Estrogen Receptor Downregulator Research Articles

Abstract P3-05-03: Reversal of endocrine therapy resistance with inhibitors of AKT, mTOR, or MEK as single agents or in combination

Abstract Background: Crosstalk between estrogen receptor (ER) and growth factor receptor (GFR) downstream signaling pathways [PI3K/AKT/mTOR and MEK\p42\44MAPK (MAPK)] has been associated with endocrine resistance. Single downstream inhibitors like everolimus partially reverse endocrine resistance. However, more than one downstream escape pathway may contribute to endocrine resistance. Furthermore, disruption by a single downstream inhibitor of the negative feedback loops that balance the amplified signals from GFRs may result in compensatory pathway activation. Therefore we investigated whether dual downstream signaling inhibitions are required to more effectively overcome tamoxifen-resistant growth, using in vitro and in vivo models. Materials and Methods: The effects of different kinase inhibitors (i) AZD2014 (mTORi), AZD5363 (AKTi), and AZD6244 (MEKi) on endocrine therapy [tamoxifen (Tam) and fulvestrant (Fulv)] were tested in ER+ MCF7 and T47D Tam-resistant derivatives (TamR). In vitro growth and apoptosis were assessed using methylene blue and c-PARP, respectively. Western blot analysis was used to analyze the effect of each inhibitor or combination on their respective pathway substrates. Nude mice with transplantable MCF7 TamR xenografts at a size of 200 mm3 were randomized to continued Tam, continued Tam + (mTORi, AKTi, MEKi, mTORi+MEKi, AKTi+MEKi) or Fulv ± the inhibitor combinations. Results: We found that in two ER+ models MCF7 TamR and T47D TamR in vitro, both mTORi and AKTi were effective in restoring growth inhibition, and effective in inhibiting their respective pathways. Interestingly, inhibition of mTOR and AKT resulted in upregulation of pMAPK. However, while MEKi did inhibit its pathway; it did not restore growth inhibition by the antiestrogens. On the other hand, dual inhibition, adding the MEKi to either mTORi or AKTi, resulted in a more potent reduction of cell growth as well as of downstream signaling. In the TamR derivative of MCF7, ER is still maintained and plays a role in resistance, unlike the T47D model, where there is little to no ER expression after TamR develops. Thus, as might be expected, combining downstream inhibitors with potent ER blockade by Fulv enhances the effect of single and dual downstream signaling inhibitors mainly in the MCF7 TamR model. Finally, Fulv in addition to dual downstream inhibitions (mTORi+MEKi or AKTi+MEKi) delayed MCF7 TamR xenograft growth significantly more than Fulv with single downstream inhibitors. Conclusion: This study provides evidence that dual inhibition of GFR downstream pathways is needed to overcome activation of escape mechanisms that are up-regulated with acquired endocrine resistance and after resistance to single pathway inhibitors. Although the downstream inhibitors alone significantly inhibit TamR growth, combination with Fulv robustly slowed growth of TamR tumors in vivo. Based on these results further studies combining MEK inhibition with inhibitors of the PI3K pathway and ER downregulators are warranted. Citation Format: Agostina Nardone, Gladys Morrison, Xiaoyong Fu, Martin Shea, Tamika Mitchell, Teresa Klinowska, C Kent Osborne, Rachel Schiff. Reversal of endocrine therapy resistance with inhibitors of AKT, mTOR, or MEK as single agents or in combination [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-03.

Abstract OT2-1-01: Fulvestrant alone versus fulvestrant and everolimus versus fulvestrant, everolimus and anastrozole: A Phase III randomized, placebo-controlled trial in postmenopausal patients with hormone-receptor-positive stage IV breast cancer: SWOG-Clinical Trials Ini

Abstract BACKGROUND: The median survival for women with hormone-receptor-positive (HR+) stage IV metastatic breast cancer (MBC) is 36 months. Strategies to improve outcome for postmenopausal women, include combinations of aromatase inhibitors (AIs) with either the estrogen receptor downregulator fulvestrant, or with the PI3kinase/AKT/mTOR signal inhibitor everolimus, as this pathway has been implicated as a mediator of resistance to endocrine therapies. However,fulvestrant 500 mg has not been tested in combination with everolimus alone, or in combination with everolimus and an AI. SPECIFIC AIMS/TRIAL DESIGN: S1222 (NCT02137837) is a randomized phase III double-blinded, placebo-controlled clinical trial and is currently accruing. Patients receive fulvestrant (500 mg IM every 2 weeks for 3 doses, then monthly) on all arms, everolimus 10 PO daily (Arms 2 and 3, placebo Arm 1), and anastrozole 1 mg daily (Arm 3), or placebo (Arms 2 and 1). The co-primary objectives are to compare progression-free survival (PFS) between fulvestrant and everolimus (Arm 2) vs. fulvestrant (Arm 1), and fulvestrant, everolimus, and anastrozole (Arm 3) vs. fulvestrant (Arm 1). Additional objectives include comparison of PFS between Arms 2 and 3, and overall survival (OS), response and clinical benefit rates, toxicities, feasibility, compliance, and OS among the study arms. Translational research goals are to test molecular determinants of response in circulating tumor cells (CTCs), assess a previously piloted CTC-Endocrine Therapy Index (CTC ETI), perform CTC-Next Generation Sequencing Analysis (CTC-NGS), and NGS on cancer tissue and germ line DNA. ELIGIBILITY CRITERIA: Postmenopausal women with histologically confirmed de novo or newly relapsed stage IV MBC, with HER2-negative and HR-positive (>1% positive nuclear staining) features, with either measurable, or evaluable disease are eligible. Previous neoadjuvant/adjuvant therapy allowed, but adjuvant anti-HR therapy must have been completed ≥ 12 months prior to enrollment. STATISTICAL METHODS/TARGET ACCRUAL: This is a parallel randomized design with equal allocation to the three arms: (1) fulvestrant + placebo for everolimus + placebo for anastrozole; (2) fulvestrant + everolimus + placebo for anastrozole; (3) fulvestrant, everolimus, and anastrozole. PFS is the primary outcome. There are two coprimary hypotheses: (1) Arm 2 versus Arm 1; and (2) Arm 3 versus Arm 1, each will be tested at α = 0.025 (2-sided) .Arm 2 versus Arm 3 is a secondary comparison. Sample size computations are based on accrual of 33 patients per month for 24 months resulting in a computed accrual total of 792. We estimate that the final analysis will occur 24 months after the last patient is accrued, an average followup of 36 months at the time of the final analysis with an expected trial duration of 4 years. *SWOG-CTI manages the non-federally funded components of SWOG under The Hope Foundation. The study is supported by AstraZeneca and Novartis Pharmaceuticals Corp. Citation Format: George Somlo, William Barlow, Halle Moore, Julie Gralow, Anne Schott, Daniel Hayes, Peter Kuhn, James Hicks, Danika Lew, Debu Tripathy, Gabriel Hortobagyl. Fulvestrant alone versus fulvestrant and everolimus versus fulvestrant, everolimus and anastrozole: A Phase III randomized, placebo-controlled trial in postmenopausal patients with hormone-receptor-positive stage IV breast cancer: SWOG-Clinical Trials Ini [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT2-1-01.

Abstract S6-03: Randomized phase II trial of fulvestrant alone or in combination with bortezomib in hormone receptor-positive metastatic breast cancer resistant to aromatase inhibitors: A New York cancer consortium trial

Abstract Purpose: Fulvestrant (F) is a selective estrogen receptor downregulator (SERD) with activity in aromatase-inhibitor (AI) resistant estrogen receptor (ER)-positive metastatic breast cancer (MBC). In preclinical studies, the proteasome inhibitor bortezomib (B) enhances the antineoplastic effects of F, in part by promoting accumulation of large ER-aggregates that lead to cell death (Ishii et al. Clin Cancer Res 2011 17:2292). The objective of this study was to determine if the combination of F+B was more efficacious than F alone in MBC after AI progression. Patients and Methods: Postmenopausal women with ER-positive MBC who had progressive disease after prior AI therapy were eligible. They were randomized to F alone (500 mg IM days -15, 1, 15 in cycle 1, and day 1 of each subsequent cycle) or in combination with B (1.6 mg/m2 IV on days 1, 8, 15). The primary endpoint was progression free survival (PFS), measured from cycle 1, day 1 of starting F. A sample size of 118 was pre-specified in order to provide sufficient power to detect an improvement in median PFS from 5.4 to 9.0 months, and compare PFS rates after 6 and 12 months (1-sided alpha=0.10, beta=0.10). Patients with progression on F could cross over to the F+B combination. Results: Of 118 patients enrolled, 59 received F alone (arm A), 57 received F+B (arm B), and 2 assigned to arm B never initiated protocol therapy. There were no significant differences in patient characteristics between arms with regard to median age (57 vs. 59 years), ECOG performance status (0 and 1, 64% and 36%, respectively), prior chemotherapy for metastasis (25%), or liver metastases (37%), although patients in arm A had longer median interval between diagnosis and metastasis (49 vs. 28 months) and were more likely to present with metastasis (32% vs. 26%). Patients in arm B had more adverse events (all grades), including nausea (63% vs. 29%), diarrhea (47% vs. 8%), sensory neuropathy (46% vs. 29%), and limb edema (37% vs. 19%), although grade 3-4 events were uncommon, and only 11% discontinued B due to toxicity. At 12 months, the PFS proportion in Arm A and Arm B was 13.6% vs. 28.1%, respectively (P=0.03, 1-sided chi-square test) (95% CI for difference [14.5%] = -0.06%, 29.1%). Although median PFS was similar in the two arms (2.69 vs. 2.73 months, respectively), the hazard ratio for Arm B vs. Arm A (referent) was 0.73 (95% CI = 0.49, 1.09, P=0.06, 1-sided log rank test). Both results were significant at the pre-specified 1-sided 0.10 alpha level. Of 27 patients on arm A who crossed over to F+B at progression, 4 (15%) were progression-free for at least 24 weeks and had periods of disease control that were longer than when treated with F alone. Conclusion: Adding bortezomib to fulvestrant in AI-resistant ER-positive MBC enhances its effectiveness by delaying acquired fulvestrant resistance. These results support additional evaluation of proteasome inhibitors in combination with SERDs. Acknowledgement: Supported by contract N01-CM-62204 to the New York Cancer Consortium (P.I. J. Sparano) and grant P30 CA013330 (P.I. D. Goldman) from the National Institutes of Health, and by a grant from Millennium, Inc. Citation Format: Kerin B Adelson, Bhuvaneswari Ramaswamy, Joseph A Sparano, Paul J Christos, John J Wright, George Raptis, Miguel C Villalona, Cynthia X Ma, Dawn Hershman, Joseph Baar, Paula Klein, Tessa Cigler, G Thomas Budd, Yelena Novik, Antoinette R Tan, Susan Tannenbaum, Anupama Goel, Ellis Levine, Charles L Shapiro, Eleni Andreopoulou, Michael Naughton, Kevin Kalinsky, Samuel Waxman, Doris Germain. Randomized phase II trial of fulvestrant alone or in combination with bortezomib in hormone receptor-positive metastatic breast cancer resistant to aromatase inhibitors: A New York cancer consortium trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S6-03.

Aberrant plasma levels of circulatingmiR-16, miR-107, miR-130aandmiR-146aare associated with lymph node metastasis and receptor status of breast cancer patients

Within the multicenter SUCCESS trial, we investigated the association of plasma microRNAs with different subtypes of invasive breast cancer.Six miRs (miR-16, miR-27a, miR-107, miR-130a, miR-132 and miR-146a) were selected from microarray profiling and further validated in plasma of 111 breast cancer patients before and after chemotherapy and 46 healthy women by quantitative real-time PCR.Plasma levels of miR-16 (p = 0.0001), miR-27a (p = 0.039) and miR-132 (p = 0.020) were higher in breast cancer patients before chemotherapy than healthy women. With the exception of miR-16, the increased levels of miR-27a (p = 0.035) and miR-132 (p = 0.025) decreased after chemotherapy to those observed in healthy women. Levels of miR-16 (p = 0.019), miR-107 (p = 0.036), miR-130a (p = 0.027) and miR-146a (p = 0.047) were different between lymph node -positive and -negative patients, while the levels of miR-130a (p = 0.001) and miR-146a (p = 0.025) also differed between HER2-positive and -negative status. Estrogen-receptor negative tumors displayed higher concentrations of circulating miR-107 than their counterparts (p = 0.035). However, overexpression of miR-107 in MCF-7 cells did not downregulate estrogen receptor protein. Altered expression levels of miR-107 influenced the migration and invasion behavior of MCF-7 and MDA-MB-231 cells.Our data indicate differential concentrations of plasma miR-16, miR-107, miR-130a and miR-146a in different breast cancer subtypes, suggesting a potential role of these miRs in breast cancer biology and tumor progression.

Investigation of (E)-3-[4-(2-Oxo-3-aryl-chromen-4-yl)oxyphenyl]acrylic Acids as Oral Selective Estrogen Receptor Down-Regulators.

A novel estrogen receptor down-regulator, 7-hydroxycoumarin (5, SS5020), has been reported with antitumor effects against chemically induced mammary tumors. Here, we report on our own investigation of 7-hydroxycoumarins as potential selective estrogen receptor down-regulators, which led us to the discovery of potent down-regulating antagonists, such as 33. Subsequent optimization and removal of the 7-hydroxy group led to coumarin 59, which had increased potency and improved rat bioavailability relative to SS5020.

PI3K/mTOR mediate mitogen-dependent HDAC1 phosphorylation in breast cancer: a novel regulation of estrogen receptor expression.

Histone deacetylase 1 (HDAC1) is an important epigenetic controller involved in transcriptional regulation through modification of chromatin structure. Genetic and epigenetic changes and deregulation of signal transduction pathways have been implicated in the development of breast cancer. Downregulation of estrogen receptor α (ERα) expression is one of the mechanisms behind the acquisition of endocrine resistance. Sustained and increased hormone and growth factor receptor signaling in breast cancer cells contribute to resistance to endocrine therapy. Both HDACs and the PI3K/mTOR signaling pathway are becoming promising targets in breast cancer, reversing also acquired hormone resistance. Here we show how mitogens, activating the PI3K/mTOR pathway, trigger the phosphorylation of HDAC1 in breast cancer cells, which is completely dependent on the activity of the p70 S6 kinase (S6K1). Our findings show that S6K1, overexpressed in many breast cancers, controls HDAC1-dependent transcriptional regulation of ERα levels upon mitogenic stimuli, controlling HDAC1 recruitment to the ERα promoter. Furthermore, cell treatment with both mTOR and HDACs inhibitors shows an additive effect in inhibiting breast cancer proliferation. This confirms the novel cross-talk between the HDAC1 and PI3K pathways with clinical implications towards the treatment of this malignant disease.

GPR30 is a Potential Therapeutic Target in Human Carcinoma in situ and Seminomas

The G protein-coupled estrogen receptor (GPR30) is suggested to exert a role in non-nuclear estrogen signalling and is over-expressed in a variety of hormone dependent cancer entities. It is well established that oestrogens are involved in testicular germ cell tumours. In a recent paper published in Journal of Cellular Physiology, we show that down regulation of estrogen receptor β (ERβ) associates with GPR30 over-expression both in human testicular carcinoma in situ (CIS) and seminomas. In addition, we demonstrate that 17b-oestradiol induces the ERK1/2 activation through GPR30. The results suggested that exposure to oestrogens or oestrogen-mimics, in some as of yet undefined manner, diminishes the ERb-mediated growth restraint in CIS and in human testicular seminoma, indicating that GPR30 could be a potential therapeutic target to design specific inhibitors.

Revisiting the estrogen receptor pathway and its role in endocrine therapy for postmenopausal women with estrogen receptor-positive metastatic breast cancer

Endocrine therapy (ET) is the most commonly administered first-line systemic therapy for estrogen receptor-positive (ER+) metastatic breast cancer (MBC). Manipulation of hormone levels was one of the earliest ET approaches. However, treatment modalities have since evolved with the growing understanding of estrogen biosynthesis and ER biology. The current armamentarium of ET includes selective estrogen receptor modulation, aromatase inhibition, and selective estrogen receptor downregulation. However, intrinsic or acquired resistance to ET is frequently observed. Significant strides have been made in recent years in our understanding of the mechanisms of resistance to ET, and several targeted approaches including inhibitors against the phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway and cyclin-dependent kinase 4/6 (CDK4/6) have shown great promise. The mTOR inhibitor, everolimus, is already in clinical use for the treatment of resistant ER+MBC. However, multiple levels of evidence indicate that ER signaling remains as an important therapeutic target even in the resistance setting, providing the rationale for sequencing multiple lines and combinations of ET. In addition, recurrent mutations in estrogen receptor 1 (ESR1), the gene that encodes the ER, have been identified in the genomic studies of metastatic ER+ breast cancer. ESR1 mutations are an important mechanism for acquired resistance, and effective ER targeting in this setting is particularly important.

Dyssynchronous secretory endometrial glands often show sporadically acquired progesterone nonresponsiveness.

Primary sporadic gene-inactivating events within the progesterone response cascade might explain the presence of individual dyssynchronous (outlier) glands commonly observed in a secretory background. We queried morphologically dyssynchronous glands in mid-secretory endometrium with a series of markers normally downregulated by progesterone. Seventy-nine mid-secretory endometrial biopsies were stained with hematoxylin and eosin, MIB-1, PAX2, estrogen and progesterone receptors, and PTEN. Aberrant staining of glands was independently scored for each marker. Outlier glands overlapping between stains were enumerated. A total of 63% of cases had hematoxylin and eosin stained outlier glands (average 9), which often demonstrated failed progesterone-mediated downregulation of PAX2 (43%), estrogen (40%), and/or progesterone receptors (28%). Aberrations of progesterone response was seen in 70% to 85% of cases overall, averaging 10 to 30 glands/affected case. The frequency and burden of affected glands was similar to that seen for primary inactivating events of the PAX2 and PTEN genes (35% and 41% of cases, respectively, averaging 32 and 38 glands per affected patient). Sporadic gene-inactivating events are common during endometrial regeneration, and may cause morphologic changes unmasked by the hormonal context. Some of these dyssynchronous "outlier" glands, whether evident on hematoxylin and eosin stain or not, have an interrupted progesterone response.

Cross-talk between ER and HER2 regulates c-MYC-mediated glutamine metabolism in aromatase inhibitor resistant breast cancer cells

Resistance to endocrine therapies in hormone receptor (HR)-positive breast cancer is a significant clinical problem for a considerable number of patients. The oncogenic transcription factor c-MYC (hereafter referred to as MYC), which regulates glutamine metabolism in cancer cells, has been linked to endocrine resistance. We were interested in whether MYC-mediated glutamine metabolism is also associated with aromatase inhibitor (AI) resistant breast cancer. We studied the expression and regulation of MYC and the effects of inhibition of MYC expression in both AI sensitive and resistant breast cancer cells. Considering the role of MYC in glutamine metabolism, we evaluated the contribution of glutamine to the proliferation of AI sensitive and resistant cells, and performed RNA-sequencing to investigate mechanisms of MYC-mediated glutamine utilization in AI resistance. We found that glutamine metabolism was independent of estrogen but still required estrogen receptor (ER) in AI resistant breast cancer cells. The expression of MYC oncogene was up-regulated through the cross-talk between ER and human epidermal growth factor receptor 2 (HER2) in AI resistant breast cancer cells. Moreover, the glutamine transporter solute carrier family (SLC) 1A5 was significantly up-regulated in AI resistant breast cancer cells. ER down-regulator fulvestrant inhibited MYC, SLC1A5, glutaminase (GLS) and glutamine consumption in AI resistant breast cancer cells. Inhibition of MYC, SLC1A5 and GLS decreased AI resistant breast cancer cell proliferation. Our study has uncovered that MYC expression is up-regulated by the cross-talk between ER and HER2 in AI resistant breast cancer cells. MYC-mediated glutamine metabolism is associated with AI resistance of breast cancer.

Reduction of no-reflow and reperfusion injury with the synthetic 17β-aminoestrogen compound Prolame is associated with PI3K/Akt/eNOS signaling cascade.

A high proportion of primary percutaneous coronary interventions performed in the setting of acute myocardial infarction, concur with inadequate myocardial perfusion at the microvascular level. This phenomenon, known as "no-reflow" contributes to reperfusion injury, poor prognosis and to unfavorable clinical outcome. In this study, we evaluated the hypothesis that the synthetic 17β-aminoestrogen Prolame, may confer cardioprotection and prevent against no-reflow. In an open-chest model of 30-min ischemia and 90-min reperfusion, male Wistar rats were randomly assigned to different groups: Control, Prolame, Prolame followed by the nitric oxide synthase inhibitor (L-NAME), and 17β-estradiol. Areas of risk, infarct size and no-reflow were determined by planimetry with triphenyltetrazolium chloride and thioflavin-S stains. Structural damage of the vasculature was measured as capillary compression in clarified tissue after intra-atrial injection of Microfil. Hemodynamic function was obtained at the end of stabilization, ischemia and reperfusion; nitric oxide (NO·) content was determined indirectly using the Griess reaction. Activation of the eNOS signaling cascade was determined by western blot. Prolame reduced the infarcted area, decreased the zones of no-reflow and capillary compression by activating the PI3K/Akt/eNOS signaling pathway in correlation with NO· increase. Prolame also activated endothelial cells augmenting NO· production, which was inhibited by ICI182780 (a selective estrogen receptor down-regulator), supporting the notion that the cardioprotective effect of Prolame involves the preservation of endothelium through the activation of estrogen receptor downstream signaling. Our results provide evidence that Prolame has potential therapeutic application in patients with AMI, as it prevents from both vascular and cardiac tissue damage.

Current medical treatment of estrogen receptor-positive breast cancer.

Approximately 80% of breast cancers (BC) are estrogen receptor (ER)-positive and thus endocrine therapy (ET) should be considered complementary to surgery in the majority of patients. The advantages of oophorectomy, adrenalectomy and hypophysectomy in women with advanced BC have been demonstrated many years ago, and currently ET consist of (1) ovarian function suppression (OFS), usually obtained using gonadotropin-releasing hormone agonists (GnRHa); (2) selective estrogen receptor modulators or down-regulators (SERMs or SERDs); and (3) aromatase inhibitors (AIs), or a combination of two or more drugs. For patients aged less than 50 years and ER+ BC, there is no conclusive evidence that the combination of OFS and SERMs (i.e., tamoxifen) or chemotherapy is superior to OFS alone. Tamoxifen users exhibit a reduced risk of BC, both invasive and in situ, especially during the first 5 years of therapy, and extending the treatment to 10 years further reduced the risk of recurrences. SERDs (i.e., fulvestrant) are especially useful in the neoadjuvant treatment of advanced BC, alone or in combination with either cytotoxic agents or AIs. There are two types of AIs: type I are permanent steroidal inhibitors of aromatase, while type II are reversible nonsteroidal inhibitors. Several studies demonstrated the superiority of the third-generation AIs (i.e., anastrozole and letrozole) compared with tamoxifen, and adjuvant therapy with AIs reduces the recurrence risk especially in patients with advanced BC. Unfortunately, some cancers are or became ET-resistant, and thus other drugs have been suggested in combination with SERMs or AIs, including cyclin-dependent kinase 4/6 inhibitors (palbociclib) and mammalian target of rapamycin (mTOR) inhibitors, such as everolimus. Further studies are required to confirm their real usefulness.

Measuring residual estrogen receptor availability during fulvestrant therapy in patients with metastatic breast cancer.

It is unknown whether the current dose of fulvestrant, an estrogen receptor (ER) antagonist, is sufficient for maximal ER downregulation in patients with metastatic breast cancer. We performed a feasibility study to assess ER availability before and during fulvestrant. Sixteen patients with ER-positive metastatic breast cancer underwent positron emission tomography/computed tomography (PET/CT) at baseline (scan 1), day 28 (scan 2), and day 84 (scan 3) to monitor tumor [(18)F]fluoroestradiol (FES) uptake. Incomplete reduction in ER availability was predefined as <75% decrease in median tumor FES uptake and a residual standardized uptake value (SUVmax) of ≥1.5. In total, 131 FES-positive lesions were identified (median SUVmax of 2.9; range, 1.7-6.5). The median change in patients during fulvestrant treatment was -85% at scan 2, but varied widely (-99% to +60%). Fulvestrant reduced tumor FES uptake incompletely at scan 2 in 6 (38%) of the 16 patients, which was associated with early progression. Serial imaging of tumor estrogen uptake by FES-PET can give insight into the dose needed for ER antagonists to completely abolish ER. FES-PET showed significant residual ER availability in tumors during fulvestrant therapy in 38% of patients, which was associated with early progression.

The aryl hydrocarbon receptor (AhR) mediates resistance to apoptosis induced in breast cancer cells

The aryl hydrocarbon receptor (AhR) is well known as a ligand binding transcription factor regulating various biological effects. Previously we have shown that long-term exposure to estrogen in breast cancer cells caused not only down regulation of estrogen receptor (ER) but also overexpression of AhR. The AhR interacts with several cell signaling pathways associated with induction of tyrosine kinases, cytokines and growth factors which may support the survival roles of AhR escaping from apoptosis elicited by a variety of apoptosis inducing agents in breast cancer. In this study, we studied the anti-apoptotic role of AhR in different breast cancer cells when apoptosis was induced by exposure to UV light and chemotherapeutic agents. Activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in AhR overexpressing breast cancer cells effectively suppressed the apoptotic response induced by UV-irradiation, doxorubicin, lapatinib and paclitaxel. The anti-apoptotic response of TCDD was uniformly antagonized by the treatment with 3′methoxy-4′nitroflavone (MNF), a specific antagonist of AhR. TCDD's survival action of apoptosis was accompanied with the induction of well-known inflammatory genes, such as cyclooxygenase-2 (COX-2) and NF-κB subunit RelB. Moreover, TCDD increased the activity of the immunosuppressive enzyme indoleamine 2, 3-dioxygenase (IDO), which metabolizes tryptophan to kynurenine (Kyn) and mediates tumor immunity. Kyn also acts as an AhR ligand like TCDD, and kyn induced an anti-apoptotic response in breast cancer cells. Accordingly, our present study suggests that AhR plays a pivotal role in the development of breast cancer via the suppression of apoptosis, and provides an idea that the use of AhR antagonists with chemotherapeutic agents may effectively synergize the elimination of breast cancer cells.

Altered radiation responses of breast cancer cells resistant to hormonal therapy.

Endocrine therapy agents (the selective estrogen receptor (ER) modulators such as tamoxifen or the selective ER down-regulators such as ICI 182,780) are key treatment regimens for hormone receptor-positive breast cancers. While these drugs are very effective in controlling ER-positive breast cancer, many tumors that initially respond well to treatment often acquire drug resistance, which is a major clinical problem. In clinical practice, hormonal therapy agents are commonly used in combination or sequence with radiation therapy. Tamoxifen treatment and radiotherapy improve both local tumor control and patient survival. However, tamoxifen treatment may render cancer cells less responsive to radiation therapy. Only a handful of data exist on the effects of radiation on cells resistant to hormonal therapy agents. These scarce data show that cells that were resistant to tamoxifen were also resistant to radiation. Yet, the existence and mechanisms of cross-resistance to endocrine therapy and radiation therapy need to be established. Here, we for the first time examined and compared radiation responses of MCF-7 breast adenocarcinoma cells (MCF-7/S0.5) and two antiestrogen resistant cell lines derived from MCF-7/S0.5: the tamoxifen resistant MCF-7/TAMR-1 and ICI 182,780 resistant MCF-7/182R-6 cell lines. Specifically, we analyzed the radiation-induced changes in the expression of genes involved in DNA damage, apoptosis, and cell cycle regulation. We found that the tamoxifen-resistant cell line in contrast to the parental and ICI 182,780-resistant cell lines displayed a significantly less radiation-induced decrease in the expression of genes involved in DNA repair. Furthermore, we show that MCF-7/TAMR-1 and MCF-7/182R-6 cells were less susceptible to radiation-induced apoptosis as compared to the parental line. These data indicate that tamoxifen-resistant breast cancer cells have a reduced sensitivity to radiation treatment. The current study may therefore serve as a roadmap to the future analysis of the mechanisms of cross-resistance between hormonal therapy and radiation.

Abstract 2101: Role of CDK8 in estrogen receptor signaling in breast cancers

Abstract The majority of breast cancers (BrCa) overexpress estrogen receptor (ER)α, which regulates transcription and drives estrogen-stimulated proliferation of ER+ tumor cells. ER+ patients usually receive adjuvant anti-estrogen therapy based on ER modification, downregulation, or estrogen depletion. Tumors frequently develop resistance to anti-estrogen therapy through various mechanisms, which may involve stimulation of ER itself or of downstream mediators of ER-driven transcription, as well as activation of alternative proliferation pathways, in particular those driven by HER2/EGFR. The treatment efficacy of hormone-resistant BrCa could be greatly augmented by targeting new druggable mediators of ER activity. We have now identified a transcription-regulating oncogenic kinase CDK8 as a potentiator of ER signaling and ER-driven BrCa cell proliferation. Immunohistochemical staining of breast tissue arrays and bioinformatics analysis of gene expression microarray data of breast cancer patients revealed that CDK8 is overexpressed in BrCa and that higher CDK8 expression correlates with the failure of systemic therapy. Among systemically untreated patients, higher CDK8 expression was correlated with shorter relapse-free survival in a subset of ER+ tumors that expressed the lowest levels of ERα. This correlation suggested that CDK8 could play a role in the progression of ER+ breast cancers with reduced levels of ER, possibly by stimulating the mitogenic effects of ER-mediated transcription. Indeed, a selective small-molecule CDK8 inhibitor (Senexin A) decreased estrogen-induced ER-dependent transcription and inhibited estrogen-stimulated proliferation of ER+ BrCa cell lines. Microarray analysis revealed that CDK8 inhibition diminished the induction of genes that show rapid and sustained activation by estrogen in ER+ cells. CDK8 inhibition had an additive effect in combination with anti-estrogens in ER+ BrCa and a synergistic effect with fulvestrant in BrCa cells resistant to estrogen deprivation. Some of these cell lines also express HER2/EGFR, and CDK8 inhibition in combination with a HER2/EGFR inhibitor lapatinib synergistically inhibited the growth of these cells. These results suggest that combining anti-estrogen and anti-CDK8 therapy may be more effective than conventional hormone therapy for ER+ BrCa. Citation Format: Martina McDermott, Balazs Gyorffy, Chang-uk Lim, Alexander Chumanevich, Zhengguan Yang, Mengqian Chen, James F. Catroppo, Igor Roninson, Eugenia V. Broude. Role of CDK8 in estrogen receptor signaling in breast cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2101. doi:10.1158/1538-7445.AM2014-2101

Abstract 5568: CYP17A1 and abiraterone: Implications for breast cancer endocrine therapy

Abstract The cytochrome P450 17A1 (CYP17A1) inhibitor abiraterone, which blocks synthesis of all steroid hormones including androgens and estrogens, is an effective treatment for castrate resistant prostate cancer. Its utility for treating hormone receptor-positive breast cancer has yet to be established. In prostate cancer, the anti-proliferative effects of abiraterone are mediated by inhibiting CYP17A1 activity resulting in a significant reduction in circulating concentrations of the sex steroids dehydroepiandrosterone (DHEA), androstenedione, testosterone, and dihydrotestosterone (DHT). Recently, it has been proposed that the reduction in aromatizable androgens by abiraterone may make this drug useful for the management of hormone-dependent breast cancer. However, data from our lab suggests that abiraterone has estrogenic properties and can induce proliferation in breast cancer cells expressing the estrogen receptor (ER). Crystal violet assays were used to assess abiraterone-induced proliferation in the ER-positive, estrogen-dependent breast cancer cell lines MCF-7 and T47D. We show that abiraterone induced a dose dependent increase in cell proliferation with an IC50 of 3.7 μM and maximal stimulation (200%) observed at 8μM, compared to vehicle treated cells. Abiraterone also induced the expression of the ER response gene, GREB1. Abiraterone-induced proliferation and gene expression was blocked by increasing doses of the selective estrogen receptor down-regulator (SERD) fulvestrant. Abiraterone induced the dose-dependent expression of luciferase in MCF-7 cells transfected with an estrogen responsive luciferase reporter construct. In conclusion, our data suggest that abiraterone can induce ER-positive breast cancer cell proliferation in vitro by acting as a weak ER agonist. Further studies are underway to determine whether estrogenic effects of abiraterone are also observed using in vivo models of ER response. If abiraterone exhibits estrogenic effects in vivo then these data would suggest that abiraterone should be combined with an ER antagonist if used for the clinical management of women with ER+ tumors. Citation Format: Cameron P. Capper, Michael D. Johnson, José M. Larios, James M. Rae. CYP17A1 and abiraterone: Implications for breast cancer endocrine therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5568. doi:10.1158/1538-7445.AM2014-5568

Abstract 1266: Racial differences in the association of cruciferous vegetable intake with breast cancer hormone receptor status and tumor subtype

Abstract Background: Dietary isothiocyanates (ITCs) are chemopreventive phytochemicals from cruciferous vegetables (CV). ITCs down-regulate estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) in breast cancer cells, and inhibit development of breast cancer in animal models. CV intake was found to be inversely associated with breast cancer risk. However, the association of CV intake with breast cancer subtype is unknown. Further, racial differences exist in dietary habits and polymorphic genes in ITC-metabolizing pathway. In a prospective cohort, we examined the association of prior CV intake with breast cancer hormone receptor status and subtype by race. Methods: Among 2908 breast cancer patients diagnosed 2005-2013 at Kaiser Permanente Northern California (KPNC), data on CV intake in the past 6 months were obtained from a validated food frequency questionnaire as part of a comprehensive baseline interview about two months post-diagnosis. Breast cancer characteristics came from the KPNC Cancer Registry. CV intake and its association with hormone receptor status (ER+/-, PR+/-, and HER2+/-) and subtype (luminal A/luminal B/triple negative/HER2-overexpressing) were examined among Whites, African Americans (AA), Asians, and Hispanics. Logistic regression was used adjusting for age at diagnosis, race, menopausal status, parity, energy intake, and dietary fiber from vegetables. Results: CV intake (grams/day) differed across racial groups (p&amp;lt;0.01), with 46.9, 35.6, 30.7, and 27.8 for Asians, AA, Whites, and Hispanics, respectively. Except for broccoli, intake of other individual CVs differed across racial groups (p&amp;lt;0.01). Total CV intake was not associated with breast cancer subtypes in any racial group. However, compared with PR+ disease, coleslaw intake was associated with reduced odds of PR- disease in Asians (high vs. low: OR=0.63, 95%CI=0.41-0.97), while intake of greens including collard, kale, turnip and mustard was associated with increased odds of PR- disease in Whites (high vs. low: OR=1.34, 95%CI=1.09-1.66). Broccoli and cabbage intake were associated with reduced odds of HER2+ disease in Asians, compared with HER2- disease (high vs. low: OR=0.46, 95%CI=0.23-0.94 for broccoli, and OR=0.48, 95%CI=0.24-0.95 for cabbage). No associations were observed for Hispanics and AAs. In all racial groups combined, total CV intake, specifically broccoli intake, was associated with increased odds of luminal B, compared with luminal A subtype (OR=1.83, 95%CI= 1.19- 2.79). However, the association became null with increasing broccoli intake. Discussion: The association of CV intake with breast cancer characteristics varied by race and by individual CV. The differences may be attributable to diverse genetic background among racial groups, and specific ITCs in individual CVs. Funded by K07 CA148888 and R01 CA105274 Citation Format: Li Tang, Marilyn L. Kwan, Song Yao, Janise M. Roh, Cecile A. Laurent, Dawn L. Hershman, Schicha Kumar, Gregory E. Wilding, Christine B. Ambrosone, Lawrence H. Kushi. Racial differences in the association of cruciferous vegetable intake with breast cancer hormone receptor status and tumor subtype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1266. doi:10.1158/1538-7445.AM2014-1266

EFFECT OF AROMATASE INIBITOR IN OVULATION INDUCTION IN FETILE WOMEN WITH POLYCYSTIC OVARY SYNDROME

Backgrounds: Polycystic Ovary Syndrome (PCOS) is one of the most common causes of female infertility due to ovulation disorders. Clomiphene citrate (CC) is a first choice to restore ovulation but it has some side effects by estrogen receptor down-regulation. Aromatase inhibitor (AI) is a newer class of drugs which increases the production of endogenous FSH to stimulate ovulation. Subjects and methods: randomized control trial to compare 64 cases of infertile women with PCOS examined at the Hue University Hospital, alternately used AI (group I) or CC (group II) for ovulation induction from day 2 cycle. Follow-up follicle growth, endometrium and ovulation via ultrasound. Evaluation were done on 10th day cycle, day of hCG trigger and after administration of hCG. Results: Total of 64 PCOS cases distributed into 2 groups using alternatively AI and CC had similar characteristics with average age of 28.8 ± 4.6, the majority were primary infertility (84.4%), infertility duration was 2.6 ± 2.4 years, 85.9% had oligomenorrhrea or amenorrhea, normal body mass index accounts for 60.9% and 21.9% was lean. Evaluation of both groups on day 10 revealed no differences in the dominant follicle and endometrial thickness. Number of days until the follicle mature appears to be shorter in AI group (15.1 ± 2.9) compared to the CC group (16.5 ± 2.8) with statistical significance. The number of mature follicles in 2 groups were not different at a rate of 81.3% (AI) and 84.4% (CC) but a higher proportion of single mature follicle in the AI ​​group (71.9%) compared with the CC group (65.7%) and There is no case with 3-4 mature follicles in the AI group. The rate of thin endometrium (&lt;8 mm) in the AI group (25%) was lower than the CC group (53.1%) with statistically significance and higher ovulation rate (68.8%) compared with the CC group (56.3%) but have not found statistically significant. Conclusion: Two drugs AI and CC potentially induce follicle development and ovulation similarly, but AI has the potential to be more effective than CC on factors such as the shorter stimulation duration, increasing rate of single follicle, limiting multiple pregnancies, improve endometrial thickness and higher ovulation rate. More researches are needed with a larger sample size to clarify the statistical significance of differences.

Preferential estrogen receptor β ligands reduce Bcl-2 expression in hormone-resistant breast cancer cells to increase autophagy.

Acquired resistance to selective estrogen receptor (ER) modulators (SERM) and downregulators (SERD) is a significant clinical problem in the treatment of estrogen (E2) receptor-positive (ER(+)) breast cancers. There are two ER subtypes, ERα and ERβ, which promote and inhibit breast cancer cell proliferation, respectively. Although ER(+) breast cancers typically express a high ratio of ERα to ERβ, the acquisition of SERM resistance in vitro and in vivo is associated with increased relative expression of the ERβ. On some gene enhancers, ERβ has been shown to function in opposition to the ERα in the presence of E2. Here, we demonstrate that two different ERβ agonists, WAY-20070 and a novel "A-CD" estrogen called L17, produce a marked reduction in G(2)-M phase correlated with effects on cyclin D1 and cyclin E expression in a SERM/SERD-resistant breast cancer cell line. ERβ agonists recruited both the ERα and ERβ to the Bcl-2 E2-response element strongly reducing Bcl-2 mRNA and protein in an ERβ-dependent manner. L17 recruited RIP140 to the Bcl-2 promoter in cells overexpressing ERβ. Exposure to the ERβ ligands also resulted in increased processing of LC3-I to LC3-II, indicative of enhanced autophagic flux. The coaddition of ERβ agonist and the autophagy inhibitor chloroquine resulted in a significant accumulation of sub-G1 DNA which was completely prevented by the addition of the caspase inhibitor Z-VAD-FMK. We propose that combined therapies with an ERβ agonist and an inhibitor of autophagy may provide the basis for a novel approach to the treatment of SERM/SERD-resistant breast cancers.