Endocrine-resistant Breast Cancer Cells Research Articles

Abstract B1-23: Early growth response (EGR1) is a critical regulator of cellular metabolism and predicts increased responsiveness to antiestrogens in breast cancer

Abstract Breast cancer is the most commonly diagnosed cancer in women and about 1 million new cases per year are diagnosed worldwide. About 70% of all breast cancers are estrogen receptor alpha positive (ER+). Antiestrogens (e.g., Tamoxifen or Faslodex) or aromatase inhibitors (e.g., Letrozole) are often used to treat ER+ breast cancers. However, resistance to these therapies (endocrine resistance) is prevalent in the clinic and the underlying mechanisms remain unclear. We have recently shown that the oncogene MYC is overexpressed in ER+ breast cancer and up-regulates glucose and glutamine uptake in endocrine resistant breast cancer cells, which suggests that the metabolomic profile of endocrine resistant breast cancer cells may contain features that are distinct from sensitive cells. In this study, to identify the biochemical pathways that are differentially regulated in endocrine resistance in breast cancer cells, we have analyzed gene expression data and untargeted metabolite profiles of ER+ MCF7-derived breast cancer cells that are antiestrogen sensitive (LCC1) or antiestrogen resistant (LCC9) under basal conditions. Glycolysis and glutamine-dependent pathways were increased in endocrine resistant cells. Integration of the transcriptomics and metabolomics data predicted an essential role for a gene-metabolite network associated with early growth response (EGR1) and glutamine metabolism in endocrine resistant cells. EGR1 is an immediate-early gene induced by E2, growth factors, or stress signals, and has been reported to exhibit both tumor suppressor and promoter activities, based on cellular context. While EGR1 mediated signaling is important for the normal development of female reproductive organs, its precise role in breast cancer remains unknown. EGR1 gene expression and protein levels were significantly higher in LCC1 cells compared with LCC9 cells. Kaplan-Meier survival curves with gene expression data obtained from ER+ human breast tumors treated with endocrine therapy show that higher EGR1 expression is associated with a more favorable prognosis: GSE17705 [HR=0.38 (0.21-0.69); p=0.00083], GSE6532 (ER+ samples on GPL96 platform) [HR=0.55 (0.34-0.9); p=0.017]. In GSE20181, pre-treatment vs 90 days post-treatment comparisons show significantly increased levels of EGR1 expression (p<0.0001) only in the responder group. Interestingly, in both LCC1 and LCC9 cells, EGR1 overexpression increased ER protein levels and cell proliferation while EGR1 knockdown decreased ER protein levels and cell proliferation. Therefore, to understand the precise role of down-regulated EGR1 in regulating cellular metabolism and survival in endocrine resistance, we compared metabolite profiles in LCC9 cells following knockdown or overexpression of EGR1. Several major biochemical pathways such as glycolysis, lipid metabolism, glutathione, and polyamine metabolism were shown to be regulated by EGR1 in LCC9 cells. Collectively, these findings indicate that down-regulated EGR1 is an important regulator of the aberrant cellular metabolic pathways specific to endocrine resistance. Furthermore, high levels of EGR1 may serve as a favorable prognostic marker in endocrine treatment strategies in breast cancer. Citation Format: Ayesha N. Shajahan-Haq, Lu Jin, Amrita K. Cheema, Simina M. Boca, Yuriy Gusev, Krithika Bhuvaneshwar, Diane M. Demas, Habtom Ressom, Ryan Michalek, Xi Chen, Jianhua Xuan, Subha Madhavan, Robert Clarke. Early growth response (EGR1) is a critical regulator of cellular metabolism and predicts increased responsiveness to antiestrogens in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-23.

Blockade of voltage-gated sodium channels inhibits invasion of endocrine-resistant breast cancer cells.

Voltage-gated Na+ channels (VGSCs) are membrane proteins which are normally expressed in excitable cells but have also been detected in cancer cells, where they are thought to be involved in malignancy progression. In this study we examined the ion current and expression profile of VGSC (Nav1.5) in estrogen receptor (ER)-positive (MCF-7) and silenced (pII) breast cancer cells and its possible influence on their proliferation, motility and invasion. VGSC currents were analysed by whole cell patch clamp recording. Nav1.5 expression and localization, in response to EGF stimulation, was examined by western blotting and immunofluorescence respectively. Cell invasion (under-agarose and Matrigel assays), motility (wound healing assay) and proliferation (MTT assay) were assessed in pII cells in response to VGSC blockers, phenytoin (PHT) and tetrodotoxin (TTX), or by siRNA knockdown of Nav1.5. The effect of PHT and TTX on modulating EGF-induced phosphorylation of Akt and ERK1/2 was determined by western blotting. Total matrix metalloproteinase (MMP) was determined using a fluorometric-based activity assay. The level of various human proteases was detected by using proteome profiler array kit. VGSC currents were detected in pII cells, but were absent in MCF-7. Nav1.5 showed cytoplasmic and perinuclear expression in both MCF-7 and pII cells, with enhanced expression upon EGF stimulation. Treatment of pII cells with PHT, TTX or siRNA significantly reduced invasion towards serum components and EGF, in part through reduction of P-ERK1/2 and proteases such as cathepsin E, kallikrein-10 and MMP-7, as well as total MMP activity. At high concentrations, PHT inhibited motility while TTX reduced cell proliferation. Pharmacological or genetic blockade of Nav1.5 may serve as a potential anti-metastatic therapy for breast cancer.

Investigating the functional role of the oestrogen receptor in LY2 endocrine resistant breast cancer cells

Background The issue of acquired resistance to breast cancer regimes such as tamoxifen continues to negatively affect clinical outcomes. While many mechanisms of resistance have been discovered [1,2], there is evidence now of acquired resistance through adaptation of the oestrogen receptor itself leading to tumour progression [3]. A thorough understanding of the processes involved in the receptor's adaptation remains unclear. This study gives evidence of the gene signalling which shed light on the mechanism of adaptation in an LY2 endocrine resistant cell line oestrogen receptor knockdown model.

Breast Cancer Anti-Estrogen Resistance 4 (BCAR4) Drives Proliferation of IPH-926 lobular Carcinoma Cells.

BackgroundMost breast cancers depend on estrogenic growth stimulation. Functional genetic screenings in in vitro cell models have identified genes, which override growth suppression induced by anti-estrogenic drugs like tamoxifen. Using that approach, we have previously identified Breast Cancer Anti-Estrogen Resistance 4 (BCAR4) as a mediator of cell proliferation and tamoxifen-resistance. Here, we show high level of expression and function of BCAR4 in human breast cancer.Methods BCAR4 mRNA expression was evaluated by (q)RT-PCR in a panel of human normal tissues, primary breast cancers and cell lines. A new antibody raised against C78-I97 of the putative BCAR4 protein and used for western blot and immunoprecipitation assays. Furthermore, siRNA-mediated gene silencing was implemented to study the function of BCAR4 and its downstream targets ERBB2/3.ResultsExcept for placenta, all human normal tissues tested were BCAR4-negative. In primary breast cancers, BCAR4 expression was comparatively rare (10%), but associated with enhanced proliferation. Relative high BCAR4 mRNA expression was identified in IPH-926, a cell line derived from an endocrine-resistant lobular breast cancer. Moderate BCAR4 expression was evident in MDA-MB-134 and MDA-MB-453 breast cancer cells. BCAR4 protein was detected in breast cancer cells with ectopic (ZR-75-1-BCAR4) and endogenous (IPH-926, MDA-MB-453) BCAR4 mRNA expression. Knockdown of BCAR4 inhibited cell proliferation. A similar effect was observed upon knockdown of ERBB2/3 and exposure to lapatinib, implying that BCAR4 acts in an ERBB2/3-dependent manner.Conclusion BCAR4 encodes a functional protein, which drives proliferation of endocrine-resistant breast cancer cells. Lapatinib, a clinically approved EGFR/ERBB2 inhibitor, counteracts BCAR4-driven tumor cell growth, a clinical relevant observation.

Abstract 1097: Knockdown of splicing factor ESRP1 affects multiple splicing factors

Abstract Introduction: Tissue-specific alternative splicing (AS) is an important mechanism for regulating gene expression. Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2), two RNA-binding proteins (RBPs) that promote splicing, are potential candidates that contribute to breast cancer recurrence and resistance to therapies. In our prior studies, we have shown that ESRPs are associated with endocrine resistance. In this study, we seek to investigate the impact of the ESRP1 knockdown in endocrine resistant breast cancer cells. Methods: Expression of ESRP1 was analyzed in endocrine resistant-LCC2 and LCC9 cells. Knockdown of ESRP1 expression in LCC2 and LCC9 cells was achieved using lenti-viral based shRNA approach (Mission TRC human shRNA constructs, Sigma). We next performed paired-end RNA-sequencing of LCC2 and LCC9 cells transfected with control and two clones representative of shRNA knockdown (Illumina HiSeq platform, SeqWright Genomic Services) according to the manufacturer's instructions. After processing and quality check of raw FASTQ data (Illumina CASAVA, FASTQ and FASTX toolkits), reads were aligned, and quantified using bioinformatics software tools (Tophat/Cufflinks and Sailfish). Results: Expression of ESRP1 is significantly elevated in ERα-positive endocrine -resistant cells (LCC2 and LCC9 cells). ESRP1 knockdown was confirmed using qRT-PCR analysis in LCC2 cell lines (clone1 - 80% reduction and clone 3- 95% reduction) and in LCC9 cells (clone 2- 90% reduction and clone 3- 50% reduction). RNA-seq analysis identified 5153 differentially regulated genes between ESRP1 knockdown and control LCC2 cell lines. Of the 5153 genes, 1117 and 2109 genes were unique to clone 1 and clone 3, respectively, while 1927 genes were common in both clones. Targeted RNA-seq analysis also revealed that ESRP1 regulates the expression of several genes including other splicing factors. Furthermore, a functional enrichment analysis of these genes revealed a possible role of altering important pathways such as cell-cell adhesion and cell signaling. Additionally, it was observed that neither ESR1 nor any of its downstream genes were affected by the knockdown of ESRP1. This data was similar to that obtained in knockdown of LCC9 cells. Conclusions: ESRP1 regulates splicing of multiple genes in ER+ breast cancer. The process is independent of ER pathway. Targeting splicing could be a novel modality for treating endocrine therapy resistant breast cancer. Citation Format: Yesim Gökmen-Polar, Yaseswini Neelamraju, Xiaoping Gu, Gouthami Nallamothu, Sarath C. Janga, Sunil Badve. Knockdown of splicing factor ESRP1 affects multiple splicing factors. [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 1097. doi:10.1158/1538-7445.AM2015-1097

DMXL2 drives epithelial to mesenchymal transition in hormonal therapy resistant breast cancer through Notch hyper-activation.

The acquisition of endocrine therapy resistance in estrogen receptor α (ERα) breast cancer patients represents a major clinical problem. Notch signalling has been extensively linked to breast cancer especially in patients who fail to respond to endocrine therapy. Following activation, Notch intracellular domain is released and enters the nucleus where activates transcription of target genes. The numerous steps that cascade after activation of the receptor complicate using Notch as biomarker. Hence, this warrants the development of reliable indicators of Notch activity. DMXL2 is a novel regulator of Notch signalling not yet investigated in breast cancer. Here, we demonstrate that DMXL2 is overexpressed in a subset of endocrine therapy resistant breast cancer cell lines where it promotes epithelial to mesenchymal transition through hyper-activation of Notch signalling via V-ATPase dependent acidification. Following DMXL2 depletion or treatment with Bafilomycin A1, both EMT targets and Notch signalling pathway significantly decrease. We show for the first time that DMXL2 protein levels are significantly increased in ERα positive breast cancer patients that progress after endocrine therapy. Finally, we demonstrate that DMXL2 is a transmembrane protein with a potential extra-cellular domain. These findings identify DMXL2 as a novel, functional biomarker for ERα positive breast cancer.

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.

Abstract P4-06-02: Insulin receptor targeting in breast cancer through yeast surface display

Abstract Selective estrogen receptor modulators (SERMs) such as tamoxifen have been a conventional therapy for breast cancer patients expressing estrogen receptor, representing the most common breast cancer type. The development of secondary resistance to SERM is an unsolved clinical dilemma, leading to the exploration of more targeted therapies. The insulin-like growth factor (IGF) system is well documented and is implicated as a contributor to endocrine resistance. Unfortunately, antibodies directed against type I IGF receptor (IGF1R) failed to demonstrate efficacy in endocrine resistant breast cancer. We have previously shown that tamoxifen resistant (TamR) breast cancer cells lose IGF1R yet retain expression of insulin receptor (InsR), a closely related receptor to IGF1R. Thus, we hypothesized that InsR may serve as a compensatory pathway to the loss of IGF1R in TamR cells and InsR may be a target in the therapy of endocrine resistant breast cancer. Indeed, our preliminary results show that TamR breast cancer cells were more sensitive to insulin stimulation. As compared to the parental cells, TamR cells showed stronger PI3K/AKT and MAPK/ERK activation, greater MTT proliferation growth and anchorage-independent growth upon insulin treatment. Knocking down InsR in TamR cells with shRNA was able to attenuate their sensitivity towards insulin-mediated PI3K/MAPK activation and growth, suggesting InsR targeting may be necessary in endocrine resistant breast cancer. To develop new agents to target InsR, we used yeast surface display to develop an InsR-selective protein scaffold – the 10th type III domain of human fibronectin (Fn3). This technique has arisen as an alternative method for the development of specific binders of cell surface proteins. Compared to antibodies, the Fn3 proteins are smaller (∼10kDa), thermally more stable, lack disulfide bonds and have the ability to bind a large variety of proteins. Using error-prone polymerase mutagenesis, we identified improved Fn3 proteins with increased InsR affinity, specificity, and stability. Our current results show that InsR is an important target in endocrine resistant breast cancer cells. Development of engineered Fn3 peptides with increased InsR binding specificity and affinity for InsR compared to IGF1R could be used to disrupt signaling through this pathway. Engineered Fn3 will further be evaluated as a potential imaging and therapeutic tool. Citation Format: Jie Ying Chan, Benjamin Hackel, Douglas Yee. Insulin receptor targeting in breast cancer through yeast surface display [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 P4-06-02.

Bleb formation is induced by alkaline but not acidic pH in estrogen receptor silenced breast cancer cells.

De novo and acquired resistance to endocrine-based therapies in breast cancer occurs in parallel with epithelial to mesenchymal transition (EMT), which is associated with enhanced proliferative and metastatic potential, and poor clinical outcome. We have established several endocrine insensitive breast cancer lines by shRNA-induced depletion of estrogen receptor (ER) by transfection of MCF7 cells. All of these exhibit EMT. We have previously reported that brief exposure of specifically ER- breast cancer cells, to extracellular alkaline pH, results in cell rounding and segregation, and leads to enhanced invasive potential. In this study we describe more detailed morphological changes and compare these with cell exposure to acidic pH. Morphological changes and localization of various molecules critical for cell adhesion and motility, associated with pH effects, were assessed by live cell microscopy, electron microscopy, and immunofluorescence. Exposure of either ER- or ER+ breast cancer cells to extracellular acidic pH did not induce significant changes in morphological appearance. Conversely, brief exposure of specifically ER silenced cells, to alkaline pH, resulted in cell contractolation and formation of bleb-like actin-rich structures which were evenly distributed on the outer membrane. Integrin α2, FAK, and JAM-1 were found in the cytoplasm streaming into the newly formed blebs. These blebs appear to be related to cell polarity and movement. Pre-treatment with cytochalasin-D or inhibitors of Rho or MLCK prevented both contractolation and bleb formation. Our data suggest that the effect of pH on the microenvironment of endocrine resistant breast cancer cells needs to be more extensively investigated. Alkaline, rather than acidic pH, appears to induce dramatic morphological changes, and enhances their invasive capabilities, through re-organization of cortical actin.

Endocrine therapy resistance and epithelial to mesenchymal transition are driven by Nicastrin and Notch4 cooperation in MCF7 breast cancer cells

Endocrine therapy resistant (ETR) tumors often display mesenchymal features, associated with aggressive and enhanced motility behaviour. Notch signalling is over-activated in ETR cells. By blocking it, it is possible to interfere with the cell growth. Notch is also implicated in regulating epithelial to mesenchymal transition (EMT) affecting both migration and invasion in breast cancer cells. We know that Nicastrin is the major component of the multi-subunit protease complex Gamma Secretase (GS) which executes intramembrane proteolysis of integral proteins such Notch. We used two models of Endocrine Therapy Resistant MCF7 breast cancer cell lines which acquired EMT feature and showed higher levels of Nicastrin and Notch targets. Moreover, they displayed higher Notch4 levels but lower Notch1 and Notch2, indicating a possible switch of the Notch signalling when cells acquire resistance. We demonstrated that Gamma Secretase inhibitor PF03084014 and anti-Nicastrin MAbs were particularly effective in partially inhibiting the EMT process by blocking Nicastrin-Notch4 contribution to resistance. Importantly, we also demonstrated that the stem cells-like population was reduced upon these treatments. Both Nicastrin and Notch4 genetic silencing lead to similar effects. Finally, stably overexpressing Nicastrin, was sufficient to activate Notch4 in MCF7 and render them unresponsive to tamoxifen. Here we highlight our recent study.

A771726, an anti-inflammatory drug, exerts an anticancer effect and reverses tamoxifen resistance in endocrine-resistant breast cancer cells.

A771726, an orally available anti-inflammatory agent, has been approved for the treatment of multiple sclerosis by diminishing entire inflammatory responses through multiple signaling pathways. Recently, a few emerging studies have focused on the potential application of A771726 in cancer therapy, less on the treatment of breast cancer and particularly on overcoming drug resistance in breast cancer. We report here for the first time the cytotoxic activity and drug resistance reversal of A771726 in acquired tamoxifen-resistant breast cancer cell line MCF-7/LCC9. We discovered that A771726 treatment showed antiproliferative activities in MCF-7/LCC9 cells, which were even more sensitive to A771726 than their parental tamoxifen-sensitive cells (MCF‑7). A771726 also exerted pro-apoptotic activities and induced cell cycle arrest at the G1 phase. Notably, treatment of A771726 restored the sensitivity of MCF-7/LCC9 cells to tamoxifen. Western blot analysis revealed that A771726 decreased the phosphorylation level of Src, one key driver of tamoxifen resistance. Moreover, in order to comprehensively clarify the mechanisms of A771726 in anti-estrogen-resistant cells, we explored a genome-wide transcriptomic analysis, and showed that A771726 could modulate multiple signaling pathways (e.g. cell cycle, apoptosis, MAPK, metabolism and p53 signaling pathway) and cellular processes (e.g. signal transduction, transcription and cell cycle). Overall, our results indicate that A771726 alone and the combination of A771726 with anti-estrogens may be of therapeutic benefit for ER-positive and endocrine-resistant breast cancer.

Global Gene Repression by the Steroid Receptor Coactivator SRC-1 Promotes Oncogenesis

Transcriptional control is the major determinant of cell fate. The steroid receptor coactivator (SRC)-1 enhances the activity of the estrogen receptor in breast cancer cells, where it confers cell survival benefits. Here, we report that a global analysis of SRC-1 target genes suggested that SRC-1 also mediates transcriptional repression in breast cancer cells. Combined SRC-1 and HOXC11 ChIPseq analysis identified the differentiation marker, CD24, and the apoptotic protein, PAWR, as direct SRC-1/HOXC11 suppression targets. Reduced expression of both CD24 and PAWR was associated with disease progression in patients with breast cancer, and their expression was suppressed in metastatic tissues. Investigations in endocrine-resistant breast cancer cell lines and SRC-1(-/-)/PyMT mice confirmed a role for SRC-1 and HOXC11 in downregulation of CD24 and PAWR. Through bioinformatic analysis and liquid chromatography/mass spectrometry, we identified AP1 proteins and Jumonji domain containing 2C (JMD2C/KDM4C), respectively, as members of the SRC-1 interactome responsible for transcriptional repression. Our findings deepen the understanding of how SRC-1 controls transcription in breast cancers.

β-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression

Endocrine therapies have been successfully used for breast cancer patients with estrogen receptor α (ERα) positive tumors, but ∼40% of patients relapse due to endocrine resistance. β-glucans are components of plant cell walls that have immunomodulatory and anticancer activity. The objective of this study was to examine the activity of β-D-glucan, purified from barley, in endocrine-sensitive MCF-7 versus endocrine-resistant LCC9 and LY2 breast cancer cells. β-D-glucan dissolved in DMSO but not water inhibited MCF-7 cell proliferation in a concentration-dependent manner as measured by BrdU incorporation with an IC50 of ∼164±12 μg/ml. β-D-glucan dissolved in DMSO inhibited tamoxifen/endocrine-resistant LCC9 and LY2 cell proliferation with IC50 values of 4.6±0.3 and 24.2±1.4 μg/ml, respectively. MCF-10A normal breast epithelial cells showed a higher IC50 ∼464 μg/ml and the proliferation of MDA-MB-231 triple negative breast cancer cells was not inhibited by β-D-glucan. Concentration-dependent increases in the BAX/BCL2 ratio and cell death with β-D-glucan were observed in MCF-7 and LCC9 cells. PCR array analysis revealed changes in gene expression in response to 24-h treatment with 10 or 50 μg/ml β-D-glucan that were different between MCF-7 and LCC9 cells as well as differences in basal gene expression between the two cell lines. Select results were confirmed by quantitative real-time PCR demonstrating that β-D-glucan increased RASSF1 expression in MCF-7 cells and IGFBP3, CTNNB1 and ERβ transcript expression in LCC9 cells. Our data indicate that β-D-glucan regulates breast cancer-relevant gene expression and may be useful for inhibiting endocrine-resistant breast cancer cell proliferation.

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.

COUP-TFII inhibits NFkappaB activation in endocrine-resistant breast cancer cells

Reduced COUP-TFII expression contributes to endocrine resistance in breast cancer cells. Endocrine-resistant breast cancer cells have higher NFkappa B (NFκB) activity and target gene expression. The goal of this study was to determine if COUP-TFII modulates NFκB activity. Endocrine-resistant LCC9 cells with low endogenous COUP-TFII displayed ∼5-fold higher basal NFκB activity than parental endocrine-sensitive MCF-7 breast cancer cells. Transient transfection of LCC9 cells with COUP-TFII inhibited NFκB activation and reduced NFκB target gene expression. COUP-TFII and NFκB were inversely correlated in breast cancer patient samples. Endogenous COUP-TFII coimmunoprecipitated with NFκB subunits RelB and NFκB1 in MCF-7 cells. COUP-TFII inhibited NFκB-DNA binding in vitro and impaired coactivator induced NFκB transactivation. LCC9 cells were growth-inhibited by an NFκB inhibitor and 4-hydroxytamoxifen compared to MCF-7 cells. Together these data indicate a novel role for COUP-TFII in suppression of NFκB activity and explain, in part, why decreased COUP-TFII expression results in an endocrine-resistant phenotype.

Extracellular Alkaline pH Leads to Increased Metastatic Potential of Estrogen Receptor Silenced Endocrine Resistant Breast Cancer Cells

IntroductionEndocrine resistance in breast cancer is associated with enhanced metastatic potential and poor clinical outcome, presenting a significant therapeutic challenge. We have established several endocrine insensitive breast cancer lines by shRNA induced depletion of estrogen receptor (ER) by transfection of MCF-7 cells which all exhibit enhanced expression profile of mesenchymal markers with reduction of epithelial markers, indicating an epithelial to mesenchymal transition. In this study we describe their behaviour in response to change in extracellular pH, an important factor controlling cell motility and metastasis.MethodsMorphological changes associated with cell exposure to extracellular alkaline pH were assessed by live cell microscopy and the effect of various ion pumps on this behavior was investigated by pretreatment with chemical inhibitors. The activity and expression profile of key signaling molecules was assessed by western blotting. Cell motility and invasion were examined by scratch and under-agarose assays respectively. Total matrix metalloproteinase (MMP) activity and specifically of MMP2/9 was assessed in conditioned medium in response to brief alkaline pH exposure.ResultsExposure of ER –ve but not ER +ve breast cancer cells to extracellular alkaline pH resulted in cell shrinkage and spherical appearance (termed contractolation); this was reversed by returning the pH back to 7.4. Contractolation was blocked by targeting the Na+/K+ and Na+/H+ pumps with specific chemical inhibitors. The activity and expression profile of key signaling molecules critical for cell adhesion were modulated by the exposure to alkaline pH. Brief exposure to alkaline pH enhanced MMP2/9 activity and the invasive potential of ER –ve cells in response to serum components and epithelial growth factor stimulation without affecting unhindered motility.ConclusionsEndocrine resistant breast cancer cells behave very differently to estrogen responsive cells in alkaline pH, with enhanced invasive potential; these studies emphasise the crucial influence of extracellular pH and caution against indiscriminate application of alkalinising drug therapy.

Crosstalk between PKCα and Notch-4 in endocrine-resistant breast cancer cells.

The Notch pathway is functionally important in breast cancer. Notch-1 has been reported to maintain an estrogen-independent phenotype in estrogen receptor α (ERα)+ breast cancer cells. Notch-4 expression correlates with Ki67. Notch-4 also plays a key role in breast cancer stem-like cells. Estrogen-independent breast cancer cell lines have higher Notch activity than estrogen-dependent lines. Protein kinase Cα (PKCα) overexpression is common in endocrine-resistant breast cancers and promotes tamoxifen (TAM)-resistant growth in breast cancer cell lines. We tested whether PKCα overexpression affects Notch activity and whether Notch signaling contributes to endocrine resistance in PKCα-overexpressing breast cancer cells.Analysis of published microarray data from ERα+ breast carcinomas shows that PKCα expression correlates strongly with Notch-4. Real-time reverse transcription PCR and immunohistochemistry on archival specimens confirmed this finding. In a PKCα-overexpressing, TAM-resistant T47D model, PKCα selectively increases Notch-4, but not Notch-1, expression in vitro and in vivo. This effect is mediated by activator protein-1 (AP-1) occupancy of the Notch-4 promoter. Notch-4 knockdown inhibits estrogen-independent growth of PKCα-overexpressing T47D cells, whereas Notch-4IC expression stimulates it. Gene expression profiling shows that multiple genes and pathways associated with endocrine resistance are induced in Notch-4IC- and PKCα-expressing T47D cells. In PKCα-overexpressing T47D xenografts, an orally active γ-secretase inhibitor at clinically relevant doses significantly decreased estrogen-independent tumor growth, alone and in combination with TAM. In conclusion, PKCα overexpression induces Notch-4 through AP-1. Notch-4 promotes estrogen-independent, TAM-resistant growth and activates multiple pathways connected with endocrine resistance and chemoresistance. Notch inhibitors should be clinically evaluated in PKCα- and Notch-4-overexpressing, endocrine-resistant breast cancers.

Abstract 4762: Anti-Nicastrin antibodies for the treatment of endocrine resistant breast cancer .

Abstract Background: Resistance to endocrine therapy occurs in at least 30% of patients with breast cancer (BC) and remains a major clinical problem. Among the mechanisms underpinning endocrine resistance is the enrichment of epithelial-to-mesenchymal transition (EMT) features and signalling pathways such as EGFR and Notch. Notch receptors are activated by the gamma-secretase (GS) enzyme composed of nicastrin, presenilin, PEN2 and Aph-1. Gamma secretase inhibitors are being tested as novel therapeutics for cancer treatment, predominantly in combination with anti-hormonal agents as well as chemotherapy, however treatment related toxicities are apparent. Nicastrin is upregulated in BC and we have developed anti-nicastrin monoclonal antibodies (MAbs) to target its functions and block GS. Here, we show that nicastrin may be a relevant therapeutic target in endocrine resistance and demonstrate the effects of anti-nicastrin MAbs. Materials and Methods: We used two cell line models of MCF7 cells which developed endocrine resistance: TamR cells (Hiscox et al) and MLET2/MLET5 developed in our laboratory. We raised anti-nicastrin MAbs (MAb1 and MAb2) against nicastrin extracellular domain. Western blotting, RT-qPCR, transwell invasion and 3D matrigel growth assays were used to demonstrate their molecular and functional effects. Gamma secreatse inhibitor RO4929097 was used for comparison. Results: We demonstrate that nicastrin is overexpressed at both mRNA and protein level in endocrine resistant cell lines (> 4-fold). These cells concomitantly exhibit elevated molecular markers of EMT: Vimentin (3-fold), CD44 (> 2-fold), EGFR (> 4-fold), RhoGTPase Cdc42 target genes IQGAP1 (2-fold) and IQGAP2 (>100-fold); as well as Notch4 (>2-fold), Notch-targets Hey1 and Hes1 (4-fold), while Notch1 is suppressed (> 2-fold). Anti-nicastrin MAb1, MAb2 and RO4929097 were equally potent in inhibiting Notch target genes Hey1 and Hes1, as well as growth of TAMR and MLET acini in 3D Matrigel. Interestingly, effective inhibition of vimentin, CD44 and IQGAPs was observed only upon treatment with anti-nicastrin MAbs, while RO4929097 had no effect. Interestinly, a clear distinction was noted between anti-nicastrin MAbs and RO4929097 in terms of their effect on Notch4. MAb1 and MAb2 attenuated Notch4 transcription and reduced activated Notch4 and nicastrin levels, while RO4929097 achieved an opposite effect. Furthermore, only anti-nicastrin MAbs were also potent inhibitors of TAMR cell invasion (by >50%, p<0.01). Conclusion: Nicastrin is overexpressed in endocrine resistant breast cancer cells. Anti-nicastrin monoclonal antibodies inhibit Notch4 signalling, attenuate expression of pro-invasive molecules such as vimentin, IQGAPs and CD44 and reduce cellular invasiveness and growth in 3D Matrigel. Therefore, targeting nicastrin may be a valid therapeutic strategy in breast cancer patients that relapse of endocrine therapy. Citation Format: Monica Faronato, Ylenia Lombardo, Aleksandra Filipovic, Simak Ali, R.Charles Coombes. Anti-Nicastrin antibodies for the treatment of endocrine resistant breast cancer . [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 4762. doi:10.1158/1538-7445.AM2013-4762

Abstract P4-07-03: Rapamycin and Dalotuzumab in combination inhibit parental and endocrine resistant breast cancer cells.

Abstract The PI3K/Akt/mTOR pathway is involved in breast cancer resistance to endocrine therapy. Inhibitors of mTOR have been shown to have benefit for patients with ER positive tumors that have developed endocrine therapy resistance. Inhibition of mTOR triggers a negative feedback loop resulting in enhanced phosphorylation of Akt and subsequent breast cancer cell proliferation via the IGF signaling pathway. Therefore, blockade of both IGF-1R and mTOR may be necessary to completely suppress this pathway. Our laboratory studied the effect of dual inhibition of mTOR and IGF-1R with rapamycin and dalotuzumab respectively on parental and endocrine resistant MCF-7 breast cancer cell tumorigenesis. Immunoblotting demonstrates parental MCF-7L cells treated with rapamycin alone demonstrate increased phosphorylation of Akt. Inhibition of phosphorylation of Akt can be achieved with combined treatment with rapamycin and dalotuzumab at concentrations of 5 nM and 2 µg/mL respectively. In addition to inhibition of pAkt, combined treatment inhibits phosphorylation of S6K1 and the translational repressor protein, 4eBP1. Furthermore, MTT proliferation assay and soft agar assay demonstrate inhibition of parental cell proliferation and colonization respectively with combined rapamycin and dalotuzumab treatment. In order to examine the effect of combination therapy in endocrine resistant cell lines, we established tamoxifen resistant (TamR) and long term estrogen deprived (LTED) cell lines. TamR cells were generated by culturing MCF-7L cells in the presence of tamoxifen for more than 1 year. LTED cells were generated by culturing MCF-7L cells in the absence of estrogen for more than 6 months. Proliferation of TamR and LTED cells treated with rapamycin at a concentration of 5nM and dalotuzumab at a concentration of 2µg/mL in combination was significantly inhibited. We conclude that combination of IGF1R and mTOR inhibition might be necessary to inhibit endocrine resistant breast cancers. Ongoing clinical trials will test this combination of drugs. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-07-03.

Abstract P6-04-30: COUP-TFII suppresses NFκB activation in endocrine-resistant breast cancer cells

Abstract COUP-TFII is an orphan nuclear receptor that functions as either a transcriptional activator or repressor. We previously reported that COUP-TFII expression is reduced in endocrine-resistant LCC9 breast cancer cells and that overexpression of COUP-TFII restored the ability of the antiestrogen tamoxifen to inhibit cell proliferation. An increase in NFκB activity and p65/RELA expression has been reported in endocrine-resistant breast cancer cells. Because NFκB activation results in increased expression of anti-apoptotic and pro-proliferative genes, increased NFκB signaling promotes the survival of cancer cells. The goal of the current study was to further elucidate the mechanism by which COUP-TFII enables cells to maintain endocrine sensitivity and to determine if COUP-TFII can modulate NFκB activity in tamoxifen-resistant breast cancer cells. To examine whether COUP-TFII can suppress NFκB activity, MCF-7 (tamoxifen-sensitive, TAM-S) and LCC9 (tamoxifen-resistant, TAM-R) breast cancer cells were transfected with a NFκB luciferase reporter and treated with TNFα to activate NFκB. NFκB activity was elevated in LCC9 TAM-R cells and co-transfection with COUP-TFII reduced NFκB activity. COUP-TFII overexpression led to a statistically significant reduction in the expression of NFκB subunits RELA, RELB, NFKB1, and REL in LCC9 cells. Significantly reduced expression of NFκB target genes IL6, ICAM1, TNFAIP3, and CCL2 was also evident upon COUP-TFII overexpression, reflecting the suppression of the NFκB pathway by COUP-TFII. Inhibition of NFκB activation by COUP-TFII also led to a reduction in cell viability in TAM-R cells in response to tamoxifen treatment. Together these data indicate a novel role for COUP-TFII in suppression of NFκB activity which may explain the ability of COUP-TFII to promote tamoxifen-sensitivity. COUP-TFII may be both a useful biomarker to predict tamoxifen-sensitivity as well as a target to restore endocrine sensitivity to resistant cells. Supported by NIEHS T32 ES011564 predoctoral fellowship to L.M.L. and Susan G. Komen for the Cure grant KG080365 to C.M.K. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-30.