Estrogen-stimulated Growth Research Articles

Abstract OT2-01-10: WinPro: A window of opportunity study of endocrine therapy with and without prometrium in postmenopausal women with early-stage hormone receptor-positive breast cancer

Abstract WinPro: A window of opportunity study of endocrine therapy with and without prometrium in postmenopausal women with early-stage hormone receptor-positive breast cancer Authors Teesha Downton1,2,3, Davendra Segara3, Andrew Ong4, Janne Bingham5, Emma-Kate Carson4, Julia Chen1,2,3, Kate Middleton3, Geoffrey Lindeman6, Andrew Parker3, Elgene Lim1,2,3. Affiliations 1Garvan Institute of Medical Research, Darlinghurst NSW, Australia; 2School of Clinical Medicine, St Vincent’s Healthcare Clinical Campus, Faculty of Medicine and Health, University of New South Wales Sydney, Australia; 3St Vincent’s Hospital Sydney, Darlinghurst NSW, Australia; 4Campbelltown Hospital, Campbelltown NSW, Australia; 5Royal Adelaide Hospital, Adelaide SA, Australia; 6Walter & Eliza Hall Institute of Medical Research, Parkville VIC, Australia Disclosures T. Downton: None. D. Segara: None. A. Ong: None. J. Bingham: None. E. Carson: None. J. Chen: None. K. Middleton: None. G. Lindeman: None. A. Parker: None. E. Lim: Advisory Board for Pfizer, Astra Zeneca, Lilly, Roche, Novartis, Gilead Australia. Research Funding from Pfizer, Novartis, Bayer. Abstract Background: Preclinical studies have observed that progesterone has inhibitory effects on the estrogen-stimulated growth of estrogen receptor (ER)-positive, progesterone receptor (PR)-positive breast cancer. Mohammed H et al. (Nature 2015) identified that activated PR associates with the ER and modulates the interactions of the ER with chromatin, with a shift towards the transcription of genes associated with apoptosis and differentiation, and away from genes associated with proliferation. We hypothesize that the addition of prometrium, a microionized progesterone, may enhance the anti-proliferative effects of standard endocrine therapy in women with ER-positive PR-positive breast cancer. Trial Design: WinPro (NCT03906669) is an ongoing multicenter, phase II, randomized, open-label, window of opportunity study comparing the effect on standard endocrine therapy with or without prometrium on breast cancer cell proliferation. The study population is postmenopausal women with early-stage operable breast cancer where the tumor is ≥5mm on imaging, ER ≥10%, PR≥10%, and HER2-negative. Patients currently on hormone replacement therapy or the oral contraceptive pill, or who have a history of endometrial cancer or venous thromboembolism are not eligible. Patients are randomized 1:1:1 to letrozole 2.5mg daily, letrozole 2.5mg + prometrium 300mg daily, or tamoxifen 20mg + prometrium 300mg daily. Allocated treatment is taken for 14 days prior to surgery. Primary surgery and adjuvant treatment is as per standard of care. Objectives: The primary objective is to compare the geometric mean suppression of centrally assessed Ki67 between the diagnostic biopsy sample (pre-treatment) and the surgical sample (post-treatment). The secondary objective is to evaluate safety and tolerability. Tertiary objectives include defining genes predictive of a reduction in Ki67, and evaluating the changes in ER, PR, AR, FoxA1, Cyclin D1, and apoptotic markers in breast tumors post-intervention. Accrual: This study opened in February 2018 and as of 13 July 2022, 164 patients have been enrolled. Target accrual is 200 patients. Contact information: This study is led at St Vincent’s Hospital Sydney, Australia, and funded by the Cancer Council of NSW and the NHMRC Translational Breast Cancer Project grant. Contact: Elgene Lim MBBS FRACP PhD at e.lim@garvan.org.au. Citation Format: Teesha Downton, Davendra Segara, Andrew Ong, Janne Bingham, Emma-Kate Carson, Julia Chen, Kate Middleton, Geoffrey Lindeman, Andrew Parker, Elgene Lim. WinPro: A window of opportunity study of endocrine therapy with and without prometrium in postmenopausal women with early-stage hormone receptor-positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-01-10.

Suppression of Insulin Receptor Substrate 1 Inhibits Breast Cancer Growth In Vitro and in Female Athymic Mice.

Targeting the type I insulin-like growth factor receptor (IGF-IR) has not been successful in breast cancer. Data suggest the highly homologous insulin receptor (IR) may be an alternate growth stimulatory pathway used by cancer cells. Since both receptors phosphorylate the insulin receptor substrate 1 (IRS-1) protein as an immediate consequence of ligand binding, disruption of both receptors could be accomplished by suppression of IRS-1. IRS-1 gene deletion by CRISPR/Cas9 editing resulted in suppression of IGF-I, insulin, and estrogen-stimulated growth in hormone-dependent MCF-7L breast cancer cells. A doxycycline-inducible IRS-1 shRNA lentiviral construct was also used to infect MCF-7L breast cancer cells. IRS-1 shRNA downregulation resulted in decreased responses to IGF-I, insulin, and estradiol in monolayer and anchorage-independent growth assays. Decreased IRS-1 levels also suppressed estradiol-stimulated gene expression and estrogen receptor binding to DNA. Xenograft growth was also inhibited by induction of IRS-1 shRNA. These data show that IRS-1 is a critical regulator of endocrine responsive breast cancer. Efforts to target this adaptor protein could have broader growth inhibitory effects and receptor targeting.

OR05-06 The Androgen Receptor Is a Tumour Suppressor in Estrogen Receptor Positive Breast Cancer

There is strong interest in targeting the androgen receptor (AR) in estrogen receptor (ER) positive breast cancer, but widespread confusion exits as to what therapeutic strategy - agonism or antagonism - is appropriate. Current understanding of AR predominantly stems from the field of prostate cancer, where AR is the key oncogenic driver and therapeutic target. An ensuing assumption is that AR promotes malignancy in breast cancer and should be therapeutically antagonised. However, compelling pre-clinical data to support this assumption is lacking. Since estrogen stimulates and androgen inhibits the development of normal breast tissue, we hypothesized that AR acts as a tumour suppressor in the breast and that AR agonism is the appropriate therapeutic strategy for ER-driven breast cancer. We tested this hypothesis using a large suite of cell line and patient-derived explant (PDE) and xenograft (PDX) models of breast cancer, including those that were resistant to current therapies and those harbouring genomic anomalies of ESR1 associated with treatment-resistant disease. Across the diverse models we found compelling evidence that AR agonism, but not antagonism, potently and durably inhibited tumour growth. A signature of AR activity derived from the xenograft models positively predicted disease survival in multiple large clinical cohorts of ER+ breast cancer, out-performing other breast cancer-specific prognostic signatures. We also show that an AR agonist can be combined with current ER target therapies such as Tamoxifen or a CDK4/6 inhibitor to maximize growth inhibition. Mechanistically, agonist-bound AR opposed ER signalling by repositioning ER and the co-activator p300 in the chromatin landscape, resulting in down-regulation of cell cycle genes. Introduction of an AR DNA binding mutant had no effect on ER signalling or estrogen-stimulated growth in breast cancer cells. As part of this study, we have generated consensus AR cistromes representing ER+ breast cancer cell lines and ER+ tumours that provide a new understanding of AR activity and clearly show differences to those associated with prostate cancer cell lines and tumours. In conclusion, our data provides a compelling biological rationale for AR agonism as a therapeutic strategy in multiple, clinically relevant contexts of ER-positive breast cancer. These findings should dispel widespread confusion over the role of AR in ER-driven breast cancer, an issue that currently hinders progress in leveraging modern AR-targeted therapies (e.g. selective androgen receptor modulators) that lack the undesirable side-effects of androgens for clinical benefit.

Effects of Metformin on Cellular Proliferation and Steroid Hormone Receptors in Patient-Derived, Low-Grade Endometrial Cancer Cell Lines.

Endometrial cancer (EC) is the most common gynecologic malignancy and is the result of disruption of the balance between estrogen-stimulated growth and progesterone-induced growth modulation. Metformin has been shown to inhibit EC proliferation; however, its role in early-stage EC and its effects on steroid hormone receptors have not been adequately explored. Our aim was to examine the effects of metformin on cellular proliferation in patient-derived, low-grade EC cell lines and to determine whether it directly modulates steroid hormone receptor expression. Two novel EC cell lines were produced (EM2 and 3) from endometrial tumor tissue obtained from women undergoing surgery. Cellular proliferation was determined by the 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay, and in both cell lines, metformin decreased cell proliferation in a dose-dependent (10-200 µmol/L) manner and induced apoptosis as measured by cleaved PARP. Furthermore, metformin abrogated the effects of E2 on cell proliferation. Using quantitative real-time polymerase chain reaction and Western immunoblotting, metformin significantly decreased estrogen receptor (ER) α messenger RNA abundance but did not consistently affect the expression of progesterone receptor. Estrogen receptor α protein levels significantly decreased across all metformin doses tested, which resulted in a significant decrease in the expression of the ER targets genes Keratin-19 and Wnt-1 inducible signaling pathway 2. In addition, metformin increased phosphorylation of AMPK in a dose-dependent manner (10-200 µmol/L) indicating an effect on mammalian target of rapamycin (mTOR) signaling. Our data suggest that metformin therapy represents a potential fertility-sparing option for women with early-stage EC, given its capacity to inhibit EC cell proliferation, ERα expression, and the mTOR cell proliferation pathway.

Abstract P5-05-13: Quantitative combinatory indexed ChIP-seq reveals distinct transcriptional complexes containing estrogen receptor and GREB1 at chromatin in breast cancer cells

Abstract GREB1 is an estrogen-activated gene in estrogen receptor a (ER)-positive breast cancer cells. GREB1 is also required for estrogen-stimulated breast cancer cell growth and its level is highly correlated to ER level in breast cancer cells and tumor samples. In endocrine resistant diseases, GREB1 is often dysregulated. GREB1 has been shown to interact with ER and bind to the same ER binding sites throughout the genome. There is no identified functional domain in GREB1 and it is not completely known how GREB1 exerts its function to regulate the transcription of ER target genes. In order to demonstrate whether GREB1 is present in the ER-containing transcriptional complex at chromatin, we have adapted and developed a quantitative combinatory indexed ChIP-seq assay suitable for dissecting components in a transcriptional cofactor complex in a genome-wide scale. In ER-positive MCF-7 breast cancer cells, we found that almost all GREB1 binding sites are the same sites bound by ER or its bona fide coactivator SRC-3. We further found that GREB1 and SRC-3 are both present in the same ER-containing complex at chromatin. Thus, both GREB1 and SRC-3 are integral members of the ER transcriptional complex at chromatin. Moreover, we discovered that only a portion of GREB1 at chromatin is present in the ER complex while the other portion of GREB1 is present in a different complex lacking ER or SRC-3 at the same genomic loci. Thus, two distinct GREB1-containing complexes are identified in equilibrium at chromatin: one contains ER/SRC-3 and the other one lacks ER/SRC-3. Our results suggest a non-traditional role of GREB1 in transcriptional regulation of ER target genes. The method used in our study can be widely applied for probing components of transcriptional complexes at chromatin. Citation Format: Sun J, Lippman ME. Quantitative combinatory indexed ChIP-seq reveals distinct transcriptional complexes containing estrogen receptor and GREB1 at chromatin in breast cancer cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-13.

Abstract 1926: Npy1r as a prognostic marker and a novel drug target in estrogen receptor-positive breast cancer

Abstract Despite the crucial role of some G protein-coupled receptors (GPCRs) in cancer progression and metastases reported in recent studies, the function of majority of GPCRs, as a class, remains largely unexplored in breast cancer. In this study, we evaluated transcriptome, proteome and phosphoproteome data for 398 non-sensory GPCRs (IUPHAR v1.0) using the landmark TCGA breast cancer proteogenomics dataset and Illumina HiSeq dataset. Neuropeptide Y Receptor Y1 (NPY1R) was the top most hypo-phosphorylated (at S368) GPCR in 92 out of 99 evaluable samples. Furthermore, NPY1R gene expression was found to be significantly lower in tumor samples than tumor-matched normal (n=112). Among tumor samples, the LumA subtype had significantly higher NPY1R expression than LumB, Basal and Her2 subtype (p<0.05, One-way ANOVA, Tukey test). Interestingly, the trend of NPY1R gene expression, protein expression and phosphosite expression was decreasing in the order of LumA, LumB, Basal and Her2 subtype (p<0.01, Jonckheere trend test). Using publicly available dataset of 22,277 genes on survival in 2,422 patients, we found that higher NPY1R expression predicted better overall survival and recurrence-free survival in estrogen receptor-positive (ER+) breast cancer patients. In light of this, we interrogated NPY1R gene expression in endocrine sensitive and resistant cell line models and the impact of NPY1R antagonist in ER+ breast cancer cell lines. NPY1R expression was increased in response to estradiol in various ER+ breast cancer cell line models (MCF7, T47D, and BT474) in publicly available datasets. Conversely, we found that tamoxifen or estrogen deprivation (ED) treatment reduced NPY1R expression in MCF7 and T47D cells. In our endocrine-resistant (tamoxifen-resistant, estrogen deprivation-resistant, and fulvestrant-resistant) cell line models, NPY1R expression remained significantly lower compared to the parental cells in vitro and in vivo (p<0.05, One-way ANOVA, Tukey test). Treatment of MCF7 parental cells with 100 nM NPY significantly reduced estrogen-stimulated cell growth (p<0.05, One-way ANOVA, Tukey test). Chronic treatment of cells with 1 μM BIBP3226, an NPY1R antagonist, for 2 weeks reversed the effect of NPY on the estradiol-stimulated cell growth. Estradiol-stimulated cell growth was modest (~40%) in ED-resistant MCF7 cells compared to that (~250%) in parental MCF7 cells. While 100 nM NPY treatment did not affect the estrogen-stimulated growth in ED-resistant MCF7 cells, which can be explained by the undetectable expression of NPY1R, it caused significant inhibition of the estrogen-stimulated growth in the same cells treated chronically with BIBP3226. Molecular mechanisms to explain these effects of chronic NPY1R antagonism are underway. Our ongoing studies will further elucidate the role of NPY1R as a novel drug target in ER+ breast cancer. Citation Format: Raksha Bhat, Suhas Vasaikar, Leon Bae, De Angelis Carmine, Maria Letizia Cataldo, Sarmistha Nanda, Bing Zhang, Rachel Schiff, Meghana V. Trivedi. Npy1r as a prognostic marker and a novel drug target in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1926.

ZBTB7A governs estrogen receptor alpha expression in breast cancer.

ZBTB7A, a member of the POZ/BTB and Krüppel (POK) family of transcription factors, has been shown to have a context-dependent role in cancer development and progression. The role of ZBTB7A in estrogen receptor alpha (ERα)-positive breast cancer is largely unknown. Approximately 70% of breast cancers are classified as ERα-positive. ERα carries out the biological effects of estrogen and its expression level dictates response to endocrine therapies and prognosis for breast cancer patients. In this study, we find that ZBTB7A transcriptionally regulates ERα expression in ERα-positive breast cancer cell lines by binding to the ESR1 promoter leading to increased transcription of ERα. Inhibition of ZBTB7A in ERα-positive cells results in decreased estrogen responsiveness as demonstrated by diminished estrogen-response element-driven luciferase reporter activity, induction of estrogen target genes, and estrogen-stimulated growth. We also report that ERα potentiates ZBTB7A expression via a post-translational mechanism, suggesting the presence of a positive feedback loop between ZBTB7A and ERα, conferring sensitivity to estrogen in breast cancer. Clinically, we find that ZBTB7A and ERα are often co-expressed in breast cancers and that high ZBTB7A expression correlates with improved overall and relapse-free survival for breast cancer patients. Importantly, high ZBTB7A expression predicts a more favorable outcome for patients treated with endocrine therapies. Together, these findings demonstrate that ZBTB7A contributes to the transcriptional program maintaining ERα expression and potentially an endocrine therapy-responsive phenotype in breast cancer.

Pharmacodynamic and Pharmacokinetic Properties of AFPep, a Novel Peptide for the Treatment of Breast Cancer

AFPep is a small synthetic cyclized peptide derived from alpha-fetoprotein that has been shown to have anti-estrogenic and anti-breast cancer activity. The purpose of this study was to establish blood levels of AFPep that are associated with biological activity. Blood levels of AFPep were measured by LC/MS/MS. Once daily treatment of mice with 4 mg/kg i.p. AFPep yielded a Cmax of 7 µg/ml and was sufficient to inhibit estrogen-stimulated growth of mouse uterus and of human breast cancer xenografts even though the half-life of this drug was only 11 min in mice, suggesting that its biological effects last much longer than its chemical half-life. In dose de-escalation studies, blood levels of 100 ng/ml of AFPep were still found to be biologically active. AFPep was effective by parenteral routes and with dose escalation also by the oral route. Blood levels of AFPep that were biologically effective in mice were readily achieved in dogs through parenteral as well as oral routes with no apparent evidence of host toxicity. In conclusion, AFPep blood levels can be measured by LC/MS/MS with accuracy into the ng/ml range. Blood levels in the 100 ng/ml range are associated with efficacious biological activity. This drug shows great promise for the treatment as well as prevention of breast cancer.

Insulin and IGF-1, but not 17β-estradiol, alter the subcellular localization of MIER1α in MCF7 breast carcinoma cells.

BackgroundMIER1α is a transcriptional regulator that interacts with estrogen receptor α and inhibits estrogen-stimulated growth of breast carcinoma cells. Interestingly, analysis of MIER1α subcellular localization in breast samples revealed a stepwise shift from the nucleus to the cytoplasm during progression to invasive carcinoma. Previously, we demonstrated that MIER1α is nuclear in MCF7 cells yet it does not contain a nuclear localization signal. Instead MIER1α is targeted to the nucleus through interaction and co-transport with HDAC 1 and 2.ResultsIn this study, we demonstrate that treatment of MCF7 breast carcinoma cells with either insulin or insulin-like growth factor affects the subcellular localization of MIER1α. Both factors reduce the percentage of cells with nuclear MIER1α from 81 and 89 to 41 and 56 %, respectively. Treatment with 17β-estradiol, on the other hand, had no effect and MIER1α remained nuclear.ConclusionsOur data demonstrate that insulin and IGF-1 can contribute to loss of nuclear MIER1α in the MCF7 breast carcinoma cell line.

Pharmacological relevance of endoxifen in a laboratory simulation of breast cancer in postmenopausal patients.

Tamoxifen is metabolically activated via a CYP2D6 enzyme system to the more potent hydroxylated derivatives 4-hydroxytamoxifen and endoxifen. This study addresses the pharmacological importance of endoxifen by simulating clinical scenarios in vitro. Clinical levels of tamoxifen metabolites in postmenopausal breast cancer patients previously genotyped for CYP2D6 were used in vitro along with clinical estrogen levels (estrone and estradiol) in postmenopausal patients determined in previous studies. The biological effects on cell growth were evaluated in a panel of estrogen receptor-positive breast cancer cell lines via cell proliferation assays and real-time polymerase chain reaction (PCR). Data were analyzed with one- and two-way analysis of variance and Student's t test. All statistical tests were two-sided. Postmenopausal levels of estrogen-induced proliferation of all test breast cancer cell lines (mean fold induction ± SD vs vehicle control: MCF-7 = 11 ± 1.74, P < .001; T47D = 7.52 ± 0.72, P < .001; BT474 = 1.75 ± 0.23, P < .001; ZR-75-1 = 5.5 ± 1.95, P = .001. Tamoxifen and primary metabolites completely inhibited cell growth regardless of the CYP2D6 genotype in all cell lines (mean fold induction ± SD vs vehicle control: MCF-7 = 1.57 ± 0.38, P = .54; T47D = 1.17 ± 0.23, P = .79; BT474 = 0.96 ± 0.2, P = .98; ZR-75-1 = 0.86 ± 0.67, P = .99). Interestingly, tamoxifen and its primary metabolites were not able to fully inhibit the estrogen-stimulated expression of estrogen-responsive genes in MCF-7 cells (P < .05 for all genes), but the addition of endoxifen was able to produce additional antiestrogenic effect on these genes. The results indicate that tamoxifen and other metabolites, excluding endoxifen, completely inhibit estrogen-stimulated growth in all cell lines, but additional antiestrogenic action from endoxifen is necessary for complete blockade of estrogen-stimulated genes. Endoxifen is of supportive importance for the therapeutic effect of tamoxifen in a postmenopausal setting.

ER-α36-Mediated Rapid Estrogen Signaling Positively Regulates ER-Positive Breast Cancer Stem/Progenitor Cells

The breast cancer stem cells (BCSC) play important roles in breast cancer occurrence, recurrence and metastasis. However, the role of estrogen signaling, a signaling pathway important in development and progression of breast cancer, in regulation of BCSC has not been well established. Previously, we identified and cloned a variant of estrogen receptor α, ER-α36, with a molecular weight of 36 kDa. ER-α36 lacks both transactivation domains AF-1 and AF-2 of the 66 kDa full-length ER-α (ER-α66) and mediates rapid estrogen signaling to promote proliferation of breast cancer cells. In this study, we aim to investigate the function and the underlying mechanism of ER-α36-mediated rapid estrogen signaling in growth regulation of the ER-positive breast cancer stem/progenitor cells. ER-positive breast cancer cells MCF7 and T47D as well as the variants with different levels of ER-α36 expression were used. The effects of estrogen on BCSC's abilities of growth, self-renewal, differentiation and tumor-seeding were examined using tumorsphere formation, flow cytometry, indirect immunofluorence staining and in vivo xenograft assays. The underlying mechanisms were also studied with Western-blot analysis. We found that 17-β-estradiol (E2β) treatment increased the population of ER-positive breast cancer stem/progenitor cells while failed to do so in the cells with knocked-down levels of ER-α36 expression. Cells with forced expression of recombinant ER-α36, however, responded strongly to E2β treatment by increasing growth in vitro and tumor-seeding efficiency in vivo. The rapid estrogen signaling via the AKT/GSK3β pathway is involved in estrogen-stimulated growth of ER-positive breast cancer stem/progenitor cells. We concluded that ER-α36-mediated rapid estrogen signaling plays an important role in regulation and maintenance of ER-positive breast cancer stem/progenitor cells.

Endometrial Cancer: Molecular and Cellular Basis of Tumor Development, Novel Biomarkers and Therapeutic Agents, and Innovative Research Approaches

Endometrial Cancer: Molecular and Cellular Basis of Tumor Development, Novel Biomarkers and Therapeutic Agents, and Innovative Research Approaches

Nuclear Localization of the Transcriptional Regulator MIER1α Requires Interaction with HDAC1/2 in Breast Cancer Cells

MIER1α is a transcriptional regulator that functions in gene repression through its ability to interact with various chromatin modifiers and transcription factors. We have also shown that MIER1α interacts with ERα and inhibits estrogen-stimulated growth. While MIER1α is localized in the nucleus of MCF7 cells, previous studies have shown that it does not contain a nuclear localization signal. In this report, we investigate the mechanism involved in transporting MIER1α into the nucleus. We explored the possibility that MIER1α is transported into the nucleus through a ‘piggyback’ mechanism. One obvious choice is via interaction with ERα, however we demonstrate that nuclear targeting of MIER1α does not require ERα. Knockdown of ERα reduced protein expression to 22% of control, but did not alter the percentage of cells with nuclear MIER1α (98% nuclear with scrambled shRNA vs. 95% with ERα shRNA). Further evidence was obtained using two stable transfectants derived from the ER-negative MDA231 cell line: MC2 (ERα+) and VC5 (ERα-). Confocal analysis showed no difference in MIER1α localization (86% nuclear in MC2 vs. 89% in VC5). These data demonstrate that ERα is not involved in nuclear localization of MIER1α. To identify the critical MIER1α sequence, we performed a deletion analysis and determined that the ELM2 domain was necessary and sufficient for nuclear localization. This domain binds HDAC1 & 2, therefore we investigated their role. Confocal analysis of an MIER1α containing an ELM2 point mutation previously shown to abolish HDAC binding revealed that this mutation results in almost complete loss of nuclear targeting: 10% nuclear vs. 97% with WT-MIER1α. Moreover, double knockdown of HDAC1 and 2 caused a reduction in percent nuclear from 86% to 44%. The results of this study demonstrate that nuclear targeting of MIER1α requires an intact ELM2 domain and is dependent on interaction with HDAC1/2.

Abstract LB-265: Patient-derived xenografts from advanced luminal-type breast cancer: insights into endocrine therapy resistance.

Abstract Deeper understanding the mechanisms by which luminal-type breast cancer develops resistance to endocrine therapy and development of novel strategies to treat these patients requires model systems recapitulate human breast cancer as accurately as possible. An increasing body of work suggests patient derived xenografts (PDX) may represent an informative model for development of novel therapeutics. We therefore established seven xenograft tumor lines from late-stage breast cancer patients with estrogen positive (ER+) disease. To date five ER+ PDX lines have been tested for responses to estradiol treatment in overiectomized NOD/SCID mice. Three showed estradiol independent-growth, one estrogen-stimulated growth and in one estradiol-induced a regression. These patterns mimicked the clinical phenotypes of each patient, tracking survival and responses to serial endocrine treatments. To define new mechanisms for resistance, whole genome DNA sequencing, RNA sequencing and Reverse Phase Protein Assay analysis was conducted. These studies identified an ESR1/YAP1 balanced translocation in a PDX model and tumor of origin showing low levels of ER, paradoxical high level expression form luminal genes and extreme ET resistance. The ESR1 YAP1 fusion maintained the N terminal DNA binding motif of ESR1, but the hormone binding and AF2 motifs were replaced with the C terminal transactivation domain of YAP1. Expression ESR1 YAP1 in ER+ breast cancer models down-regulated ER and induced estrogen independent growth. PDX endocrine phenotypes parallel tumor of origin responses to endocrine therapy and revel novel mechanism for endocrine therapy resistance. Citation Format: Matthew J. Ellis, Shunqiang Li, Dong Shen, Li Ding, Robert Crowder, Jeiya Shao, Rodrigo Goncalves, Yu Tao, Jingqin Luo, Aleix Prat, Wenbin Liu, Ana Maria Gonzalez-Angulo, Shuying Liu, Joshua F. McMichael, Chris Miller, Dave Larson, Robert S. Fulton, Tom Mooney, Jeremy Hoog, Li Lin, Therese Giuntoli, Caroline Bumb, Crystal Cooper, Rebecca Aft, Robert T. Kitchens, Stephen N. Johnson, Chanpheng Phommaly, Megha Shiyam Kavuri, Katherine DeSchryver, Austin Lin, YiYu Dong, Cynthia X. Ma, Timothy Pluard, Michael Naughton, Ron Bose, Rama Suresh, Reida G. McDowell, Loren Michel, Richard Wilson, Shaomeng Wang, Christopher Maher, Gordon B. Mills, Charles Perou, Elaine R. Mardis. Patient-derived xenografts from advanced luminal-type breast cancer: insights into endocrine therapy resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-265. doi:10.1158/1538-7445.AM2013-LB-265

Protein expression pattern of human MIER1 alpha, a novel estrogen receptor binding protein

MIER1 is a transcriptional regulator that exists as several isoforms. Of particular interest is the MIER1α isoform, which contains in its unique C-terminus an LXXLL motif for interaction with nuclear hormone receptors. Indeed, MIER1α has been shown to interact with ERα and inhibit estrogen-stimulated growth of breast carcinoma cells. Moreover, the subcellular localization of MIER1α changes dramatically, from nuclear to cytoplasmic, during progression to invasive breast carcinoma. While human MIER1 RNA and protein expression pattern data have been posted on several websites, none of these studies use probes or antibodies that distinguish between the α and β isoforms. We report here the first immunohistochemical study of the MIER1α protein expression pattern in human tissues. Our analysis revealed intense staining of specific cell types within virtually every endocrine and reproductive tissue except for the thyroid gland. In particular, we detected intense staining of ovarian follicles and germinal epithelium, ductal epithelial cells of the breast, pancreatic islet cells, all areas of the anterior pituitary and all zones of the adrenal cortex; moderate staining of germ cells and Leydig cells within the testis, patches of chromaffin cells in the adrenal medulla and weak staining of the fibromuscular stroma within the prostate. Immunoreactivity was limited to the cytoplasm in all positive cells except for oocytes and germinal epithelial cells in which the nucleus was also stained and in ductal epithelial cells of the breast in which staining was exclusively nuclear. In general, non-endocrine tissues were negative, however a few exceptions were noted. These included hepatocytes, myocardial fibers and neurons in all regions of the brain examined, with the exception of the thalamus. Neuronal staining was restricted to the cell bodies and dendrites, as most axons were negative. These data suggest that human MIER1α functions specifically in endocrine tissues and in a limited number of non-endocrine organs.

Abstract P2-13-09: Pharmacological impact of endoxifen in a laboratory simulation of tamoxifen therapy in postmenopausal breast cancer patients

Abstract Tamoxifen is a well-established adjuvant drug for the treatment and prevention of Estrogen Receptor (ER) positive breast cancer, effective in both pre- and postmenopausal women. However, the metabolic activation of tamoxifen to endoxifen by the CYP2D6 enzyme system still remains controversial to plan the treatment of patients with breast cancer. Since the discovery and description of the pharmacological properties of endoxifen, clinical trials were undertaken to determine the pharmacological relevance of endoxifen. The results of these trials, however, vary. In this study we demonstrate the pharmacological impact of endoxifen in a laboratory simulation of breast cancer therapy with tamoxifen in postmenopausal women in vitro in a panel of human breast cancer cell lines (MCF-7, T47D, ZR-75-1 and BT474). The concentrations of estrogens (E1/E2) used to simulate postmenopausal women treated with tamoxifen, were obtained from published studies, as well as the concentrations of tamoxifen and its metabolites (N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen) based on the CYP2D6 genotype in postmenopausal breast cancer patients (Murdter TE et al, Clin Pharmacol Ther, 2011). Our results demonstrate that irrespective of CYP2D6 genotype, tamoxifen and its primary metabolites (N-desmethyltamoxifen and 4-hydroxytamoxifen) are able to inhibit completely the estrogen-stimulated growth of breast cancer cells in vitro. Additional endoxifen in any concentration corresponding to CYP2D6 genotype was not able to increase the antiestrogenic effect of tamoxifen and its primary metabolites. Moreover, we demonstrate that 4-hydroxytamoxifen is absolutely essentialforinhibition of estrogen action. Based on our results, we can conclude that endoxifen is pharmacologically supportive but not essential for any genotype of CYP2D6 in a postmenopausal setting. This study (PYM) was supported by Susan G. Komen for the Cure International Postdoctoral Fellowship under Award number SAC100009(VCJ), and the Department of Defense Breast Program under Award number W81XWH-06-1-0590 (VCJ). Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-13-09.

Trans-resveratrol boronic acid exhibits enhanced anti-proliferative activity on estrogen-dependent MCF-7 breast cancer cells

Resveratrol (RSV), a natural compound present in the skin and seeds of red grapes, is considered a phytoestrogen and has structural similarity to the synthetic estrogen diethylstilbestrol. RSV inhibits tumor cell growth in estrogen receptor-positive (ER+) and negative (ER-) breast cancer cell lines resulting in cell specific regulation of the G1/S and G2/M stages of the cell cycle. However apoptotic cell death was only observed in ER+ MCF-7 cells. In this study, we designed and synthesized boronic acid derivative of RSV and evaluated their biological effects on ER+ MCF-7 breast cancer cells. The trans-4 analog inhibited the growth of MCF-7 cells and is not a substrate for p-glycoprotein. The trans-4 analog induces G1 cell cycle arrest, which coincides with marked inhibition of G1 cell cycle proteins and a greater pro-apoptotic effect. Finally, the trans-4 analog had no effect on the estrogen-stimulated growth of MCF-7 cells. Our results demonstrate that the trans-4 analog inhibits MCF-7 breast cancer cells by a different mechanism of action than that of RSV (S-phase arrest), and provides a new class of novel boronic acids of RSV that inhibit breast cancer cell growth.

Abstract 5682: Modeling the pharmacological importance of endoxifen for the treatment of ER-positive breast cancer in premenopausal patients

Abstract Long term adjuvant tamoxifen therapy for five years is the antiestrogenic standard of care for ER-positive breast cancer in premenopausal patients. The metabolic activation of tamoxifen by CYP2D6 to endoxifen remains controversial to plan the treatment of patients with breast cancer. However, all retrospective studies focus entirely on postmenopausal patients and no studies have been undertaken in the relevant premenopausal treatment population. We have addressed the issue of the pharmacological importance of endoxifen to control the estrogen-stimulated growth of four ER-positive breast cancer cell lines (MCF-7, T47-D, BT-474 and ZR-75). We have modeled the actual estrogen environment in the laboratory (estradiol plus estrone) based on previous data from premenopausal patients taking tamoxifen (Jordan et al, JNCI 2001; 83:1488-91). Our strategy was to evaluate the anti-proliferative actions of actual concentrations of tamoxifen, N-desmethyltamoxifen and 4-hydroxytamoxifen combined, based on the actual measurements reported for these metabolites in patients who were extensive metabolizers (EM), intermediate metabolizers (IM) or poor metabolizers (PM) (Mürdter et al, Clin Pharmacol Ther 2011; 89:708-17). These data were then compared with similar anti-proliferative experiments in the four cell lines, using endoxifen obtained from four sources (Mayo Clinic, University of Indiana, Institut für Klinische Pharmakologie (IKP) and the Fox Chase Cancer Center). Each cell line was evaluated with the clinically relevant circulating levels of estrogen, with and without the inclusion of the different endoxifens at the published concentrations from EM, IM and PM. It is important to note that all synthetic endoxifen currently available is an impure mixture of geometric isomers. Therefore, results obtained are only an estimate of the efficacy of endoxifen in the patient. To address this issue, we have specifically synthesized the appropriate isomer of endoxifen as a fixed ring compound that can no longer isomerize. We have previously used this strategy to discover the actual pharmacology of the two geometric isomers of 4-hydroxytamoxifen (Murphy et al, Mol Pharm 1990; 38:737-43). The results demonstrate the essential requirement with the appropriate concentration of endoxifen necessary to block estrogen-mediated cell replication. Acknowledgements: This work (VCJ) was supported the subcontract under the SU2C (AACR) Grant number SU2C-AACR-DT0409; the Susan G Komen For The Cure Foundation under Award number SAC100009. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5682. doi:1538-7445.AM2012-5682

Abstract 2882: Penta-O-galloyl-beta-D-glucose induces DNA replicative S arrest and G1 arrest independent of p21Cip1, p27Kip1 and p53 in human breast cancer cells and is orally active against breast cancer xenograft

Abstract Natural herbal compounds with novel actions different from existing breast cancer targeting modalities are attractive for improving treatment and prevention efficacy and safety. We have recently shown that penta-1,2,3,4,6-O-galloyl-beta-D-glucose (PGG), a naturally occurring gallotannin polyphenol compound, induced S-phase arrest in prostate cancer cells through inhibiting DNA replicative synthesis, in addition to the induction of G1 arrest and cell deaths at higher levels of exposure. We and others have shown that PGG through i.p. injection exerts a strong in vivo growth suppression of human prostate cancer xenograft models in athymic nude mice. We hypothesize that the novel DNA replication targeting action and the cellular arrest and death effects of PGG are applicable to breast cancer cells and PGG could be a novel agent for treatment or prevention of breast cancer, especially those without proven drugable targets such as triple-negative breast cancer. In cell culture studies, exposure to serum achievable level of PGG induced rapid S arrest in p53-wild type ER-dependent MCF-7 breast cancer cells and in p53-mutant ER-/PR- and Her2-negative (triple-negative) MDA-MB-231 breast cancer cells as indicated by the lack of BrdU incorporation into S-phase cells. Higher levels of PGG induced more caspase-mediated apoptosis in MCF-7 than in MDA-MB-231 cells. In addition to S-arrest, PGG induced G1 arrest in both cell lines. Contrary to a study by others with questionable design, we have found no induction of CDK inhibitory proteins p21Cip1 and p27Kip1 with G1 arrest. PGG treatment led to decreased cyclin D1 in both cell lines and over-expressing cyclin D1 abolished G1 arrest and hastened S arrest. In serum-starvation synchronized MCF-7 cells, we observed a close association of down-regulation of cyclin D1 and de-phosphorylation of Rb by PGG shortly before G1-S transition stimulated by serum. Though PGG inhibited estrogen-stimulated cell growth in MCF-7 cells, it had little effect on estrogen receptor-alpha level. To test the in vivo efficacy of PGG, we treated female athymic mice inoculated with MDA-MB-231 xenograft with 10 mg/kg body weight by daily gavage and compared with weekly paclitaxel treatment at the same dosage. Oral administration of PGG led to a better growth inhibitory efficacy than the taxane drug. Together, our in vitro and in vivo data support PGG as a potential chemopreventve or treatment agent for breast cancer with novel targeting actions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2882.

Tamoxifen Resistance in Breast Tumors Is Driven by Growth Factor Receptor Signaling with Repression of Classic Estrogen Receptor Genomic Function

Not all breast cancers respond to tamoxifen, and many develop resistance despite initial benefit. We used an in vivo model of estrogen receptor (ER)-positive breast cancer (MCF-7 xenografts) to investigate mechanisms of this resistance and develop strategies to circumvent it. Epidermal growth factor receptor (EGFR) and HER2, which were barely detected in control estrogen-treated tumors, increased slightly with tamoxifen and were markedly increased when tumors became resistant. Gefitinib, which inhibits EGFR/HER2, improved the antitumor effect of tamoxifen and delayed acquired resistance, but had no effect on estrogen-stimulated growth. Phosphorylated levels of p42/44 and p38 mitogen-activated protein kinases (both downstream of EGFR/HER2) were increased in the tamoxifen-resistant tumors and were suppressed by gefitinib. There was no apparent increase in phosphorylated AKT (also downstream of EGFR/HER2) in resistant tumors, but it was nonetheless suppressed by gefitinib. Phosphorylated insulin-like growth factor-IR (IGF-IR), which can interact with both EGFR and membrane ER, was elevated in the tamoxifen-resistant tumors compared with the sensitive group. However, ER-regulated gene products, including total IGF-IR itself and progesterone receptor, remained suppressed even at the time of acquired resistance. Tamoxifen's antagonism of classic ER genomic function was retained in these resistant tumors and even in tumors that overexpress HER2 (MCF-7 HER2/18) and are de novo tamoxifen-resistant. In conclusion, EGFR/HER2 may mediate tamoxifen resistance in ER-positive breast cancer despite continued suppression of ER genomic function by tamoxifen. IGF-IR expression remains dependent on ER but is activated in the tamoxifen-resistant tumors. This study provides a rationale to combine HER inhibitors with tamoxifen in clinical studies, even in tumors that do not initially overexpress EGFR/HER2.