE estrogen (EtOXTPE) is able to induce the ER-mediated apoptosis only at a later time compared with planar estradiol in these cells. Using real-time polymerase chain reaction, chromatin immunoprecipitation, western blotting, molecular modeling, and X-ray crystallography techniques, we report novel conformations of the ER complex with an angular estrogen EtOXTPE and endoxifen. We propose that alteration of the conformation of the ER complexes, with changes in coactivator binding, governs estrogen-induced apoptosis through the protein kinase regulated by RNA-like endoplasmic reticulum kinase sensor system to trigger an unfolded protein response.

Chemoprevention of breast cancer with selective oestrogen-receptor modulators.

Twenty years ago, a new therapeutic dimension was conceived that not only had the potential to treat and prevent osteoporosis, but to prevent breast and endometrial cancer at the same time. As osteoporosis was known to be caused by oestrogen withdrawal after menopause, whereas breast and endometrial cancer are caused by unopposed oestrogen action, the new tissue-selective oestrogens and anti-oestrogens, or selective oestrogen-receptor modulators (SERMs), had to recruit new networks to activate or suppress target tissues selectively. New medicines now promise to provide chemoprevention strategies for women at risk for the development of many diseases.

Differential DNase I hypersensitivity reveals factor-dependent chromatin dynamics.

Transcription factor cistromes are highly cell-type specific. Chromatin accessibility, histone modifications, and nucleosome occupancy have all been found to play a role in defining these binding locations. Here, we show that hormone-induced DNase I hypersensitivity changes (ΔDHS) are highly predictive of androgen receptor (AR) and estrogen receptor 1 (ESR1) binding in prostate cancer and breast cancer cells, respectively. While chromatin structure prior to receptor binding and nucleosome occupancy after binding are strikingly different for ESR1 and AR, ΔDHS is highly predictive for both. AR binding is associated with changes in both local nucleosome occupancy and DNase I hypersensitivity. In contrast, while global ESR1 binding is unrelated to changes in nucleosome occupancy, DNase I hypersensitivity dynamics are also predictive of the ESR1 cistrome. These findings suggest that AR and ESR1 have distinct modes of interaction with chromatin and that DNase I hypersensitivity dynamics provides a general approach for predicting cell-type specific cistromes.

Inhibition of BET proteins impairs estrogen-mediated growth and transcription in breast cancers by pausing RNA polymerase advancement.

Estrogen (E2)-induced transcription requires coordinated recruitment of estrogen receptor α (ER) and multiple factors at the promoter of activated genes. However, the precise mechanism by which this complex stimulates the RNA polymerase II activity required to execute transcription is largely unresolved. We investigated the role of bromodomain (BRD) containing bromodomain and extra-terminal (BET) proteins, in E2-induced growth and gene activation. JQ1, a specific BET protein inhibitor, was used to block BET protein function in two different ER-positive breast cancer cell lines (MCF7 and T47D). Real-time PCR and ChIP assays were used to measure RNA expression and to detect recruitment of various factors on the genes, respectively. Protein levels were measured by Western blotting. JQ1 suppressed E2-induced growth and transcription in both MCF7 and T47D cells. The combination of E2 and JQ1 down-regulated the levels of ER protein in MCF7 cells but the loss of ER was not responsible for JQ1-mediated inhibition of E2 signaling. JQ1 did not disrupt E2-induced recruitment of ER and co-activator (SRC3) at the E2-responsive DNA elements. The E2-induced increase in histone acetylation was also not altered by JQ1. However, JQ1 blocked the E2-induced transition of RNA polymerase II from initiation to elongation by stalling it at the promoter region of the responsive genes upstream of the transcription start site. This study establishes BET proteins as the key mediators of E2-induced transcriptional activation. This adds another layer of complexity to the regulation of estrogen-induced gene activation that can potentially be targeted for therapeutic intervention.

Obesity and male breast cancer: provocative parallels?

While rare compared to female breast cancer the incidence of male breast cancer (MBC) has increased in the last few decades. Without comprehensive epidemiological studies, the explanation for the increased incidence of MBC can only be speculated. Nevertheless, one of the most worrying global public health issues is the exponential rise in the number of overweight and obese people, especially in the developed world. Although obesity is not considered an established risk factor for MBC, studies have shown increased incidence among obese individuals. With this observation in mind, this article highlights the correlation between the increased incidence of MBC and the current trends in obesity as a growing problem in the 21(st) century, including how this may impact treatment. With MBC becoming more prominent we put forward the notion that, not only is obesity a risk factor for MBC, but that increasing obesity trends are a contributing factor to its increased incidence.

Defining the conformation of the estrogen receptor complex that controls estrogen-induced apoptosis in breast cancer.

Development of acquired antihormone resistance exposes a vulnerability in breast cancer: estrogen-induced apoptosis. Triphenylethylenes (TPEs), which are structurally similar to 4-hydroxytamoxifen (4OHT), were used for mechanistic studies of estrogen-induced apoptosis. These TPEs all stimulate growth in MCF-7 cells, but unlike the planar estrogens they block estrogen-induced apoptosis in the long-term estrogen-deprived MCF7:5C cells. To define the conformation of the TPE:estrogen receptor (ER) complex, we employed a previously validated assay using the induction of transforming growth factor α (TGFα) mRNA in situ in MDA-MB 231 cells stably transfected with wild-type ER (MC2) or D351G ER mutant (JM6). The assays discriminate ligand fit in the ER based on the extremes of published crystallography of planar estrogens or TPE antiestrogens. We classified the conformation of planar estrogens or angular TPE complexes as "estrogen-like" or "antiestrogen-like" complexes, respectively. The TPE:ER complexes did not readily recruit the coactivator steroid receptor coactivator-3 (SRC3) or ER to the PS2 promoter in MCF-7 and MCF7:5C cells, and molecular modeling showed that they prefer to bind to the ER in an antagonistic fashion, i.e., helix 12 not sealing the ligand binding domain (LBD) effectively, and therefore reduce critical SRC3 binding. The fully activated ER complex with helix 12 sealing the LBD is suggested to be the appropriate trigger to initiate rapid estrogen-induced apoptosis.

Cyclin dependent kinase-9 mediated transcriptional de-regulation of cMYC as a critical determinant of endocrine-therapy resistance in breast cancers.

Endocrine therapy resistance in estrogen receptor alpha positive (ERα+) breast cancers remains a major obstacle for maintaining efficacy of targeted therapies. We investigated the significance and the mechanisms involved in cMYC over-expression in a MCF7 derived panel of ERα+ breast cancer cells which can proliferate in the absence of estrogen with different sensitivities to anti-hormone therapies. We show that all the resistant cell lines tested over-express cMYC as compared to parental MCF7 cells and its inhibition lead to the differential blocking of estrogen-independent proliferation in resistant cells. Further investigation of the resistant cell line, MCF7:5C, suggested transcriptional de-regulation of cMYC gene was responsible for its over-expression. Chromatin immuno-precipitation assay revealed markedly higher recruitment of phosphorylated serine-2 carboxy-terminal domain (CTD) of RNA polymerase-II at the proximal promoter of cMYC gene, which is responsible for transcriptional elongation of the cMYC RNA. The level of CDK9, a factor responsible for the phosphorylation of serine-2 of RNA polymerase II CTD, was found to be elevated in all the resistant cell lines. Pharmacological inhibition of CDK9 not only reduced the transcripts and the protein levels of cMYC in MCF7:5C cells but also selectively inhibited the estrogen-independent growth of all the resistant cell lines. This study describes the up-stream molecular events involved in the transcriptional over-expression of cMYC gene in breast cancer cells proliferating estrogen-independently and identifies CDK9 as a potential novel drug target for therapeutic intervention in endocrine-resistant breast cancers.

Estrogen induces apoptosis in estrogen deprivation-resistant breast cancer through stress responses as identified by global gene expression across time.

In laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with either 17ß-estradiol (E(2)) or tamoxifen. Phase II resistance develops after 5 y of therapy, and tamoxifen still stimulates growth; however, E(2) paradoxically induces apoptosis. This finding is the basis for the clinical use of estrogen to treat advanced antihormone-resistant breast cancer. We interrogated E(2)-induced apoptosis by analysis of gene expression across time (2-96 h) in MCF-7 cell variants that were estrogen-dependent (WS8) or resistant to estrogen deprivation and refractory (2A) or sensitive (5C) to E(2)-induced apoptosis. We developed a method termed differential area under the curve analysis that identified genes uniquely regulated by E(2) in 5C cells compared with both WS8 and 2A cells and hence, were associated with E(2)-induced apoptosis. Estrogen signaling, endoplasmic reticulum stress (ERS), and inflammatory response genes were overrepresented among the 5C-specific genes. The identified ERS genes indicated that E(2) inhibited protein folding, translation, and fatty acid synthesis. Meanwhile, the ERS-associated apoptotic genes Bcl-2 interacting mediator of cell death (BIM; BCL2L11) and caspase-4 (CASP4), among others, were induced. Evaluation of a caspase peptide inhibitor panel showed that the CASP4 inhibitor z-LEVD-fmk was the most active at blocking E(2)-induced apoptosis. Furthermore, z-LEVD-fmk completely prevented poly (ADP-ribose) polymerase (PARP) cleavage, E(2)-inhibited growth, and apoptotic morphology. The up-regulated proinflammatory genes included IL, IFN, and arachidonic acid-related genes. Functional testing showed that arachidonic acid and E(2) interacted to superadditively induce apoptosis. Therefore, these data indicate that E(2) induced apoptosis through ERS and inflammatory responses in advanced antihormone-resistant breast cancer.

DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination.

OBJECTIVES: Cell lines derived from human ovarian and endometrial cancers, and their immortalized non-malignant counterparts, are critical tools to investigate and characterize molecular mechanisms underlying gynecologic tumorigenesis, and facilitate development of novel therapeutics. To determine the extent of misidentification, contamination and redundancy, with evident consequences for the validity of research based upon these models, we undertook a systematic analysis and cataloging of endometrial and ovarian cell lines. METHODS: Profiling of cell lines by analysis of DNA microsatellite short tandem repeats (STR), p53 nucleotide polymorphisms and microsatellite instability was performed. RESULTS: Fifty-one ovarian cancer lines were profiled with ten found to be redundant and five (A2008, OV2008, C13, SK-OV-4 and SK-OV-6) identified as cervical cancer cells. Ten endometrial cell lines were analyzed, with RL-92, HEC-1A, HEC-1B, HEC-50, KLE, and AN3CA all exhibiting unique, uncontaminated STR profiles. Multiple variants of Ishikawa and ECC-1 endometrial cancer cell lines were genotyped and analyzed by sequencing of mutations in the p53 gene. The profile of ECC-1 cells did not match the EnCa-101 tumor, from which it was reportedly derived, and all ECC-1 isolates were genotyped as Ishikawa cells, MCF-7 breast cancer cells, or a combination thereof. Two normal, immortalized endometrial epithelial cell lines, HES cells and the hTERT-EEC line, were identified as HeLa cervical carcinoma and MCF-7 breast cancer cells, respectively. CONCLUSIONS: Results demonstrate significant misidentification, duplication, and loss of integrity of endometrial and ovarian cancer cell lines. Authentication by STR DNA profiling is a simple and economical method to verify and validate studies undertaken with these models.

Selective estrogen receptor modulation: concept and consequences in cancer.

Extended exposure to the selective estrogen receptor modulators (SERMs) such as raloxifene to prevent osteoporosis and tamoxifen or the aromatase inhibitors to treat or prevent breast cancer are established therapeutic strategies. However, there are now clearly defined consequences of exhaustive antihormonal therapy in breast cancer. Ultimately, drug resistance to SERMs and aromatase inhibitors enhances cancer cell survival but a paradoxical supersensitivity to estrogen action develops that causes cancer cell apoptosis. The future exploitation of these novel data will allow selective killing of cancer with fewer side effects for patients.