Antiestrogen-resistant Breast Cancer Cells Research Articles

Protein Aggregation Patterns Inform about Breast Cancer Response to Antiestrogens and Reveal the RNA Ligase RTCB as Mediator of Acquired Tamoxifen Resistance.

Simple SummaryAcquired resistance to antiestrogenic therapy remains the major obstacle to curing luminal subtype breast cancer. While current treatment in acquired endocrine-resistant settings includes combined therapy with receptor tyrosine kinase or cyclin-dependent kinase inhibitors, progression to incurable disease remains possible. In recent years, the antioxidant system and the protein quality control network have been associated with the enhanced resistance of breast cancer cells to hormonal therapy. In this work, we raise the hypothesis that antiestrogen treatment induces the accumulation of protein aggregates in sensitive cells, which in turn could hinder the activation of survival pathways. We present evidence concerning a novel way to identify antiestrogen response and disclose a novel protein, RTBC, that controls acquired antiestrogen resistance. This work opens a new avenue for research towards finding breast cancer prognostic markers and therapeutic targets, where the identification of proteins prone to aggregate could help to identify antiestrogen response and understand mechanisms of disease.The protein quality control network, including autophagy, the proteasome and the unfolded protein response (UPR), is triggered by stress and is overactive in acquired antiestrogen therapy resistance. We show for the first time that the aggresome load correlates with apoptosis and is increased in antiestrogen-sensitive cells compared to endocrine-resistant variants. LC-MS/MS analysis of the aggregated proteins obtained after 4OH-tamoxifen and Fulvestrant treatment identified proteins with essential function in protein quality control in antiestrogen-sensitive cells, but not in resistant variants. These include the UPR modulators RTCB and PDIA6, as well as many proteasome proteins such as PSMC2 and PSMD11. RTCB is a tRNA and XBP1 ligase and its aggregation induced by antiestrogens correlated with impaired XBP1s expression in sensitive cells. Knock down of RTCB was sufficient to restore sensitivity to tamoxifen in endocrine-resistant cells and increased the formation of aggresomes, leading to apoptotic cell death. Analysis of primary human breast cancer samples and their metastases appearing after endocrine treatment showed that RTCB is only localized to aggresomes in the primary tumors, while total aggresomes, including aggregated RTCB, were significantly reduced in the metastases. Therefore, different protein aggregation patterns may indicate loss of function of essential proteins resulting in enhanced protein aggregation that can be used to identify antiestrogen-resistant breast cancer cells and improve the response to antiestrogenic therapy.

Abstract PS17-43: Sco-101 is a novel oral drug that reverses antiestrogen resistance in breast cancer cells

Abstract Breast cancer is the most frequent cancer diagnosed in women and 80% of all cases of breast cancer patients present with estrogen receptor (ER)-positive disease. Treatment with antiestrogens most notable tamoxifen and faslodex are effective for a large proportion of ER-positive breast cancer. However, antiestrogen resistance eventually arises in all patients with advanced disease. Thus, antiestrogen resistance represents a major problem in the clinical management of breast cancer patients and there is currently no treatment to overcome resistance to antiestrogens. SCO-101 is an oral drug currently being tested in a Phase II clinical trial enrolling metastatic colorectal cancer patients with drug resistant disease (ClinicalTrials.gov Identifier: NCT04247256). In the current work, we investigated the potential of SCO-101 to act in combination with the antiestrogens tamoxifen or fulvestrant (faslodex) in antiestrogen resistant breast cancer cell lines (MCF-7/LCC-2, MCF-7/LCC-9, T47D/TR1) and - as a negative control - the ER negative MDA-MB-231. Treatment effects were investigated by MTT cell viability assays, siRNA knock-down experiments and western blots. SCO-101 only had minor inhibitory effects on cell viability when administered alone. Interestingly, when combining SCO-101 with tamoxifen or fulvestrant in MCF-7 or T47D antiestrogen resistant breast cancer cells, an additive to synergistic effect on cell viability was observed. In contrast to these results, SCO-101 in combination with antiestrogens had no effects on the triple negative MDA-MB-231 cell line, indicating the ER and or PR (progesterone receptor) is important for the effect of SCO-101 in antiestrogen resistant breast cancer cells. As SCO-101 has been described to targets the volume-regulated anion channel (VRAC), in which LRRC8A is the essential subunit, we investigated whether knockdown of LRRC8A would impact the treatment outcome. No apparent changes in response to SCO-101 and antiestrogens were observed upon the knockdown. Additionally, the protein level of LRRC8A was examined upon treatment with anti-estrogens +/- SCO-101 and the treatment did not alter LRRC8A protein expression. Our findings strongly suggest that SCO-101 interferes with antiestrogen resistance in breast cancer and we have recently received an EURECA Eurostars grant to further investigate the molecular mechanisms of action for SCO-101 in reversing antiestrogen resistance, to investigate drug response in 3D breast cancer models, and to conduct a Phase Ib dose escalation clinical trial with SCO-101 and faslodex in patients with antiestrogen resistant ER positive breast cancer. Citation Format: Jan Stenvang, Ida CB Madsen, Katrine Hartfelt, Haatisha Jandu, Signe L Nielsen, Palle Christophersen, Nils Brünner. Sco-101 is a novel oral drug that reverses antiestrogen resistance in breast cancer cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-43.

The Structure-Function Relationship of Angular Estrogens and Estrogen Receptor Alpha to Initiate Estrogen-Induced Apoptosis in Breast Cancer Cells.

High-dose synthetic estrogen therapy was the standard treatment of advanced breast cancer for three decades until the discovery of tamoxifen. A range of substituted triphenylethylene synthetic estrogens and diethylstilbestrol were used. It is now known that low doses of estrogens can cause apoptosis in long-term estrogen deprived (LTED) breast cancer cells resistant to antiestrogens. This action of estrogen can explain the reduced breast cancer incidence in postmenopausal women over 60 who are taking conjugated equine estrogens and the beneficial effect of low-dose estrogen treatment of patients with acquired aromatase inhibitor resistance in clinical trials. To decipher the molecular mechanism of estrogens at the estrogen receptor (ER) complex by different types of estrogens—planar [17β-estradiol (E2)] and angular triphenylethylene (TPE) derivatives—we have synthesized a small series of compounds with either no substitutions on the TPE phenyl ring containing the antiestrogenic side chain of endoxifen or a free hydroxyl. In the first week of treatment with E2 the LTED cells undergo apoptosis completely. By contrast, the test TPE derivatives act as antiestrogens with a free para-hydroxyl on the phenyl ring that contains an antiestrogenic side chain in endoxifen. This inhibits early E2-induced apoptosis if a free hydroxyl is present. No substitution at the site occupied by the antiestrogenic side chain of endoxifen results in early apoptosis similar to planar E2. The TPE compounds recruit coregulators to the ER differentially and predictably, leading to delayed apoptosis in these cells.SIGNIFICANCE STATEMENTIn this paper we investigate the role of the structure-function relationship of a panel of synthetic triphenylethylene (TPE) derivatives and a novel mechanism of estrogen-induced cell death in breast cancer, which is now clinically relevant. Our study indicates that these TPE derivatives, depending on the positioning of the hydroxyl groups, induce various conformations of the estrogen receptor’s ligand-binding domain, which in turn produces differential recruitment of coregulators and subsequently different apoptotic effects on the antiestrogen-resistant breast cancer cells.

Inhibition of histone methyltransferase DOT1L silences ERα gene and blocks proliferation of antiestrogen-resistant breast cancer cells.

Breast cancer (BC) resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor α (ERα) signaling, and ways to block ERα pathway in these tumors are sought after. We identified the H3K79 methyltransferase DOT1L as a novel cofactor of ERα in BC cell chromatin, where the two proteins colocalize to regulate estrogen target gene transcription. DOT1L blockade reduces proliferation of hormone-responsive BC cells in vivo and in vitro, consequent to cell cycle arrest and apoptotic cell death, with widespread effects on ER-dependent gene transcription, including ERα and FOXA1 gene silencing. Antiestrogen-resistant BC cells respond to DOT1L inhibition also in mouse xenografts, with reduction in ERα levels, H3K79 methylation, and tumor growth. These results indicate that DOT1L is an exploitable epigenetic target for treatment of endocrine therapy-resistant ERα-positive BCs.

A suppressive role of guanine nucleotide-binding protein subunit beta-4 inhibited by DNA methylation in the growth of anti-estrogen resistant breast cancer cells

BackgroundBreast cancer is the most common malignancy in women worldwide. Although the endocrine therapy that targets estrogen receptor α (ERα) signaling has been well established as an effective adjuvant treatment for patients with ERα-positive breast cancers, long-term exposure may eventually lead to the development of acquired resistance to the anti-estrogen drugs, such as fulvestrant and tamoxifen. A better understanding of the mechanisms underlying antiestrogen resistance and identification of the key molecules involved may help in overcoming antiestrogen resistance in breast cancer.MethodsThe whole-genome gene expression and DNA methylation profilings were performed using fulvestrant-resistant cell line 182R-6 and tamoxifen-resistant cell line TAMR-1 as a model system. In addition, qRT-PCR and Western blot analysis were performed to determine the levels of mRNA and protein molecules. MTT, apoptosis and cell cycle analyses were performed to examine the effect of either guanine nucleotide-binding protein beta-4 (GNB4) overexpression or knockdown on cell proliferation, apoptosis and cell cycle.ResultsAmong 9 candidate genes, GNB4 was identified and validated by qRT-PCR as a potential target silenced by DNA methylation via DNA methyltransferase 3B (DNMT3B). We generated stable 182R-6 and TAMR-1 cell lines that are constantly expressing GNB4 and determined the effect of the ectopic GNB4 on cell proliferation, cell cycle, and apoptosis of the antiestrogen-resistant cells in response to either fulvestrant or tamoxifen. Ectopic expression of GNB4 in two antiestrogen resistant cell lines significantly promoted cell growth and shortened cell cycle in the presence of either fulvestrant or tamoxifen. The ectopic GNB4 induced apoptosis in 182R-6 cells, whereas it inhibited apoptosis in TAMR-1 cells. Many regulators controlling cell cycle and apoptosis were aberrantly expressed in two resistant cell lines in response to the enforced GNB4 expression, which may contribute to GNB4-mediated biologic and/or pathologic processes. Furthermore, knockdown of GNB4 decreased growth of both antiestrogen resistant and sensitive breast cancer cells.ConclusionGNB4 is important for growth of breast cancer cells and a potential target for treatment.

Abstract 3318: A critical role for c-Jun N-terminal kinase in autophagy and cell survival of breast cancer cells

Antiestrogen resistance is the major impediment to the eradication of estrogen receptor positive (ER+) breast cancer. Approximately 30% of ER+ breast tumors initially responsive to antiestrogen therapy will acquire resistance. One approach to reducing the occurrence of acquired antiestrogen resistance is to identify and target key signaling nodes that specifically attenuate the ability of antiestrogens to kill cancer cells. Toward this goal, we identified MEK/MAPK1/2 as a key molecular target based on the general inability of antiestrogens to effectively block MEK1/MAPK1/2-mediated phosphorylation of BimEL in breast cancer cells. BimEL is a pro-apoptotic member of the BH3 family of proteins that is inactivated (degraded by the proteasome) when phosphorylated by MAPK1/2. The combination of an antiestrogen and MEK1 inhibitor robustly-induced BimEL-dependent breast cancer cell apoptosis (Periyasamy-Thandavan et al., 2012). However, a subpopulation of breast cancer cells survive this combined treatment via an autophagy-dependent mechanism. Based on the key role of JNK in regulating autophagy in cancer cells via BimEL phosphorylation, we hypothesized that JNK was a key effector of autophagy in breast cancer cells surviving antiestrogen treatment when used as a single agent or in combination with a MEK inhibitor. To test this hypothesis, we utilized the selective JNK inhibitor SP600125 as a single agent or in combination with estradiol (E2), 4-hydroxytamoxifen (4-OHT), and/or U0126 (a selective MEK1 inhibitor). Treatments were conducted with the antiestrogen-sensitive cell lines MCF-7 and T-47D and the antiestrogen resistant cell line TR5, previously derived in our laboratory by a step-wise 4-OHT selection. Effects on cell death (determination of apoptosis) and autophagy (determination of autophagy levels and flux) were evaluated for the various hormonal treatments conducted in the presence or absence of JNK inhibition. These studies showed that: (1) targeting JNK in antiestrogen sensitive breast cancer cells induces apoptosis with caspase-dependent cleavage of PARP as a surrogate marker of apoptosis; (2) JNK activity is elevated in antiestrogen resistant TR5 cells and JNK inhibition induces TR5 cell death; and (3) antiestrogen sensitive and resistant breast cancer cells dying as a result of JNK inhibition can show extensive cytosolic vacuolization with the autophagy protein LC3 (ATG8) localized to the aberrant vacuoles. Overall, our results support the conclusion that JNK plays a key autophagy-dependent survival role in breast cancer cells. Ongoing studies aim to identify the specific JNK protein(s) that disrupt autophagy and enhance death of breast cancer cells toward the goal of identifying specific molecular targets to block autophagy in breast cancer cells. Citation Format: Rohit Munagala, Carol Joseph, Annie Liu, Kebin Liu, Muthusamy Thangaraju, Patricia V. Schoenlein. A critical role for c-Jun N-terminal kinase in autophagy and cell survival of 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 3318. doi:10.1158/1538-7445.AM2017-3318

Abstract 1005: Dual targeting of MEK/MAPK1/2 and pro-survival autophagy to optimize antiestrogen treatment toward the eradication of antiestrogen resistant breast cancer

Abstract In past studies, we showed that pro-survival autophagy mediates resistance to antiestrogen-induced apoptosis and facilitates the emergence of antiestrogen resistant breast cancer cells (Sammadar et al., Molecular Cancer Ther. 9, 2008). We also identified the pro-apoptotic BimEL protein as a key death effector of antiestrogen treatment and determined that BimEL pro-apoptotic activity is abrogated by a MEK1/MAPK1/2-dependent phosphorylation (Periyasamy-Thandavan et al., Breast Cancer Res. 14, 2012). In this study, we tested the hypothesis that the MEK1/MAPK1/2/BimEL signaling node simultaneously regulates antiestrogen- induced pro-survival autophagy and apoptosis in ER+ breast cancer cells. In our approach, we utilized T47-D and MCF-7 breast cancer cells and modulated the activity of MEK1/MAPK1/2/ BimEL expression with U0126, a small molecule inhibitor of MEK1, BimEL cDNA over-expression, or siRNA targeting under a variety of endocrine treatments. We also conducted studies in the presence or absence of the pan-caspase inhibitor ZVAD-fmk so that autophagy levels/function could be analyzed independent of effects mediated by BimEL-dependent caspase activation. Autophagic flux was measured by comparing the levels of lipidated LC3II in cell populations undergoing the different treatments in the presence or absence of chloroquine (CQ), a lysosomotropic agent that prevents autolysosomal turnover. These studies determined that: 1) dephosphorylated BimEL significantly attenuates autophagic flux, but primarily as a consequence of BimEL-induced caspase-activation; (2) BimEL-dependent apoptotic death induced in antiestrogen-treated ER+ breast cancer cells by MEK1 inhibition is not dependent on autophagy; and (3) agents that selectively target autophagy (i.e. CQ and spautin) enhance apoptosis induced by MEK1 blockade in breast cancer cells undergoing endocrine treatment. Based on these pre-clinical in vitro studies, we postulate that the eradication of antiestrogen resistant breast cancer will require the targeting of MEK1/MAPK1/2 to activate BimEL and pro-survival autophagy to eradicate cells surviving BimEL-dependent apoptosis. Citation Format: Mathew Manning, Suchreet Takhar, Sudharsan Periyasamy-Thandavan, Michael Cheng, Thomas Barrett, William Hill, Darren Browning, Muthusamy Thangaraju, Meghan McGee-Lawrence, Patricia V. Schoenlein. Dual targeting of MEK/MAPK1/2 and pro-survival autophagy to optimize antiestrogen treatment toward the eradication of antiestrogen resistant breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1005. doi:10.1158/1538-7445.AM2015-1005

Aurora kinase B is important for antiestrogen resistant cell growth and a potential biomarker for tamoxifen resistant breast cancer.

BackgroundResistance to antiestrogen therapy is a major clinical challenge in the treatment of estrogen receptor α (ER)-positive breast cancer. The aim of the study was to explore the growth promoting pathways of antiestrogen resistant breast cancer cells to identify biomarkers and novel treatment targets.MethodsAntiestrogen sensitive and resistant T47D breast cancer cell lines were used as model systems. Parental and fulvestrant resistant cell lines were subjected to a kinase inhibitor library. Kinase inhibitors preferentially targeting growth of fulvestrant resistant cells were identified and the growth inhibitory effect verified by dose–response cell growth experiments. Protein expression and phosphorylation were investigated by western blot analysis. Cell cycle phase distribution and cell death were analyzed by flow cytometry. To evaluate Aurora kinase B as a biomarker for endocrine resistance, immunohistochemistry was performed on archival primary tumor tissue from breast cancer patients who have received adjuvant endocrine treatment with tamoxifen.ResultsThe selective Aurora kinase B inhibitor barasertib was identified to preferentially inhibit growth of fulvestrant resistant T47D breast cancer cell lines. Compared with parental cells, phosphorylation of Aurora kinase B was higher in the fulvestrant resistant T47D cells. Barasertib induced degradation of Aurora kinase B, caused mitotic errors, and induced apoptotic cell death as measured by accumulation of SubG1 cells and PARP cleavage in the fulvestrant resistant cells. Barasertib also exerted preferential growth inhibition of tamoxifen resistant T47D cell lines. Finally, high percentage of Aurora kinase B positive tumor cells was significantly associated with reduced disease-free and overall survival in 261 ER-positive breast cancer patients, who have received tamoxifen as first-line adjuvant endocrine treatment.ConclusionsOur results indicate that Aurora kinase B is a driving factor for growth of antiestrogen resistant T47D breast cancer cell lines, and a biomarker for reduced benefit of tamoxifen treatment. Thus, inhibition of Aurora kinase B, e.g. with the highly selective kinase inhibitor barasertib, could be a candidate new treatment for breast cancer patients with acquired resistance to antiestrogens.

SRC drives growth of antiestrogen resistant breast cancer cell lines and is a marker for reduced benefit of tamoxifen treatment.

The underlying mechanisms leading to antiestrogen resistance in estrogen-receptor α (ER)-positive breast cancer is still poorly understood. The aim of this study was therefore to identify biomarkers and novel treatments for antiestrogen resistant breast cancer. We performed a kinase inhibitor screen on antiestrogen responsive T47D breast cancer cells and T47D-derived tamoxifen and fulvestrant resistant cell lines. We found that dasatinib, a broad-spectrum kinase inhibitor, inhibited growth of the antiestrogen resistant cells compared to parental T47D cells. Furthermore western blot analysis showed increased expression and phosphorylation of Src in the resistant cells and that dasatinib inhibited phosphorylation of Src and also signaling via Akt and Erk in all cell lines. Immunoprecipitation revealed Src: ER complexes only in the parental T47D cells. In fulvestrant resistant cells, Src formed complexes with the Human Epidermal growth factor Receptor (HER)1 and HER2. Neither HER receptors nor ER were co-precipitated with Src in the tamoxifen resistant cell lines. Compared to treatment with dasatinib alone, combined treatment with dasatinib and fulvestrant had a stronger inhibitory effect on tamoxifen resistant cell growth, whereas dasatinib in combination with tamoxifen had no additive inhibitory effect on fulvestrant resistant growth. When performing immunohistochemical staining on 268 primary tumors from breast cancer patients who had received tamoxifen as first line endocrine treatment, we found that membrane expression of Src in the tumor cells was significant associated with reduced disease-free and overall survival. In conclusion, Src was identified as target for treatment of antiestrogen resistant T47D breast cancer cells. For tamoxifen resistant T47D cells, combined treatment with dasatinib and fulvestrant was superior to treatment with dasatinib alone. Src located at the membrane has potential as a new biomarker for reduced benefit of tamoxifen.

Abstract B50: MEK inhibitors mount a two-pronged attack to kill estrogen receptor positive (ER+) breast cancer cells undergoing hormonal therapy: Attenuated autophagy and induction of apoptosis

Abstract In a recent study, we identified the dephosphorylated form of BimEL as a key death effector of antiestrogen treatment of ER+ breast cancer cells and further showed that MEK1/MAPK1/2 blockade was required to produce high levels of dephosphorylated BimEL, particularly under conditions of insulin like growth factor 1 (IGF1) stimulation (Periyasamy-Thandavan et al., Breast Cancer Res. 14, 2012). Studies by others have identified MEK1/MAPK1/2 activation as essential to autophagy, a catabolic process induced by multiple stresses including ROS, ceramide accumulation, and nutrient deprivation. Autophagy induction results in autophagosome formation, trafficking of damaged proteins and mitochondria to the autophagosomes, and ultimately fusion with the lysosomes resulting in autolysosome formation. The autolysosome and its contents are degraded by the hydrolytic enzymes of the lysosome. Of particular interest to antiestrogen treatment of breast cancer, we and others have shown that pro-survival autophagy facilitates the emergence of antiestrogen resistant breast cancer cells. Thus, we are keenly interested in how MEK1/MAPK1/2 signaling affects pro-survival autophagy and if MEK blockade would be an effective approach toward blocking pro-survival autophagy in ER+ breast cancer cells undergoing hormonal treatment. In this study, we hypothesized that the requirement of MEK1/MAPK1/2 for pro-survival autophagy is due, in part, to its role in blocking the intracellular accumulation of dephosphorylated BimEL. To test this hypothesis, we modulated the expression of dephosphorylated BimEL with either a BimEL cDNA expression vector, siRNA targeting of BimEL, or MEK1 blockade with the small molecule inhibitor U0126 and determined the levels of the autophagic flux in ER+ breast cancer cells undergoing antiestrogen treatment. The determination of autophagic flux was made by comparing the levels of two proteins involved in autophagy -the LC3 /Atg8 and p62 (SQSTM1) proteins- in cell populations undergoing the different treatments in the presence or absence of chloroquine (CQ). The lipidated form of LC3, designated LC3II, is typically increased in cells undergoing autophagy, facilitates the formation of the mature autophagosomal membranes, and is subsequently degraded in the autolysosome. The p62 protein is required for the delivery of ubiquitinated protein complexes to the autophagosome and is degraded along with the ubiquitinated complex of proteins. CQ is a lysosomotrophic agent routinely used in autophagic flux assays because it blocks the turnover of autolysosomes with accumulation of LC3 II and p62, allowing the total levels of LC3II and p62 to be ascertained under all treatment conditions. These studies showed that siRNA targeting of BimEL increased basal and tamoxifen-induced autophagy in ER+ MCF-7 breast cancer cells. In contrast, the overexpression of dephosphorylated BimEL led to an increase in LC3 II and p62 levels due to a significant attenuation of autophagic flux (approximately 50%) in antiestrogen-treated cell populations. Current studies are focused on the mechanism of BimEL-mediated blockade of pro-survival autophagy, with the long term goal of optimizing this “downstream effector” function of MEK1/MAPK1/2 blockade in ER+ breast cancer cells for improved therapeutic outcome. Citation Format: S. Takhar, M. Manning, A. Eason, M. Dix, S. Periyasamy-Thandavan, R. Padi, E. Bieberich, W. Hill, D. Browning, V. Ganapathy, M. Thangaraju, P. V. Schoenlein. MEK inhibitors mount a two-pronged attack to kill estrogen receptor positive (ER+) breast cancer cells undergoing hormonal therapy: Attenuated autophagy and induction of apoptosis. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B50. doi: 10.1158/1557-3125.RASONC14-B50

Small-molecule "BRCA1-mimetics" are antagonists of estrogen receptor-α.

Resistance to conventional antiestrogens is a major cause of treatment failure and, ultimately, death in breast cancer. The objective of the study was to identify small-molecule estrogen receptor (ER)-α antagonists that work differently from tamoxifen and other selective estrogen receptor modulators. Based on in silico screening of a pharmacophore database using a computed model of the BRCA1-ER-α complex (with ER-α liganded to 17β-estradiol), we identified a candidate group of small-molecule compounds predicted to bind to a BRCA1-binding interface separate from the ligand-binding pocket and the coactivator binding site of ER-α. Among 40 candidate compounds, six inhibited estradiol-stimulated ER-α activity by at least 50% in breast carcinoma cells, with IC50 values ranging between 3 and 50 μM. These ER-α inhibitory compounds were further studied by molecular and cell biological techniques. The compounds strongly inhibited ER-α activity at concentrations that yielded little or no nonspecific toxicity, but they produced only a modest inhibition of progesterone receptor activity. Importantly, the compounds blocked proliferation and inhibited ER-α activity about equally well in antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. Representative compounds disrupted the interaction of BRCA1 and ER-α in the cultured cells and blocked the interaction of ER-α with the estrogen response element. However, the compounds had no effect on the total cellular ER-α levels. These findings suggest that we have identified a new class of ER-α antagonists that work differently from conventional antiestrogens (eg, tamoxifen and fulvestrant).

A kinase inhibitor screen identifies Mcl-1 and Aurora kinase A as novel treatment targets in antiestrogen-resistant breast cancer cells.

Antiestrogen resistance is a major problem in breast cancer treatment. Therefore, the search for new therapeutic targets and biomarkers for antiestrogen resistance is crucial. In this study, we performed a kinase inhibitor screen on antiestrogen responsive MCF-7 cells and a panel of MCF-7-derived tamoxifen- and fulvestrant-resistant cell lines. Our focus was to identify common and distinct molecular mechanisms involved in tamoxifen- and fulvestrant-resistant cell growth. We identified 18 inhibitors, of which the majority was common for both tamoxifen- and fulvestrant-resistant cell lines. Two compounds, WP1130 and JNJ-7706621, exhibiting prominent preferential growth inhibition of antiestrogen-resistant cell lines, were selected for further studies. WP1130, a deubiquitinase inhibitor, induced caspase-mediated cell death in both tamoxifen- and fulvestrant-resistant cell lines by destabilization of the anti-apoptotic protein Mcl-1. Mcl-1 expression was found upregulated in the antiestrogen-resistant cell lines and depletion of Mcl-1 in resistant cells caused decreased viability. JNJ-7706621, a dual Aurora kinase and cyclin-dependent kinase inhibitor, specifically inhibited growth and caused G2 phase cell cycle arrest of the tamoxifen-resistant cell lines. Knockdown studies showed that Aurora kinase A is essential for growth of the tamoxifen-resistant cells and inhibition of Aurora kinase A resensitized tamoxifen-resistant cells to tamoxifen treatment. Preferential growth inhibition by WP1130 and JNJ-7706621 was also found in T47D-derived tamoxifen-resistant cell lines, pointing at Mcl-1 and Aurora kinase A as potential treatment targets. In addition, tumor samples from 244 estrogen receptor-positive breast cancer patients treated with adjuvant tamoxifen showed that higher expression level of Aurora kinase A was significantly associated with shorter disease-free and overall survival, demonstrating the potential of Aurora kinase A as a biomarker for tamoxifen resistance.

Abstract 679: Glutamine metabolism in MYC-driven antiestrogen resistant breast cancer cells confers metabolic flexibility through the unfolded protein response

Abstract Antiestrogens are used to treat estrogen receptor positive (ER+) breast tumors that constitute 70% of all breast cancer cases. Unfortunately, acquired resistance to antiestrogen therapy remains a critical clinical obstacle. Here we show that human breast cancer cells and rat mammary tumors that have acquired resistance to antiestrogens express increased levels of MYC, a major regulator of both glutamine and glucose. Glutamine metabolism and glucose uptake were elevated in ER+ antiestrogen resistant cells (LCC9) compared with sensitive cells (LCC1). Inhibition of MYC, with siRNA or small molecule inhibitor, reduced cell viability and uptake of both glutamine and glucose in resistant cells. In resistant cells, MYC expression controlled protein levels of glutamine, glutamate and glucose transporters as well as GLUL and GLS, two enzymes that promote glutamate-glutamine inter-conversion. Increased MYC function in resistant cells correlated with increased cellular sensitivity to deprivation of, and also inhibitors of, both glutamine and glucose. While apoptosis eliminated all resistant cells in glucose-only conditions beyond 72 h, in glutamine-only conditions, the unfolded protein response (UPR) via GRP78-IRE1α and activating JNK and increased CHOP, induced apoptosis in majority of the cells but promoted survival in some. The antiestrogen faslodex (FAS; ICI 182,780) significantly reduced glucose uptake in antiestrogen resistant cells compared with sensitive cells. Thus, our findings reveal unique roles for MYC in promoting metabolic flexibility in and promoting survival in antiestrogen resistant breast cancer cells via the UPR. Targeting glutamine and glucose metabolism pathways, therefore, may provide novel strategies in treating endocrine resistant breast cancers. Citation Format: Ayesha N. Shajahan-Haq, Katherine L. Cook, Jessica L. Schwartz-Roberts, Ahreej E. Eltayeb, Diane M. Demas, Anni M. Warri, Leena A. Hilakivi-Clarke, Robert Clarke. Glutamine metabolism in MYC-driven antiestrogen resistant breast cancer cells confers metabolic flexibility through the unfolded protein response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 679. doi:10.1158/1538-7445.AM2014-679

Abstract 2443: Novel role of glucose-regulated protein 78 mediating lipid metabolism through controlling stearoyl-CoA desaturase 1

Abstract Over 230,000 new cases of invasive breast cancer are diagnosed annually within the United States. Of these cases, 70% express the estrogen receptor-α and may be treated with antiestrogens or aromatase inhibitors. While these drugs exhibit clinical benefits, over 50% will develop or not respond to antiestrogen therapy, making understanding the molecular mechanism of antiestrogen resistance critical to preventing and treating endocrine resistant breast cancer. Recently, we have determined a key role of the unfolded protein response endoplasmic reticulum (EnR) protein chaperone, glucose regulated protein 78 (GRP78), in mediating antiestrogen resistance. A sole designation of GRP78 as a protein chaperone controlling UPR signaling is limiting because GRP78 is multifunctional. We now expand the role of GRP78 to include its novel actions regulating lipid metabolism. Metabolic analysis of antiestrogen sensitive LCC1 and antiestrogen resistant LCC9 breast cancer cells indicate that inhibition of GRP78 through RNAi results in an increase in the intracellular concentrations of the polyunsaturated fats (PUFA) linoleate (18:2n6), linolenate [alpha or gamma; (18:3n3 or 6)], dihomo-linolenate (20:3n3 or n6), and dihomo-linoleate (20:2n6). GRP78 knockdown results in an increase in delta-6 desaturase in the LCC9 cells corresponding with the observed increase in PUFA. Moreover, knockdown of GRP78 decreased stearoyl-CoA desaturase 1 (SCD1) and fatty acid synthase (FASN) protein levels, further supporting a role for GRP78 in lipid metabolism. Treatment of breast cancer cells with increasing doses of PUFA linoleic acid results in the inhibition of SCD1, suggesting that GRP78 regulation of PUFA metabolism mediates GRP78 actions on SCD1 and lipid biogenesis. Antiestrogen resistant cells express higher levels of SCD1. Moreover, elevated SCD1 is associated with breast cancer reoccurrence and reduced survival in breast cancer patient-derived data sets, suggesting a critical role of SCD1 in mediating antiestrogen resistance. LCC9 orthotopic tumors treated with GRP78 targeting morpholino were re-sensitized to tamoxifen. Targeting GRP78 in vivo resulted in reduced SCD1 protein in GRP78 morpholino+tamoxifen treated tumors, demonstrating a key role of GRP78/SCD1 interaction in drug resistance. Inhibition of GRP78 also increased short-chained acyl-CoA dehydrogenase (ACADS) protein expression and decreased the fatty acid metabolite butyrylcarnitine, indicating a stimulation of beta-oxidation. These data suggest that the effect of GRP78 knockdown on fatty acid metabolism and biogenesis may represent a new homeostatic ability of GRP78 to modify fatty acid metabolism. The consequences of these activities may help reduce EnR stress by providing an additional energy source for protein folding/refolding and so help alleviate the accumulation of unfolded/misfolded proteins accumulation within the EnR. Citation Format: Katherine L. Cook, Pamela A.G. Clarke, Margaret Axelrod, Anni Wärri, Robert Clarke. Novel role of glucose-regulated protein 78 mediating lipid metabolism through controlling stearoyl-CoA desaturase 1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2443. doi:10.1158/1538-7445.AM2014-2443

Glutamine metabolism and the unfolded protein response in MYC-driven breast cancer

Antiestrogens are used to treat estrogen receptor positive (ER+) breast tumors that constitute 70% of all breast cancer cases. Unfortunately, acquired resistance to antiestrogen therapy remains a critical clinical obstacle. Here we show that human breast cancer cells and rat mammary tumors that have acquired resistance to antiestrogens express increased levels of MYC, a major regulator of both glutamine and glucose. Glutamine metabolism and glucose uptake were elevated in ER+ antiestrogen resistant cells (LCC9) compared with sensitive cells (LCC1). Inhibition of MYC, with siRNA or small molecule inhibitor, reduced cell viability and uptake of both glutamine and glucose in resistant cells. In resistant cells, MYC expression controlled protein levels of glutamine, glutamate and glucose transporters as well as GLUL and GLS, two enzymes that promote glutamate-glutamine inter-conversion. Increased MYC function in resistant cells correlated with increased cellular sensitivity to deprivation of, and also inhibitors of, both glutamine and glucose. While apoptosis eliminated all resistant cells in glucose-only conditions beyond 72 h, in glutamine-only conditions, the unfolded protein response (UPR) via GRP78-IRE1a and activating JNK and increased CHOP, induced apoptosis in majority of the cells but promoted survival in some. The antiestrogen faslodex (FAS; ICI 182,780) significantly reduced glucose uptake in antiestrogen resistant cells compared with sensitive cells. Thus, our findings reveal unique roles for MYC in promoting metabolic flexibility in and promoting survival in antiestrogen resistant breast cancer cells via the UPR. Targeting glutamine and glucose metabolism pathways, therefore, may provide novel strategies in treating endocrine resistant breast cancers.

LRH-1 Governs Vital Transcriptional Programs in Endocrine-Sensitive and -Resistant Breast Cancer Cells

Tumor characteristics are decisive in the determination of treatment strategy for patients with breast cancer. Patients with estrogen receptor α (ERα)-positive breast cancer can benefit from long-term hormonal treatment. Nonetheless, the majority of patients will develop resistance to these therapies. Here, we investigated the role of the nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) in antiestrogen-sensitive and -resistant breast cancer cells. We identified genome-wide LRH-1-binding sites using ChIP-seq (chromatin immunoprecipitation sequencing), uncovering preferential binding to regions distal to transcriptional start sites. We further characterized these LRH-1-binding sites by integrating overlapping layers of specific chromatin marks, revealing that many LRH-1-binding sites are active and could be involved in long-range enhancer-promoter looping. Combined with transcriptome analysis of LRH-1-depleted cells, these results show that LRH-1 regulates specific subsets of genes involved in cell proliferation in antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. Furthermore, the LRH-1 transcriptional program is highly associated with a signature of poor outcome and high-grade breast cancer tumors in vivo. Herein, we report the genome-wide location and molecular function of LRH-1 in breast cancer cells and reveal its therapeutic potential for the treatment of breast cancers, notably for tumors resistant to treatments currently used in therapies.

5-Aza-2-deoxycytidine and trichostatin A increase COUP-TFII expression in antiestrogen-resistant breast cancer cell lines

COUP-TFII is reduced in endocrine-resistant breast cancer cells and is negatively associated with tumor grade. Transient re-expression of COUP-TFII restores antiestrogen sensitivity in resistant LCC2 and LCC9 cells and repression of COUP-TFII results in antiestrogen-resistance in MCF-7 endocrine-sensitive cells. We addressed the hypothesis that reduced COUP-TFII expression in endocrine-resistant breast cancer cells results from epigenetic modification. The NR2F2 gene encoding COUP-TFII includes seven CpG islands, including one in the 5′ promoter and one in exon 1. Treatment of LCC2 and LCC9 endocrine-resistant breast cancer cells with 5-aza-2′-deoxycytidine (AZA), a DNA methyltransferase (DNMT) inhibitor, +/− trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, increased COUP-TFII suggesting that the decrease in COUP-TFII is mediated by epigenetic changes. Methylation-specific PCR (MSP) revealed higher methylation of NR2F2 in the first exon in LCC2 and LCC9 cells compared to MCF-7 cells and AZA reduced this methylation. Translational importance is suggested by Cancer Methylome System (CMS) analysis revealing that breast tumors have increased COUP-TFII (NR2F2) promoter and gene methylation versus normal breast.

Abstract 5369: Methylation profile of markers for epithelial-mesenchymal transition and stem cell in tamoxifen-resistant MCF-7 breast cancer cell line.

Abstract DNA methylation plays a key role in the epigenetic-based regulation of gene expression. Several genes that showed increased hypomethylations of gene promotors in antiestrogen-resistant breast cancer cells were found to be increased in tumor cells and possess oncogenic activity. The purpose of this study is to investigate the differences of methylation profiles, especially EMT markers, between tamoxifen-sensitive breast cancer cell lines and tamoxifen-resistant cell lines using MCF7 and tamoxifen-resistant MCF7 (MCF7-R) cell lines, and then to indentify the presence of the identified markers in the breast tissues of recurred patients with antiestrogen treatments. Bisulphite-converted genomic DNA was assayed on Infinium Human Methylation 450K Beadchips using the Illumina Infinium HD methylation assay kit using four groups of the cells including MCF7, MCF7-R, MCF+E2 and MCF7-R+E2. . In MCF7-R cells, 31023 CpG sites showed altered methylations, with 13617 hypomethylated and 17406 hypermethylated, compared with MCF7. The numbers of hypomethylated and hypermethylated CpG sites in the promoter regions were 4719 and 3347, respectively. Among the well-known EMT markers, the genes meeting the results of methylation status selected were CTNNB1, SNAIL2, TWIST1, LEF1, GSC and ETS1. Although the results of the current study suggest that aberrant DNA methylations of EMT markers are not the main force driving the molecular biology of resistance against antiestrogen therapy, these will contribute and result in to some extent. Citation Format: Youngseok Lee, Aeree Kim, Han-Kyeom Kim. Methylation profile of markers for epithelial-mesenchymal transition and stem cell in tamoxifen-resistant MCF-7 breast cancer cell line. [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 5369. doi:10.1158/1538-7445.AM2013-5369

Abstract 1677: Inhibition of autophagy proteins ATG7 and BECN1 correlates with increased interferon regulatory factor-1 (IRF1) protein expression through STAT1 phosphorylation.

Abstract Approximately two-thirds of newly diagnosed invasive breast tumors express the estrogen receptor-α protein (ER+) and most will be treated with an endocrine therapy such as an antiestrogen or aromatase inhibitor. Despite the widespread clinical efficacy of antiestrogens in the treatment of ER+ breast cancers, approximately half of these women will exhibit de novo or acquired resistance to endocrine therapies. One mechanism by which breast cancer cells evade antiestrogen-induced cell death is through stimulation of autophagy. Autophagy is a catabolic process in which cells digest subcellular organelles in an attempt to maintain homeostasis. Evidence for increased autophagy has been shown to protect cancer cells against various therapies, including radiotherapy and the antiestrogen, tamoxifen. Previous studies from our lab have also established interferon regulatory factor-1 (IRF1) as a tumor suppressor gene and mediator of antiestrogen sensitivity. High mRNA levels of IRF1 in breast cancer core biopsies correlated significantly with a pathologic complete response after neoadjuvant systemic treatment. However, the link between autophagy and this immune function gene is largely unknown. Here, we show that reduced expression of autophagy-related protein, ATG7, correlates with increased nuclear IRF1 protein in 107 human breast cancer core biopsies. Knockout of a single ATG7 allele in mice induced IRF1 activation in both mammary and spleen tissue compared to wild-type mice. In antiestrogen sensitive and resistant breast cancer cells, knockdown of ATG7 and BECN1, but not ATG5, using siRNA resulted in increased IRF1 protein expression. We go on to show that increased IRF1 expression is an effect of increased phosphorylation of STAT1, suggesting that autophagy inhibits the interferon signaling pathway. Using the contrary approach, we found that inhibition of IRF1 using siRNA inhibits the lipidation of LC3 and autophagosome formation. Furthermore, antiestrogen sensitive LCC1 cells become resistant to the growth inhibitory effects of faslodex (Fulvestrant; ICI182780;ICI), 3-methyladenine, and doxorubicin following knockdown of IRF1. Overexpression of IRF1 in antiestrogen resistant LCC9 cells inhibits ATG7 protein expression and induces mitochondrial outer membrane permeability. Thus, the disruption of autophagy machinery in breast cancer cells activates the interferon signaling pathway to induce apoptosis and breast cancer cell death. Citation Format: Jessica L. Schwartz-Roberts, Katherine L. Cook, Ayesha N. Shajahan, Margaret Axelrod, Anni Warri, Robert Clarke. Inhibition of autophagy proteins ATG7 and BECN1 correlates with increased interferon regulatory factor-1 (IRF1) protein expression through STAT1 phosphorylation. [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 1677. doi:10.1158/1538-7445.AM2013-1677

GX15-070 (Obatoclax) Induces Apoptosis and Inhibits Cathepsin D- and L–Mediated Autophagosomal Lysis in Antiestrogen-Resistant Breast Cancer Cells

In estrogen receptor-positive (ER+) breast cancer cells, BCL2 overexpression contributes to antiestrogen resistance. Direct targeting of the antiapoptotic BCL2 members with GX15-070 (obatoclax), a BH3-mimetic currently in clinical development, is an attractive strategy to overcome antiestrogen resistance in some breast cancers. Recently, GX15-070 has been shown to induce both apoptosis and autophagy, yet the underlying cell death mechanisms have yet to be elucidated. Here, we show that GX15-070 is more effective in reducing the cell density of antiestrogen-resistant breast cancer cells versus sensitive cells and that this increased sensitivity of resistant cells to GX15-070 correlates with an accumulation of autophagic vacuoles. Formation of autophagosomes in GX15-070-treated cells was verified by changes in expression of the lipidation of microtubule-associated protein-1 light chain-3 and both confocal and transmission electron microscopy. While GX15-070 treatment promotes autophagic vacuole and autolysosome formation, p62/SQSTM1, a marker for autophagic degradation, levels accumulate. Moreover, GX15-070 exposure leads to a reduction in cathepsin D (CTSD) and L (CTSL1) protein expression that would otherwise digest autolysosome cargo. Thus, GX15-070 has dual roles in promoting cell death: (i) directly inhibiting antiapoptotic BCL2 family members, thereby inducing apoptosis; and (ii) inhibiting downstream CTSD and CTSL1 protein expression to limit the ability of cells to use degraded material to fuel cellular metabolism and restore homeostasis. Our data highlight a new mechanism of GX15-070-induced cell death that could be used to design novel therapeutic interventions for antiestrogen resistant breast cancer.