Effect of a tissue selective estrogen complex on breast cancer: Role of unique properties of conjugated equine estrogen.

The Women's Health Initiative studies reported that the menopausal hormone therapy (MHT) regimen containing conjugated equine estrogen (CEE) and medroxyprogesterone acetate increased, whereas CEE alone reduced breast cancer incidence. These observations suggest the possibility that CEE might exert unique actions on breast and also suggest the need to eliminate the progestogen from MHT regimens. A MHT regimen called a tissue selective estrogen complex (TSEC), containing CEE plus bazedoxifene (BZA), to avoid the need for a progestogen, was developed and FDA approved. Our study addressed two questions regarding this TSEC: (i) whether CEE exert effects on breast cancer which differ from those of estradiol (E2 ) and (ii) whether BZA antagonize the effects of E2 and CEE on breast cancer? Two rodent models (NMU and ACI) were used to compare the effect of CEE with E2 on mammary tumor formation, proliferation and apoptosis. In both the NMU and ACI models, E2 significantly increased tumor incidence and multiplicity whereas in striking contrast CEE did not, even though the estrogenic effects of CEE and E2 on uterine weight were identical. Mechanistically E2 blocked whereas CEE stimulated apoptosis (cleaved caspase-3) in ACI animals and only E2 stimulated proliferation (Ki67). BZA exerted highly potent anti-estrogenic effects on tumors by completely blocking palpable tumor formation. These data suggest that the CEE/BZA TSEC may be a safer, breast-antagonistic, MHT agent for women and might have potential to prevent breast cancer while relieving menopausal symptoms.

Adenosine monophosphate activated protein kinase (AMPK), a mediator of estradiol-induced apoptosis in long-term estrogen deprived breast cancer cells.

Estrogens stimulate growth of hormone-dependent breast cancer but paradoxically induce tumor regress under certain circumstances. We have shown that long-term estrogen deprivation (LTED) enhances the sensitivity of hormone dependent breast cancer cells to estradiol (E2) so that physiological concentrations of estradiol induce apoptosis in these cells. E2-induced apoptosis involve both intrinsic and extrinsic pathways but precise mechanisms remain unclear. We found that exposure of LTED MCF-7 cells to E2 activated AMP activated protein kinase (AMPK). In contrast, E2 inhibited AMPK activation in wild type MCF-7 cells where E2 prevents apoptosis. As a result of AMPK activation, the transcriptional activity of FoxO3, a downstream factor of AMPK, was up-regulated in E2 treatment of LTED. Increased activity of FoxO3 was demonstrated by up-regulation of three FoxO3 target genes, Bim, Fas ligand (FasL), and Gadd45α. Among them, Bim and FasL mediate intrinsic and extrinsic apoptosis respectively and Gadd45α causes cell cycle arrest at the G2/M phase. To further confirm the role of AMPK in apoptosis, we used AMPK activator AICAR in wild type MCF-7 cells and examined apoptosis, proliferation and expression of Bim, FasL, and Gadd45α. The effects of AICAR on these parameters recapitulated those observed in E2-treated LTED cells. Activation of AMPK by AICAR also increased expression of Bax in MCF-7 cells and its localization to mitochondria, which is a required process for apoptosis. These results reveal that AMPK is an important factor mediating E2-induced apoptosis in LTED cells, which is implicative of therapeutic potential for relapsing breast cancer after hormone therapy.

Breast-related effects of selective estrogen receptor modulators and tissue-selective estrogen complexes.

A number of available treatments provide relief of menopausal symptoms and prevention of postmenopausal osteoporosis. However, as breast safety is a major concern, new options are needed, particularly agents with an improved mammary safety profile. Results from several large randomized and observational studies have shown an association between hormone therapy, particularly combined estrogen-progestin therapy, and a small increased risk of breast cancer and breast pain or tenderness. In addition, progestin-containing hormone therapy has been shown to increase mammographic breast density, which is an important risk factor for breast cancer. Selective estrogen receptor modulators (SERMs) provide bone protection, are generally well tolerated, and have demonstrated reductions in breast cancer risk, but do not relieve menopausal symptoms (that is, vasomotor symptoms). Tissue-selective estrogen complexes (TSECs) pair a SERM with one or more estrogens and aim to blend the positive effects of the components to provide relief of menopausal symptoms and prevention of postmenopausal osteoporosis without stimulating the breast or endometrium. One TSEC combination pairing conjugated estrogens (CEs) with the SERM bazedoxifene (BZA) has completed clinical development and is now available as an alternative option for menopausal therapy. Preclinical evidence suggests that CE/BZA induces inhibitory effects on breast tissue, and phase 3 clinical studies suggest breast neutrality, with no increases seen in breast tenderness, breast density, or cancer. In non-hysterectomized postmenopausal women, CE/BZA was associated with increased bone mineral density and relief of menopausal symptoms, along with endometrial safety. Taken together, these results support the potential of CE/BZA for the relief of menopausal symptoms and prevention of postmenopausal osteoporosis combined with breast and endometrial safety.

Role of ERα and Aromatase in Juvenile Gigantomastia.

CONTEXT: Approximately 150 patients with juvenile gigantomastia have been reported in the literature but the underlying biologic mechanisms remain unknown. OBJECTIVE: To conduct extensive clinical, biochemical, immunochemical, and genetic studies in 3 patients with juvenile gigantomastia to determine causative biologic factors. METHODS: We examined clinical effects of estrogen by blockading estrogen synthesis or its action. Breast tissue aromatase expression and activity were quantitated in 1 patient and 5 controls. Other biochemical markers, including estrogen receptor α (ERα), cyclin D1 and E, p-RB, p-MAPK, p-AKT, BCL-2, EGF-R, IGF-IR ß, and p-EGFR were assayed by Western blot. Immunohistochemical analyses for aromatase, ERα and ß, PgR, Ki67, sulfotransferase, estrone sulfatase, and 17ßHD were performed in all 3 patients. The entire genomes of the mother, father, and patient in the 3 families were sequenced. RESULTS: Blockade of estrogen synthesis or action in patients resulted in demonstrable clinical effects. Biochemical studies on fresh frozen tissue revealed no differences between patients and controls, presumably due to tissue dilution from the large proportion of stroma. However, immunohistochemical analysis of ductal breast cells in the 3 patients revealed a high percent of ERα (64.1% ± 7.8% vs reference women 9.6%, range 2.3-15%); aromatase score of 4 (76%-100% of cells positive vs 30.4% ± 5.6%); PgR (69.5% ± 15.2% vs 6.0%, range 2.7%-11.9%) and Ki67 (23.7% ± 0.54% vs 4.2%). Genetic studies were inconclusive although some intriguing variants were identified. CONCLUSION: The data implicate an important biologic role for ERα to increase tissue sensitivity to estrogen and aromatase to enhance local tissue production as biologic factors involved in juvenile gigantomastia.

Induction of apoptosis in hormone refractory breast cancer: horizontal modulation is superior to vertical.

Women with estrogen receptor positive (ER+) breast cancer receive treatment with tamoxifen or aromatase inhibitors as adjuvant hormone therapy, but their tumors frequently exhibit de novo or acquired resistance. Current strategies being developed to overcome resistance involve a combination of growth factor pathway inhibitors in addition to hormone therapy. Unfortunately, prolongation of responses with these new approaches is measured only in months. We reasoned that a pro-apoptotic strategy would be preferable since cell death would abrogate the process of adaptive reprogramming and eliminate the resistant cells rather than inhibiting their growth. Our hypothesis was that combinations of pro-apoptotic agents could be designed that would act synergistically as opposed to a merely additively. We examined two model strategies to determine which would result in the greatest synergy: a vertical approach that involved the targeting of two or more steps in a single pathway and a horizontal one, targeting steps in more than one parallel pathway. We found that combinations involving horizontal activation resulted in greater synergy than vertical. Combination index and isobologram analyses revealed that the horizontal combination of the small molecule 4-(4-chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH) along with T-DM1 displayed the strongest synergy for inducing apoptosis in hormone refractory breast cancer cells. Both the reprogrammed, hormone resistant cells and the wild type responded to certain combinations with synergistic enhancement of apoptosis. These data suggest that combinations using T-DM1 are promising for further in vivo studies both in xenografts and in patients.

Intensive, telemedicine-based, self-management program for rural, underserved patients with diabetes mellitus: Re-entry of retired endocrinologists into practice.

INTRODUCTION: Endocrinology workforce data demonstrate a substantial gap in the number of practicing endocrinologists, a phenomenon particularly affecting patients with diabetes in rural, financially challenged, underserved areas. We evaluated the concept that retired endocrinologists could re-enter practice part time and utilize telemedicine in collaboration with personnel in Federally Qualified Community Health Center clinics to conduct an intensive self-management program and provide 6-month concurrent care for patients with diabetes. METHODS: The program involved intensive glucose control measures and education in diabetes, nutrition, and lifestyle changes over a 6-month period. Key elements included comprehensive initial telehealth evaluations, frequent phone calls, and collaboration with certified diabetes care and education specialists, referring providers, referring-clinic staff, and the University of Virginia Telehealth Center. RESULTS: The mean A1C in the 139 patients completing the 6-month self-management program decreased from 10.3 ± 1.94% to 7.78 ± 1.51% p < 0.0001. The number of treatment modalities per patient ranged from one to five with several different regimens utilized. The majority of patients maintained the reduction in A1C levels without recidivism over a mean follow-up of 16 months after discharge. Strategies using meal replacements are being implemented to facilitate weight loss. DISCUSSION: This program resulted in improved A1C levels of patients with diabetes in rural, financially challenged, underserved areas; met recidivism goals; and provided a practical template to reduce the workforce gap of endocrinologists in those areas.

Mechanistic Effects of Estrogens on Breast Cancer.

PURPOSE: Current concepts regarding estrogen and its mechanistic effects on breast cancer in women are evolving. This article reviews studies that address estrogen-mediated breast cancer development, the prevalence of occult tumors at autopsy, and the natural history of breast cancer as predicted by a newly developed tumor kinetic model. METHODS: This article reviews previously published studies from the authors and articles pertinent to the data presented. RESULTS: We discuss the concepts of adaptive hypersensitivity that develops in response to long-term deprivation of estrogen and results in both increased cell proliferation and apoptosis. The effects of menopausal hormonal therapy on breast cancer in postmenopausal women are interpreted based on the tumor kinetic model. Studies of the administration of a tissue selective estrogen complex in vitro, in vivo, and in patients are described. We review the various clinical studies of breast cancer prevention with selective estrogen receptor modulators and aromatase inhibitors. Finally, the effects of the underlying risk of breast cancer on the effects of menopausal hormone therapy are outlined. DISCUSSION: The overall intent of this review is to present data supporting recent concepts, discuss pertinent literature, and critically examine areas of controversy.

Approach to the Patient With New-Onset Secondary Amenorrhea: Is This Primary Ovarian Insufficiency?

Menstrual cyclicity is a marker of health for reproductively mature women. Absent menses, or amenorrhea, is often the initial sign of pregnancy-an indication that the system is functioning appropriately and capable of generating the intended evolutionary outcome. Perturbations of menstrual regularity in the absence of pregnancy provide a marker for physiological or pathological disruption of this well-orchestrated process. New-onset amenorrhea with duration of 3 to 6 months should be promptly evaluated. Secondary amenorrhea can reflect structural or functional disturbances occurring from higher centers in the hypothalamus to the pituitary, the ovary, and finally, the uterus. Amenorrhea can also be a manifestation of systemic disorders resulting in compensatory inhibition of reproduction. Identifying the point of the breakdown is essential to restoring reproductive homeostasis to maintain future fertility and reestablish reproductive hormonal integrity. Among the most challenging disorders contributing to secondary amenorrhea is primary ovarian insufficiency (POI). This diagnosis stems from a number of possible etiologies, including autoimmune, genetic, metabolic, toxic, iatrogenic, and idiopathic, each with associated conditions and attendant medical concerns. The dual assaults of unanticipated compromised fertility concurrently with depletion of the normal reproductive hormonal milieu yield multiple management challenges. Fertility restoration is an area of active research, while optimal management of estrogen deficiency symptoms and the anticipated preventive benefits of hormone replacement for bone, cardiovascular, and neurocognitive health remain understudied. The state of the evidence for an optimal, individualized, clinical management approach to women with POI is discussed along with priorities for additional research in this population.

Isolated and combined action of tamoxifen and metformin in wild-type, tamoxifen-resistant, and estrogen-deprived MCF-7 cells.

Resistance to tamoxifen (TAM) and aromatase inhibitors represents a major drawback to the treatment of hormone-dependent breast cancer, and strategies to overcome this problem are urgently needed. The anti-diabetic biguanide metformin (MF) exerts pleiotropic effects which could enhance the effectiveness of available hormonal therapies. This study modeled several aspects of hormonal therapy in women and examined the effectiveness of MF under those conditions. For cell growth evaluation, wild-type (wt), TAM-resistant (TAM-R), and long-term estradiol-deprived (LTED) MCF-7 cells, as a model of aromatase inhibitor resistance, were grown in the presence or absence of TAM or MF for 5 days. For immunoblot analysis and aromatase activity measurements, these cells were grown for 48 h. Wild-type and LTED cells were equally sensitive to the growth inhibitory effects of TAM and MF, while TAM-R cells were less sensitive to TAM than to MF. Partial additive effects on cell number of TAM combined with MF were greatest (if compared with isolated TAM action) in TAM-R and LTED cells. In contrast to the decrease in PCNA values in TAM-resistant cells treated with the TAM and MF combination, no other changes were found in the levels of this proliferation marker. These findings suggested a major component of apoptosis in the growth inhibitory effect. This was confirmed with Western blot analysis of PARP and caspase 7 as well as with apoptosis ELISA assay. MF also altered signaling pathways. AMP-kinase was stimulated by MF approximately equally in MCF-7, TAM-R, and LTED cells, while inhibition by biguanide of p-S6K as a downstream target of mTOR was strongest in TAM-R cells. Under the influence of MF, expression of ER-α was decreased in wt MCF-7 cells suggesting possible involvement of this compound in estrogen signaling. Metformin interacts additively with TAM to reduce neoplastic cells growth. The cellular context (including loss of sensitivity to TAM and estrogen deprivation) is of importance in influencing breast cancer responses to MF and to a combination of MF and TAM.

Tetra-methoxystilbene modulates ductal growth of the developing murine mammary gland.

Extensive data suggest that estradiol contributes to the development of breast cancer by acting as a mitogen and exerting direct genotoxic effects after enzymatic conversion to 4-hydroxyestradiol (4-OHE2) via cytochrome P450 1B1 (CYP1B1). The mammary gland, ovary, and uterus all express CYP1B1. Overexpression of this enzyme has been associated with an increased risk of breast cancer and blockade might reduce this carcinogenic effect. For this reason, we conducted systematic in vitro and in vivo studies of a CYP1B1 inhibitor, TMS (2,3',4,5'-tetramethoxystilbene). We found that TMS blocked the enzymatic conversion of radiolabeled estradiol to both 2-hydroxyestradiol (2-OHE2) and 4-OHE2, but did not inhibit Cyp1b1 message formation. In vivo studies using mass spectrometry showed that TMS inhibited formation of 2-OHE2 and 4-OHE2 and the resulting estrogen-DNA adducts. To examine its biologic actions in vivo, we investigated whether TMS could block the hyperplastic changes that occur in the developing breast of aromatase-transfected mice. We found that TMS induced a significant reduction of ductal structures in mice less than 6 months in age. In older mice, no reduction in breast morphology occurred. These latter studies uncovered unexpected estrogen agonistic actions of TMS at high doses, including a paradoxical stimulation of breast ductal structures and the endometrium. These studies suggest that the enzyme inhibitory properties of TMS, as well as the effects on developing breast, could implicate a role for TMS in breast cancer prevention, but only in low doses and on developing breast.

Aromatase immunoreactivity is increased in mammographically dense regions of the breast.

Mammographic breast density (MBD) is one of the strongest risk factors for breast cancer. Unfortunately, the biologic basis underlying this association is unknown. This study compared aromatase expression or immunoreactivity (IR) in core biopsies from mammographically dense versus non-dense regions of the breast to examine whether estrogen synthesis in the breast is associated with MBD and one possible mechanism through which MBD may influence breast cancer. Eligible participants were 40+ years, had a screening mammogram with visible MBD and no prior cancer or current endocrine therapy. Mammograms were used to identify dense and non-dense regions and ultrasound-guided core biopsies were performed to obtain tissue from these regions. Immunostaining for aromatase employed the streptavidin-biotin amplification method and #677 mouse monoclonal antibody. Aromatase IR was scored in terms of extent and intensity of staining for each cell type (stroma, epithelium, adipocytes) on histologic sections. A modified histological H-score provided quantitation of aromatase IR in each cell type and overall. Repeated measure analyses evaluated average differences (ß(H)) in H-score in dense versus non-dense tissue within and across cell types. Forty-nine women with mean age 50 years (range: 40-82), participated. Aromatase IR was increased in dense (vs. non-dense) tissue in both the stroma (ß(H) = 0.58) and epithelium (ß(H) = 0.12) (P < 0.01). Adipocytes from non-dense tissue, however, had a greater IR compared to those from dense tissue (ß(H) = -0.24, P < 0.01). An overall H-score which integrated results from all cell types demonstrated that aromatase IR was twice as great for dense (mean H-score = 0.90, SD = 0.53) versus non-dense (mean H-score = 0.45, SD = 0.39) breast tissue (ß(H) = 0.45; P < 0.001). Overall, aromatase IR was greater for mammographically dense versus non-dense tissue and may partly explain how MBD influences breast cancer.

TMS, a chemically modified herbal derivative of resveratrol, induces cell death by targeting Bax.

Breast cancer recurrence after an initial favorable response to treatment is a major concern for patients who receive hormonal therapies. Additional therapies are necessary to extend the time of response, and ideally, these therapies should exhibit minimal toxicity. Our study described herein focuses on a non-toxic pro-apoptotic agent, TMS (2,4,3',5'-tetramethoxystilbene), which belongs to the Resveratrol family of stilbenes. Prior study demonstrated that TMS was more effective than Resveratrol for inducing apoptosis. Additionally, TMS was effective for invoking death of relapsing breast cancer cells. As TMS was effective for reducing tumor burden, we sought to determine the mechanism by which it achieved its effects. Microarray analysis demonstrated that TMS treatment increased tubulin genes as well as stress response and pro-apoptotic genes. Fractionation studies uncovered that TMS treatment causes cleavage of Bax from the p21 form to a truncated p18 form which is associated with the induction of potent apoptosis. Co-localization analysis of immunofluorescent studies showed that Bax moved from the cytosol to the mitochondria. In addition, the pro-apoptotic proteins Noxa and Bim (EL, L, and S) were increased upon TMS treatment. Cell lines reduced for Bax, Bim, and Noxa are compromised for TMS-mediated cell death. Electron microscopy revealed evidence of nuclear condensation, formation of apoptotic bodies and DAPI staining showed evidence of DNA fragmentation. TMS treatment was able to induce both caspase-independent and caspase-dependent death via the intrinsic death pathway.

Systemic estradiol levels with low-dose vaginal estrogens.

OBJECTIVES: To critically evaluate published systemic estradiol levels during use of low-dose vaginal estrogens considering detection method and estrogen dose; describe challenges with accurately measuring estradiol; and determine the normal estradiol level range in postmenopausal women. METHODS: PubMed was searched for studies reporting systemic estradiol levels with lower-dose vaginal estrogens (≤25 µg estradiol or 0.3 mg conjugated equine estrogens). Estradiol levels at baseline and during treatment, area under the curve, and maximum estradiol concentrations were summarized by dose within assay type. A proposed range of systemic estradiol in normal, untreated, postmenopausal women was estimated by conservatively pooling means and standard deviations from published studies. RESULTS: Mean basal estradiol levels were 3.1 to 4.9 pg/mL using liquid or gas chromatography/mass spectroscopy (LC or GC/MS/MS) with a range of undetectable to 10.5 pg/mL using radioimmunoassay. Systemic estradiol levels with vaginal estrogens reflected their doses as measured with LC or GC/MS/MS in different studies: 7.1 to 9.1 pg/mL and 16.7 to 22.7 pg/mL with a 25-µg softgel capsule insert and a tablet insert, respectively; 4.6 to 7.4 pg/mL and 6.6 to 14.8 pg/mL with a 10-µg softgel capsule and a tablet insert, respectively; and 3.6 to 3.9 pg/mL with a 4-µg softgel capsule insert. A mean systemic estradiol concentration ranging from undetectable to 10.7 pg/mL is proposed as an estimate for basal estradiol levels in normal, untreated, postmenopausal women. Systemic estradiol absorption may be influenced by the placement of estradiol higher (as with an applicator) versus lower (as without an applicator) in the vagina, as estradiol transport to the uterus would be more likely further away than closer to the introitus. CONCLUSION: Serum estradiol concentrations were generally lower when measured with more specific and sensitive assays. Estradiol absorption was dose-dependent, and may be influenced by dose, formulation, and positioning in the vagina. Very low systemic estradiol absorption with low/ultralow-dose vaginal estrogens may potentially decrease any adverse events that may be associated with higher doses of vaginal estrogens used for treating moderate to severe VVA due to less estradiol exposure.

Approach to Managing a Postmenopausal Patient.

CASE AND PRINCIPLES OF MANAGEMENT: The case of a symptomatic, postmenopausal woman is presented and a full discussion of the approach to her management is discussed. Pertinent guidelines and scientific evidence are emphasized as support for the recommendations.

Workshop on normal reference ranges for estradiol in postmenopausal women, September 2019, Chicago, Illinois.

The North American Menopause Society (NAMS) organized the Workshop on Normal Ranges for Estradiol in Postmenopausal Women from September 23 to 24, 2019, in Chicago, Illinois. The aim of the workshop was to review existing analytical methodologies for measuring estradiol in postmenopausal women and to assess existing data and study cohorts of postmenopausal women for their suitability to establish normal postmenopausal ranges. The anticipated outcome of the workshop was to develop recommendations for establishing normal ranges generated with a standardized and certified assay that could be adopted by clinical and research communities. The attendees determined that the term reference range was a better descriptor than normal range for estradiol measurements in postmenopausal women. Twenty-eight speakers presented during the workshop.

Underlying Breast Cancer Risk and Menopausal Hormone Therapy.

The recent Collaborative Group on Hormonal Factors in Breast Cancer (CGHFBC) publication calculated the attributable risk of breast cancer from use of estrogen alone and estrogen plus a synthetic progestogen for less than 5 to 15 or more years of use. This CGHFB report calculated attributable risk based on their findings of relative risk from pooled data from 58 studies. Notably, neither the CGHFBC nor other previous studies have examined the effect of underlying risk of breast cancer on attributable risk. This omission prompted us to determine the magnitude of the effect of underlying risk on attributable risk in this perspective. Meaningful communication of the potential risk of menopausal hormonal therapy requires providing women with the estimated risk above their existing underlying risk (ie, attributable risk). Therefore, we have estimated attributable risks from the data published by the CGHFBC, taking into account varying degrees of underlying risk. Based on the Endocrine Society Guideline on Menopausal Hormone Therapy (MHT), we divided groups into 3 categories of risk: low (1.5%), intermediate (3.0%), and high (6.0%) underlying risk of breast cancer over 5 years. In women taking estrogen plus a synthetic progestogen for 5 to 9 years, the attributable risks of MHT increased from 12, to 42, to 85 additional women per 1000 in the low-, intermediate-, and high-risk groups, respectively. The attributable risks for estrogen alone were lower but also increased based on underlying risk. Notably, the attributable risks were amplified with duration of MHT use, which increased both relative risk and breast cancer incidence.

Estrogen signals via an extra-nuclear pathway involving IGF-1R and EGFR in tamoxifen-sensitive and -resistant breast cancer cells.

Activation of IGF-1R can activate metalloproteinases which release heparin-binding EGF (Hb-EGF) and lead to EGFR-dependent MAPK activation in certain tissues. We postulated that this pathway is operative in E(2)-induced MAPK activation in breast cancer tissues. As evidence, we showed that E(2) rapidly induced the phosphorylation of both IGF-1R and EGFR and that siRNA knockdown or selective inhibitors against either growth factor receptor inhibited E(2)-induced MAPK activation. The selective inhibitors or knockdown of either IGF-1R or EGFR significantly inhibited cell growth and reversed cell death protection induced by E(2) in MCF-7 cells. Our data support the conclusion that the IGF-1R acts upstream of EGFR in a linear pathway which mediates E(2) action on MAPK activation, cell growth stimulation and anti-apoptosis in breast cancer cells. During the process of development of tamoxifen resistance this pathway is up-regulated with increased sensitivity to activate EGFR for cell growth and protection against apoptosis. Surprisingly, translocation of ERalpha out of the nucleus into the cytoplasm, mediated by c-Src, occurs during development of resistance. This effect can be abrogated by administration of the c-Src inhibitor, PP2 which also restores sensitivity to tamoxifen.

Long-term treatment with tamoxifen facilitates translocation of estrogen receptor alpha out of the nucleus and enhances its interaction with EGFR in MCF-7 breast cancer cells.

The therapeutic benefit of tamoxifen in patients with hormone-dependent breast cancer is limited by acquired resistance to this drug. To investigate the biological alterations responsible for tamoxifen resistance, an in vitro model was established. After 6-month continuous exposure to tamoxifen (10(-7) mol/L), growth of MCF-7 breast cancer cells was no longer inhibited by this antiestrogen. Although there was no significant increase in the basal levels of activated mitogen-activated protein kinase (MAPK), tamoxifen-resistant (TAM-R) cells exhibited enhanced sensitivity to epidermal growth factor (EGF) and estradiol stimulated activation of MAPK. Tamoxifen elicited rapid phosphorylation of MAPK, in contrast to its antagonistic activity in control cells. Blockade of the EGF receptor (EGFR)/MAPK pathway caused more dramatic inhibition of growth of TAM-R cells than the control cells. An increased amount of estrogen receptor alpha (ERalpha) was coimmunoprecipitated with EGFR from TAM-R cells although the total levels of these receptors were not increased. Notably, ERalpha seemed to redistribute to extranuclear sites in TAM-R cells. Increased ERalpha immunoreactivity in the cytoplasm and plasma membrane of TAM-R cells was shown by fluorescent microscopy and by Western analysis of isolated cellular fractions. In TAM-R cells, an increased amount of c-Src was coprecipitated with EGFR or ERalpha. Blockade of c-Src activity resulted in redistribution of ERalpha back to the nucleus and in reduction of its interaction with EGFR. Prolonged blockade of c-Src activity restored sensitivity of TAM-R cells to tamoxifen. Our results suggest that enhanced nongenomic function of ERalpha via cooperation with the EGFR pathway is one of the mechanisms responsible for acquired tamoxifen resistance.