Tamoxifen-sensitive Breast Cancer Cells Research Articles

Correlating NAD(P)H lifetime shifts to tamoxifen resistance in breast cancer cells: A metabolic screening study with time-resolved flow cytometry

Time-resolved flow cytometry (TRFC) was used to measure metabolic differences in estrogen receptor-positive breast cancer cells. This specialty cytometry technique measures fluorescence lifetimes as a single-cell parameter thereby providing a unique approach for high-throughput cell counting and screening. Differences in fluorescence lifetime were detected and this was associated with sensitivity to the commonly prescribed therapeutic tamoxifen. Differences in fluorescence lifetime are attributed to the binding states of the autofluorescent metabolite NAD(P)H. The function of NAD(P)H is well described and in general involves cycling from a reduced to oxidized state to facilitate electron transport for the conversion of pyruvate to lactate. NAD(P)H fluorescence lifetimes depend on the bound or unbound state of the metabolite, which also relates to metabolic transitions between oxidative phosphorylation and glycolysis. To determine if fundamental metabolic profiles differ for cells that are sensitive to tamoxifen compared to those that are resistant, large populations of MCF-7 breast cancer cells were screened and fluorescence lifetimes were quantified. Additionally, metabolic differences associated with tamoxifen sensitivity were measured with a Seahorse HS mini metabolic analyzer (Agilent Technologies Inc. Santa Clara, CA) and confocal imaging. Results show that tamoxifen-resistant breast cancer cells have increased utilization of glycolysis for energy production compared to tamoxifen-sensitive breast cancer cells. This work is impacting because it establishes an early step toward developing a reliable screening technology in which large cell censuses can be differentiated for drug sensitivity in a label-free fashion.

USP46 enhances tamoxifen resistance in breast cancer cells by stabilizing PTBP1 to facilitate glycolysis

Tamoxifen (TAM) is the primary drug for treating estrogen receptor alpha-positive (ER+) breast cancer (BC). However, resistance to TAM can develop in some patients, limiting its therapeutic efficacy. The ubiquitin-specific protease (USP) family has been associated with the development, progression, and drug resistance of various cancers. To explore the role of USPs in TAM resistance in BC, we used qRT-PCR to compare USP expression between TAM-sensitive (MCF-7 and T47D) and TAM-resistant cells (MCF-7R and T47DR). We then modulated USP46 expression and examined its impact on cell proliferation, drug resistance (via CCK-8 and EdU experiments), glycolysis levels (using a glycolysis detection assay), protein interactions (confirmed by co-IP), and protein changes (analyzed through Western blotting). Our findings revealed that USP46 was significantly overexpressed in TAM-resistant BC cells, leading to the inhibition of the ubiquitin degradation of polypyrimidine tract-binding protein 1 (PTBP1). Overexpression of PTBP1 increased the PKM2/PKM1 ratio, promoted glycolysis, and intensified TAM resistance in BC cells. Knockdown of USP46 induced downregulation of PTBP1 protein by promoting its K48-linked ubiquitination, resulting in a decreased PKM2/PKM1 ratio, reduced glycolysis, and heightened TAM sensitivity in BC cells. In conclusion, this study highlights the critical role of the USP46/PTBP1/PKM2 axis in TAM resistance in BC. Targeted therapy against USP46 may represent a promising strategy to improve the prognosis of TAM-resistant patients.

An Angiogenesis-Related lncRNA Signature Is Associated with Prognosis and Tumor Immune Microenvironment in Breast Cancer.

Angiogenesis is crucial in the development and progression of tumors. This study examined the relationship between angiogenesis-related lncRNAs (AR-lncRNAs) and breast cancer (BC) immunity and prognosis. We used univariate Cox regression analysis to obtain AR-lncRNAs closely related to BC prognosis. Cluster analysis of BC patients was performed using non-negative matrix factorization (NMF) analysis according to the expression of AR-lncRNAs that were prognostically relevant. An AR-lncRNA risk model (AR-lncM) was created using LASSO regression analysis to predict the prognosis and survival of BC patients. Subsequently, the effect of LINC01614 on cell migration and invasion was verified by Transwell and Western blot assays, and the CCK-8 assay detected its impact on cell sensitivity to tamoxifen. Finally, we obtained 17 AR-lncRNAs from the TCGA database that were closely associated with the prognosis of BC patients. Based on the expression of these AR-lncRNAs, BC patients were divided into five clusters using NMF analysis. Cluster 1 was found to have a better prognosis, higher expression of immune checkpoints, and higher levels of immune cell infiltration. Furthermore, an AR-LncM model was created using ten prognostic-related AR-lncRNAs. The model's risk predictive performance was validated using survival analysis, timeROC curves, and univariate and multivariate Cox analysis. The most interesting gene in the model, LINC01614, was found to regulate epithelial-mesenchymal transition (EMT) and tamoxifen sensitivity in BC cells, implying that LINC01614 could be a potential therapeutic target for BC patients.

YBX1/lncRNA SBF2-AS1 interaction regulates proliferation and tamoxifen sensitivity via PI3K/AKT/MTOR signaling in breast cancer cells.

Y-box binding protein 1 (YBX1) is a multifunctional oncoprotein that can interact with several long non-coding RNAs (lncRNAs)to regulate metastasis in malignancies including breast cancer (BC). In the present study, we demonstrated the association of YBX1 with oncogenic lncRNA SBF2-AS1 (SET-binding factor 2 antisense RNA 1) via PI3K/AKT/mTOR signaling to regulate BC cell proliferation. We further explored the involvement of the YBX1/SBF2-AS1/PI3K/AKT/mTOR axis in the restoration of tamoxifen (TAM) sensitivity. YBX1-SBF2-AS1 association was predicted in silico and verified by RNA immunoprecipitation (RIP)-qPCR assay. Transfection experiments, Real-time RT PCR, Western blots, Phospho AKT/mTOR antibody array kit, and cell proliferation/apoptosis assays were employed to detect the YBX1/SBF2-AS1/ PI3K/AKT/mTOR axis and its effects upon TAM treatment in vitro. We identified that the YBX1 protein specifically binds to lncRNA SBF2-AS1. Our transfection experiments in MCF-7 and MDA-MB-468 cells with SBF2-AS1 silenced or overexpressed YBX1 plasmids, and their negative controls revealed that YBX1 regulates the expression of SBF2-AS1 by forming a positive feedback loop for its activation. We further demonstrated YBX1-SBF2-AS1 association exerts its effects on cell proliferation via PI3K/AKT/mTOR signaling pathway. Furthermore, we observed an increase in TAM sensitivity in BC cells after the knockdown of YBX1-SBF2-AS1 marked by decreased cell proliferation through disruption of the PI3K/AKT/mTOR axis. Our study has identified a novel YBX1/SBF2-AS1/PI3K/AKT/mTOR regulatory axis which may serve as a potential target to improve the effectiveness and efficacy of TAM treatment in BC.

Identification of key genes involved in tamoxifen-resistant breast cancer using bioinformatics analysis.

BackgroundThe purpose of the present study was to investigate the molecular mechanisms of tamoxifen resistance in breast cancer and to identify potential targets for antitamoxifen resistance.MethodsDifferentially expressed genes (DEGs) in tamoxifen-resistant and tamoxifen-sensitive breast cancer cells were assessed using the GSE67916 dataset acquired from the Gene Expression Omnibus database. Gene ontology (GO) and pathway enrichment analyses were applied to investigate the functions and pathways of the DEGs. Subsequently, the protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING), and subnetworks were further analyzed by Molecular Complex Detection (MCODE). The PPI network and subnetworks were visualized using Cytoscape software.ResultsIn total, 438 DEGs were identified, of which 300 were upregulated and 138 were downregulated. The DEGs were significantly enriched in the protein binding, cellular response to estradiol stimulus, and immune response GO terms while the most significant pathways included the mitogen-activated protein kinase (MAPK) signaling pathway in cancer. The PPI network of DEGs was constructed with 288 nodes and 629 edges, and 2 subnetworks were screened out from the entire network.ConclusionsA number of significant hub DEGs were identified based on their degree of connectivity in the PPI network, , included MAPK1 (node degree 36), ESR1 (node degree 27), SMARCA4 (node degree 27), RANBP2 (node degree 25), and PRKCA (node degree 21). These critical hub genes were found to be related to tamoxifen resistance in breast cancer. The results of this study further the understanding of tamoxifen resistance at the molecular level and identify potential therapeutic targets for tamoxifen-resistant breast cancer.

High expression of TRAF4 predicts poor prognosis in tamoxifen-treated breast cancer and promotes tamoxifen resistance.

Tamoxifen is the main adjuvant endocrine therapeutic agent for patients with estrogen receptor positive breast cancer. However, the resistance to tamoxifen has become a serious clinical challenge and the underlying mechanisms are still poorly understood. TRAF4 is a member of tumor necrosis factor receptor-associated factor family and its role in tamoxifen resistance has not been found. In this study, we aimed to explore the roles of TRAF4 in tamoxifen-treated breast cancer and tamoxifen resistance. Through high-throughput sequencing and differential gene expression analyses, TRAF4 was identified as the research object in this study. The prognosis significance of TRAF4 was studied based on 155 tamoxifen-treated breast cancer patients obtained from Gene Expression Omnibus (GEO) database. We then investigated the TRAF4 expression level in tamoxifen-resistant and the tamoxifen-sensitive breast cancer cell lines with western blot and real-time quantitative PCR. The loss- and gain-of-function assay of TRAF4 in a tamoxifen-resistant cell line was evaluated using colony formation experiments and cell count kit-8 assay. We identified that TRAF4 was overexpressed in tamoxifen-resistant breast cancer cell line and TRAF4 overexpression was associated with worse overall survival (hazard ratio = 2.538, P = 0.017) and cancer-specific survival (hazard ratio = 2.713, P = 0.036) in tamoxifen-treated patients. Knockdown of TRAF4 reversed tamoxifen resistance, while overexpression of TRAF4 increased tamoxifen resistance, which confirmed the role of TRAF4 in tamoxifen resistance. Taken together, our study demonstrated that TRAF4 could be a novel prognostic biomarker for tamoxifen-treated breast cancer patients and a potential therapeutic target for tamoxifen resistance.

Abstract P6-04-17: Role of HOXB13 in modulating estrogen signaling in breast cancer cells

Abstract Background: The ratio of HOXB13 (H) and IL17BR (I) gene expression levels in hormone receptor-positive (HR+) primary early stage breast tumors define a predictive index (H/I) that, combined with the Molecular Grade Index (MGI), constitute the Breast Cancer Index (BCI). The BCI score (MGI+H/I prognostic model) provides individualized risk of overall (0-10 years) and specifically late (5-10 years) distant recurrence, and BCI (H/I) predicts the benefit of extended endocrine therapy (EET). Hormone receptor responses are dependent on genome-wide hormone receptor binding patterns. In hormone-dependent cancers, nuclear receptor binding patterns are frequently reprogrammed by other transcription factors. For example, in breast cancer cells, GATA3 has been shown to reprogram estrogen receptor (ER) binding and to alter gene expression. Similarly, HOXB13 was previously shown to reprogram genome-wide binding of the androgen receptor (AR) during prostate cancer tumorigenesis. To assess whether HOXB13 expression is associated with regulation of ER binding, global ER binding data in highly HOXB13-expressing, tamoxifen-resistant breast cancer cells to the ER binding pattern in HOXB13-low, tamoxifen-sensitive breast cancer cells were compared. The study objective was to understand whether HOXB13 contributes to ER reprogramming in early-stage breast cancer. Methods: Global ER, HOXB13 and FOXA1 DNA binding patterns and the enhancer activation mark histone H3 lysine 27 acetylation (H3K27ac) were characterized in BT474 cells by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq). To compare ER binding in highly HOXB13-expressing, tamoxifen-resistant BT474 cells to published ER binding data in HOXB13-low, tamoxifen-sensitive MCF-7 breast cancer cells, we downloaded published ER, FOXA1, GATA3 and H3K27ac ChIP-seq data from MCF-7 cells. After aligning reads with Bowtie2, peak calling and data integration was performed using HOMERv4.10. Results: Analysis of highly HOXB13-expressing BT474 cells identified a large number of HOXB13 binding sites, which overlapped with approximately half of the ER binding sites in these cells. Many of these binding sites were co-bound by FoxA1, a general pioneer factor that has been shown to also colocalize with HoxB13 at reprogrammed AR binding sites in human prostate tumors. Interestingly, most of these locations are distinct from the genomic binding sites of ER in HOXB13-low MCF-7 cells, where ER frequently binds to MCF-7-specific sites in conjunction with GATA3. Conclusion: Our preliminary evidence suggests that HOXB13 influences the estrogen receptor binding pattern in human breast cancer cells and that ER binding sites differ between HOXB13 high- and low-expressing cell lines, which may underlie their differential responses to endocrine therapy. These results will be compared to ongoing experiments assessing the effects of conditional expression or loss of HOXB13 on ER binding and function in HOXB13-negative and HOXB13-positive cells, respectively. Citation Format: Kai Treuner, Michael Hayes, Christopher Benner, Catherine Schnabel, Sven Heinz. Role of HOXB13 in modulating estrogen signaling in breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-17.

AhR ligand aminoflavone suppresses α6-integrin-Src-Akt signaling to attenuate tamoxifen resistance in breast cancer cells.

More than 40% of patients with luminal breast cancer treated with endocrine therapy agent tamoxifen demonstrate resistance. Emerging evidence suggests tumor initiating cells (TICs) and aberrant activation of Src and Akt signaling drive tamoxifen resistance and relapse. We previously demonstrated that aryl hydrocarbon receptor ligand aminoflavone (AF) inhibits the expression of TIC gene α6-integrin and disrupts mammospheres derived from tamoxifen-sensitive breast cancer cells. In the current study, we hypothesize that tamoxifen-resistant (TamR) cells exhibit higher levels of α6-integrin than tamoxifen-sensitive cells and that AF inhibits the growth of TamR cells by suppressing α6-integrin-Src-Akt signaling. In support of our hypothesis, TamR cells and associated mammospheres were found to exhibit elevated α6-integrin expression compared with their tamoxifen-sensitive counterparts. Furthermore, tumor sections from patients who relapsed on tamoxifen showed enhanced α6-integrin expression. Gene expression profiling from the TCGA database further revealed that basal-like breast cancer samples, known to be largely unresponsive to tamoxifen, demonstrated higher α6-integrin levels than luminal breast cancer samples. Importantly, AF reduced TamR cell viability and disrupted TamR mammospheres while concomitantly reducing α6-integrin messenger RNA and protein levels. In addition, AF and small interfering RNA against α6-integrin blocked tamoxifen-stimulated proliferation of TamR MCF-7 cells and further sensitized these cells to tamoxifen. Moreover, AF reduced Src and Akt signaling activation in TamR MCF-7 cells. Our findings suggest elevated α6-integrin expression is associated with tamoxifen resistance and AF suppresses α6-integrin-Src-Akt signaling activation to confer activity against TamR breast cancer.

MiR-449a Suppresses Tamoxifen Resistance in Human Breast Cancer Cells by Targeting ADAM22

Background/Aims: Most of estrogen receptor positive breast cancer patients respond well initially to endocrine therapies, but often develop resistance during treatment with selective estrogen receptor modulators (SERMs) such as tamoxifen. Altered expression and functions of microRNAs (miRNAs) have been reportedly associated with tamoxifen resistance. Thus, it is necessary to further elucidate the function and mechanism of miRNAs in tamoxifen resistance. Methods: Tamoxifen sensitivity was validated by using Cell Counting Kit-8 in tamoxifen-sensitive breast cancer cells (MCF-7, T47D) and tamoxifen-resistant cells (MCF-7/TAM, T47D/ TAM). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression level of miR-449a in tamoxifen-sensitive/-resistant cells and patient serums. Dual-luciferase assay was used to identify the binding of miR-449a and predicted gene ADAM22. The expression level of ADAM22 was determined by qRT-PCR and western blotting in miR-449a +/- breast cancer cells. Subsequently, rescue experiments were carried out to identify the function of ADAM22 in miR-449a-reduced tamoxifen resistance. Finally, Gene ontology (GO) and Protein-protein interaction analyses were performed to evaluate the potential mechanisms of ADAM22 in regulating tamoxifen resistance. Results: MiR-449a levels were downregulated significantly in tamoxifen-resistant breast cancer cells when compared with their parental cells, as well as in clinical breast cancer serum samples. Overexpression of miR-449a re-sensitized the tamoxifen-resistant breast cancer cells, while inhibition of miR-449a conferred tamoxifen resistance in parental cells. Luciferase assay identified ADAM22 as a direct target gene of miR-449a. Additionally, silencing of ADAM22 could reverse tamoxifen resistance induced by miR-449a inhibition in ER-positive breast cancer cells. GO analysis results showed ADAM22 was mainly enriched in the biological processes of cell adhesion, cell differentiation, gliogenesis and so on. Protein-protein interaction analyses appeared that ADAM22 might regulate tamoxifen resistance through PPARG, LGI1, KRAS and LYN. Conclusion: Decreased miR-449a causes the upregulation of ADAM22, which induces tamoxifen resistance of breast cancer cells. These results suggest that miR-449a, functioning by targeting ADAM22, contributes to the mechanisms underlying breast cancer endocrine resistance, which may provide a potential therapeutic strategy in ER-positive breast cancers.

Knockdown of Long Non-Coding RNA UCA1 Increases the Tamoxifen Sensitivity of Breast Cancer Cells through Inhibition of Wnt/β-Catenin Pathway.

Acquired resistance to tamoxifen remains a major obstacle in breast cancer (BC) treatment, since the underlying mechanism has not been fully elucidated. The long non-coding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) has been recently shown to be dysregulated and plays important roles in progression of breast cancer. In the present study, we aimed to investigate the biological role and clinical significance of UCA1 in BC drug resistance. Hence, we used quantitative PCR assay to evaluate the UCA1 expression in tissues from patients with BC as well as established tamoxifen-resistant BC cell lines in vitro. We tested the viability, invasive ability and apoptosis rate in MCF-7 and T47D cells using MTT assay, transwell assay and flow cytometry assay, respectively. The influence of UCA1 on tumorigenesis was monitored by in vivo mice xenograft model. The activation of Wnt/β-catenin signaling pathway was evaluated by immunofluorescence assay, western blot assay and luciferase reporter assay, respectively. We found that the expression of UCA1 positively correlated with the pathological grade and mortality of breast cancer patients, moreover, expressions of UCA1 was increased significantly in the tamoxifen-resistant cell lines compared with the wild type parental cells. Ectopic expression of UCA1 promoted cell survival and resistance to tamoxifen treatment, whereas inhibition of UCA1 enhanced tamoxifen sensitivity of BC cells and induced more apoptotic cells. In addition, tamoxifen-resistant cells exhibited increased Wnt signaling activation as measured by the TOP/FOP Wnt luciferase reporter assay and β-catenin protein level compared with parental MCF-7 and T47D cells, respectively. In line with these data, UCA1 depletion attenuated the activity of Wnt/β-catenin pathway activation and the tumorigenicity of the tamoxifen-resistant BC cells. Taken together, our data highlights the pivotal role of UCA1-Wnt/β-catenin signaling pathway in the tamoxifen resistance in breast cancer, which could be targeted to improve the effectiveness and efficacy of tamoxifen treatment in breast cancer.

A new selective fluorescent probe based on tamoxifen

Developing targeted validation probes that can interrogate biology is of interest for both chemists and biologists. The synthesis of suitable compounds provides a means for avoiding the costly labeling of cells with specific antibodies and the bias associated with the interpretation of biological validation experiments. The chemotherapeutic agent, tamoxifen has been routinely used in the treatment of breast cancer for decades. Once metabolized, the active form of tamoxifen (4-hydroxytamoxifen) competes with the binding of estrogens to the estrogen receptors (ER). Its selectivity in ER modulation makes it an ideal candidate for the development of materials to be used as chemical probes. Here we report the synthesis of a fluorescent BODIPY®FL conjugate of tamoxifen linked through an ethylene glycol moiety, and present proof-of-principle results in ER positive and ER negative cell lines. Optical microscopy indicates that the fluorescent probe binds selectively to tamoxifen sensitive breast cancer cell lines. The compound showed no affinity for the tamoxifen resistant breast cancer lines. The specificity of the new compound make it a valuable addition to the chemical probe tool kit for estrogen receptors.

P21-activated kinase group II small compound inhibitor GNE-2861 perturbs estrogen receptor alpha signaling and restores tamoxifen-sensitivity in breast cancer cells.

Estrogen receptor alpha (ERα) is highly expressed in most breast cancers. Consequently, ERα modulators, such as tamoxifen, are successful in breast cancer treatment, although tamoxifen resistance is commonly observed. While tamoxifen resistance may be caused by altered ERα signaling, the molecular mechanisms regulating ERα signaling and tamoxifen resistance are not entirely clear. Here, we found that PAK4 expression was consistently correlated to poor patient outcome in endocrine treated and tamoxifen-only treated breast cancer patients. Importantly, while PAK4 overexpression promoted tamoxifen resistance in MCF-7 human breast cancer cells, pharmacological treatment with a group II PAK (PAK4, 5, 6) inhibitor, GNE-2861, sensitized tamoxifen resistant MCF-7/LCC2 breast cancer cells to tamoxifen. Mechanistically, we identified a regulatory positive feedback loop, where ERα bound to the PAK4 gene, thereby promoting PAK4 expression, while PAK4 in turn stabilized the ERα protein, activated ERα transcriptional activity and ERα target gene expression. Further, PAK4 phosphorylated ERα-Ser305, a phosphorylation event needed for the PAK4 activation of ERα-dependent transcription. In conclusion, PAK4 may be a suitable target for perturbing ERα signaling and tamoxifen resistance in breast cancer patients.

Protein kinase C-α downregulates estrogen receptor-α by suppressing c-Jun phosphorylation in estrogen receptor-positive breast cancer cells

Protein kinase C (PKC) activity is elevated in malignant compared with that in normal human breast tissue. In the present study, we investigated the regulatory mechanism and the co-relationship between PKC-α and estrogen receptor-α (ER-α) in ER-α-positive and tamoxifen-resistant (TAMR) breast cancer cells. Our results showed that the level of ER-α expression was significantly decreased in TAMR when compared with that in tamoxifen-sensitive (TAMS) breast cancer cells. However, PKC-α phosphorylation was increased in TAMR breast cancer cells when compared to that in TAMS breast cancer cells. Additionally, ER-α expression was significantly decreased due to the overexpression of constitutively active PKC-α (CA-PKC-α). Next, we investigated the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a reversible activator of PKC, on ER-α expression in ER-α-positive breast cancer cells. TPA decreased the levels of ER-α expression in a time- and dose-dependent manner. In contrast, the TPA-induced downregulation of ER-α was prevented by Go6983, a specific PKC inhibitor. Notably, we found that CA-PKC-α suppressed c-JUN phosphorylation, which is a major activating protein-1 factor, and TPA-induced downregulation of ER-α was prevented by SR11302, a specific activator protein-1 inhibitor. Taken together, we demonstrated that PKC-α activity suppressed the level of ER-α expression by inhibiting c-JUN phosphorylation in ER-α-positive breast cancer cells. Therefore, we suggest that PKC-α may be a potential therapeutic target for treating ER-positive and TAMR breast cancer.

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 P6-04-16: The role of RIP140 and FOXA1 in breast cancer endocrine sensitivity and resistance

Abstract Background: The estrogen receptor-α (ERα) is implicated in the initiation and progression of breast cancer. Endocrine therapeutics, including tamoxifen (OHT) and fulvestrant (ICI182780; ICI) improve disease-free survival. Despite positive effects, de novo or acquired resistance to endocrine therapies frequently occurs. It has recently been published that nuclear receptor–interacting protein 1, NRIP1, also called RIP140, encodes an ER corepressor RIP140 and has a role in the regulation of breast cancer cell growth in response to anti-estrogens. FOXA1 has previously been shown to function as a ‘pioneer factor’ and dictate ER dependent gene expression in both normal and malignant breast epithelial cells. In this study we investigated the expression of ERα cofactors RIP140 and FOXA1 and their roles in hormonal therapy and endocrine resistance. Methods: Tamoxifen sensitive or resistant breast cancer cell lines were analysed for protein and mRNA expression by western blotting and quantitative real-time PCR with or without the inhibition of ERα with tamoxifen or ICI. To determine the functional role of RIP140 and FOXA1 as well as their interaction in breast cancer, tamoxifen sensitive breast cancer cells (eg. MCF-7 and ZR-75-1) were transfected with either RIP140 or FOXA1 expression vector and examined for the expression of ER-target genes by Western blot and RT-qPCR analysis. The consequence of depletion of RIP140 and FOXA1 by RNA interference (RNAi) was also studied in these breast cancer cells. The expression of RIP140 and FOXA1 and potential target genes were also validated in breast cancer patient samples by Immunohistochemistry. Results: We first examined the basal expression levels of RIP140 and FOXA1 in tamoxifen sensitive as well as resistant breast cancer cell lines. The expression levels of RIP140 were low in the resistance cells compared with their parental sensitive counterparts, while that of FOXA1 had no significant difference between the cell lines. The degradation of ERα by ICI treatment resulted in the down-regulation of RIP140 and FOXA1 expression in the endocrine sensitive. Our experiments also demonstrated the functional interaction between RIP140 and FOXA1 and showed that depletion of RIP140 by RNA interference (RNAi) in sensitive cells resulted in reduction of FOXA1 expression. We also indentified from this work, a number of ER-regulated genes which are activated by FOXA1 and repressed by RIP140 expression. The regulation of these target genes by ERα, FOXA1 and RIP140 were also confirmed in the breast cancer patient samples. Conclusion: The results from this study demonstrated that RIP140 and FOXA1 play a pivotal role in dictating hormonal sensitivity and resistance in breast cancer. Moreover, this work also identified a number of ERα-regulated genes which are downstream targets of FOXA1 and RIP140, and as such, they may be reliable prognostic markers for endocrine sensitivity in breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-16.

Identification and Characterization of Nucleolin as a COUP-TFII Coactivator of Retinoic Acid Receptor β Transcription in Breast Cancer Cells

IntroductionThe orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine- resistant versus sensitive breast cancer cell lines. The identification of COUP-TFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer.ResultsFLAG-affinity purification and multidimensional protein identification technology (MudPIT) identified nucleolin among the proteins interacting with COUP-TFII in MCF-7 tamoxifen-sensitive breast cancer cells. Interaction of COUP-TFII and nucleolin was confirmed by coimmunoprecipitation of endogenous proteins in MCF-7 and T47D breast cancer cells. In vitro studies revealed that COUP-TFII interacts with the C-terminal arginine-glycine repeat (RGG) domain of nucleolin. Functional interaction between COUP-TFII and nucleolin was indicated by studies showing that siRNA knockdown of nucleolin and an oligonucleotide aptamer that targets nucleolin, AS1411, inhibited endogenous COUP-TFII-stimulated RARB2 expression in MCF-7 and T47D cells. Chromatin immunoprecipitation revealed COUP-TFII occupancy of the RARB2 promoter was increased by all-trans retinoic acid (atRA). RARβ2 regulated gene RRIG1 was increased by atRA and COUP-TFII transfection and inhibited by siCOUP-TFII. Immunohistochemical staining of breast tumor microarrays showed nuclear COUP-TFII and nucleolin staining was correlated in invasive ductal carcinomas. COUP-TFII staining correlated with ERα, SRC-1, AIB1, Pea3, MMP2, and phospho-Src and was reduced with increased tumor grade.ConclusionsOur data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII.

CtIP, a BRCA1 binding protein, is a transcriptional corepressor of estrogen receptor α.

Abstract Abstract #606 The role of estrogen and estrogen receptor (ER) transcriptional regulation in breast carcinogenesis is indisputable. In the last few years there has been dramatic progress in understanding how receptor transcriptional coactivator and corepressor complexes operate.
 We demonstrated that CtIP (CtBP interacting protein) is downregulated in tamoxifen resistant breast cancer lines. Furthermore, CtIP protein expression correlates with response to neo-adjuvant endocrine therapy and patients with progressive disease express lower CtIP protein in their primary breast carcinomas than those who respond. CtIP expression directly correlates with ER alpha expression, and inversely correlates with disease free survival and breast cancer metastasis. Importantly, silencing endogenous CtIP confers tamoxifen resistance in tamoxifen sensitive breast cancer cells and re-expression restored tamoxifen sensitivity in resistant breast cancer cells (Wu et al. Molec. Cancer Res. 2007; 5: 1285-1295). Our findings indicate that CtIP silencing is a novel mechanism for the development of tamoxifen resistance in breast cancer.
 CtIP interacts with the BRCT domains of BRCA1 where most mutations occur in familial BRCA1 breast cancers. CtIP also interacts with the transcriptional corepressor CtBP. Emerging evidence suggests that CtIP is involved in transcriptional repression and may be a tumor suppressor gene. We hypothesize that CtIP plays a key role in repression of ER transcription and such repressive function is linked to the inhibitory growth effects exerted by tamoxifen. We further speculate that CtIP inhibition of ER transcription is mediated by its ability to bridge BRCA1 with CtBP to form a transcriptional corepressor complex able to modulate ER activity via the interaction between BRCA1 and ER and that CtIP is in fact required for BRCA1 to exert its transcriptional repression activity on ER. We will present our work showing that CtIP, BRCA1 and CtBP form a multiprotein complex, that requires CtIP, and associates with ER in breast cancer cells. More importantly, we will show that BRCA1, CtIP and CtBP associate with ER on the ER-dependent promoter of the pS2 gene in the absence of ligand. Stimulation by ligand leads to dissociation of the complex and ER-dependent gene expression. Furthermore, silencing CtIP expression using RNAi in E2-starved MCF-7 cells led to an increase of basal transcription of the pS2 gene. CtIP was also observed to repress E2 stimulation of ERE-luciferase in transfected MCF-7 cells. We are currently characterizing the role of the CtIP corepressor complex in ER-transcriptional activity. We will present the results of our efforts to identify specific ER-dependent promoters repressed by the CtIP corepressor. In addition, we will present results of studies aimed at characterizing CtIP-specific changes in chromatin remodeling and ER-cofactor recruitment at ER-dependent promoters. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 606.

CtIP Silencing as a Novel Mechanism of Tamoxifen Resistance in Breast Cancer

Acquired resistance to the antiestrogen tamoxifen constitutes a major clinical challenge in breast cancer therapy. However, the mechanisms involved are still poorly understood. Using serial analysis of gene expression, we identified CtIP, a BRCA1- and CtBP-interacting protein, as one of the most significantly down-regulated transcripts in estrogen receptor alpha-positive (ER+) MCF-7 tamoxifen-resistant breast cancer cells. We further confirmed the association of CtIP down-regulation with tamoxifen resistance in an additional ER+ breast cancer line (T47D), strengthening the relevance of the phenomenon observed. In additional studies, we found CtIP protein expression in a majority of ER+ breast cancer cell lines that we tested, but no or very little CtIP expression in ER-negative lines. Furthermore, CtIP protein expression status correlates with clinical response to neoadjuvant endocrine therapy, and patients with progressive disease express significantly lower CtIP protein in their primary breast carcinomas than those who respond. Meta-analysis of seven publicly available gene expression microarray data sets showed that CtIP expression is significantly associated with ER, disease-free survival, and breast cancer metastasis status. Importantly, we found that silencing endogenous CtIP in tamoxifen-sensitive breast cancer cells confers tamoxifen resistance. On the other hand, reexpression of CtIP in tamoxifen-resistant breast cancer cells restores sensitivity to the inhibitory growth effects of tamoxifen. Together, our findings indicate that CtIP silencing might be a novel mechanism for the development of tamoxifen resistance in breast cancer, suggesting that CtIP is likely associated with ER function, and that CtIP gene and protein expression may be useful biomarkers for breast cancer prognosis and clinical management.

Identification of eight genes that are potentially involved in tamoxifen sensitivity in breast cancer cells

Although the antiestrogen agent tamoxifen has long been used to treat women with hormone receptor positive invasive breast carcinoma, the mechanisms of its action and acquired resistance to tamoxifen during treatment are largely unknown. A number of studies have revealed that over-activation of some signaling pathways can cause tamoxifen resistance; however, very little information is available regarding the genes whose loss-of-function alternation contribute to tamoxifen resistance. Here we used a forward genetic approach in vitro to generate tamoxifen resistant cells from the tamoxifen sensitive breast cancer cell line ZR-75-1, and further identified the disrupted gene in different tamoxifen resistant clones. Retinol binding protein 7, DNA polymerase-transactivated protein 3, g-glutamyltransferase-like activity 1, slit-robo RhoGTPase-activating protein, tetraspan NET-4, HSPC194, amiloride-sensitive epithelial sodium channel gene, and Notch2, were the eight mutated genes identified in different tamoxifen resistant clones, suggesting their requirement for tamoxifen sensitivity in ZR-75-1 cells. Since the functions of these genes are not related to each other, it suggests that multiple pathways can influence tamoxifen sensitivity in breast cancer cells.

Cyclin D1 expression is dependent on estrogen receptor function in tamoxifen-resistant breast cancer cells

The development of resistance to tamoxifen, the most common antiestrogen used in the treatment of breast cancer, is a frequent and severe clinical problem. Tamoxifen-resistant tumors are still capable of responding to other hormonal therapies such as those that downregulate estrogen receptor expression. Mechanisms leading to acquisition of tamoxifen-resistant but hormone-sensitive growth are not completely understood. In tamoxifen-sensitive breast cancer cells, tamoxifen inhibits, whereas estrogen induces, expression of cyclin D1, a key cell cycle regulatory protein. Ectopic expression of cyclin D1 can lead to antiestrogen resistance. Thus, to determine whether cyclin D1 is involved in the growth of tamoxifen-resistant cells, we developed several tamoxifen-resistant variants from MCF-7 cells. These variants grow in the absence of estrogen or in the presence of tamoxifen, but their growth is inhibited by estrogen receptor downregulators. We show here that cyclin D1 expression is maintained at comparable levels in all tamoxifen-resistant variants, whereas pS2, another estrogen-regulated protein, is not. The addition of physiological levels of estrogen further stimulates cyclin D1 expression and proliferation. In contrast, treatment with estrogen receptor downregulators decreases cyclin D1 expression and proliferation. Thus, changes in cyclin D1 expression upon second-line hormonal therapy may predict hormonal sensitivity of tamoxifen-resistant tumors. These studies suggest that estrogen receptor mediates cyclin D1 expression and growth of tamoxifen-resistant tumors.