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BACKGROUND: Reduction of immunosuppression (IS) upon detection of Polyomavirus (BK) viremia is widely used to prevent BK virus nephropathy. This retrospective case-control study assesses the frequency of de novo donor-specific antibodies (dnDSA) in renal transplant recipients with IS modulation due to BK viremia and the associated risk of antibody mediated rejection. METHODS: Our cohort included recipients of kidney transplantation between 2007 and 2017 with clinical, HLA antibody, and biopsy data. BK positivity was defined as viremia >10 000 c/ml or biopsy proven BK nephropathy. A total of 190 BK cases matched our inclusion criteria, each case was matched with two controls based on gender, donor type, and transplant within 1 year (N = 396). RESULTS: Despite lower number of HLA antigen mismatches (mean = 3.5 vs. 4.4, p < .001), dnDSA rates were higher in BK cases than in control group (22.1% vs. 13.9%, p = .02), with the majority detected following IS reduction for BK infection, and arising earlier posttransplant compared with no BK infection (294d vs. 434d, p < .001). Antibody mediated rejection rates were similar between cases and controls (8.9% and 8.3%, respectively), but rejection was more likely to occur earlier posttransplant in the BK cases (354d vs. 602d, p = .03). CONCLUSION: Our data suggest a link between IS reduction and the generation of dnDSA and/or rejection, supporting close monitoring for DSA in patients with reduced IS due to BK infection given their increased risk to develop dnDSA.
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Virus BK , Trasplante de Riñón , Infecciones por Polyomavirus , Infecciones Tumorales por Virus , Humanos , Trasplante de Riñón/efectos adversos , Estudios Retrospectivos , Estudios de Casos y Controles , Viremia , Terapia de Inmunosupresión/efectos adversos , Receptores de Trasplantes , Rechazo de Injerto/prevención & controlRESUMEN
Despite improvements in the treatment of endocrine-resistant metastatic disease using combination therapies in patients with estrogen receptor α (ERα) primary tumors, the mechanisms underlying endocrine resistance remain to be elucidated. Non-coding RNAs (ncRNAs), including microRNAs (miRNA) and long non-coding RNAs (lncRNA), are targets and regulators of cell signaling pathways and their exosomal transport may contribute to metastasis. Previous studies have shown that a low expression of miR-29a-3p and miR-29b-3p is associated with lower overall breast cancer survival before 150 mos. Transient, modest overexpression of miR-29b1-3p or miR-29a-3p inhibited MCF-7 tamoxifen-sensitive and LCC9 tamoxifen-resistant cell proliferation. Here, we identify miR-29b-1/a-regulated and non-regulated differentially expressed lncRNAs in MCF-7 and LCC9 cells using next-generation RNA seq. More lncRNAs were miR-29b-1/a-regulated in LCC9 cells than in MCF-7 cells, including DANCR, GAS5, DSCAM-AS1, SNHG5, and CRND. We examined the roles of miR-29-regulated and differentially expressed lncRNAs in endocrine-resistant breast cancer, including putative and proven targets and expression patterns in survival analysis using the KM Plotter and TCGA databases. This study provides new insights into lncRNAs in endocrine-resistant breast cancer.
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BACKGROUND: For many laboratories, autoimmune encephalopathy (AE) panels are send-out tests. These tests are expensive, and ordering patterns vary greatly. There is also a lack of consensus on which panel to order and poor understanding of the clinical utility of these panels. These challenges were presented to our newly formed, multidisciplinary, diagnostic stewardship committee (DSC). Through this collaboration, we developed an algorithm for ordering AE panels; combining diagnostic criteria with practice guidelines. METHODS: We analyzed test-ordering patterns in 2018 and calculated a true-positive rate based on clinical presentation and panel interpretation. An evidence-based approach was combined with input from the Department of Neurology to synthesize our algorithm. Efficacy of the algorithm (number of panels ordered, cost, and true positives) was assessed before and after implementation. RESULTS: In 2018, 77 AE-related panels were ordered, costing $137 510. The true-positive rate was 10%, although ordering multiple, similar panels for the same patient was common. Before implementing the algorithm (January 1-July 31, 2019), 55 panels were ordered, costing $105 120. The total true-positive rate was 3.6%. After implementation, 23 tests were ordered in a 5-month period, totaling $50 220. The true-positive rate was 13%. CONCLUSION: With the DSC-directed mandate, we developed an algorithm for ordering AE panels. Comparison of pre- and postimplementation data showed a higher true-positive rate, indicating that our algorithm was able to successfully identify the at-risk population for AE disorders. This was met with a 43% decrease in the number of tests ordered, with total cost savings of $25 000 over 5 months.
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Encefalitis , Enfermedad de Hashimoto , Algoritmos , Ahorro de Costo , Humanos , LaboratoriosRESUMEN
BACKGROUND: κ and λ free light chains (FLCs) are monitored to aid in the diagnosis of plasma cell disorders. Our goal was to validate the Diazyme Human κ and λ assays on Beckman Coulter UniCel DxC 800 Synchron and compare to Freelite κ and λ assays on Roche Cobas Integra. METHODS: Linearity verification, within- and between-run precision, method comparison, and reference range (RR) verification were conducted using CLSI guidelines. Statistical analysis was performed using EP Evaluator®. Mean, SD, CV, and bias were determined. RESULTS: Diazyme κ FLC assay was linear within 0.00-191.00 mg/L. Diazyme λ FLC assay was linear within 0.00-205.30 mg/L. Diazyme κ FLC QC1 had a mean of 16.70 mg/L, CV of 7.0%. QC2 had a mean of 33.37 mg/L, CV of 2.6%. Diazyme λ FLC QC1 had a mean of 21.73 mg/L, CV of 2.3%. QC2 had a mean of 42.05 mg/L, CV of 1.5%. Bias of DxC-Diazyme FLCs compared to Integra-Freelite FLCs was -2.55 mg/L (κ FLC), and 4.54 mg/L (λ FLC). Qualitative comparison of κ FLC assays indicated 100% agreement for both normal and abnormal values. For λ FLC assay, agreement was 95% for normal values and 75% for abnormal values. For κ/λ ratio there was 50% agreement for normal values, and 100% for abnormal values. For RR verification, 1 sample was outside the Diazyme κ RR. For λ, all samples were within the manufacturer's RR. CONCLUSIONS: Diazyme assays for FLCs have excellent precision and accuracy and are comparable to Freelite assays.
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Aprobación de Pruebas de Diagnóstico , Cadenas kappa de Inmunoglobulina/sangre , Cadenas lambda de Inmunoglobulina/sangre , Pruebas Inmunológicas/métodos , Pruebas Inmunológicas/normas , Humanos , Juego de Reactivos para Diagnóstico , Reproducibilidad de los Resultados , Estados Unidos/epidemiología , United States Food and Drug AdministrationRESUMEN
Liquid biopsy is a noninvasive dynamic approach for monitoring disease over time. It offers advantages including limited risks of blood sampling, opportunity for more frequent sampling, lower costs and theoretically non-biased sampling compared with tissue biopsy. There is a high degree of concordance between circulating tumor DNA mutations versus primary tumor mutations. Remote sampling of circulating tumor DNA can serve as viable option in clinical diagnostics. Here, we discuss the progress toward broad adoption of liquid biopsy as a diagnostic tool and discuss knowledge gaps that remain to be addressed.
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Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/genética , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Biopsia Líquida/métodos , Neoplasias/diagnóstico , Neoplasias/genética , Ácidos Nucleicos Libres de Células/orina , ADN de Neoplasias/orina , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Biopsia Líquida/tendencias , Mutación , Neoplasias/sangre , Pruebas de Farmacogenómica , Reacción en Cadena de la Polimerasa/métodos , PronósticoRESUMEN
Anacardic acid (AnAc), a potential dietary agent for preventing and treating breast cancer, inhibited the proliferation of estrogen receptor α (ERα) positive MCF-7 and MDA-MB-231 triple negative breast cancer cells. To characterize potential regulators of AnAc action, MCF-7 and MDA-MB-231 cells were treated for 6 h with purified AnAc 24:1n5 congener followed by next generation transcriptomic sequencing (RNA-seq) and network analysis. We reported that AnAc-differentially regulated miRNA transcriptomes in each cell line and now identify AnAc-regulated changes in mRNA and lncRNA transcript expression. In MCF-7 cells, 80 AnAc-responsive genes were identified, including lncRNA MIR22HG. More AnAc-responsive genes (886) were identified in MDA-MB-231 cells. Only six genes were commonly altered by AnAc in both cell lines: SCD, INSIG1, and TGM2 were decreased and PDK4, GPR176, and ZBT20 were increased. Modeling of AnAc-induced gene changes suggests that AnAc inhibits monounsaturated fatty acid biosynthesis in both cell lines and increases endoplasmic reticulum stress in MDA-MB-231 cells. Since modeling of downregulated genes implicated NFκB in MCF-7, we confirmed that AnAc inhibited TNFα-induced NFκB reporter activity in MCF-7 cells. These data identify new targets and pathways that may account for AnAc's anti-proliferative and pro-apoptotic activity.
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Ácidos Anacárdicos/farmacología , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Apoptosis , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Proliferación Celular , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Células Tumorales CultivadasRESUMEN
MicroRNAs are biomarkers and potential therapeutic targets for breast cancer. Anacardic acid (AnAc) is a dietary phenolic lipid that inhibits both MCF-7 estrogen receptor α (ERα) positive and MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation with IC50s of 13.5 and 35 µM, respectively. To identify potential mediators of AnAc action in breast cancer, we profiled the genome-wide microRNA transcriptome (microRNAome) in these two cell lines altered by the AnAc 24:1n5 congener. Whole genome expression profiling (RNA-seq) and subsequent network analysis in MetaCore Gene Ontology (GO) algorithm was used to characterize the biological pathways altered by AnAc. In MCF-7 cells, 69 AnAc-responsive miRNAs were identified, e.g., increased let-7a and reduced miR-584. Fewer, i.e., 37 AnAc-responsive miRNAs were identified in MDA-MB-231 cells, e.g., decreased miR-23b and increased miR-1257. Only two miRNAs were increased by AnAc in both cell lines: miR-612 and miR-20b; however, opposite miRNA arm preference was noted: miR-20b-3p and miR-20b-5p were upregulated in MCF-7 and MDA-MB-231, respectively. miR-20b-5p target EFNB2 transcript levels were reduced by AnAc in MDA-MB-231 cells. AnAc reduced miR-378g that targets VIM (vimentin) and VIM mRNA transcript expression was increased in AnAc-treated MCF-7 cells, suggesting a reciprocal relationship. The top three enriched GO terms for AnAc-treated MCF-7 cells were B cell receptor signaling pathway and ribosomal large subunit biogenesis and S-adenosylmethionine metabolic process for AnAc-treated MDA-MB-231 cells. The pathways modulated by these AnAc-regulated miRNAs suggest that key nodal molecules, e.g., Cyclin D1, MYC, c-FOS, PPARγ, and SIN3, are targets of AnAc activity.
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Ácidos Anacárdicos/farmacología , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Estudio de Asociación del Genoma Completo , MicroARNs/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Interferencia de ARN , ARN Mensajero/genética , TranscriptomaRESUMEN
Aberrant microRNA expression contributes to breast cancer progression and endocrine resistance. We reported that although tamoxifen stimulated miR-29b-1/a transcription in tamoxifen (TAM)-resistant breast cancer cells, ectopic expression of miR-29b-1/a did not drive TAM-resistance in MCF-7 breast cancer cells. However, miR-29b-1/a overexpression significantly repressed TAM-resistant LCC9 cell proliferation, suggesting that miR-29b-1/a is not mediating TAM resistance but acts as a tumor suppressor in TAM-resistant cells. The target genes mediating this tumor suppressor activity were unknown. Here, we identify miR-29b-1 and miR-29a target transcripts in both MCF-7 and LCC9 cells. We find that miR-29b-1 and miR-29a regulate common and unique transcripts in each cell line. The cell-specific and common downregulated genes were characterized using the MetaCore Gene Ontology (GO) enrichment analysis algorithm. LCC9-sepecific miR-29b-1/a-regulated GO processes include oxidative phosphorylation, ATP metabolism, and apoptosis. Extracellular flux analysis of cells transfected with anti- or pre- miR-29a confirmed that miR-29a inhibits mitochondrial bioenergetics in LCC9 cells. qPCR,luciferase reporter assays, and western blot also verified the ATP synthase subunit genes ATP5G1 and ATPIF1 as bone fide miR29b-1/a targets. Our results suggest that miR-29 repression of TAM-resistant breast cancer cell proliferation is mediated in part through repression of genes important in mitochondrial bioenergetics.
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Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Tamoxifeno/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Proliferación Celular , Antagonistas de Estrógenos/farmacología , Femenino , Humanos , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Transcriptoma , Células Tumorales CultivadasRESUMEN
miR-29b and miR-29a transcript levels were reported to increase in exponentially growing CHO-K1 cells. Here, we examine the regulation of miR-29b-1/a in CHO-K1 cells. We observed that 4-hydroxytamoxifen (4-OHT) increased pri-miR-29b-1 and pri-miR-29a transcription in CHO-K1 cells by activating endogenous estrogen receptor α (ERα). DICER, an established, bona fide target of miR-29b-1/a, was shown to be regulated by 4-OHT in CHO-K1 cells. We showed that miR-29b-1 and miR-29a serve a repressive role in cell proliferation, migration, invasion, and colony formation in CHO-K1 cells. To identify other targets of miR-29b-1 and miR-29a, RNA sequencing was performed by transfecting cells with anti-miR-29a, which inhibits both miR-29a and miR-29b-1, pre-miR-29b-1, and/or pre-miR-29a. In silico network analysis in MetaCore™ identified common and unique putative gene targets of miR-29b-1 and miR-29a. Pathway analysis of identified putative miR-29 targets were related to cell adhesion, cytoskeletal remodeling, and development. Further inquiry revealed regulation of pathways mediating responses to growth factor stimulus and cell cycle regulation.
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Regulación de la Expresión Génica , MicroARNs/genética , Transcripción Genética , Animales , Células CHO , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Cricetinae , Cricetulus , Receptor alfa de Estrógeno/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , MicroARNs/metabolismo , ARN Mensajero/metabolismo , Ribonucleasa III/metabolismo , Análisis de Secuencia de ARN , Tamoxifeno/farmacología , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Therapies targeting estrogen receptor α (ERα) including tamoxifen, a selective estrogen receptor modulator (SERM) and aromatase inhibitors (AI), e.g., letrozole, have proven successful in reducing the death rate for breast cancer patients whose initial tumors express ERα. However, about 40% of patients develop acquired resistance to these endocrine treatments. There is a critical need to develop sensitive circulating biomarkers that accurately identify signaling pathways altered in breast cancer patients resistant to endocrine therapies. Serum miRNAs have the potential to serve as biomarkers of the progression of endocrine-resistant breast cancer due to their cancer-specific expression and stability. Exosomal transfer of miRNAs has been implicated in metastasis and endocrine-resistance. This review focuses on miRNAs in breast tumors and in serum, including exosomes, from breast cancer patients that are associated with resistance to tamoxifen since it is best-studied.
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Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Antineoplásicos Hormonales/uso terapéutico , Inhibidores de la Aromatasa/uso terapéutico , Biomarcadores de Tumor/sangre , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Receptor alfa de Estrógeno/metabolismo , Exosomas/química , Exosomas/metabolismo , Exosomas/patología , Femenino , Humanos , Letrozol , MicroARNs/sangre , Invasividad Neoplásica , Metástasis de la Neoplasia , Nitrilos/uso terapéutico , Transducción de Señal , Tamoxifeno/uso terapéutico , Triazoles/uso terapéuticoRESUMEN
Endocrine-resistance develops in â¼40% of breast cancer patients after tamoxifen (TAM) therapy. Although microRNAs are dysregulated in breast cancer, their contribution to endocrine-resistance is not yet understood. Previous microarray analysis identified miR-29a and miR-29b-1 as repressed by TAM in MCF-7 endocrine-sensitive breast cancer cells but stimulated by TAM in LY2 endocrine-resistant breast cancer cells. Here we examined the mechanism for the differential regulation of these miRs by TAM in MCF-7 versus TAM-resistant LY2 and LCC9 breast cancer cells and the functional role of these microRNAs in these cells. Knockdown studies revealed that ERα is responsible for TAM regulation of miR-29b-1/a transcription. We also demonstrated that transient overexpression of miR-29b-1/a decreased MCF-7, LCC9, and LY2 proliferation and inhibited LY2 cell migration and colony formation but did not sensitize LCC9 or LY2 cells to TAM. Furthermore, TAM reduced DICER1 mRNA and protein in LY2 cells, a known target of miR-29. Supporting this observation, anti-miR-29b-1 or anti-miR-29a inhibited the suppression of DICER by 4-OHT. These results suggest miR-29b-1/a has tumor suppressor activity in TAM-resistant cells and does not appear to play a role in mediating TAM resistance.
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Neoplasias de la Mama/tratamiento farmacológico , Antagonistas de Estrógenos/uso terapéutico , MicroARNs/genética , Tamoxifeno/uso terapéutico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Diferenciación Celular , Proliferación Celular , Antagonistas de Estrógenos/farmacología , Femenino , Humanos , Tamoxifeno/farmacología , TransfecciónRESUMEN
Acquired tamoxifen (TAM) resistance is a significant clinical problem in treating patients with estrogen receptor α (ERα)+ breast cancer. We reported that ERα increases nuclear respiratory factor-1 (NRF-1), which regulates nuclear-encoded mitochondrial gene transcription, in MCF-7 breast cancer cells and NRF-1 knockdown stimulates apoptosis. Whether NRF-1 and target gene expression is altered in endocrine resistant breast cancer cells is unknown. We measured NRF-1and metabolic features in a cell model of progressive TAM-resistance. NRF-1 and its target mitochondrial transcription factor A (TFAM) were higher in TAM-resistant LCC2 and LCC9 cells than TAM-sensitive MCF-7 cells. Using extracellular flux assays we observed that LCC1, LCC2, and LCC9 cells showed similar oxygen consumption rate (OCR), but lower mitochondrial reserve capacity which was correlated with lower Succinate Dehydrogenase Complex, Subunit B in LCC1 and LCC2 cells. Complex III activity was lower in LCC9 than MCF-7 cells. LCC1, LCC2, and LCC9 cells had higher basal extracellular acidification (ECAR), indicating higher aerobic glycolysis, relative to MCF-7 cells. Mitochondrial bioenergetic responses to estradiol and 4-hydroxytamoxifen were reduced in the endocrine-resistant cells compared to MCF-7 cells. These results suggest the acquisition of altered metabolic phenotypes in response to long term antiestrogen treatment may increase vulnerability to metabolic stress.
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Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Metabolismo Energético , Factor Nuclear 1 de Respiración/metabolismo , Tamoxifeno/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Respiración de la Célula/efectos de los fármacos , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Estradiol/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ontología de Genes , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Subunidades de Proteína/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Tamoxifeno/análogos & derivados , Factores de Transcripción/metabolismoRESUMEN
Therapies targeting estrogen receptor alpha (ERα), including selective ER modulators such as tamoxifen, selective ER downregulators such as fulvestrant (ICI 182â780), and aromatase inhibitors such as letrozole, are successfully used in treating breast cancer patients whose initial tumor expresses ERα. Unfortunately, the effectiveness of endocrine therapies is limited by acquired resistance. The role of microRNAs (miRNAs) in the progression of endocrine-resistant breast cancer is of keen interest in developing biomarkers and therapies to counter metastatic disease. This review focuses on miRNAs implicated as disruptors of antiestrogen therapies, their bona fide gene targets and associated pathways promoting endocrine resistance.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Antagonistas de Estrógenos/uso terapéutico , MicroARNs/genética , Neoplasias de la Mama/patología , Femenino , HumanosRESUMEN
INTRODUCTION: The role of miRNAs in acquired endocrine-resistant breast cancer is not fully understood. One hallmark of tumor progression is epithelial-to-mesenchymal transition (EMT), characterized by a loss of cell adhesion resulting from reduced E-cadherin and increased cell mobility. miR-200 family members regulate EMT by suppressing expression of transcriptional repressors ZEB1/2. Previously we reported that the expression of miR-200a, miR-200b, and miR-200c was lower in LY2 endocrine-resistant, mesenchymal breast cancer cells compared to parental, endocrine sensitive, epithelial MCF-7 breast cancer cells. Here we investigated the regulation of miR-200 family members and their role in endocrine-sensitivity in breast cancer cells. RESULTS: miR-200 family expression was progressively reduced in a breast cancer cell line model of advancing endocrine/tamoxifen (TAM) resistance. Concomitant with miR-200 decrease, there was an increase in ZEB1 mRNA expression. Overexpression of miR-200b or miR-200c in LY2 cells altered cell morphology to a more epithelial appearance and inhibited cell migration. Further, miR-200b and miR-200c overexpression sensitized LY2 cells to growth inhibition by estrogen receptor (ER) antagonists TAM and fulvestrant. Knockdown of ZEB1 in LY2 cells recapitulated the effect of miR-200b and miR-200c overexpression resulting in inhibition of LY2 cell proliferation by TAM and fulvestrant, but not the aromatase inhibitor exemestane. Demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) in combination with histone deacetylase inhibitor trichostatin A (TSA) increased miR-200b and miR-200c in LY2 cells. Concomitant with the increase in miR-200b and miR-200c, ZEB1 expression was decreased and cells appeared more epithelial in morphology and were sensitized to TAM and fulvestrant inhibition. Likewise, knockdown of ZEB1 increased antiestrogen sensitivity of LY2 cells resulting in inhibition of cell proliferation. CONCLUSIONS: Our data indicate that reduced miRNA-200b and miR-200c expression contributes to endocrine resistance in breast cancer cells and that the reduced expression of these miR-200 family members in endocrine-resistant cells can be reversed by 5-aza-dC+TSA.