Long-term Estrogen-deprived Breast Cancer Research Articles

2-Methoxyestradiol as an Antiproliferative Agent for Long-Term Estrogen-Deprived Breast Cancer Cells.

To identify effective treatment modalities for breast cancer with acquired resistance, we first compared the responsiveness of estrogen receptor-positive breast cancer MCF-7 cells and long-term estrogen-deprived (LTED) cells (a cell model of endocrine therapy-resistant breast cancer) derived from MCF-7 cells to G-1 and 2-methoxyestradiol (2-MeO-E2), which are microtubule-destabilizing agents and agonists of the G protein-coupled estrogen receptor 1 (GPER1). The expression of GPER1 in LTED cells was low (~0.44-fold), and LTED cells displayed approximately 1.5-fold faster proliferation than MCF-7 cells. Although G-1 induced comparable antiproliferative effects on both MCF-7 and LTED cells (IC50 values of >10 µM), 2-MeO-E2 exerted antiproliferative effects selective for LTED cells with an IC50 value of 0.93 μM (vs. 6.79 μM for MCF-7 cells) and induced G2/M cell cycle arrest. Moreover, we detected higher amounts of β-tubulin proteins in LTED cells than in MCF-7 cells. Among the β-tubulin (TUBB) isotype genes, the highest expression of TUBB2B (~3.2-fold) was detected in LTED cells compared to that in MCF-7 cells. Additionally, siTUBB2B restores 2-MeO-E2-mediated inhibition of LTED cell proliferation. Other microtubule-targeting agents, i.e., paclitaxel, nocodazole, and colchicine, were not selective for LTED cells. Therefore, 2-MeO-E2 can be an antiproliferative agent to suppress LTED cell proliferation.

Estrogen for the Treatment and Prevention of Breast Cancer: A Tale of 2 Karnofsky Lectures.

In 1971, Sir Alexander Haddow et al. delivered the inaugural David A. Karnofsky lecture at the American Society for Clinical Oncology. This award was designated American Society for Clinical Oncology's highest, as he had used translational research to identify the first clinical therapy, that is, synthetic estrogens to treat breast cancer. His lecture was entitled "Thoughts on Chemical Therapy." For 40 years, high-dose synthetic estrogens were used as palliative therapy, for some advanced breast cancer patients 5 years following menopause. Mechanisms were unknown. Tamoxifen, a failed "morning-after pill," is an antiestrogen in estrogen receptor-positive breast cancer, which was subsequently used to treat all stages of breast cancer and to prevent breast cancer. In 2008, Jordan was selected to present the 38th Karnofsky lecture entitled: "The Paradoxical Action of Estrogen in Breast Cancer-Survival or Death?" Unexpectedly, through a study of acquired resistance to long-term tamoxifen therapy, estrogen-induced apoptosis in long-term estrogen-deprived breast cancer was deciphered in Jordan's laboratory. These data and the biological rules established under laboratory conditions provided molecular mechanisms to aid in the interpretation of the Women's Health initiative in the United States and the Million Women Study in the United Kingdom. In addition, by establishing laboratory models to understand mechanisms of estrogen-induced apoptosis, new estrogen derivatives were successfully evaluated in the laboratory and tested as candidates for women after the therapeutic failure of antiestrogenic strategies to treat breast cancer. For the future, the knowledge obtained about estrogen-induced apoptosis in cancer holds the promise of discovering new therapies to control or cure cancer in general.

Estrogen Receptor Complex to Trigger or Delay Estrogen-Induced Apoptosis in Long-Term Estrogen Deprived Breast Cancer.

Antiestrogen therapy of breast cancer has been a “gold standard” of treatment of estrogen receptor (ER)-positive breast cancer for decades. Resistance to antiestrogen therapy may develop, however, a vulnerability in long-term estrogen deprived (LTED) breast cancer cells was discovered. LTED breast cancer cells may undergo estrogen-induced apoptosis within a week of treatment with estrogen in vitro. This phenomenon has been also validated in vivo and in the clinic. The molecular ER-mediated mechanism of action of estrogen-induced apoptosis was deciphered, however, the relationship between the structure of estrogenic ligands and the activity of the ER in LTED breast cancer cells remained a mystery until recently. In this review we provide an overview of the structure-activity relationship of various estrogens with different chemical structures and the modulation of estrogen-induced apoptosis in LTED breast cancer cells resistant to antihormone therapy. We provide analysis of evidence gathered over more than a decade of structure-activity relationship studies by our group on the role of the change in the conformation of the estrogen receptor and the biological activities of different classes of estrogens and the receptor as well in LTED breast cancer.

Acquired mutations and transcriptional remodeling in long-term estrogen-deprived locoregional breast cancer recurrences

BackgroundEndocrine therapy resistance is a hallmark of advanced estrogen receptor (ER)-positive breast cancer. In this study, we aimed to determine acquired genomic changes in endocrine-resistant disease.MethodsWe performed DNA/RNA hybrid-capture sequencing on 12 locoregional recurrences after long-term estrogen deprivation and identified acquired genomic changes versus each tumor’s matched primary.ResultsDespite being up to 7 years removed from the primary lesion, most recurrences harbored similar intrinsic transcriptional and copy number profiles. Only two genes, AKAP9 and KMT2C, were found to have single nucleotide variant (SNV) enrichments in more than one recurrence. Enriched mutations in single cases included SNVs within transcriptional regulators such as ARID1A, TP53, FOXO1, BRD1, NCOA1, and NCOR2 with one local recurrence gaining three PIK3CA mutations. In contrast to DNA-level changes, we discovered recurrent outlier mRNA expression alterations were common—including outlier gains in TP63 (n = 5 cases [42%]), NTRK3 (n = 5 [42%]), NTRK2 (n = 4 [33%]), PAX3 (n = 4 [33%]), FGFR4 (n = 3 [25%]), and TERT (n = 3 [25%]). Recurrent losses involved ESR1 (n = 5 [42%]), RELN (n = 5 [42%]), SFRP4 (n = 4 [33%]), and FOSB (n = 4 [33%]). ESR1-depleted recurrences harbored shared transcriptional remodeling events including upregulation of PROM1 and other basal cancer markers.ConclusionsTaken together, this study defines acquired genomic changes in long-term, estrogen-deprived disease; highlights the importance of longitudinal RNA profiling; and identifies a common ESR1-depleted endocrine-resistant breast cancer subtype with basal-like transcriptional reprogramming.

Rapid Induction of the Unfolded Protein Response and Apoptosis by Estrogen Mimic TTC-352 for the Treatment of Endocrine-Resistant Breast Cancer.

Patients with long-term estrogen-deprived breast cancer, after resistance to tamoxifen or aromatase inhibitors develops, can experience tumor regression when treated with estrogens. Estrogen's antitumor effect is attributed to apoptosis via the estrogen receptor (ER). Estrogen treatment can have unpleasant gynecologic and nongynecologic adverse events; thus, the development of safer estrogenic agents remains a clinical priority. Here, we study synthetic selective estrogen mimics (SEM) BMI-135 and TTC-352, and the naturally occurring estrogen estetrol (E4), which are proposed as safer estrogenic agents compared with 17β-estradiol (E2), for the treatment of endocrine-resistant breast cancer. TTC-352 and E4 are being evaluated in breast cancer clinical trials. Cell viability assays, real-time PCR, immunoblotting, ERE DNA pulldowns, mass spectrometry, X-ray crystallography, docking and molecular dynamic simulations, live cell imaging, and Annexin V staining were conducted in 11 biologically different breast cancer models. Results were compared with the potent full agonist E2, less potent full agonist E4, the benchmark partial agonist triphenylethylene bisphenol (BPTPE), and antagonists 4-hydroxytamoxifen and endoxifen. We report ERα's regulation and coregulators' binding profiles with SEMs and E4 We describe TTC-352's pharmacology as a weak full agonist and antitumor molecular mechanisms. This study highlights TTC-352's benzothiophene scaffold that yields an H-bond with Glu353, which allows Asp351-to-helix 12 (H12) interaction, sealing ERα's ligand-binding domain, recruiting E2-enriched coactivators, and triggering rapid ERα-induced unfolded protein response (UPR) and apoptosis, as the basis of its anticancer properties. BPTPE's phenolic OH yields an H-Bond with Thr347, which disrupts Asp351-to-H12 interaction, delaying UPR and apoptosis and increasing clonal evolution risk.

Non-canonical AR activity facilitates endocrine resistance in breast cancer.

The role of androgen receptor (AR) in endocrine-resistant breast cancer is controversial and clinical trials targeting AR with an AR antagonist (e.g., enzalutamide) have been initiated. Here, we investigated the consequence of AR antagonism using in vitro and in vivo models of endocrine resistance. AR antagonism in MCF7-derived tamoxifen-resistant (TamR) and long-term estrogen-deprived breast cancer cell lines were achieved using siRNA-mediated knockdown or pharmacological inhibition with enzalutamide. The efficacy of enzalutamide was further assessed in vivo in an estrogen-independent endocrine-resistant patient-derived xenograft (PDX) model. Knockdown of AR inhibited the growth of the endocrine-resistant cell line models. Microarray gene expression profiling of the TamR cells following AR knockdown revealed perturbations in proliferative signaling pathways upregulated in endocrine resistance. AR loss also increased some canonical ER signaling events and restored sensitivity of TamR cells to tamoxifen. In contrast, enzalutamide did not recapitulate the effect of AR knockdown in vitro, even though it inhibited canonical AR signaling, which suggests that it is the non-canonical AR activity that facilitated endocrine resistance. Enzalutamide had demonstrable efficacy in inhibiting AR activity in vivo but did not affect the growth of the endocrine-resistant PDX model. Our findings implicate non-canonical AR activity in facilitating an endocrine-resistant phenotype in breast cancer. Unlike canonical AR signaling which is inhibited by enzalutamide, non-canonical AR activity is not effectively antagonized by enzalutamide, and this has important implications in the design of future AR-targeted clinical trials in endocrine-resistant breast cancer.

Pro-Apoptotic Effects of Estetrol on Long-Term Estrogen-Deprived Breast Cancer Cells and at Low Doses on Hormone-Sensitive Cells.

Purpose:Postmenopausal women with estrogen receptor-positive breast cancers often respond initially to tamoxifen or aromatase inhibitor therapy. Resistance to these treatments usually develops within 12 to 18 months. Clinical studies have demonstrated that high-dose estrogen can induce regression of these endocrine-resistant tumors. However, side-effects of high-dose estradiol (E2) or diethylstilbestrol (DES) limit their usage. Estetrol (E4) is the most abundant estrogen during pregnancy and has a long half-life and a low potential for side-effects. Estetrol might then provide benefits similar to DES on tumor regression but with lesser toxicity.Methods:In this study, we systematically evaluated the effects of E4 on cell proliferation and apoptosis in wild-type MCF-7 and long-term estrogen-deprived (LTED) MCF-7 cells and compared its effects with E2 and estriol (E3).Results:Estetrol induced apoptosis in LTED cells but stimulated growth of MCF-7 cells at concentrations from 10−11 to 10−8 M. These effects of E4 are similar to those of E2 but require much higher doses. Differing from E2, E4 at 10−12 M induced apoptosis in MCF-7 cells and another pregnancy estrogen, E3, acted similarly. No antagonistic effect of E4 or E3 against E2 occurred when they were combined.Conclusions:The pro-apoptotic effects of E4 and E3 on LTED cells and at low doses on MCF-7 cells indicate that these steroids could be used as therapeutic agents for endocrine-resistant or sensitive breast cancer.

Abstract 3432: Evolving genomes of breast cancer aromatase inhibitor resistance models

Abstract Acquired endocrine resistance is a severe barrier in the anti-hormonal treatment of breast cancer and is responsible for its failure in about half of the treated patients. In vitro models of long-term estrogen deprived breast cancer cells serve as laboratory tools to explore new therapeutic strategies. The models MCF-7:5C and MCF-7:2A mimic clinical AI resistance but regress with E2 due to the reconfiguration of survival signaling known as E2-inducible apoptosis (5C rapid, 2A delayed reaction). Alterations of stress related pathways including the accumulation of endoplasmic reticulum stress, oxidative stress, and inflammatory stress that occur prior to E2-induced apoptosis (Ariazi et al. 2011; Fan et al. 2013, 2015; Sweeney et al. 2014) and specific miRNA expression profiles (Hoppe et al Oncotarget 2016) have been linked with these resistance and E2-inducible apoptosis phenotypes. Little is known on their underlying genetic variability. Here we compare the mutational profiles of 5C and 2A clones with those of the estradiol stimulated growth sensitive MCF-7:WS8 and standard MCF-7 (ATCC) cells. Whole genome libraries were sequenced on the Illumina HiSeq platform (2x150bp reads). Alignments were manually inspected (bcftools mpileup) to avoid false negative variant calling in poorly (<20x) covered regions. Loci with at least 10x coverage and absence of any variant reads were classified as wildtype. The coding genome positions defined as mutant or wildtype in all four cell types served as a basis to select candidate mutations for the verification of MCF-7 variants via comparison to the COSMIC database (http://cancer.sanger.ac.uk/cosmic) and identification of novel mutations by variant calling (GATK HaplotypeCaller). Putative protein effects of variants were predicted by the SnpEff tool. We verified 421 point mutations in MCF-7 (COSMIC) of which 203 mutations were lost in our models, with additional specific losses of 4, 4, and 18 mutations in WS8, 2A and 5C, respectively. In 5C cells new mutations affected 261 genes with 31 high impact mutations based on splice site alterations in 7 genes (i.e. CERS2, SEMA3A, TDRD7, ATP5H) and gain of stop codons in 24 genes (i.e. CACNA1S, NOTCH4, SUN1, JPH3, SERPINB2). Similarly, in 2A cells there were 12 high impact mutations among 122 affected genes (e.g. ARHGEF5, ARHGAP35, ARAP2). We will report on the integrative genomic analysis including larger structural variants (short indels and CNVs) towards the evolution of these models for a comprehensive understanding of the biology of E2-inducible apoptosis. Citation Format: Reiner Hoppe, Siarhei Kandabarau, Ping Fan, V Craig Jordan, Hiltrud B. Brauch. Evolving genomes of breast cancer aromatase inhibitor resistance models [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 3432.

Abstract 5633: Unique modulation of estrogen-induced apoptosis with structural analogues of endoxifen in long-term estrogen-deprived (LTED) breast cancer cells

Abstract Endoxifen, a non-steroidal anti-estrogen is the active hydroxylated metabolite of N-desmethyltamoxifen. The structure is based on a synthetic triphenylethylene-type angular estrogen. Endoxifen is in clinical trials as a therapeutic agent to treat aromatase resistant breast cancer. Paradoxically, LTED breast cancer is known to respond to synthetic and natural estrogens through estrogen-induced apoptosis. A series of synthetic endoxifen analogs, were used to modulate estrogen-induced apoptosis in LTED breast cancer cells (MCF7:5C). X-ray crystallography was performed on endoxifen, bisphenol, and diethylstillbestrol (DES), bound to the ligand-binding domain (LBD) of the human estrogen receptor (ER). Cellular studies established that (MCF-7:5C) of estrogen-induced apoptosis was dependent on the LBD of the ER. Similarly, endoxifen blocked estrogen-induced apoptosis in a concentration and time-dependent manner. Apoptosis triggered by DES was blocked by endoxifen and was ER-dependent. X-ray crystallography of both endoxifen and DES illustrate the two extremes of estrogen-induced apoptosis, which is dependent upon the closure of helix 12 over the LBD to generate estrogenic activity. Despite the fact that bisphenol, an analog of endoxifen without the anti-estrogenic monomethylamino ethyl side chain, is an estrogen that causes breast cancer cell replication; bisphenol initially inhibits estrogen-induced apoptosis, but triggers estrogen-induced apoptosis itself after 7 days of treatment of LTED breast cancer cells. X-ray crystallography demonstrates that bisphenol can produce a unique complex with the ER:LBD, which is similar to the DES:LBD estrogenic complex. In both cases, the ligands are sealed into the LBD by helix 12. This delay in apoptosis caused by an analog of endoxifen, that is an angular estrogen, possess unique biological features. We hypothesize that in LTED cells the complex passes through an anti-estrogenic conformation to evolve into an estrogenic complex with helix 12 sealing the LBD. We describe a unique property of a ligand:receptor complex. For the first time we demonstrate that a ligand:receptor complex is slowly activated from an antagonist to an agonist complex over a period of days. Citation Format: Balkees Abderrahman, Sean W. Fanning, Daniela Quintana Rincon, Geoffrey Greene, Philipp Y. Maximov, V. Craig Jordan. Unique modulation of estrogen-induced apoptosis with structural analogues of endoxifen in long-term estrogen-deprived (LTED) breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5633. doi:10.1158/1538-7445.AM2017-5633

Abstract P3-05-11: The evolution of the estrogen receptor (ER) complex conformation governs estrogen-induced apoptosis in antihormone-resistant breast cancer cells

Abstract Over the past decade new insights have been gained into the acquired resistance to tamoxifen and the Aromatase Inhibitors (AIs) with the discovery of the new biology of estrogen-induced apoptosis. However, it has also been learned that estrogens can be classified into planar class I and angular class II estrogens. Using model systems of long-term estrogen-deprived breast cancer cells in vitro (MCF-7:5C), it was previously shown that class I estrogens cause immediate apoptosis over a 3-4 day period. Paradoxically, class II estrogens actually block apoptosis caused by planar class I estrogens. To gain insight into this paradox we have successfully crystallized an angular class II triphenylethylene (TPE) estrogen bound to the ligand binding domain (LBD) of the ER and derived a new conformation for the TPE:ER complex (code 3Q97 in the PDB). Surprisingly, Helix 12 seals the LBD with the class II estrogen, but not the same conformation is observed with the planar class I estrogen 17β-estradiol (E2). There would seem to be no reason why the 3Q97 complex would not cause immediate apoptosis. To address this issue we have used Western blot analysis for protein and qRT-PCR for mRNA levels for the ER. ER parameters were monitored for up to 72 hours and results compared and contrasted between E2, the Class II estrogens, 4-hydroxytamoxifen (4OHT) and endoxifen (Endox). ER protein and mRNA levels with 4OHT or Endox accumulated and remained high throughout the study period. In contrast, the planar estrogen E2 produced a rapid decline in the protein and mRNA levels for the ER complex. The angular class II estrogens initially produced an accumulation of the ER protein complex, which decreased with time. Using a chromatin immunoprecipitation (ChIP) technique we demonstrated that the class II estrogens recruit only half of the ER to the estrogen-responsive genes promoters (TFF1 And BREB1) and less than half co-activator binding compared to E2. The TPEs were only partial agonists compared to planar estrogen. These results explain why the Class II estrogens induce delayed apoptosis. We conclude that, for the first time, we have observed the binding of a ligand to a receptor that changes conformation against time and evolves from an antagonist to an agonist conformation to trigger apoptosis. These observations have current relevance to the decryption of the protective effects of estrogen alone therapy against breast cancer incidence in the Women's Health Initiative (WHI) trial. This work was supported by the Susan G. Komen for the Cure Foundation award SAC100009. Citation Format: Maximov PY, Sengupta S, Fernandes DJ, Fan P, Curpan RF, Rajan SS, Greene GL, Jordan VC. The evolution of the estrogen receptor (ER) complex conformation governs estrogen-induced apoptosis in antihormone-resistant breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-11.

Abstract OT2-1-08: A phase II open label study of everolimus in combination with anti-estrogen therapy in hormone receptor-positive HER2-negative advanced breast cancer

Abstract Background: Hormone receptor positive (HR+) breast cancer, defined as estrogen receptor and/or progesterone receptor positive, accounts for 70% of invasive breast cancers. While most HR+ tumors initially respond to anti-estrogen therapy, resistance, linked to crosstalk between cell signaling and signal transduction pathways, subsequently develops in almost 100%. Preclinical data has shown that the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway is activated in long-term estrogen-deprived and aromatase inhibitor-resistant breast cancer cells. It is hypothesized that in this group of endocrine-resistant patients (pts), resistance to anti-estrogen therapy is driven by the PI3K/Akt/mTOR pathway and hence the inhibition of this pathway may reverse resistance. This study will evaluate the efficacy of adding everolimus (an inhibitor of mTOR) to anti-estrogen therapy in pts with HR+ metastatic breast cancer (MBC) who have progressed on anti-estrogen therapy. Study Objectives: This multi-center, open-label Phase II trial is designed to determine the efficacy (primary endpoint is PFS), safety and tolerability of everolimus in combination with anti-estrogen therapy in pts with HR+, HER2-negative MBC with disease progression on prior anti-estrogen therapy. An exploratory objective of this study is to evaluate the prognostic value of the VeriStrat assay in this population. VeriStrat testing has been used to predict treatment efficacy in clinical trials of non small cell lung cancer, head and neck squamous carcinoma, and MBC. Eligibility: Pts ≥18 years with HR+, HER2-negative breast cancer that is either unresectable, locally recurrent, or metastatic are eligible. Pts must have progressed on anti-estrogen therapy (recurrence while on/within 12 months of treatment for early stage breast cancer, or progressed while on/within 1 month of treatment for advanced/MBC), but anti-estrogen therapy is not required to be the last therapy prior to enrollment. No washout for anti-estrogen therapy is required, and ≤1 previous chemotherapy regimen is allowed. Post menopausal or pre-/ peri-menopausal women on tamoxifen are eligible and ovarian function suppression is permitted. Prior treatment with a mTOR inhibitor is not allowed. Additional eligibility requirements include: measurable or evaluable disease, ECOG ≤2, adequate bone marrow and organ function and an adequate lipid profile (fasting serum cholesterol ≤300 mg/dL or ≤7.75 mmol/L and fasting triglyceride ≤2.5 x ULN). Trial Design: Everolimus will be administered at a dose of 10 mg PO daily in combination with the FDA prescribed doses of the most recent anti-estrogen therapy on which the pt progressed. Treatment cycles will be 4 weeks, with response evaluations by CT scans every 2 cycles. Pts will be treated until disease progression or unacceptable toxicity occurs. With an alpha=0.5, a sample size of 42 will provide 80% power to detect improvement in median PFS. Blood samples for the VeriStrat assay will be collected at baseline, on Cycle 3 Day 1, at disease progression and at the first follow-up visit post disease progression. Archival tumor tissue samples will be collected for comprehensive genomic profiling. This trial has a total accrual goal of 46 pts. Citation Format: Denise A Yardley, Mythili Shastry, Laura M DeBusk, Howard A Burris III, John D Hainsworth. A phase II open label study of everolimus in combination with anti-estrogen therapy in hormone receptor-positive HER2-negative advanced breast cancer [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 OT2-1-08.

Abstract P4-06-06: Growth hormone induction of oncogenic signaling promotes survival of endocrine resistant breast cancer cells

Abstract Targeting endocrine resistant breast cancer cells has been successfully accomplished by the use of mTORC1 inhibitors in combination with either aromatase inhibitors or tamoxifen. While there was preclinical data suggesting that targeting of the type I IGF receptor (IGF1R) might also be a target for endocrine resistant cells, clinical trials using IGF1R monoclonal antibodies showed no advantage and even suggested potential harm for the combination. IGF1R inhibition results in the disruption of an endocrine feedback loop and results in elevation of the pituitary growth factor growth hormone (GH). GH is involved in normal human growth, development, metabolism, and longevity. Growth hormone released from the pituitary stimulates production of IGF-I from the liver. The GH/IGF-1 axis is known to promote mammary gland hyperplasia and breast cancer carcinogenesis. Previous studies have shown that GH promotes carcinogenesis independently of insulin-like growth factor. The role of GH in specific subtypes of breast carcinoma remains to be defined. Our laboratory has found that estrogen receptor positive breast cancer cell lines up regulated oncogenic STAT5, Akt, and MAPK signaling pathways in response to GH. Tamoxifen resistant (TamR) and long-term estrogen deprived (LTED) breast cancer cell lines derived from MCF-7 and T47D cells demonstrated increased oncogenic signaling in response to GH compared to the corresponding parental cell lines from which they were derived. In contrast, GH stimulation of a triple negative breast cancer cell line (MDA-MB-231) failed to induce signaling via these oncogenic pathways. Although GH treatment of parental, TamR, and LTED breast cancer cells had minimal effect on cell proliferation, cell cycle progression, or migration; GH signaling protected cells from cytotoxic chemotherapy induced apoptosis. This data shows that GH signaling is found in endocrine sensitive and resistant cells. Furthermore, GH may function in protecting cells from cell death induced by either endocrine or cytotoxic drugs. GH receptor blockade may be a valid treatment option for endocrine resistant breast cancer. Citation Format: Heather C Beckwith, Douglas Yee. Growth hormone induction of oncogenic signaling promotes survival of endocrine resistant breast cancer cells [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 P4-06-06.

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.

Mechanisms underlying differential response to estrogen-induced apoptosis in long-term estrogen-deprived breast cancer cells

Models of long-term estrogen-deprived breast cancer cells are utilized in the laboratory to mimic clinical aromatase inhibitor-resistant breast cancer and serve as a tool to discover new therapeutic strategies. The MCF-7:5C and MCF-7:2A subclones were generated through long-term estrogen deprivation of estrogen receptor (ER)-positive MCF-7 cells, and represent anti-hormone-resistant breast cancer. MCF-7:5C cells paradoxically undergo estrogen-induced apoptosis within seven days of estrogen (estradiol, E2) treatment; MCF-7:2A cells also experience E2-induced apoptosis but evade dramatic cell death until approximately 14 days of treatment. To discover and define the mechanisms by which MCF-7:2A cells survive two weeks of E2 treatment, systematic experiments were performed in this study. The data suggest that MCF-7:2A cells employ stronger antioxidant defense mechanisms than do MCF-7:5C cells, and that oxidative stress is ultimately required for MCF-7:2A cells to die in response to E2 treatment. Tumor necrosis factor (TNF) family member activation is also essential for E2-induced apoptosis to occur in MCF-7:2A cells; upregulation of TNFα occurs simultaneously with oxidative stress activation. Although the unfolded protein response (UPR) signaling pattern is similar to that in MCF-7:5C cells, it is not sufficient to cause cell death in MCF-7:2A cells. Additionally, increased insulin-like growth factor receptor β (IGF-1Rβ) confers a mechanism of growth and anti-apoptotic advantage in MCF-7:2A cells.

Abstract S3-05: Patient-derived xenograft study reveals endocrine therapy resistance of ER+ breast cancer caused by distinct ESR1 gene aberrations

Abstract Endocrine therapy resistance occurs in 50% of estrogen receptor positive (ER+) luminal breast cancers but the underlying mechanisms are poorly understood. To gain insight, we have taken advantage of whole-genome and RNA sequencing data of five late-stage hormone-resistant luminal breast tumors all of which have been successfully established as patient-derived mouse xenograft (PDX) models. Here we describe genetic alterations in ESR1-the gene coding for ERa-in three of these five tumors. They include an ESR1 point mutation (Y537S), a gene translocation causing an in-frame fusion between N-terminal ER and C-terminal Yes-associated protein 1 (YAP1), and ESR1 gene amplification. Functional characterization of the ESR1(Y537S) mutant and ESR1-YAP1 fusion in ER+ cell lines indicated that they both possess constitutive transcriptional activity and drive hormone-independent cell proliferation, mirroring the endocrine resistance of the originating tumors and the estradiol-independent growth of the PDX tumors. ESR1 (Y537S), a known gain-of-function experimental mutation in the ligand-binding domain of ER is not seen in primary breast cancer (TCGA data), suggesting it is a mutation associated with acquired resistance. Regarding the ESR1-YAP1 fusion gene, the truncated N-terminal ER fragment lacks the hormone-dependent transactivation domain (AF2) and the ligand binding domain but retains the hormone-independent transactivation domain (AF1) and therefore drives resistance to all endocrine approaches. As for ESR1 gene amplification, the tumor of origin, though resistant to aromatase inhibition, paradoxically responded to estradiol treatment, and this was recapitulated in the PDX model. Interestingly, acquired ESR1 gene amplification also occurred in long-term estrogen-deprived breast cancer MCF-7 cells which similarly regress upon estradiol exposure. Thus, ESR1 amplification may be a biomarker for paradoxical therapy with estradiol. Together, our focused study of advanced endocrine resistant luminal breast tumors revealed three distinct mutational mechanisms affecting the ESR1 gene that drive endocrine therapy resistance. Prevalence studies using RNAseq are underway to determine the frequency of somatic changes in the ESR1 gene in advanced breast cancer samples and additional ER+ PDX models. These additional data will be presented at the meeting. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S3-05.

Abstract 830: Transcriptional modulation of estrogen-induced apoptosis through activation of c-Fos/c-Jun in long-term estrogen deprived breast cancer cells.

Abstract Recent clinical trials have demonstrated that estrogen (E2) alone reduces breast cancer incidence in postmenopausal women and has therapeutic effects on aromatase inhibitor resistant patients which both are related with the effect of E2-induced apoptosis. We have shown that E2 induces apoptosis in long-term E2 deprived breast cancer cells (MCF-7:5C) through stress responses, but the molecular mechanism underlying E2-induced stress remains to be elucidated. Here, we report that E2 activated the sensors of unfolded protein response (UPR) inositol-requiring protein 1 alpha (IRE1α) and PRK-like endoplasmic reticulum kinase (PERK) within 24 hours. Knockdown of PERK and IRE-1α through small interferon RNAs (siRNA) partially prevented E2-induced apoptosis, which suggested that endoplasmic reticulum stress was involved in the E2-induced apoptosis. Further examination showed E2 activated both classical estrogen responsive element (ERE) pathway and nonclassical activating protein-1 (AP-1) pathway in MCF-7:5C cells. Classical ERE regulated genes were not directly involved in E2-induced apoptosis. However, the transcription factor c-Fos acted as a critical trigger involved in stress responses induced by E2 in MCF-7:5C cells. E2 immediately elevated c-Fos expression in MCF-7:5C cells but not in wild-type breast cancer cells and 4-hydroxytamoxifen blocked this stimulation which suggested that E2 activated c-Fos through estrogen receptor (ER). E2 increased phosphorylation of c-Jun after 24 hours treatment but did not significantly enhance the abundance of c-Jun as c-Fos. c-Fos protein forms stable heterodimers with c-Jun which preferentially bind to phorbol 12-O-tetradecanoate-13-acetate (TPA)-responsive element (TRE). Interestingly, low doses of TPA which activated TRE activity could induce apoptosis and activated apoptosis-related genes similar to E2 in MCF-7:5C cells. Knockdown of c-Fos and c-Jun with specific siRNAs blocked E2-induced apoptosis, which confirmed the activation of AP-1 played a critical role in the process of apoptosis induced by E2. Although c-Fos closely associates with c-Jun to form a stable heterodimer, they had differential functions in modulation of UPR and other nuclear transcription factors. Knockdown of c-Fos but not c-Jun could inhibit the activation of UPR by E2 in MCF-7:5C cells. Furthermore, downregulation of c-Fos abolished the expression of multiple nuclear transcription factors such as SRC-3, NF-κB, and p300 etc in MCF-7:5C cells. Overall, these data illustrate that c-Jun might provide a dimerizing site for c-Fos, whereas c-Fos was identified as a pivotal regulatory target of E2 in MCF-7:5C cells to trigger the apoptotic cascades. This study provides an important rationale for further exploration of E2-induced apoptosis in endocrine resistant breast cancer to improve clinical benefit. Citation Format: Ping Fan, Fadeke Agboke, Obi L. Griffith, Russell E. McDaniel, Xiaojun Zou, Karen Creswell, Joe W. Gray, Virgil Craig Jordan. Transcriptional modulation of estrogen-induced apoptosis through activation of c-Fos/c-Jun in long-term estrogen deprived breast cancer cells. [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 830. doi:10.1158/1538-7445.AM2013-830

Abstract 828: Mechanisms underlying differential response to estrogen-induced apoptosis in long-term estrogen-deprived breast cancer cell lines.

Abstract Models of long-term estrogen-deprived breast cancer cells are utilized in the laboratory to mimic clinical aromatase inhibitor-resistant breast cancer and serve as a tool to discover new therapeutic strategies. The MCF-7:5C and MCF-7:2A subclones are derived from the parental MCF-7 cell line through long-term estrogen deprivation (Sweeney, et al. HMBCI 2012). In addition to the different responses to antiestrogens observed between MCF-7:5C and MCF-7:2A cells, they also have different apoptotic responses to estrogen (E2); the MCF-7:5C cells undergo apoptosis and die during the first week of E2 treatment, whereas the “late-apoptosis” MCF-7:2A cells die after two weeks of E2 treatment. To discover the mechanisms by which MCF-7:2A cells survive two weeks of E2 treatment, systematic experiments were performed in this study. E2 increased the cleavages of poly (ADP-ribose) polymerase (PARP) and Caspase 9 in both cell lines within 72 hours. RT-PCR microarrays of apoptosis-related genes also showed the gene expression pattern in MCF-7:2A cells was similar to that of MCF-7:5C cells after E2 treatment. Importantly, E2 caused apoptosis in both cell lines after 72 hours of E2 treatment, detected by Annexin V binding assay through flow cytometry, but to varying degrees. The MCF-7:5C cells showed approximately double the amount of Annexin V binding (and thereby apoptosis) compared to the MCF-7:2A cells. Further experiments showed that E2 induced similar responses of the unfolded protein response (UPR), demonstrated by the markers eukaryotic initiation factor subunit alpha (eIF2α) and inositol-requiring enzyme 1 alpha (IRE1α), although MCF-7:2A cells have higher basal levels of phosphorylated eIF2α compared with MCF-7:5C. The question arises, then, of why E2 does not kill the 2A cells until the second week of treatment, when their molecular responses closely mirror the 5C cells. Our microarray database demonstrates increased superoxide dismutase 2 (SOD2) in response to E2 in MCF-7:2A but not in MCF-7:5C cells, as well as higher basal levels of glutathione. These could represent protective mechanisms in the MCF-7:2A cells to prevent E2-induced cell death. We are currently investigating whether MCF-7:2A cells are resistant to the oxidative stress induced by E2. All of these preliminary data imply that the antioxidant system may play a critical role to defend MCF-7:2A cells from apoptosis induced by E2. VCJ is supported by the Department of Defense Breast Program under Award number W81XWH-06-1-0590 Center of Excellence; subcontract under the SU2C (AACR) Grant number SU2C-AACR-DT0409; the Susan G Komen For The Cure Foundation under Award number SAC100009; GHUCCTS CTSA (Grant # UL1RR031975) and the Lombardi Comprehensive Cancer Center Support Grant (CCSG) Core Grant NIH P30 CA051008. The views and opinions of the author(s) do not reflect those of the US Army or the Department of Defense. Citation Format: Elizabeth E. Sweeney, Ping Fan, V. Craig Jordan. Mechanisms underlying differential response to estrogen-induced apoptosis in long-term estrogen-deprived breast cancer cell lines. [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 828. doi:10.1158/1538-7445.AM2013-828

Abstract 2921: Critical mediation of E2-induced apoptosis through c-Src in long-term estrogen deprived breast cancer cells

Abstract Our previous publication showed that physiological concentrations of estrogen (E2) could trigger apoptosis of long-term E2 deprived breast cancer cells (MCF-7:5C) (Lewis et al. JNCI 2005; 97:1746-59). This new targeted strategy provides novel therapeutic approaches to endocrine resistant breast cancer. A phase II clinical trial reported that E2 provided a clinical benefit for aromatase inhibitor-resistant advanced breast cancer patients. However, only 30% of patients receive clinical benefit (Ellis MJ et al. JAMA 2009;302:774-80). This prompted us to investigate the mechanisms underlying E2-induced apoptosis to find strategies to increase the therapeutic responsiveness. c-Src is currently of interest, as it is an important adapter protein of ER in breast cancer cells. Here, we found that E2 stimulated c-Src phosphorylation in MCF-7:5C cells and 4-hydroxytamoxifen (4-OHT) could block this stimulation which demonstrated that E2 activated c-Src through ER. The specific inhibitor of c-Src, PP2, could block c-Src activation induced by E2. E2 rapidly activated growth pathways within minutes in MCF-7:5C cells and PP2 could block this non-genomic pathway. E2 was also able to activate Akt/MAPK pathways after 24 hour treatment which could be blocked by inhibition of c-Src. These data showed that c-Src mediated downstream signaling pathways of ER in MCF-7:5C cells. Characteristic E2-induced apoptosis occurred around 72 hour treatment with E2, unexpectedly, inhibition of c-Src could significantly block E2-induced apoptosis which implied that c-Src played a critical role in E2-induced apoptosis. Further examination through transcriptome analysis confirmed that E2 widely induced apoptosis-related pathways. Consistent with the biological experiments, PP2 could completely abolish the apoptosis pathways induced by E2. These data illustrate that c-Src acts as a critical molecule to mediate the downstream signaling of ER (including E2-induced apoptosis) in MCF-7:5C cells. It provides an important rationale for clinical trial with c-Src inhibitors in aromatase inhibitors resistant patients, especially avoiding in combination with physiological levels of E2. Acknowledgements This work (VCJ and JWG) is supported by the SU2C (AACR) Grant (SU2C-AACR-DT0409). 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 2921. doi:1538-7445.AM2012-2921

Lapatinib or placebo plus letrozole as preoperative treatment of hormone receptor-positive HER2-negative operable breast cancer. Preliminary report of safety and activity of the double-blind randomized phase IIb LET-LOB study

623 Background: Crosstalk between hormone- and EGFR-family pathways is a possible cause of resistance to aromatase inhibitors (AI), as suggested by the increased expression of HER2, EGFR and MAPK in long-term estrogen-deprived breast cancer (BC) cells. Lapatinib (Lap) is an orally active small molecule inhibitor of EGFR and HER2 tyrosine-kinases. Combination of an AI and Lap in ER+/HER2- tumors could avoid the onset of resistance due to early stimulation of EGFR and HER2 pathways. To evaluate these effects, we designed an international multicenter phase IIb double-blind randomized trial in ER+/HER2- BC pts candidates for hormone neoadjuvant therapy. Methods: Postmenopausal pts with stage II-IIIa, HR+/HER2- BC are randomized to letrozole (Let; 2.5 mg/d) plus lapatinib (Lap; 1,500 mg/d) or placebo (P), given continuously for 24 weeks before surgery. Activity (with ultrasonographyc, US) and cardiac safety (with echocardiogram) assessment are performed after 12 and 24 weeks. Tissue biomarkers of hormone and EGFR-family related pathways are analyzed before and after treatment. An initial sample size of 42 pts will be enrolled assuming a 50% chance of cOR with Let+P and a 40% increase in cOR with the Let+Lap (equal to 70% absolute cOR). Additional 48 pts will be treated in case we observe 20 cOR in the first cohort (Simon's design). Results: 19 Italian and European centers are participating in the study. From April 2007, 28 pts have been randomized, and 15 pts were treated for at least 3 months (mo). Six pts completed the six mo treatment, and 4 underwent surgery. At diagnosis, mean US tumor size was 2.87 cm (range 2–6 cm). All pts were ER+ (73% ER+/PR+) and HER2-. No significant decrease in mean LVEF was observed at the 3-mo evaluation (60.9%-61.5%). No grade 3–4 toxicities were reported. A 34% absolute reduction in median longest US tumor diameter has been observed after 3 mo (15 pts) and 65% reduction has been obtained at 6 mo (6 pts). Updated blinded safety and activity results will be presented. Conclusions: The combination of Lap or P with Let in ER+/HER2-operable breast cancer was safe, without any early cardiac toxicity, and induced clear tumor shrinkage. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration GlaxoSmithKline GlaxoSmithKline GlaxoSmithKline

Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway Improves Response of Long-term Estrogen-Deprived Breast Cancer Xenografts to Antiestrogens

Aromatase inhibitors that block the synthesis of estrogen are proving to be superior to antiestrogens and may replace tamoxifen as first-line treatment for postmenopausal estrogen receptor (ER)-positive breast cancer patients. However, acquisition of resistance to all forms of treatments is inevitable and a major clinical concern. In this study, we have investigated the effects of long-term estrogen deprivation in the breast cancer xenograft model and whether sensitivity to antiestrogens can be restored in vivo. We also compared whether combining wortmannin with tamoxifen or fulvestrant inhibited tumor growth better than either drug alone. Long-term estrogen-deprived aromatase-transfected human ER-positive breast cancer cells (UMB-1Ca) were grown as tumors in ovariectomized athymic nude mice. Twelve weeks after inoculation, when tumors reached 300 mm(3), animals were grouped and injected with vehicle, Delta(4)A, letrozole, tamoxifen, fulvestrant, wortmannin, tamoxifen plus wortmannin, and wortmannin plus fulvestrant. Tumor volumes were measured weekly. Tumors of UMB-1Ca cells grew equally well with and without androstenedione, indicating the ability of the cells to proliferate in the absence of estrogen. The combination of wortmannin with tamoxifen or fulvestrant inhibited tumor growth better than either drug alone. The combination of wortmannin plus fulvestrant was the most effective treatment that maintained tumor regression for a prolonged time. These results suggest that blocking both ER and growth factor receptor pathways could provide effective control over tumor growth of long-term estrogen-deprived human breast cancers.