Publications by authors named "Lacey M Litchfield"

19 Publications

  • Page 1 of 1

Activation of the IFN Signaling Pathway is Associated with Resistance to CDK4/6 Inhibitors and Immune Checkpoint Activation in ER-Positive Breast Cancer.

Clin Cancer Res 2021 Feb 3. Epub 2021 Feb 3.

Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, Texas.

Purpose: Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) are highly effective against estrogen receptor-positive (ER)/HER2 breast cancer; however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes.

Experimental Design: Parental breast cancer cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen deprivation-resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes.

Results: Parental and EndoR breast cancer lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high IFN signaling and reduced CDK4/6i sensitivity; thus an "IFN-related palbociclib-resistance Signature" (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1 signaling compared with their short-term treated or untreated counterparts. In primary ER/HER2 tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis.

Conclusions: Aberrant IFN signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-4191DOI Listing
February 2021

The Genomic Landscape of Intrinsic and Acquired Resistance to Cyclin-Dependent Kinase 4/6 Inhibitors in Patients with Hormone Receptor-Positive Metastatic Breast Cancer.

Cancer Discov 2020 Aug 13;10(8):1174-1193. Epub 2020 May 13.

Center for Cancer Precision Medicine, Dana-Farber Cancer Institute, Boston, Massachusetts.

Mechanisms driving resistance to cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) in hormone receptor-positive (HR) breast cancer have not been clearly defined. Whole-exome sequencing of 59 tumors with CDK4/6i exposure revealed multiple candidate resistance mechanisms including loss, activating alterations in , and , and loss of estrogen receptor expression. experiments confirmed that these alterations conferred CDK4/6i resistance. Cancer cells cultured to resistance with CDK4/6i also acquired , or alterations, which conferred sensitivity to AURKA, ERK, or CHEK1 inhibition. Three of these activating alterations-in , and -have not, to our knowledge, been previously demonstrated as mechanisms of resistance to CDK4/6i in breast cancer preclinically or in patient samples. Together, these eight mechanisms were present in 66% of resistant tumors profiled and may define therapeutic opportunities in patients. SIGNIFICANCE: We identified eight distinct mechanisms of resistance to CDK4/6i present in 66% of resistant tumors profiled. Most of these have a therapeutic strategy to overcome or prevent resistance in these tumors. Taken together, these findings have critical implications related to the potential utility of precision-based approaches to overcome resistance in many patients with HR metastatic breast cancer..
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http://dx.doi.org/10.1158/2159-8290.CD-19-1390DOI Listing
August 2020

Combined inhibition of PIM and CDK4/6 suppresses both mTOR signaling and Rb phosphorylation and potentiates PI3K inhibition in cancer cells.

Oncotarget 2020 Apr 28;11(17):1478-1492. Epub 2020 Apr 28.

Eli Lilly and Company, Indianapolis, IN, USA.

Aberrant activation of mitogenic signaling pathways in cancer promotes growth and proliferation of cells by activating mTOR and S6 phosphorylation, and D-cyclin kinases and Rb phosphorylation, respectively. Correspondingly, inhibition of phosphorylation of both Rb and S6 is required for robust anti-tumor efficacy of drugs that inhibit cell signaling. The best-established mechanism of mTOR activation in cancer is via PI3K/Akt signaling, but mTOR activity can also be stimulated by CDK4 and PIM kinases. In this study, we show that the CDK4/6 inhibitor abemaciclib inhibits PIM kinase and S6 phosphorylation in cancer cells and concurrent inhibition of PIM, CDK4, and CDK6 suppresses both S6 and Rb phosphorylation. or mutations obviate the requirement for PIM kinase and circumvent the inhibition of S6 phosphorylation by abemaciclib. Combination with a PI3K inhibitor restored suppression of S6 phosphorylation and synergized to curtail cell growth. By combining abemaciclib with a PI3K inhibitor, three pathways (Akt, PIM, and CDK4) to mTOR activation are neutralized, suggesting a potential combination strategy for the treatment of -mutant ER+ breast cancer.
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http://dx.doi.org/10.18632/oncotarget.27539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197449PMC
April 2020

Mesothelial Cell HIF1α Expression Is Metabolically Downregulated by Metformin to Prevent Oncogenic Tumor-Stromal Crosstalk.

Cell Rep 2019 12;29(12):4086-4098.e6

Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, University of Chicago, Chicago, IL 60637, USA. Electronic address:

The tumor microenvironment (TME) plays a pivotal role in cancer progression, and, in ovarian cancer (OvCa), the primary TME is the omentum. Here, we show that the diabetes drug metformin alters mesothelial cells in the omental microenvironment. Metformin interrupts bidirectional signaling between tumor and mesothelial cells by blocking OvCa cell TGF-β signaling and mesothelial cell production of CCL2 and IL-8. Inhibition of tumor-stromal crosstalk by metformin is caused by the reduced expression of the tricarboxylic acid (TCA) enzyme succinyl CoA ligase (SUCLG2). Through repressing this TCA enzyme and its metabolite, succinate, metformin activated prolyl hydroxylases (PHDs), resulting in the degradation of hypoxia-inducible factor 1α (HIF1α) in mesothelial cells. Disruption of HIF1α-driven IL-8 signaling in mesothelial cells by metformin results in reduced OvCa invasion in an organotypic 3D model. These findings indicate that tumor-promoting signaling between mesothelial and OvCa cells in the TME can be targeted using metformin.
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http://dx.doi.org/10.1016/j.celrep.2019.11.079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218928PMC
December 2019

Unraveling the genomic determinants of response to CDK4/6 inhibitors.

Oncotarget 2018 Dec 7;9(96):36822-36823. Epub 2018 Dec 7.

Sean G. Buchanan: Eli Lilly and Company, Indianapolis, IN, USA.

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http://dx.doi.org/10.18632/oncotarget.26109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305152PMC
December 2018

The CDK4/6 Inhibitor Abemaciclib Induces a T Cell Inflamed Tumor Microenvironment and Enhances the Efficacy of PD-L1 Checkpoint Blockade.

Cell Rep 2018 03;22(11):2978-2994

Lilly Research Laboratories, Eli Lilly and Company, New York, NY 10016, USA. Electronic address:

Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6), has recently been approved for the treatment of hormone receptor-positive breast cancer. In this study, we use murine syngeneic tumor models and in vitro assays to investigate the impact of abemaciclib on T cells, the tumor immune microenvironment and the ability to combine with anti-PD-L1 blockade. Abemaciclib monotherapy resulted in tumor growth delay that was associated with an increased T cell inflammatory signature in tumors. Combination with anti-PD-L1 therapy led to complete tumor regressions and immunological memory, accompanied by enhanced antigen presentation, a T cell inflamed phenotype, and enhanced cell cycle control. In vitro, treatment with abemaciclib resulted in increased activation of human T cells and upregulated expression of antigen presentation genes in MCF-7 breast cancer cells. These data collectively support the clinical investigation of the combination of abemaciclib with agents such as anti-PD-L1 that modulate T cell anti-tumor immunity.
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http://dx.doi.org/10.1016/j.celrep.2018.02.053DOI Listing
March 2018

Genomic Aberrations that Activate D-type Cyclins Are Associated with Enhanced Sensitivity to the CDK4 and CDK6 Inhibitor Abemaciclib.

Cancer Cell 2017 Dec;32(6):761-776.e6

Eli Lilly and Company, Indianapolis, IN 46285, USA. Electronic address:

Most cancers preserve functional retinoblastoma (Rb) and may, therefore, respond to inhibition of D-cyclin-dependent Rb kinases, CDK4 and CDK6. To date, CDK4/6 inhibitors have shown promising clinical activity in breast cancer and lymphomas, but it is not clear which additional Rb-positive cancers might benefit from these agents. No systematic survey to compare relative sensitivities across tumor types and define molecular determinants of response has been described. We report a subset of cancers highly sensitive to CDK4/6 inhibition and characterized by various genomic aberrations known to elevate D-cyclin levels and describe a recurrent CCND1 3'UTR mutation associated with increased expression in endometrial cancer. The results suggest multiple additional classes of cancer that may benefit from CDK4/6-inhibiting drugs such as abemaciclib.
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http://dx.doi.org/10.1016/j.ccell.2017.11.006DOI Listing
December 2017

Metformin Targets Central Carbon Metabolism and Reveals Mitochondrial Requirements in Human Cancers.

Cell Metab 2016 11 13;24(5):728-739. Epub 2016 Oct 13.

Department of Pharmacology and Cancer Biology, Duke Cancer Institute, Duke Molecular Physiology Institute, Duke University School of Medicine, Durham, NC 27710, USA. Electronic address:

Repurposing metformin for cancer therapy is attractive due to its safety profile, epidemiological evidence, and encouraging data from human clinical trials. Although it is known to systemically affect glucose metabolism in liver, muscle, gut, and other tissues, the molecular determinants that predict a patient response in cancer remain unknown. Here, we carry out an integrative metabolomics analysis of metformin action in ovarian cancer. Metformin accumulated in patient biopsies, and pathways involving nucleotide metabolism, redox, and energy status, all related to mitochondrial metabolism, were affected in treated tumors. Strikingly, a metabolic signature obtained from a patient with an exceptional clinical outcome mirrored that of a responsive animal tumor. Mechanistically, we demonstrate with stable isotope tracing that these metabolic signatures are due to an inability to adapt nutrient utilization in the mitochondria. This analysis provides new insights into mitochondrial metabolism and may lead to more precise indications of metformin in cancer.
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http://dx.doi.org/10.1016/j.cmet.2016.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889952PMC
November 2016

Hyperglycemia-induced metabolic compensation inhibits metformin sensitivity in ovarian cancer.

Oncotarget 2015 Sep;6(27):23548-60

Department of Obstetrics and Gynecology, Gordon Center for Integrative Science, University of Chicago, Chicago, Illinois, USA.

Increasing interest in repurposing the diabetic medication metformin for cancer treatment has raised important questions about the translation of promising preclinical findings to therapeutic efficacy, especially in non-diabetic patients. A significant limitation of the findings to date is the use of supraphysiologic metformin doses and hyperglycemic conditions in vitro. Our goals were to determine the impact of hyperglycemia on metformin response and to address the applicability of metformin as a cancer therapeutic in non-diabetic patients. In normoglycemic conditions, lower concentrations of metformin were required to inhibit cell viability, while metformin treatment in hyperglycemic conditions resulted in increased glucose uptake and glycolytic flux, contributing to cell survival. Mechanistically, maintenance of c-Myc expression under conditions of hyperglycemia or via gene amplification facilitated metabolic escape from the effects of metformin. In vivo, treatment of an ovarian cancer mouse model with metformin resulted in greater tumor weight reduction in normoglycemic vs. hyperglycemic mice, with increased c-Myc expression observed in metformin-treated hyperglycemic mice. These findings indicate that hyperglycemia inhibits the anti-cancer effects of metformin in vitro and in vivo. Furthermore, our results suggest that metformin may elicit stronger responses in normoglycemic vs. hyperglycemic patients, highlighting the need for prospective clinical testing in patients without diabetes.
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http://dx.doi.org/10.18632/oncotarget.4556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4695136PMC
September 2015

Dehydroepiandrosterone Activation of G-protein-coupled Estrogen Receptor Rapidly Stimulates MicroRNA-21 Transcription in Human Hepatocellular Carcinoma Cells.

J Biol Chem 2015 Jun 11;290(25):15799-15811. Epub 2015 May 11.

Department of Biochemistry and Molecular Genetics, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, Kentucky 40292. Electronic address:

Little is known about the regulation of the oncomiR miR-21 in liver. Dehydroepiandrosterone (DHEA) regulates gene expression as a ligand for a G-protein-coupled receptor and as a precursor for steroids that activate nuclear receptor signaling. We report that 10 nm DHEA increases primary miR-21 (pri-miR-21) transcription and mature miR-21 expression in HepG2 cells in a biphasic manner with an initial peak at 1 h followed by a second, sustained response from 3-12 h. DHEA also increased miR-21 in primary human hepatocytes and Hep3B cells. siRNA, antibody, and inhibitor studies suggest that the rapid DHEA-mediated increase in miR-21 involves a G-protein-coupled estrogen receptor (GPER/GPR30), estrogen receptor α-36 (ERα36), epidermal growth factor receptor-dependent, pertussis toxin-sensitive pathway requiring activation of c-Src, ERK1/2, and PI3K. GPER antagonist G-15 attenuated DHEA- and BSA-conjugated DHEA-stimulated pri-miR-21 transcription. Like DHEA, GPER agonists G-1 and fulvestrant increased pri-miR-21 in a GPER- and ERα36-dependent manner. DHEA, like G-1, increased GPER and ERα36 mRNA and protein levels. DHEA increased ERK1/2 and c-Src phosphorylation in a GPER-responsive manner. DHEA increased c-Jun, but not c-Fos, protein expression after 2 h. DHEA increased androgen receptor, c-Fos, and c-Jun recruitment to the miR-21 promoter. These results suggest that physiological concentrations of DHEA activate a GPER intracellular signaling cascade that increases pri-miR-21 transcription mediated at least in part by AP-1 and androgen receptor miR-21 promoter interaction.
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http://dx.doi.org/10.1074/jbc.M115.641167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505488PMC
June 2015

Molecular pathways: trafficking of metabolic resources in the tumor microenvironment.

Clin Cancer Res 2015 Feb;21(4):680-6

Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, Center for Integrative Science, University of Chicago, Chicago, Illinois.

A model of tumor metabolism is proposed that describes how the complementary metabolic functions of the local stroma and the tumor cells contribute to cancer progression. Cancer cells alter the metabolism of cancer-associated fibroblasts to obtain lactate and amino acids, which are utilized for energy production, rapid growth, and resistance to chemotherapy drugs. Cancer cells use glutamine supplied by cancer-associated fibroblasts to replenish tricarboxylic acid cycle intermediates and as a nitrogen source for nucleotide synthesis. Moreover, adipocytes in the microenvironment attract cancer cells through the secretion of inflammatory cytokines and proteases. The cancer cells then induce metabolic changes in the adipocytes to acquire free fatty acids that are oxidized by cancer cells to generate energy for proliferation. Increasing knowledge about the metabolic symbiosis within the tumor has led to novel therapeutic strategies designed to restrict metabolic adaptation, including inhibiting lactate transporters and repurposing antidiabetic drugs (thiazolidinediones, metformin).
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http://dx.doi.org/10.1158/1078-0432.CCR-14-2198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333734PMC
February 2015

Metformin inhibits ovarian cancer growth and increases sensitivity to paclitaxel in mouse models.

Am J Obstet Gynecol 2015 Apr 19;212(4):479.e1-479.e10. Epub 2014 Oct 19.

Department of Obstetrics and Gynecology, Gordon Center for Integrative Science, University of Chicago, Chicago, IL. Electronic address:

Objective: There is increasing preclinical evidence indicating that metformin, a medication commonly used for type 2 diabetes mellitus, may protect against cancer. Motivated by this emerging evidence we asked 2 questions: (1) can metformin prevent ovarian cancer growth by altering metabolism and (2) will metformin increase sensitivity to chemotherapy.

Study Design: The effect of metformin in ovarian cancer was tested in vitro and with 2 different mouse models. In vitro, cell lines (n = 6) were treated with metformin (10-40 mmol/L) or phosphate-buffered saline solution and cellular proliferation and metabolic alterations (adenosine monophosphate-activated protein kinase activity, glycolysis, and lipid synthesis) were compared between the 2 groups. In mouse models, a prevention study was performed by treating mice with metformin (250 mg/kg/d intraperitoneally) or placebo for 2 weeks followed by intraperitoneal injection of the SKOV3ip1 human ovarian cancer cell line, and the mean number of tumor implants in each treatment group was compared. In a treatment study, the LSL-K-ras(G12D/+)/PTEN(floxP/floxP) genetic mouse model of ovarian cancer was used. Mice were treated with placebo, paclitaxel (3 mg/kg/wk intraperitoneally for 7 weeks), metformin (100 mg/kg/d in water for 7 weeks), or paclitaxel plus metformin, and tumor volume was compared among treatment groups.

Results: In vitro, metformin decreased proliferation of ovarian cancer cell lines and induced cell cycle arrest, but not apoptosis. Further analysis showed that metformin altered several aspects of metabolism including adenosine monophosphate-activated protein kinase activity, glycolysis, and lipid synthesis. In the prevention mouse model, mice that were pretreated with metformin had 60% fewer tumor implants compared with controls (P < .005). In the treatment study, mice that were treated with paclitaxel plus metformin had a 60% reduction in tumor weight compared with controls (P = .02), which is a level of tumor reduction greater than that resulting from either paclitaxel or metformin alone.

Conclusion: Based on these results, we conclude that metformin alters metabolism in ovarian cancer cells, prevents tumor growth, and increases sensitivity to chemotherapy in vitro and in mouse models. These preclinical findings suggest that metformin warrants further investigation for use as an ovarian cancer therapeutic.
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http://dx.doi.org/10.1016/j.ajog.2014.10.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4387077PMC
April 2015

Dehydroepiandrosterone-induces miR-21 transcription in HepG2 cells through estrogen receptor β and androgen receptor.

Mol Cell Endocrinol 2014 Jul 17;392(1-2):23-36. Epub 2014 May 17.

Department of Biochemistry & Molecular Biology, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, KY 40292, USA. Electronic address:

Although oncomiR miR-21 is highly expressed in liver and overexpressed in hepatocellular carcinoma (HCC), its regulation is uncharacterized. We examined the effect of physiologically relevant nanomolar concentrations of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEA-S) on miR-21 expression in HepG2 human hepatoma cells. 10nM DHEA and DHEA-S increase pri-miR-21 transcription in HepG2 cells. Dietary DHEA increased miR-21 in vivo in mouse liver. siRNA and inhibitor studies suggest that DHEA-S requires desulfation for activity and that DHEA-induced pri-miR-21 transcription involves metabolism to androgen and estrogen receptor (AR and ER) ligands. Activation of ERβ and AR by DHEA metabolites androst-5-ene-3,17-dione (ADIONE), androst-5-ene-3β,17β-diol (ADIOL), dihydrotestosterone (DHT), and 5α-androstane-3β,17β-diol (3β-Adiol) increased miR-21 transcription. DHEA-induced miR-21 increased cell proliferation and decreased Pdcd4 protein, a bona fide miR-21. Estradiol (E2) inhibited miR-21 expression via ERα. DHEA increased ERβ and AR recruitment to the miR-21 promoter within the VMP1/TMEM49 gene, with possible significance in hepatocellular carcinoma.
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http://dx.doi.org/10.1016/j.mce.2014.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4074919PMC
July 2014

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

Int J Oncol 2014 Apr 10;44(4):1365-75. Epub 2014 Feb 10.

Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, KY 40292, USA.

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.
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http://dx.doi.org/10.3892/ijo.2014.2294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977804PMC
April 2014

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

Cancer Lett 2014 May 7;347(1):139-50. Epub 2014 Feb 7.

Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, KY 40292, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.canlet.2014.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4021391PMC
May 2014

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

Mol Cell Endocrinol 2014 Jan 17;382(1):358-367. Epub 2013 Oct 17.

Department of Biochemistry & Molecular Biology, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, KY 40292, USA. Electronic address:

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5089806PMC
http://dx.doi.org/10.1016/j.mce.2013.10.010DOI Listing
January 2014

Reduced expression of miR-200 family members contributes to antiestrogen resistance in LY2 human breast cancer cells.

PLoS One 2013 23;8(4):e62334. Epub 2013 Apr 23.

Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, Kentucky, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062334PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633860PMC
November 2013

Multiple roles of COUP-TFII in cancer initiation and progression.

J Mol Endocrinol 2012 Dec 10;49(3):R135-48. Epub 2012 Oct 10.

Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, Kentucky, USA.

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an orphan nuclear receptor that acts as a transcriptional activator or repressor in a cell type-dependent manner. Best characterized for its role in the regulation of angiogenesis during mouse development, COUP-TFII also plays important roles in glucose metabolism and cancer. Expression of COUP-TFII is altered in various endocrine conditions. Cell type-specific functions and the regulation of COUP-TFII expression result in its varying physiological and pathological actions in diverse systems. Evidence will be reviewed for oncogenic and tumor-suppressive functions of COUP-TFII, with roles in angiogenesis, metastasis, steroidogenesis, and endocrine sensitivity of breast cancer described. The applicability of current data to our understanding of the role of COUP-TFII in cancer will be discussed.
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http://dx.doi.org/10.1530/JME-12-0144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3469769PMC
December 2012

Identification and characterization of nucleolin as a COUP-TFII coactivator of retinoic acid receptor β transcription in breast cancer cells.

PLoS One 2012 31;7(5):e38278. Epub 2012 May 31.

Department of Biochemistry & Molecular Biology and Center for Genetics and Molecular Medicine, Louisville, Kentucky, United States of America.

Introduction: The 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.

Results: FLAG-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.

Conclusions: Our data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0038278PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365040PMC
October 2012