Long-term Estrogen-deprived Breast Cancer Cells 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 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.

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 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 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.

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 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

Intrinsic Mechanism of Estradiol-Induced Apoptosis in Breast Cancer Cells Resistant to Estrogen Deprivation

We previously developed an estrogen receptor (ER)-positive breast cancer cell line (MCF-7:5C) that is resistant to long-term estrogen deprivation and undergoes rapid and complete apoptosis in the presence of physiologic concentrations of 17beta-estradiol. Here, we investigated the role of the mitochondrial apoptotic pathway in this process. Apoptosis in MCF-7:5C cells treated with estradiol, fulvestrant, or vehicle (control) was investigated by annexin V-propidium iodide double staining and 4',6-diamidino-2-phenylindole (DAPI) staining. Apoptosis was also analyzed in MCF-7:5C cells transiently transfected with small interfering RNAs (siRNAs) to apoptotic pathway components. Expression of apoptotic pathway intermediates was measured by western blot analysis. Mitochondrial transmembrane potential (psim) was determined by rhodamine-123 retention assay. Mitochondrial pathway activity was determined by cytochrome c release and cleavage of poly(ADP-ribose) polymerase (PARP) protein. Tumorigenesis was studied in ovariectomized athymic mice that were injected with MCF-7:5C cells. Differences between the treatment groups and control group were determined by two-sample t test or one-factor analysis of variance. All statistical tests were two-sided. MCF-7:5C cells treated with estradiol underwent apoptosis and showed increased expression of proapoptotic proteins, decreased psim, enhanced cytochrome c release, and PARP cleavage compared with cells treated with fulvestrant or vehicle. Blockade of Bax, Bim, and p53 mRNA expression by siRNA reduced estradiol-induced apoptosis relative to control by 76% [95% confidence interval (CI) = 73% to 79%, P < .001], 85% [95% CI = 90% to 80%, P < .001], and 40% [95% CI = 45% to 35%, P < .001], respectively, whereas blockade of FasL by siRNA had no effect. Estradiol caused complete regression of MCF-7:5C tumors in vivo. The mitochondrial pathway of apoptosis plays a critical role in estradiol-induced apoptosis in long-term estrogen-deprived breast cancer cells. Physiologic concentrations of estradiol could potentially be used to induce apoptosis and tumor regression in tumors that have developed resistance to aromatase inhibitors.