Estrogen Receptor a Promotes Breast Cancer by Reprogramming Choline Metabolism · Choline (Cho) is an essential nutrient that is necessary for cell membrane synthesis and functions

Integrated Systems and Technologies

Estrogen Receptor a Promotes Breast Cancerby Reprogramming Choline MetabolismMin Jia1, Trygve Andreassen2, Lasse Jensen3,4, Tone Frost Bathen5, Indranil Sinha1,Hui Gao1, Chunyan Zhao1, Lars-Arne Haldosen1, Yihai Cao3, Leonard Girnita6,Siver Andreas Moestue5, and Karin Dahlman-Wright1

Abstract

Estrogen receptora (ERa) is a key regulator of breast growth andbreast cancer development. Here, we report how ERa impactsthese processes by reprogramming metabolism in malignantbreast cells. We employed an integrated approach, combininggenome-wide mapping of chromatin-bound ERa with estrogen-induced transcript and metabolic profiling, to demonstrate thatERa reprogramsmetabolismupon estrogen stimulation, includingchanges in aerobic glycolysis, nucleotide and amino acid synthesis,and choline (Cho) metabolism. Cho phosphotransferase CHPT1,

identified as a direct ERa-regulated gene, was required for estro-gen-induced effects on Cho metabolism, including increasedphosphatidylcholine synthesis. CHPT1 silencing inhibited anchor-age-independent growth and cell proliferation, also suppressingearly-stage metastasis of tamoxifen-resistant breast cancer cellsin a zebrafish xenograft model. Our results showed that ERa pro-motes metabolic alterations in breast cancer cells mediated byits target CHPT1, which this study implicates as a candidate thera-peutic target. Cancer Res; 76(19); 5634–46. �2016 AACR.

IntroductionComplementary to viewing cancer as a genetic disease, cancer

can be considered a metabolic disease (1, 2). Altered metabolismin cancer directs enhanced nutrient acquisition and facilitatesassimilation of carbon into macromolecules, such as lipids,proteins, and nucleic acids. The net effect of these activities is tosupport cell growth and proliferation (3–6).

17b-Estradiol (E2) and its receptor estrogen receptor a (ERa)have been implicated in promoting proliferation, survival, andmigration of breast cancer cells through multiple mechanisms,thereby contributing to tumor growth and progression (7, 8).IndividualswithERa-positive (ERaþ) breast cancer asdeterminedby IHC account for approximately 70% of breast cancer patients.Comparative metabolomics profiling of ERaþ and ERa-negative(ERa�) breast cancer indicated clear metabolic differences corre-

lated to hormone receptor status, including differences in gluta-mine and b-alanine metabolism, as well as phospholipid metab-olism (9–11). Furthermore, estrogen stimulation enhanced therate of glucose consumption, lactate production (aerobic glycol-ysis), and glutamate synthesis and decreased the level of phos-phocholine (PCho) in breast cancer cell lines (12–14).

Choline (Cho) is an essential nutrient that is necessary for cellmembrane synthesis and functions as an important methyldonor (15, 16). Routing of Cho through its various metabolicpathways is cell and tissue specific (17). Following uptake ofCho, the intracellular metabolism of Cho is partitioned alongtwo major pathways: (i) converted to PCho for the synthesis ofphosphatidylcholine (PtdCho), a major constituent of cell mem-branes; or (ii) oxidation to produce the methyl donor betaine(16). Abnormally high synthesis of PtdCho via the cytidinediphosphate–choline (CDP-Cho) pathway, where CTP:phos-phocholine cytidylyltransferase (CCT) has been identified as therate-limiting enzyme, is generally recognized as a metabolichallmark of cancer (18, 19). PtdCho can also be synthesizedthrough methylation of phosphatidylethanolamine (PE) byphosphatidylethanolamine N-methyltransferase (PEMT; ref. 20).The PEMT gene has been shown to be induced by estrogen inhepatocytes (20).

Increased levels of PCho and total Cho-containing metabo-lites have been identified as markers for breast cancer (21).Furthermore, increased synthesis of PtdCho is one of the earliestmetabolic events associated with the initial stimulation of cellgrowth and proliferation by tumor promoters in normal cells(22–24). PtdCho has also been found to be increased in breastcancer cells by tumor promoter (25). Consistently, humanbreast cancer cells have been shown to have higher levels ofPtdCho than normal human mammary epithelial cells (26).Underlying mechanisms and potential drug targets in abnormalCho phospholipid metabolism have been widely investigated indifferent cancers, as reviewed in ref. 19. Overall, the regulation

1Department of Biosciences and Nutrition, Novum, Karolinska Institu-tet, Huddinge, Sweden. 2Department of Circulation andMedical Imag-ing, MR Core Facility, Norwegian University of Science and Technol-ogy, Trondheim, Norway. 3Department of Microbiology, Tumor, andCell Biology, Karolinska Institutet, Stockholm, Sweden. 4Departmentof Medical and Health Sciences, Unit of Cardiovascular Medicine,Link€oping University, Link€oping, Sweden. 5Department of Circulationand Medical Imaging, Norwegian University of Science and Technol-ogy, Trondheim, Norway. 6Department of Oncology and Pathology,Karolinska Institutet and Karolinska University Hospital, Stockholm,Sweden.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Min Jia, Department of Biosciences and Nutrition,Karolinska Institute, H€alsov€agen 7-9, Stockholm 14183, Sweden. Phone: 468-5248-1221; Fax: 468-5248-1130; E-mail: [email protected]; and Karin Dahlman-Wright, [email protected]

doi: 10.1158/0008-5472.CAN-15-2910

�2016 American Association for Cancer Research.

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of Cho phospholipid metabolism in breast cancer cells has beenshown to depend on breast cancer subtype with respect to geneexpression profiles and metabolic fingerprints (27).

Several studies have provided important insights into globalestrogen-regulated gene networks based on profiling ERa-bind-ing regions and estrogen-regulated expression (28–33). Toextend global estrogen-regulated networks to effects on breastcancer metabolism, we report a comprehensive analysis ofestrogen-regulated metabolic pathways in two breast cancercell lines. We integrate cistrome, transcriptome, and metabo-lome data to identify metabolic pathways regulated by estrogensignaling via ERa. We focus on effects conserved between twoERaþ breast cancer cell lines with the aim to identify generaleffectors of metabolic signaling rather than cell type-specificeffects.

Materials and MethodsAdditional and detailed methods are included in the Supple-

mentary Materials and Methods.

Cell cultureMCF7 cells developed at the Michigan Cancer Foundation

(Detroit, MI) were kindly provided by Dr. Robert P.C. Shiu(University of Manitoba, Winnipeg, Manitoba, Canada; 2012).T47D cells were purchased from the ATCC (2004). Tamoxifen-sensitive MCF7 cells and tamoxifen-resistant LCC2 cells werekindly provided by Dr. Janne Lehti€o (Karolinska Institutet, Stock-holm, Sweden; 2012). LCC2 cells originate from MCF-7 cells.These cell lines were authenticated by short tandem repeat pro-filing (Uppsala Genome Center, Uppsala, Sweden) in June 2016.MCF7 and LCC2 cells were maintained in DMEM supplementedwith 10% FBS and 1% penicillin/streptomycin (Gibco). T47Dcells were grown in RPMI1640 supplemented with 10% FBS and1%penicillin/streptomycin. DMEMandRPMI1640 culturemedi-um contain 28.57 and 21.43 mmol/L Cho, respectively. Thecontent of Cho and Cho phospholipid in bovine serum is notprovided by themanufacturer. No additional growth factors wereadded to the cell culture medium.

Chromatin immunoprecipitation followed by sequencingor qPCR

Chromatin immunoprecipitation (ChIP) was performed asdescribed previously (34).

Gene expression microarray analysisThe gene expression data for MCF7 cells has been published

previously (33). For T47D cells, Human Gene 2.1 ST Arrays wereused for analysis of global gene expression profiling. The micro-array data are deposited in GEO (accession number GSE36683and GSE74034 for MCF7 and T47D cells, respectively).

Gene ontology (GO) analysis and identification of enrichedpathways were performed using the WEB-based GEne SeT AnaL-ysis Toolkit.

Nuclear magnetic resonance spectroscopyWater-soluble metabolites were extracted using ethanol.

Nuclear magnetic resonance (NMR) spectra were recorded ona Bruker Avance III 600 MHz spectrometer. A multivariatecomparison of metabolic profiles from estrogen-stimulated andcontrol cells was performed using partial least squares discrim-

inant analysis on Pareto-scaled NMR spectra using PLS_Tool-box v7.5.2 (Eigenvector Research Inc.). This technique identi-fies linear combinations of metabolic features referred to aslatent variables (LV) that discriminate between classes of sam-ples. Quantification was performed by binning spectral regionscontaining signals from identified metabolites. To aid in theidentification of these metabolites, Chenomx NMR suite v7.7(Chenomx Inc.) was used. In addition, various 2D NMR spectra(HSQC, HMBC, COSY, TOCSY) were recorded to assureidentification.

Tissue microarray analysisCHPT1 expression in human breast cancers was analyzed in

tissue microarrays (TMA; US BioMax BR1503d) by IHC. Anti-CHPT1 antibody was from The Human Protein Atlas.

Zebrafish metastatic modelTamoxifen-sensitive MCF7 cells and tamoxifen-resistant LCC2

cells were used. The zebrafishmetastaticmodel was established asdescribed previously (35).

ResultsA core set of direct ERa-regulated genes in ERaþ breastcancer cells

To identify a core set of direct ERa-regulated genes, wecombined genomewide ERa-binding profiles with detailed tran-script profiling for the two breast cancer cell lines, MCF7 andT47D. A total of 18,040 and 12,659 ERa-binding regions wereidentified for MCF7 cells and T47D cells, respectively (Fig. 1A).General properties of the identified ERa-binding regions, suchas peak distribution and enriched motifs, are consistent withpreviously published studies (Supplementary Fig. S1A–S1C).Overlaying the MCF7 and T47D cistromes revealed 6,480 sharedbinding regions, corresponding to 36% and 51% of the MCF7and T47D cistromes, respectively (Fig. 1A and B). The sharedbinding regions overlapped with published ERa cistromes forbreast cancer cell lines (Supplementary Fig. S1D).

Global gene expression profiling revealed 2,531 and 1,800estrogen-regulated genes in MCF7 and T47D cells, respectively(Fig. 1C). Overlaying the estrogen-regulated MCF7 and T47Dtranscriptomes identified 420 common estrogen-induced and348 common estrogen-repressed genes (Fig. 1C). ERa bindingwas enriched in regions associatedwith estrogen-induced genes ascompared with repressed genes for both MCF7 and T47D cells(Fig. 1D). To further identify a core set of direct ERa target genescommon to MCF7 and T47D cells, we combined genome-wideERa-binding profiles and estrogen-regulated transcript profilesfocusing on the 4,739 ERa-binding regions within 25 kb up- anddown-stream of TSSs of their most proximal genes. Integratingthese ERa-binding regions with estrogen-regulated transcriptprofiling, we identified a core set of 207 direct ERa target genes(Supplementary Table S1). A number of well-established directERa target genes were included in the identified core set, such astrefoil factor 1 (TFF1), growth regulation by estrogen in breastcancer 1 (GREB1), and progesterone receptor (PR). A majority(71%) of the core set of direct ERa target genes were inducedby E2 treatment (Fig. 1E).

GO pathway analysis for the core set of direct ERa targetgenes showed significant enrichment of cancer pathways. Inter-estingly, metabolic pathways were also enriched (Fig. 1F).

ERa Reprograms Cell Metabolism

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Figure 1.

A core set of direct ERa-regulated genes in breast cancer cells. A, Venn diagram showing overlap of ERa cistromes between MCF7 and T47D cells. Cellswere cultured in steroid-depleted media and treated with 10 nmol/L E2 or ethanol for 45 minutes. Genome-wide ERa-binding sites were determined by ChIP-seq. B, peak intensity heatmaps of ERa-binding regions in a �5 kb, relative to the TSS, genomic window. C, overlap of estrogen-induced up- anddownregulated genes, respectively, for MCF7 and T47D cells. Cells were cultured in steroid-depleted media and treated with 10 nmol/L E2 or ethanol for 6hours. Estrogen-stimulated gene expression was assayed by microarray analysis (n ¼ 4 for MCF7 cells; n ¼ 3 for T47D cells). D, correlation of estrogen-regulated gene expression with ERa-binding intensity. Heatmaps show the expression changes of all genes that were ranked on the basis of high tolow fold change values in MCF7 or T47D cells. The line graphs represent the moving average plots (window size, 100; step size, 1), which were plotted asa function of average ERa-binding of genes. These genes were arranged according to the heatmaps. E, a core set of direct ERa up- and downregulatedgenes in breast cancer cells. Overlay of ERa cistromes with estrogen up- and downregulated genes common to MCF7 and T47D cells. F, GO networkanalysis for the core set of direct ERa-regulated genes. G, direct ERa-regulated metabolic genes. ERa binding is presented as the score obtained frompeak analysis using MACS. Estrogen-stimulated gene expression is illustrated as fold change (FC) of estrogen treatment versus vehicle control.

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Overlaying the identified core set of direct ERa target genes with1,620 metabolic enzymes extracted from the KEGG databaserevealed that 19 genes encoding metabolic enzymes were directERa target genes in the two investigated cell lines, includingestrogen upregulation of ADCY9, B4GALT1, CA12, CHPT1,CHSY1, ENTNK2, FHL2, ITPK1, MBOAT1, PISD, PTGES, andSLC27A2 and estrogen downregulation of ABCC5, ABCG1,ACSL1, CYP1A1, CYP1A2, RXRA, and ST3GAL1 (Fig. 1G).

Estrogen signaling leads to global metabolic reprogrammingin breast cancer cells

We determined the effect of estrogen signaling on levels ofintracellular and extracellular metabolites using proton NMR(1H NMR). Figure 2A demonstrates a clear effect of estrogensignaling on the intracellular metabolic profile for both celllines. Samples from estrogen-treated cells were more separatedfrom the control samples along LV1 for MCF7 cells comparedwith T47D cells, indicating a stronger metabolic response to E2stimulation in MCF7 cells. Notably, the metabolic profiles fromMCF7 and T47D cells were clearly separated along LV2 (Fig. 2A),indicating that the metabolic characteristics of MCF7 and T47Dare inherently different. Quantitative analyses of the NMRspectra normalizing to protein levels are shown in Supplemen-tary Table S2. Notably, the area-normalized spectra were usedfor identification of changed metabolites (Supplementary TableS3). For the MCF7 cell line, 29 unique metabolites were quan-tified. Levels of 19 of these metabolites were significantlymodulated (FDR-adjusted P < 0.05) upon estrogen treatment(Supplementary Table S3; Supplementary Fig. S2A). For theT47D cell line, levels of 13 of 29 metabolites were significantlymodulated upon estrogen treatment (Supplementary Table S3;Supplementary Fig. S2B). All estrogen-modulated intracellularmetabolites, seven of which were changed in both cell lines,were mapped to metabolic pathways (Fig. 2B). Estrogen mod-ulated amino acid synthesis in both cell lines (SupplementaryFig. S2A and S2B), resulting in increased phenylalanine, tyro-sine, and 1-methyl histidine levels (Fig. 2B). In addition, estro-gen modulated the Cho metabolic pathway, with PCho beingreduced in both cell lines (Fig. 2B). However, we also observeddifferential effects of estrogen on Cho-containing metabolitesbetween these two cell lines, with glycerophosphocholine(GPC) levels being reduced in MCF7 cells and Cho levelsincreased in T47D cells (Supplementary Fig. S2A and S2B).

Analysis of extracellular metabolites revealed that upon estro-gen treatment, MCF7 cells consumedmore glucose and producedmore lactate (Fig. 2C and D), leading to a higher lactate/glucoseratio (Fig. 2E). Surprisingly, T47D cells consumed significantlyhigher amounts of glucose thanMCF7 cells regardless of estrogentreatment (Fig. 2C, where the extracellular concentration of glu-cose is much lower for T47D cells compared with MCF7 cells).Similarly as observed for MCF7 cells, estrogen stimulationenhanced glucose consumption and the lactate/glucose ratio forT47D cells (Fig. 2C and E), supporting elevated aerobic glycolysisupon activation of estrogen signaling in ERaþ breast cancer cells.However, estrogen treatment did not affect lactate levels in T47Dcells (Fig. 2D).

Estrogen signaling regulates transcripts and metabolites ofthe Cho metabolic pathway in breast cancer cells

The glycerophospholipid pathway, which includes the Chometabolic pathway, was enriched for direct ERa target genes

common to MCF7 and T47D cells (Fig. 1F). Specifically, of the119 genes involved in the KEGG Homo sapiens glyceropho-spholipid pathway hsa: 00564 (27), 26 and 10 were regulatedby estrogen in MCF7 and T47D cells, respectively (Fig. 3A).Regulation of a subset of these genes was confirmed usingqPCR (Fig. 3B).

In addition, metabolic profiling confirmed alterations in Chometabolism in response to estrogen signaling for these two celllines (Fig. 2B and Supplementary Fig. S2A and S2B). Figure 3Cshows changes in the levels of metabolites in the Cho metabolicpathway upon estrogen stimulation. NMR metabolic profilingof polar extracts is not suitable for determination of the lipid-soluble metabolite PtdCho, and CDP-Cho is present in too lowconcentration for detection by NMR. To obtain a more com-plete overview of the effects of estrogen on Cho metabolism, weassayed these metabolites by alternative assays, that is, PtdCholevels by PtdCho Assay Kit and CDP-Cho levels by LC-MS. Weobserved increased levels of CDP-Cho in MCF7 cells afterestrogen stimulation, while no difference in T47D cells wasobserved (Fig. 3C). Interestingly, PtdCho levels were signifi-cantly increased 24 hours after estrogen treatment in bothMCF7 and T47D cells (Fig. 3D). Furthermore, CHPT1, the directupstream enzyme to catalyze PtdCho synthesis, was identifiedas the only direct ERa target gene in the Cho pathway, whichwas upregulated upon estrogen stimulation in both analyzedcells (Fig. 3A, E, and F).

All estrogen-regulated genes and metabolites in the Chometabolic pathway are indicated in Fig. 3G. The expression oftransmembrane Cho transporters, solute carrier family 44, mem-bers 1 and 2 (SLC44A1, SLC44A2) encoding CTL1 and CTL2,was reduced significantly after estrogen stimulation in MCF7cells (Fig. 3B). The expression of CHKB and CHKA, which areresponsible for Cho phosphorylation, was reduced by estrogenin MCF7 and T47D cells, respectively (Fig. 3B). Consistently,PCho levels were decreased in both cell lines (SupplementaryTable S4). Phospholipase A2, group VI (PLA2G6) was down-regulated upon estrogen stimulation in MCF7 cells (Fig. 3B),which may result in decreased levels of its downstream productGPC, which is consistent with reduced GPC levels in this cell linein response to estrogen stimulation. The expression of PLCD1and PLCE1 was significantly decreased in estrogen-treated MCF7cells (Fig. 3B). Another isoform of PLC, PLCB3, was down-regulated by estrogen in T47D cells (Fig. 3B). Downregulationof PLCs may result in less PCho production, consistent withwhat was observed for MCF7 and T47D cells (SupplementaryTable S4). However, it should be noted that changes in geneexpression do not necessarily translate into changes in enzymeactivity and metabolite concentrations.

GPC is formed by the deacylation of PtdCho. ElevatedPCho/GPC ratio has been observed in breast cancer cell linescomparedwith normal breast epithelial cells (36). Furthermore, ithas been proposed that this ratio can predict on breast canceraggressiveness (37, 38). Our results show that estrogen stimula-tion increased the PCho/GPC ratio inMCF7 cells (SupplementaryTable S4).On the contrary, in T47D cells, the PCho/GPC ratiowasreduced (Supplementary Table S4).

Estrogen stimulation increases the activity of CCTaCCTa is a key enzyme in the CDP–choline pathway for

de novo PtdCho biosynthesis. This enzyme is inactivated whenit is phosphorylated and activated by a phosphatase, which






Figure 2.

ERa reprograms cell metabolism. Cells were cultured in steroid-depleted media and treated with 10 nmol/L E2 or ethanol for 24 hours. Intra- and extracellularmetabolites were extracted and analyzed by 1H NMR. A, partial least squares discriminant analysis of NMR identified intracellular metabolites in MCF7and T47D cells. Data are plotted using two latent variables (LV1 and LV2; n ¼ 5). B, metabolic profile of MCF7 and T47D cells in response to estrogenstimulation. Metabolites in red, metabolic changes observed only in MCF7 cells; metabolites in green, metabolic changes observed only in T47D cells;metabolites in blue, metabolic changes observed in both MCF7 and T47D cells. Full names of the metabolites are shown in Supplementary Data. TCA,the citric acid cycle. C, decreased glucose levels in the culture medium in response to estrogen treatment for MCF7 and T47D cells. EtOH, ethanol. D,lactate levels in the culture medium in response to estrogen. Increased lactate levels are observed in MCF7 cells but not in T47D cells. E, increasedlactate/glucose ratio in the culture medium in response to estrogen treatment for MCF7 and T47D cells. C–E, data, mean � SD (n ¼ 5). Student t test wasused for calculation of statistical significance.

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Figure 3.

ERa regulates the Cho metabolic pathway. A, heatmap of estrogen-regulated genes in the Cho metabolic pathway for MCF7 and T47D cells, respectively. ETOH,ethanol. B, confirmation of a subset of estrogen-regulated genes in the Cho metabolic pathway using qRT-PCR for MCF7 and T47D cells, respectively. TFF1was used as positive control for estrogen-stimulated gene expression. The assay was performed in triplicates. C, change of CDP-Cho level in response toestrogen treatment.D, increased PtdCho levels in response to estrogen treatment for MCF7 and T47D cells. E, ERa-binding site within the CHPT1 gene locus derivedfrom ChIP-seq. F, ChIP-qPCR confirms recruitment of ERa to the CHPT1 gene. Data, fold enrichment relative to IgG. G, changes in gene expression levels andmetabolite levels in the Cho metabolic pathway upon estrogen treatment for MCF7 and T47D cells, respectively. Gene names in red, upregulated genes; genenames in green, downregulated genes; arrow, changes in metabolite levels; red arrows, upregulated metabolites; green arrows, downregulated metabolites.C, D, and F, data, means � SD (n ¼ 5 for C and n ¼ 3 for D and F). Student t test was used for calculation of statistical significance.






allows it to translocate to membranes (39). To understandwhether E2 regulates CCTa activity, we investigated thecellular distribution of CCTa. Notably, CCTa expression wasdetected in cytosol, membrane, and nuclear fractions (Fig. 4A).Interestingly, the level of CCTawas significantly decreased in thecytosol fraction, while it was increased in the membrane uponE2 stimulation inMCF7 and T47D cells (Fig. 4A), suggesting thatCCTa was recruited to the membrane and activated in responseto E2 treatment. However, no change of CCTa was observedin nuclear fraction upon E2 treatment (Fig. 4A). The separationof cytosol, membrane, and nuclear proteins was confirmedby assaying the cytosolic proteins GAPDH and a-tubulin, themembrane-associated proteins cadherin and TIM 23, and nucle-ar-associated protein lamin (Fig. 4A).

CHPT1 is critical for estrogen-induced PtdCho synthesisE2 stimulation led to upregulationof bothCHPT1 and its direct

downstream metabolite PtdCho (Fig. 3B and D). To confirm therole of CHPT1 in estrogen regulation of PtdCho, we assayedPtdCho levels upon CHPT1 depletion with and without E2treatment in MCF7 and T47D cells. Efficient knockdown ofCHPT1 was confirmed by qRT-PCR and Western blot analysis(Fig. 4B). Importantly, PtdCho levels decreased significantly afterCHPT1 knockdown, supporting a critical role of CHPT1 in reg-ulating PtdCho synthesis (Fig. 4C). Furthermore, the effect ofestrogen inpromoting PtdCho synthesiswas significantly reducedafter CHPT1 knockdown (Fig. 4C), suggesting that estrogen-induced PtdCho synthesis is dependent on CHPT1 expression.To further explore how metabolites of the Cho metabolic path-way are affected upon CHPT1 depletion, we determined levelsof Cho-containing metabolites by 1H-NMR in MCF7 cells uponCHPT1 depletion compared with control in the presence andabsence of E2. As shown in Fig. 4D, CHPT1 depletion increasedthe levels of Cho, suggesting that CHPT1 contributes significantlyto metabolic turnover in the Cho pathway in MCF7 cells. Inter-estingly, a significant reduction of the PCho/GPC ratio was ob-served upon CHPT1 depletion, and additionally, increase of thePCho/GPC ratio by E2 was abolished by CHPT1 depletion(Fig. 4E). This indicates that CHPT1 is a critical regulator ofthe PCho/GPC ratio, which previously has been suggested as apotential prognostic biomarker in breast cancer (40). To under-stand whether the PEMT pathway contributes to the increase inPtdCho levels, we assayed PEMT mRNA levels in response toE2 stimulation. However, the expression of PEMT was notinduced by estrogen in the assayed breast cancer cell lines (Sup-plementary Table S5).

CHPT1 increases anchorage-independent growth andproliferation of breast cancer cells

To further uncover the role of CHPT1 in ERaþ breast cancercells, we examined anchorage-independent growth and prolif-eration after CHPT1 knockdown. As shown in Fig. 5A and B,CHPT1 knockdown reduced the number of colonies of MCF7and T47D cells in soft agar compared with the control. Fur-thermore, we observed that knockdown of CHPT1 decreasedcell proliferation in both cell lines (Fig. 5C).

CHPT1 is overexpressed in breast cancerTo confirm CHPT1 dysregulation in breast cancer, we deter-

mined CHPT1 protein levels in tumor tissue and adjacentnormal breast tissue by IHC, using TMAs for which data were

provided regarding tumor nodes and metastasis (TNM), clini-cal stage and pathology grade, and IHC staining for HER-2, ER,and PR. Consistent with published data by The Human ProteinAtlas (http://www.proteinatlas.org/ENSG00000111666-CHPT1/tissue), we observed that almost all CHPT1 staining was local-ized to the cytoplasm (Fig. 5D). Although cytoplasmic stainingwas observed in both normal and cancerous tissue, stainingwas stronger for tumor tissues (Fig. 5D and E). Interestingly,higher CHPT1 expression was observed in ERþ breast cancercompared with ER� breast cancer (Supplementary Table S6),consistent with CHPT1 being an ERa target gene. There wasno significant correlation between CHPT1 expression andHER-2, TNM, clinical stage, and pathology grade (Supplemen-tary Table S6).

Knockdown of CHPT1 inhibits early stage of metastasis oftamoxifen-resistant breast cancer cells in vivo

To increase knowledge about the role of CHPT1 in invasionof tamoxifen-resistant breast cancer cells, we performed Trans-well cell invasion assays for both tamoxifen-sensitive MCF7cells and tamoxifen-resistant LCC2 cells upon CHPT1 knock-down (Fig. 6A). The invasion assay showed that LCC2 cellswere more invasive than MCF7 cells (Fig. 6B). Knockdown ofCHPT1 markedly inhibited invasion of both MCF7 and LCC2cells (Fig. 6B). To further study the role of CHPT1 in regulatingearly stage of metastasis of tamoxifen-resistant breast cancercells in vivo, we used a zebrafish tumor model (36). Tumor-implanted fish embryos were scored for the dissemination oftumor cells at day 4 after injection. Control siRNA–treatedLCC2 cells disseminated more widespread in the fish body ascompared with control siRNA–treated MCF7 cells. Reduceddissemination of tumor cells was observed for both MCF7 andLCC2 cells after CHPT1 knockdown (Fig. 6C). Notably, a stron-ger suppression of invasion and metastasis following CHPT1depletion was found in LCC2 cells compared with MCF7 cells(Fig. 6D).

DiscussionHere, we combine global determination of ERa-binding

regions with global determination of estrogen-induced geneexpression for two breast cancer cell lines to define a set of 207core direct ERa target genes. The two ERaþ breast cancer celllines investigated in this study, MCF7 and T47D, representdistinct molecular backgrounds for ERa activity in breastcancer (32, 41). Notably and consistent with previous findings,the number of ERa-binding regions and fold induction inexpression of estrogen-induced genes in MCF7 cells exhibitgreater sensitivity to estrogen treatment as compared withT47D cells (32, 41). This may be due to differential impactof chromatin configuration (32) and/or different ERa expres-sion levels between the cell lines (41).

The enriched pathways in the reported core set of direct ERatarget genes included cancer pathways and metabolic pathways(Fig. 1F) and combined with the metabolic profiling revealdetails of estrogen-induced metabolic reprogramming inbreast cancer cells. Metabolic profiling revealed metaboliteswith conserved modulation by estrogen between the analyzedcell lines, including higher uptake of glucose and elevatedlevels of PtdCho, tyrosine, phenylalanine, and 1-methylhisti-dine and decreased levels of PCho and ATP (Fig. 2B), which

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Figure 4.

CHPT1 is critical for estrogen-induced PtdCho synthesis. A, decreased levels of CCTa in the cytosol and increased levels of CCTa in the membrane inresponse to estrogen treatment in MCF7 and T47D cells. GAPDH is a marker for cytosol (C) proteins, cadherin is a marker for membrane (M)proteins, and lamin is a marker for nuclear (N) proteins. ETOH, ethanol. TIM23 was used as a loading control for membrane proteins. a-Tubulin wasused as a loading control for cytosol proteins, and lamin was a loading control for nuclear proteins. B, reduced mRNA and protein levels of CHPT1 72 hoursafter siRNAs knockdown. 36B4 was used for mRNA normalization, and b-actin was used as a loading control for Western blot analysis. C, estrogentreatment increases PtdCho levels dependent on CHPT1. D, Cho levels after CHPT1 depletion with and without E2 treatment in MCF7 cells. E, PCho/GPCratio after CHPT1 depletion with and without E2 treatment in MCF7 cells. B–E, cells were transfected with control or CHPT1 siRNA. Transfected cellswere cultured in steroid-depleted media and treated with 10 nmol/L E2 or ethanol for 24 hours, after which the lipid metabolites were extractedand quantified using a Phosphatidylcholine Assay Kit (Abcam), and the water-soluble metabolites were extracted and quantified by 1H-NMR. Data,means � SD (n ¼ 3). Student t test was used for calculation of statistical significance.






accounts for only 1/3 and 1/2 of E2-regulated metabolites inMCF7 and T47D cells, respectively. This complex responsecould be related to that the compositions of culture media,inherent metabolic characteristics (42), and E2-inducedchanges in gene expression differ between the two cell lines.A previous study showed that cell culture conditions, conflu-ence, serum deprivation, and acidic extracellular pH could allaffect metabolite levels (43). Furthermore, we have previouslydemonstrated that distinct metabolic profiles are associatedwith differences in gene expression for different subtypes ofbreast cancer (27). However, importantly, estrogen-regulatedmetabolites for the two breast cancer cell lines were related tofour metabolic pathways, aerobic glycolysis, nucleotide andamino acid synthesis, and glycerophospholipid metabolism,suggesting that ERa activation may increase the production of

metabolic intermediates for the synthesis of proteins, nucleicacids, and lipids to support the rapid proliferation of cancercells.

Alterations in membrane phospholipids are associated withmalignant transformation (36), tumorigenicity (19), andmetastasis (44). Interestingly, glycerophospholipid metabo-lism was one of the enriched pathways of direct ERa targetgenes, and additionally, our metabolic profiling showed altera-tions in Cho-metabolite levels in response to estrogen signal-ing. We report that E2 decreased PCho levels and increasedPtdCho levels in both MCF7 and T47D cells (SupplementaryTable S4; Fig. 3D). E2 suppression of PCho in T47D cells isconsistent with a previous report (12). Metabolite levels areregulated by rate of synthesis and consumption of the metab-olite. Reduced PCho could be attributed to decreased CHK

Figure 5.

CHPT1 knockdown inhibitsanchorage-independent growth andproliferation of breast cancer cells.A, decreased number of coloniesupon CHPT1 knockdown as assayedby anchorage-independent growth.B, quantitative analysis of theanchorage-independent assay. C,inhibition of cell proliferation uponCHPT1 knockdown as assayed bythe WST-1 assay. B and C, data,means � SD (n ¼ 3). Student t testwas used for calculation ofstatistical significance.D and E, CHPT1 expression in humanbreast cancers was analyzed inTMAs. D, representative expressionpattern of CHPT1 for normal tissueand breast cancer tissue. E,quantification of CHPT1 IHC stainingfor normal and breast cancer tissue.

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Figure 6.

Knockdown of CHPT1 inhibits early stage of metastasis of tamoxifen-resistant breast cancer cells in vivo. A, reduced mRNA and protein levels of CHPT196 hours after siRNA transfection. 36B4 was used for mRNA normalization, and b-actin was used as a loading control for Western blot analysis. B,CHPT1 depletion reduces MCF7 and LCC2 cell invasiveness. A and B, data, means� SD (n¼ 3). Student t test was used for calculation of statistical significance.C, CHPT1 knockdown results in reduced dissemination of MCF7 and LCC2 cells in the zebrafish metastasis assay. Left, 48 hours after fertilization (hpf),zebrafish embryos of the Tg(fli1:EGFP)y1 strain in which blood vessel endothelial cells express EGFP (green) were injected in the perivitelline spacewith approximately 300 DiI-labeled cells (red) transfected either with control siRNA or with siRNA complementary to CHPT1 in and imaged at 120 hpf.Regions indicated by white boxes in the upper full-embryo image were enlarged in the lower images. White arrowheads, tumor cells. Inj., injection; mets,metastases. Scale bars, 500 mm (top) and 100 mm (bottom). Right, quantification of the number of the cells in the region anterior to the intestine from theexperiments. Student t test was used for calculation of statistical significance (n ¼ 30–35 embryos). D, inhibition of invasion or metastasis after CHPT1knockdown. Data, means � SD. Student t test was used for calculation of statistical significance.






expression (Fig. 3B; Supplementary Table S4), but also toincreased CCTa activity leading to higher consumption ofPCho for CDP-Cho synthesis (Figs. 3C and 4A). Moreover,downregulation of PLC could also result in less PCho produc-tion (Fig. 3B). Furthermore, we could not exclude the possi-bility that PCho is dephosphorylated by phosphatases. How-ever, it should be noted that the relationship between transcriptlevels and metabolite concentrations in the Cho pathway ishighly complex and depends on several collateral biochemicalreactions. Furthermore, it is difficult to correlate changes inmetabolic flux through the pathway to steady-state metabolitelevels. The metabolic effects of estrogen stimulation can there-fore not be conclusively determined from changes in geneexpression.

The activity of CHPT1 is regulated by thyroid hormone (45)and by arginosuccinate. Here, we demonstrate that E2 stimu-lation led to upregulation of CHPT1 and increased PtdCholevels in breast cancer cells (Fig. 3B and D). Notably, CHPT1depletion not only decreased PtdCho levels, but also increasedCho levels in MCF7 cells (Fig. 4C and D). Accumulation of Chomay be the result of reduced flux through the pathway due toreduced CHPT1 activity. Our results suggest that CHPT1 con-tributes to regulation of PtdCho synthesis in the context of ERsignaling in breast cancer. CCT, the rate-liming enzyme forPtdCho synthesis, has been reported to display increasedexpression in cancer (46, 47). Interestingly, no induction ofPCYT, which encodes CCT, was found upon E2 stimulation(Fig. 3G). However, E2 stimulation increased the activity ofCCTa (Fig. 4A), which could result in sufficient substrateproduction for CHPT1 (CDP-Cho) to synthesize PtdCho(Fig. 3C). Hence, our study indicates that increased CHPT1

expression and increased CCTa activity was involved in theestrogen-induced increase in PtdCho synthesis (SupplementaryTable S5).

Previous studies have shown upregulation of CHPT1 mRNAlevels and activity in human breast cancer cells compared withnormal mammary epithelial cells (48, 49). Furthermore, thePCho/GPC ratio has been associated with malignant transforma-tion (36, 50). In agreement with this, CHPT1 knockdown reducedthe PCho/GPC ratio in MCF7 cells (Fig. 4E) and decreasedanchorage-independent growth (Fig. 5A and B) and proliferationof breast cancer cells (Fig. 5C).

In conclusion, our study has uncovered that ERa activa-tion reprograms metabolism in breast cancer cells. We iden-tify ERa direct target genes by integrating global ERachromatin binding with global estrogen-regulated gene pro-filing. Metabolic profiling confirms functional consequencesof these estrogen-mediated transcriptional changes. We show,for the first time, that the ERa target gene CHPT1 plays anessential role in estrogen-induced increases in PtdCho levels.Furthermore, knockdown of CHPT1 reduces malignant phe-notype and proliferation of breast cancer cells. Importantly,CHPT1 depletion greatly suppresses early stage of metastasisof tamoxifen-resistant breast cancer cells in vivo. Mechanisti-cally, estrogen-stimulated CHPT1 upregulation leading toincreased PtdCho synthesis could contribute cell membranesynthesis (Fig. 7). Finally, as CHPT1 is overexpressed inbreast cancer supports it is a potential drug target to befurther investigated.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Figure 7.

Proposed model for estrogen-regulated CHPT1-mediated promotion of anchorage-independent growth and cell proliferation. ERa activation leads to increasedCHPT1 gene expression. Overexpression of CHPT1 promotes PtdCho synthesis, which increases membrane synthesis.

Jia et al.





Authors' ContributionsConception and design: M. Jia, T.F. Bathen, H. Gao, C. Zhao, Y. Cao,S.A. Moestue, K. Dahlman-WrightDevelopment of methodology: M. Jia, T.F. Bathen, Y. CaoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Andreassen, L. Jensen, S.A. MoestueAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Jia, T. Andreassen, L. Jensen, I. Sinha, Y. Cao,L. Girnita, S.A. Moestue, K. Dahlman-WrightWriting, review, and/or revision of the manuscript: M. Jia, T. Andreassen,T.F. Bathen, I. Sinha, H. Gao, C. Zhao, L.-A. Haldosen, L. Girnita, S.A. Moestue,K. Dahlman-WrightAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): I. SinhaStudy supervision: Y. Cao, S.A. Moestue, K. Dahlman-WrightOther (has taken part in discussions of obtained data and in some casessuggested changes in experimental details): L.-A. Haldosen

AcknowledgmentsWe are grateful to the Bioinformatic and Expression Analysis core facility

at the Karolinska Institute (http://www.bea.ki.se/) for performing the Affy-

metrix and chromatin immunoprecipitation sequencing (ChIP-seq) assays.Swedish Metabolomics Centre (www.swedishmetabolomicscentre.se) isacknowledged for the method development and analysis of CDP-Cho. TheNMR analyses were performed at the MR Core Facility, Norwegian Universityof Science and Technology (NTNU). MR core facility is funded by the Facultyof Medicine at NTNU and Central Norway Regional Health Authority.

Grant SupportThis project was supported by the Swedish Cancer Society (Cancerfon-

den), the Norwegian Research Council (grant No. 239940), SwedishResearch Council, the Swedish Childhood Cancer Foundation and Stock-holm Cancer Society.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 26, 2015; revised June 27, 2016; accepted July 14, 2016;published OnlineFirst July 25, 2016.

References1. Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. Nat

Rev Cancer 2011;11:85–95.2. Boroughs LK, DeBerardinis RJ. Metabolic pathways promoting cancer cell

survival and growth. Nat Cell Biol 2015;17:351–9.3. Ganapathy-Kanniappan S, Geschwind JF. Tumor glycolysis as a target for

cancer therapy: progress and prospects. Mol Cancer 2013;12:152.4. Hensley CT, Wasti AT, DeBerardinis RJ. Glutamine and cancer: cell

biology, physiology, and clinical opportunities. J Clin Invest 2013;123:3678–84.

5. Flavin R, Peluso S, Nguyen PL, Loda M. Fatty acid synthase as a potentialtherapeutic target in cancer. Future Oncol 2010;6:551–62.

6. Galluzzi L, Kepp O, Vander Heiden MG, Kroemer G. Metabolic targets forcancer therapy. Nat Rev Drug Discov 2013;12:829–46.

7. Cicatiello L, Mutarelli M, Grober OM, Paris O, Ferraro L, Ravo M, et al.Estrogen receptor alpha controls a gene network in luminal-like breastcancer cells comprising multiple transcription factors and microRNAs.Am J Pathol 2010;176:2113–30.

8. Thomas C, Gustafsson JA. The different roles of ER subtypes in cancerbiology and therapy. Nat Rev Cancer 2011;11:597–608.

9. Budczies J, Brockmoller SF, Muller BM, Barupal DK, Richter-Ehrenstein C,Kleine-Tebbe A, et al. Comparative metabolomics of estrogen receptorpositive and estrogen receptor negative breast cancer: alterations in gluta-mine and beta-alanine metabolism. J Proteomics 2013;94:279–88.

10. Tang X, Lin CC, Spasojevic I, Iversen ES, Chi JT, Marks JR. A joint analysisof metabolomics and genetics of breast cancer. Breast Cancer Res 2014;16:415.

11. Giskeodegard GF, Grinde MT, Sitter B, Axelson DE, Lundgren S, FjosneHE, et al. Multivariate modeling and prediction of breast cancerprognostic factors using MR metabolomics. J Proteome Res 2010;9:972–9.

12. Neeman M, Degani H. Metabolic studies of estrogen- and tamoxifen-treated human breast cancer cells by nuclear magnetic resonance spectros-copy. Cancer Res 1989;49:589–94.

13. Neeman M, Degani H. Early estrogen-induced metabolic changes andtheir inhibition by actinomycin D and cycloheximide in human breastcancer cells: 31P and 13C NMR studies. Proc Natl Acad Sci U S A 1989;86:5585–9.

14. O'Mahony F, Razandi M, Pedram A, Harvey BJ, Levin ER. Estrogen mod-ulates metabolic pathway adaptation to available glucose in breast cancercells. Mol Endocrinol 2012;26:2058–70.

15. Xu X, Gammon MD, Zeisel SH, Lee YL, Wetmur JG, Teitelbaum SL, et al.Cholinemetabolism and risk of breast cancer in a population-based study.FASEB J 2008;22:2045–52.

16. Katz-Brull R, Seger D, Rivenson-Segal D, Rushkin E, Degani H. Meta-bolic markers of breast cancer: enhanced choline metabolism and

reduced choline-ether-phospholipid synthesis. Cancer Res 2002;62:1966–70.

17. Katz-Brull R, Margalit R, Degani H. Differential routing of choline inimplanted breast cancer and normal organs. Magn Reson Med 2001;46:31–8.

18. Ackerstaff E, Glunde K, Bhujwalla ZM. Choline phospholipidmetabolism:a target in cancer cells? J Cell Biochem 2003;90:525–33.

19. Glunde K, Bhujwalla ZM, Ronen SM. Choline metabolism in malignanttransformation. Nat Rev Cancer 2011;11:835–48.

20. Resseguie M, Song J, Niculescu MD, da Costa KA, Randall TA, Zeisel SH.Phosphatidylethanolamine N-methyltransferase (PEMT) gene expressionis induced by estrogen in human andmouse primary hepatocytes. FASEB J2007;21:2622–32.

21. Glunde K, Jie C, Bhujwalla ZM. Molecular causes of the aberrantcholine phospholipid metabolism in breast cancer. Cancer Res 2004;64:4270–6.

22. Grove RI, Schimmel SD. Effects of 12-O-tetradecanoylphorbol 13-acetateon glycerolipid metabolism in cultured myoblasts. Biochim Biophys Acta1982;711:272–80.

23. Rohrschneider LR, Boutwell RK. The early stimulation of phospholipidmetabolism by 12-0-tetradecanoyl-phorbol-13-acetate and its specificityfor tumor promotion. Cancer Res 1973;33:1945–52.

24. Wertz PW, Mueller GC. Rapid stimulation of phospholipidmetabolism inbovine lymphocytes by tumor-promoting phorbol esters. Cancer Res1978;38:2900–4.

25. Kiss Z, Crilly KS, Anderson WH. Phorbol ester stimulation of phos-phatidylcholine synthesis requires expression of both protein kinaseC-alpha and phospholipase D. Biochim Biophys Acta 1998;1392:109–18.

26. Ting YL, Sherr D, Degani H. Variations in energy and phospholipidmetabolism in normal and cancer human mammary epithelial cells.Anticancer Res 1996;16:1381–8.

27. Moestue SA, Borgan E, Huuse EM, Lindholm EM, Sitter B, Borresen-DaleAL, et al. Distinct choline metabolic profiles are associated with differencesin gene expression for basal-like and luminal-like breast cancer xenograftmodels. BMC Cancer 2010;10:433.

28. Nagai MA, Brentani MM. Gene expression profiles in breast cancer toidentify estrogen receptor target genes. Mini Rev Med Chem 2008;8:448–54.

29. Ross-Innes CS, Stark R, Teschendorff AE, Holmes KA, Ali HR, Dunning MJ,et al. Differential oestrogen receptor binding is associated with clinicaloutcome in breast cancer. Nature 2012;481:389–93.

30. HurtadoA,HolmesKA,Geistlinger TR,Hutcheson IR,NicholsonRI, BrownM, et al. Regulation of ERBB2 by oestrogen receptor-PAX2 determinesresponse to tamoxifen. Nature 2008;456:663–6.






31. Hua S, Kittler R,White KP. Genomic antagonismbetween retinoic acid andestrogen signaling in breast cancer. Cell 2009;137:1259–71.

32. Joseph R,Orlov YL,HussM, SunW,Kong SL,Ukil L, et al. Integrativemodelof genomic factors for determining binding site selection by estrogenreceptor-alpha. Mol Syst Biol 2010;6:456.

33. PutnikM,ZhaoC,Gustafsson JA,Dahlman-Wright K.Global identificationof genes regulated by estrogen signaling and demethylation in MCF-7breast cancer cells. Biochem Biophys Res Commun 2012;426:26–32.

34. Zhao C,Matthews J, TujagueM,Wan J, StromA, ToressonG, et al. Estrogenreceptor beta2 negatively regulates the transactivation of estrogen receptoralpha in human breast cancer cells. Cancer Res 2007;67:3955–62.

35. Rouhi P, Jensen LD, Cao Z, Hosaka K, Lanne T, Wahlberg E, et al. Hypoxia-induced metastasis model in embryonic zebrafish. Nat Protoc 2010;5:1911–8.

36. Aboagye EO, Bhujwalla ZM. Malignant transformation alters membranecholine phospholipid metabolism of human mammary epithelial cells.Cancer Res 1999;59:80–4.

37. Bhujwalla ZM, Aboagye EO,Gillies RJ, Chacko VP,Mendola CE, Backer JM.Nm23-transfected MDA-MB-435 human breast carcinoma cells formtumors with altered phospholipid metabolism and pH: a 31P nuclearmagnetic resonance study in vivo and in vitro. Magn Reson Med 1999;41:897–903.

38. Stewart JD,Marchan R, LesjakMS, Lambert J, Hergenroeder R, Ellis JK, et al.Choline-releasing glycerophosphodiesterase EDI3 drives tumor cellmigra-tion and metastasis. Proc Natl Acad Sci U S A 2012;109:8155–60.

39. Gibellini F, Smith TK. The Kennedy pathway–De novo synthesis of phos-phatidylethanolamine and phosphatidylcholine. IUBMB Life 2010;62:414–28.

40. Moestue SA, Giskeodegard GF, Cao MD, Bathen TF, Gribbestad IS. Glycer-ophosphocholine (GPC) is a poorly understood biomarker in breastcancer. Proc Natl Acad Sci U S A 2012;109:E2506.

41. Lu M, Mira-y-Lopez R, Nakajo S, Nakaya K, Jing Y. Expression of estrogenreceptor alpha, retinoic acid receptor alpha and cellular retinoic acidbinding protein II genes is coordinately regulated in human breast cancercells. Oncogene 2005;24:4362–9.

42. Radde BN, Ivanova MM, Mai HX, Salabei JK, Hill BG, Klinge CM. Bioener-getic differences between MCF-7 and T47D breast cancer cells and theirregulation by oestradiol and tamoxifen. Biochem J 2015;465:49–61.

43. Delikatny EJ, Chawla S, Leung DJ, Poptani H. MR-visible lipids and thetumor microenvironment. NMR Biomed 2011;24:592–611.

44. Dahiya R, Boyle B, Goldberg BC, Yoon WH, Konety B, Chen K, et al.Metastasis-associated alterations in phospholipids and fatty acids ofhuman prostatic adenocarcinoma cell lines. Biochem Cell Biol 1992;70:548–54.

45. Chatterjee D, Mukherjee S, Das SK. Regulation of cholinephosphotrans-ferase by thyroid hormone. Biochem Biophys Res Commun 2001;282:861–4.

46. Dueck DA, Chan M, Tran K, Wong JT, Jay FT, Littman C, et al. Themodulation of choline phosphoglyceride metabolism in human coloncancer. Mol Cell Biochem 1996;162:97–103.

47. Bell JD, Bhakoo KK. Metabolic changes underlying 31P MR spectralalterations in human hepatic tumours. NMR Biomed 1998;11:354–9.

48. Ghosh A, Akech J, Mukherjee S, Das SK. Differential expression of choli-nephosphotransferase in normal and cancerous human mammary epi-thelial cells. Biochem Biophys Res Commun 2002;297:1043–8.

49. Akech J, Sinha Roy S, Das SK. Modulation of cholinephosphotransferaseactivity in breast cancer cell lines by Ro5–4864, a peripheral benzodiaz-epine receptor agonist. Biochem Biophys Res Commun 2005;333:35–41.

50. Mimmi MC, Finato N, Pizzolato G, Beltrami CA, Fogolari F, Corazza A,et al. Absolute quantificationof choline-relatedbiomarkers in breast cancerbiopsies by liquid chromatography electrospray ionizationmass spectrom-etry. Anal Cell Pathol 2013;36:71–83.


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Editor's Note

Editor's Note: Estrogen Receptor a PromotesBreast Cancer by Reprogramming CholineMetabolismMin Jia, Trygve Andreassen, Lasse Jensen, Tone Frost Bathen,Indranil Sinha, Hui Gao, Chunyan Zhao, Lars-Arne Haldosen,Yihai Cao, Leonard Girnita, Siver Andreas Moestue, andKarin Dahlman-Wright

The editors are publishing this note to alert readers to concerns about this article (1).The editorsweremade aware of duplicatedWestern blot bands in Fig. 4A. Specifically,two sets of Western blot bands are duplicates: (i) T47D CCTa in the top right paneland T47D cadherin in the bottom right panel; and (ii) MCF7 CCTa band in the topright panel and MCF7 N in the bottom right panel. Because satisfactorily correctedfigures could not be provided, the editors are publishing this note to alert readers tothese concerns.

Reference1. Jia M, Andreassen T, Jensen L, Bathen TF, Sinha I, Gao H, et al. Estrogen receptor a promotes breast

cancer by reprogramming choline metabolism. Cancer Res 2016;76:5634–46

Published first October 15, 2019.Cancer Res 2019;79:5458doi: 10.1158/0008-5472.CAN-19-2632�2019 American Association for Cancer Research.

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Cancer Res; 79(20) October 15, 20195458

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2016;76:5634-5646. Published OnlineFirst July 25, 2016.Cancer Res Min Jia, Trygve Andreassen, Lasse Jensen, et al. Choline Metabolism

Promotes Breast Cancer by ReprogrammingαEstrogen Receptor

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Estrogen Receptor a Promotes Breast Cancer by Reprogramming Choline Metabolism · Choline (Cho) is an essential nutrient that is necessary for cell membrane synthesis and functions