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Cancer Res. Author manuscript; available in PMC 2009 Nov 1.
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PMCID: PMC2701641

ERRγ mediates Tamoxifen resistance in novel models of invasive lobular breast cancer


One-third of all ER+ breast tumors treated with endocrine therapy fail to respond, and the remainder are likely to relapse in the future. Almost all data on endocrine resistance has been obtained in models of invasive ductal carcinoma (IDC). However, invasive lobular carcinomas (ILC) comprise up to 15% of newly diagnosed invasive breast cancers diagnosed each year and, while the incidence of IDC has remained relatively constant during the last 20 years, the prevalence of ILC continues to increase among postmenopausal women. We report a new model of Tamoxifen (TAM)-resistant invasive lobular breast carcinoma cells that provides novel insights into the molecular mechanisms of endocrine resistance. SUM44 cells express ER and are sensitive to the growth inhibitory effects of antiestrogens. Selection for resistance to 4-hydroxytamoxifen led to the development of the SUM44/LCCTam cell line, which exhibits decreased expression of estrogen receptor alpha (ERα) and increased expression of the estrogen-related receptor gamma (ERRγ). Knockdown of ERRγ in SUM44/LCCTam cells by siRNA restores TAM sensitivity, and overexpression of ERRγ blocks the growth-inhibitory effects of TAM in SUM44 and MDA-MB-134 VI lobular breast cancer cells. ERRγ-driven transcription is also increased in SUM44/LCCTam, and inhibition of activator protein 1 (AP1) can restore or enhance TAM sensitivity. These data support a role for ERRγ/AP1 signaling in the development of TAM resistance, and suggest that expression of ERRγ may be a marker of poor Tamoxifen response.

Keywords: ERα, ERRγ, breast cancer, ILC, endocrine resistance


Breast cancer is the second-most common cause of cancer-related death in women (1). One of the challenges in treating breast cancer is addressing the biological heterogeneity evident in the existence of several histologic and molecular subtypes. Two of the major histologic breast cancer classifications are invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC). Currently, ILCs comprise up to 15% of invasive breast cancer diagnoses annually (2). While the incidence of IDC has remained relatively constant during the last 20 years, a significant increase in ILC diagnosis is evident among postmenopausal women in Western Europe and the United States (reviewed in (3). While the increased use of estrogen plus progestin hormone replacement therapy (HRT) for relief of peri- and post-menopausal symptoms during this same time period may have contributed to the increase in ILC incidence (3), the precise mechanism(s) remains uncertain.

The clinical and pathologic features of lobular tumors are unique. ILC typically invades in a linear pattern, creating a longer, thinner mass which is more difficult to detect by mammography, ultrasound, or breast self-exam (3). ILCs have a greater tendency to be bilateral, and women with this type of breast cancer are frequently older and have larger tumors at the time of their diagnosis (3). A higher incidence of ILC has been reported among women who initially present to the clinic with metastatic breast cancer (4). Though recent clinical studies imply that ILC is less responsive to neoadjuvant cytotoxic chemotherapy as a precursor to breast-conserving surgery (5;6), there are conflicting reports as to whether patients diagnosed with ILC have a poorer, equivalent, or improved prognosis and overall survival when compared with IDC (reviewed in (3).

Breast cancer patients whose tumors express estrogen receptor (ER) alpha (ERα) may be offered endocrine or antiestrogen therapy in addition to or in place of conventional chemotherapies. Currently, the most widely used antiestrogen is the triphenylethylene Tamoxifen (TAM), which functions as a partial antagonist by competing with estrogen for binding to the ER. TAM is known to induce a statistically significant improvement in the overall survival rate from breast cancer (7), and approximately 70% of all ER+/progesterone receptor positive (PR+) breast cancers will respond to TAM. When compared with IDC, a significantly greater percentage of ILC tumors are ER+/PR+ (discussed in (3), suggesting that women diagnosed with this tumor subtype should be ideal candidates for endocrine therapy. However, study results differ as to whether ILC patients experience a better or worse risk of mortality than IDC patients following antiestrogen treatment (8;9).

Regardless of tumor subtype, the development of endocrine resistance is a pervasive clinical problem (10-12). One-third of ER+/PR+ breast tumors treated with TAM do not respond to initial treatment, and the remaining 70% are still at risk to relapse in the future. A number of mechanisms have been proposed to control antiestrogen resistance in ER+ breast cancer (13) but many details of these mechanisms continue to be unclear. Studying endocrine resistance specifically in ILC has not been possible because of the lack of appropriate models; the most common models of resistance (notably MCF-7 cells) are derived from ductal adenocarcinomas (14).

Given the unique clinical and molecular features of lobular tumors, and the suggestion that ILC tumors may respond less well to endocrine therapy, we have developed an ILC-specific cell culture model of endocrine resistance. The SUM44 breast cancer cell line was isolated from an ILC metastasis (15), is ER+/PR+, and displays other common features of ILC such as the loss of E-cadherin (16). We show that SUM44 cells contain functional ER, and are sensitive to growth inhibition by antiestrogens. Selection of SUM44 cells against 4-hydroxytamoxifen (4HT) led to the establishment of the SUM44/LCCTam cell line, which is stably resistant to TAM. We then identified candidate genes associated with the endocrine resistant phenotype in SUM44/LCCTam cells, and found changes in the expression of ERα and the estrogen-related receptor gamma (ERRγ). Our mechanistic studies demonstrate that knockdown of ERRγ in the resistant cell line, and overexpression of ERRγ in endocrine-responsive lobular breast cancer cells, modulates TAM sensitivity. Finally, we show that ERRγ-driven transcription is increased in the resistant SUM44/LCCTam cell line, and inhibition of activator protein 1 (AP1) can restore or enhance TAM sensitivity in this model system.


Cell Culture and Reagents

All cells were shown to be free of Mycoplasma spp. contamination and maintained in a humidified incubator at 37°C in an atmosphere containing 95% air: 5% CO2. Routine tissue culture reagents (culture media and additives, phosphate-buffered saline, trypsin, etc.) were purchased from Invitrogen (Carlsbad, CA).

SUM44 cells were routinely cultured in SFIH (serum-free medium plus insulin and hydrocortisone) as described previously (15). LCCTam cells were maintained in SFIH containing 500 nM 4-hydroxytamoxifen (4HT; Sigma, St. Louis, MO). LCCTam cells were cultured in SFIH in the absence of 4HT for one week prior to all experiments. When SUM44 and LCCTam were passaged, cells were seeded in SFIH containing 2% fetal bovine serum (FBS) for the first 24 hours to neutralize trypsin and promote cell attachment. MCF-7 cells were originally obtained from Dr. Marvin Rich (Karmanos Cancer Center, Detroit, MI), and MDA-MB-134 VI breast cancer cells were purchased from ATCC (Manassas, VA); both were maintained in improved minimal essential medium (IMEM) with phenol red supplemented with 5% FBS.

17β-estradiol (estradiol, E2) was purchased from Sigma; Fulvestrant (ICI 182,780, Fulv) and the c-JUN peptide inhibitor were purchased from Tocris Bioscience (Ellisville, MO). The 3xERE-tk-luc promoter-reporter plasmid was kindly provided by Dr. Malcolm G. Parker (17), 3xSF1RE-luciferase was a gift from Dr. Jean-Marc Vanacker (18), and 3xAP1-luciferase was generously provided by Dr. Richard Pestell. The plasmid encoding wild type murine ERRγ bearing an N-terminal hemagglutinin (HA) tag (pSG5-HA-ERR3) was a gift from Dr. Michael Stallcup (19). Small inhibitory RNA (siRNA) oligonucleotide duplexes directed against ERRγ (siGENOME SMARTpool), non-silencing control oligonucleotides, and the DharmaFECT 1 reagent were purchased from Dharmacon (Lafayette, CO). The FuGene 6 transfection reagent was purchased from Roche (Indianapolis, IN).

Luciferase Promoter-Reporter Assays

Cells were seeded in SFIH at a density of 9 × 104 cells per well in 12-well plastic tissue culture dishes for 24-48 hours prior to transfection with 0.6 μg luciferase promoter-reporter construct and 0.2 μg phRL-SV40 Renilla internal control (Promega, Madison, WI). The following day transfected cells were refed with SFIH, or SFIH containing 10 nM E2, 1000 nM 4HT, 100 nM Fulv, 20 μM c-JUN peptide inhibitor, or ethanol (EtOH) vehicle as indicated in each figure for a further 24 hours prior to lysis and measurement of luciferase activity by using the Dual Luciferase Assay Kit (Promega) as described previously (20). Luminescence was quantified using a Lumat LB 9501 luminometer (EG&G Berthold, Bundoora VIC, Australia).

Proliferation Assays

Cells were seeded in SFIH at a density of 2-3 × 104 per well in 24-well in 6-well plastic tissue culture dishes one day prior to the addition of the indicated concentrations of drug or ethanol (EtOH) vehicle. Cells were cultured for 6 days with two media changes prior to being trypsinized, resuspended in phosphate-buffered saline (PBS), and counted using a Z1 Single Coulter Counter (Beckman/Coulter, Miami, FL). At least three independent assays were performed in triplicate or quadruplicate, and the data were normalized to vehicle-treated cells.


Cells were seeded in SFIH at a density of 1 × 104 cells/well in 96-well plastic tissue culture dishes one day prior to the addition of drug or EtOH vehicle as indicated. Cells were then cultured for approximately 54 hours prior to the addition of 5-bromo-2′-deoxyuridine (BrdU) (final concentration 10 μM) for an additional 18 hours (total incubation in drug = 72 hours) before performing the Cell Proliferation ELISA, BrdU (colorimetric) assay as directed by the manufacturer (Roche). At least three independent assays were performed with five replicate wells per treatment group, and data were normalized to vehicle-treated cells.

BrdU Immunofluorescence Assays

These assays were performed as described above (drug treatment and BrdU addition) and in (21) (cell seeding and staining procedures) with the following modifications: ERRγ expression was detected using the HA.11 monoclonal antibody from Covance (Princeton, NJ; 1:500) followed by AlexaFluor594-conjugated goat anti-mouse secondary antibody (Invitrogen; 1:500), and BrdU incorporation was detected using the AlexaFluor488-conjugated anti-BrdU antibody (1:10) (BD Biosciences; San Jose, CA). Cells were visualized on a Nikon E600 epifluorescence microscope at 20X magnification (Melville, NY).

Cell Cycle Analysis

Cells were seeded in SFIH at a density of 5 × 104 cells/well in 6-well plastic tissue culture dishes one day prior to the addition of 1000 nM 4HT or EtOH vehicle. Cells were then cultured for 72 hours prior to harvesting and cell cycle analysis by the Vindelov method (22).

Derivation of SUM44/LCCTam cells

A Tamoxifen-resistant SUM44 variant was established according to previously published procedures (23). Sub-confluent T-25 cm2 tissue culture flasks of SUM44 cells were selected against increasing concentrations of 4HT, beginning with 1 nM. After three passages of the cells at each dose, the drug concentration was increased (1→5→10→50→100→500 nM), terminating at a concentration of 500 nM 4HT. Cells proliferating in 500 nM 4HT were designated SUM44/LCCTam (hereafter abbreviated as LCCTam). LCCTam cells were cultured in SFIH in the absence of 4HT for one week prior to all experiments.

Comparative Genomic Hybridization (CGH)

Normal control DNA was prepared from peripheral blood lymphocytes of a normal donor and test DNA was extracted from the cultured cell lines (SUM44 and the Tamoxifen-resistant LCCTam variant) using standard protocols, and CGH was performed as previously described (24). Gray scale images from at least 10 metaphases from each hybridization were acquired with a cooled charge-coupled device CCD camera (CH250, Photometrics, Tucson, AZ) connected to a Leica DMRBE microscope equipped with fluorochrome specific optical filters TR1, TR2, TR3 (Chroma Technology, Brattleboro, VT). Quantitative evaluation of the hybridization was done using commercially available software (Applied Imaging, Pittsburgh, PA). Average ratio profiles were calculated as the mean value of at least 8 ratio images to identify chromosomal copy number changes in all cases (see Supplementary Figure 1).

RNA Isolation, Gene Expression Microarray Preprocessing, and Data Analysis

Total RNA was extracted from sub-confluent T-25 cm2 tissue culture flasks of SUM44 and LCCTam cells, then processed and arrayed as described in (25). Microarray data quality was then assessed using several tools, including those recommended by Affymetrix and a series of additional QC measures under development in our laboratory (26). The Robust Multiple-Array Average (RMA) method was used to preprocess the raw gene expression data, as implemented in the Bioconductor project (http://bioconductor.org). We then isolated a reduced dimension dataset that included genes that exhibit ≥2 fold change, p<0.05 and genes with intensity ≥log2(10) in both SUM44 and SUM44/LCCTam groups. Data visualization before and after dimensionality reduction was facilitated by multidimensional scaling as estimated using Principal Component Analysis (PCA) and Discriminant Component Analysis (DCA) (27), to ensure that the global structure of the data was not altered by dimensionality reduction procedures (see Supplementary Figure 2).

Real Time qPCR

Total RNA from independent cultures (not RNA from cultures used for microarray analysis) was isolated, cleaned, quantified, and reverse-transcribed as described in (25). qPCR reactions for each cDNA sample and a standard curve were performed using TaqMan Universal PCR Master Mix and the following TaqMan Gene Expression Assay primers (Applied Biosystems): ESR1 = Hs00174860_m1; ESRRG = Hs00155006_m1; and the housekeeping gene RPLP0 = Hs99999902_m1) as in (25). Expression data for each gene was estimated relative to the housekeeping control and these data were used to calculate the ratio of expression relative to that in the parental SUM44 cell line.

Cell Lysis and Western Blot Analysis

Sub-confluent monolayers of cells were harvested, lysed, and analyzed by Western blot as in (28). Primary antibodies for ERRγ (1:1000), ERRα (1:500), and ERRβ (1:500) were purchased from GenWay (San Diego, CA). Antibodies for ERα (1:500) and ERβ (1:1000) were purchased from NovoCastra (Newcastle upon Tyne, UK) and Affinity Bioreagents (Golden, CO), respectively. Antibodies for FASN (1:500) and HMGCS2 (1:2000) were purchased from Abcam (Cambridge, MA). To confirm equal loading, membranes were reprobed using a β-actin monoclonal antibody (1:5000) purchased from Sigma, or a GAPDH goat polyclonal antibody (1:5000) purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies conjugated to horseradish peroxidase were purchased from GE Healthcare (Piscataway, NJ) and Santa Cruz Biotechnology (Santa Cruz, CA). Densitometry was performed using NIH ImageJ software (http://rsb.info.nih.gov/ij) and images were compiled using Adobe Photoshop CS2 (San Jose, CA).


LCCTam cells were seeded in 96-well plastic tissue culture dishes in SFIH at 1 × 104 cells per well one day prior to transfection with 100 nM ERRγ (siERRγ) or non-silencing control siRNA oligonucleotides (siC) using DharmaFECT1 (Dharmacon) according to manufacturer’s specifications. The next day, cells were treated with 1000 nM 4HT or EtOH vehicle prior to addition of BrdU for an additional 18 hours (total incubation in drug = 48 hours). Cell Proliferation ELISA, BrdU (colorimetric) assays were performed as described above. In parallel, cells were seeded in 12-well dishes at a density of 9×104 cells per well, transfected with 100 nM siC or siERRγ, and cells were lysed on the same day that BrdU ELISAs were performed (total transfection time = 72 hours) for Western blot analysis.


All statistical calculations were performed using SigmaStat version 3.0 (Systat; San Jose, CA). Luciferase promoter-reporter, cell proliferation, BrdU, real time RT-PCR, cell size, and microarray data from in vitro studies were compared using either Student’s t-test or one-way analysis of variance (ANOVA) followed by post-hoc t test, as appropriate, and indicated in the text and figure legends. Statistical significance is defined at ≥95% confidence level, or a p value of ≤0.05.


SUM44 cells have functional ER and are sensitive to growth inhibition by 4-hydroxytamoxifen

The SUM44 breast cancer cell line was derived from an invasive lobular carcinoma (ILC) and a high percentage of ILC tumors are estrogen receptor-positive (ER+) (29). While this cell line is also ER+ (15), ER functional status is unknown and SUM44 responsiveness to estrogens and antiestrogens has not previously been determined. Therefore, SUM44 cells were transfected with the 3xERE-tk-luc reporter construct and stimulated with estrogen, antiestrogen, or ethanol (EtOH) control (Figure 1A). Estrogen (E2) modestly but significantly induces, while 4-hydroxytamoxifen (4HT) significantly decreases, ERE-luciferase activity (p<0.001). We also observed that the steroidal antiestrogen Fulvestrant (Fulv) decreases ERE-luciferase activity, and that both 4HT and Fulv block the E2-induced stimulation of ERE-luciferase activity (p<0.001). These data suggest that the SUM44 estrogen receptor responds appropriately to estrogenic and antiestrogenic stimuli.

Fig 1
SUM44 cell proliferation and ER transcriptional activity are inhibited by antiestrogens, and LCCTam cells have acquired resistance to TAM

To determine whether SUM44 cells are sensitive to growth inhibition by 4HT, cells were treated with antiestrogen as indicated for 6 days (Figure 1B, closed circles). 4HT significantly inhibit the proliferation of SUM44 cells (ANOVA p<0.001). The observed reduction in cell number is also reflected in an inhibition of DNA synthesis as shown by reduced BrdU incorporation following 72 hours of 4HT treatment (Figure 1C, closed circles, ANOVA p<0.001), consistent with the known cytostatic effect of 4HT (12).

Generation of a TAM-resistant SUM44 variant

Since ILCs are predominantly ER+ and TAM has been the most widely used endocrine agent for the treatment of ER+ breast cancer, we sought to develop a TAM-resistant ILC model using the SUM44 cell line. Cells were selected against increasing concentrations of 4HT, and the cell population proliferating in 500 nM 4HT (within the range of clinically relevant concentrations; ref. (10) was designated SUM44/LCCTam (hereafter referred to as LCCTam).

The basal growth rate of LCCTam is identical to that of the parental SUM44 cell line and as expected, LCCTam cells are no longer responsive to the anti-proliferative effects of 4HT (Figure 1B, open triangles, N.S.), and LCCTam DNA synthesis is no longer inhibited by 4HT (Figure 1C, open triangles, ANOVA p=0.212). To further confirm that differences in SUM44 and LCCTam cell proliferation in response to antiestrogen reflect changes in sensitivity to the cytostatic effects of 4HT, we performed cell cycle analysis. SUM44 cells treated with 1 μM 4HT show a significantly greater fraction of cells arrested in the G1 phase as compared to EtOH-treated controls (p≤0.001, data not shown), while 4HT no longer induces an accumulation of LCCTam cells in G1 (p=0.722, data not shown). Together, these findings show that SUM44 cell growth and cell cycle progression are efficiently inhibited by 4HT, but that LCCTam cells have acquired resistance to the inhibitory effects of this antiestrogen.

Changes in the transcriptome of LCCTam cells are not associated with chromosomal aberrations

To characterize further this novel ILC cell model, we determined the pattern of, and differences in, genomic alterations and gene expression between SUM44 and LCCTam cells using comparative genomic hybridization (CGH) and Affymetrix gene expression microarray analysis, respectively. The genetic lineage of the two cell lines was confirmed to be identical by DNA fingerprinting using genetic markers at 9 different loci. CGH analysis revealed changes in the DNA copy number (gains, losses and amplifications) in both SUM44 and LCCTam (Supplementary Figure 1). Importantly, a comparison between our CGH findings and a previously-reported CGH analysis of SUM44 show a similar pattern of aberrations (30). We found no significant difference in the pattern of chromosomal alterations between the two cell lines; acquired estrogen independence also is not associated with changes in the amplification of DNA sequences (31).

In marked contrast, microarray analysis reveals a large number of changes in gene expression. We used principal component analysis (PCA) (27) to visualize the high-dimensional data set in two dimensions; SUM44 and LCCTam are linearly separable in this 2-dimensional PCA projection based on the top two principal components that capture 95% of the cumulative variance in the data (Supplementary Figure 2). Using a final cut-off of ≥2-fold change with p≤0.05 (univariate, uncorrected, T-statistic), we find that 380 genes are likely to be significantly altered: expression of 91 genes are increased and 289 genes are decreased in LCCTam vs. SUM44 controls (Supplementary Table 1).

To maintain focus on the TAM-resistant phenotype observed in LCCTam cells, we first chose to investigate gene expression changes in estrogen receptors and other members of the nuclear receptor superfamily. Expression of estrogen receptor alpha (ERα; HUGO symbol ESR1) is decreased 3.1-fold in LCCTam as compared to SUM44 cells by microarray (p=0.0013), which was subsequently confirmed by qPCR analysis (Figure 2A, white bars, ↓2.98-fold, p<0.001). In contrast, expression of the orphan nuclear receptor estrogen-related receptor gamma (ERRγ; HUGO symbol ESRRG) is 4.4-fold increased in the resistant LCCTam cells by microarray (p=0.01) and 10-fold increased by qPCR (Figure 2A, black bars, p=0.03).

Fig 2
ERα and ERRγ mRNA and protein expression are significantly altered during the acquisition of TAM resistance

To confirm that differences in the mRNA expression of these receptors are maintained at the protein level, cell lysates were collected and analyzed for ERRγ and ERα expression by Western blot (Figure 2B, inset). As observed for mRNA, ERRγ protein expression is increased (↑2.5-fold, p=0.03) and ERα expression is decreased (↓2-fold, p=0.03) in LCCTam cells. We also examined the protein levels of all other ERs and ERRs (ERβ, ERRα, and ERRβ) and find no differences in their expression between SUM44 and LCCTam cells (Figure 2C).

ERRγ plays a functional role in TAM resistance in LCCTam cells

ERRγ is an orphan nuclear receptor with no known natural ligand that has been shown to have constitutive transcriptional activity at several DNA response elements (reviewed in (32;33). ERRγ and its family members ERRα1 and ERRβ bear some structural similarity to the estrogen receptor (32;34). Although ERRα1 has previously been shown to activate or repress estrogen response element (ERE)-mediated transcription depending on cellular context (34) and to participate in HER2-dependent signaling in BT474 breast cancer cells (35), the role of ERRγ in breast cancer therapeutic response is under-explored (36).

We hypothesized that if increased expression of ERRγ in LCCTam cells performs a functional role in the acquired TAM resistance phenotype, knockdown of receptor expression should restore TAM sensitivity. LCCTam cells were transiently transfected with small inhibitory RNA (siRNA) oligonucleotides directed against ERRγ (siERRγ) or a non-silencing control (siC) prior to treating the cells with 4HT and assessing DNA synthesis as measured by BrdU incorporation. A two to three-fold decrease in ERRγ expression is attained by siRNA (Figure 3A, p<0.001). Importantly, ERRγ knockdown also partially restores sensitivity to 4HT in the LCCTam cells (Figure 3B, p=0.03 vs. siERRγ EtOH and p<0.001 vs. siC in 1000 nM 4HT) with no effect on the expression of ERα (Figure 3A, inset, bottom panel). These data suggest that ERRγ plays a key functional role in the LCCTam TAM resistance phenotype.

Fig 3
siRNA knock-down of ERRγ in LCCTam cells restores TAM sensitivity

Overexpression of ERRγ induces 4HT resistance

Next, we sought to determine whether ERRγ overexpression could induce TAM resistance in endocrine-responsive breast cancer cells. SUM44 cells grown on fibronectin-coated coverslips were transiently transfected with the pSG5-HA-ERR3 plasmid, encoding the murine homolog of ERRγ which is 100% identical to human ERRγ at the amino acid level (19), or the empty vector (pSG5). Cells were then treated with 1 μM 4HT or EtOH vehicle and immunostained for BrdU incorporation (green) and ERRγ expression (HA, red) (21). In agreement with our results in Figure 1C, 4HT significantly reduces BrdU incorporation in SUM44 cells transfected with the empty vector pSG5 (Figure 4A, panel ii vs. iv, 48.9% vs. 16.9% BrdU incorporation, p<0.001). However 4HT can no longer inhibit DNA synthesis when ERRγ is overexpressed (Figure 4A, panel iv vs. viii, 16.9% vs. 53.9% BrdU incorporation, p<0.001). The effect of ERRγ overexpression is particularly striking when comparing BrdU incorporation in transfected vs. untransfected cells in the presence of 4HT within the same field of view. In Figure 4A, most ERRγ-positive (red) cells incorporate BrdU (panel viii, arrowheads), while ERRγ-negative cells show little-to-no BrdU incorporation (panel viii, asterisks).

Fig 4
ERRγ overexpression in SUM44 and MDA-MB-134 VI breast cancer cells induces TAM resistance

To confirm that ERRγ can regulate TAM resistance in breast cancer cell lines other than SUM44, we performed the same study in MDA-MB-134 VI cells, which are ER+ and TAM-sensitive (37) and are also considered to be of lobular origin (38). When transfected with the pSG5 empty vector, DNA synthesis in MDA-MB-134 VI cells is inhibited by 4HT by nearly 2-fold (Figure 4B, 49.9% vs. 27.3% BrdU incorporation, p<0.001). However, when ERRγ is overexpressed, these cells become significantly less responsive to the inhibitory effects of 4HT (Figure 4B, 27.3% vs. 44.7% BrdU incorporation, p=0.001). Together, these data show that increased expression of ERRγ can induce TAM resistance in several ER+ lobular breast cancer cell lines.

ERRγ-associated transcriptional activity is increased in resistant LCCTam cells.

A crucial difference between ERRγ and liganded nuclear receptors like ERα is the regulation of their transcriptional activities. While ERα is dependent upon ligand for full activation, ERRγ and the other members of this orphan family exhibit constitutive transcriptional activity. The ERRγ DNA binding domain (DBD) is ∼ 64% identical to that of ERα (34). Consequently ERRγ can bind to the same estrogen response elements (EREs) as ERα, but it can also potently activate the steroidogenic factor-1 response element (SF1RE) (32). While none of the ERR family members are affected by E2 stimulation because their ligand binding domains cannot accommodate E2 binding (discussed in (34), ERRγ transcriptional activity at EREs and SF1REs can be inhibited by 4HT (39;40). In contrast, 4HT-bound ERRγ acquires the ability to positively regulate transcription at activator protein 1 (AP1) sites (reviewed in (34).

To begin to understand the mechanism by which ERRγ upregulation confers resistance to LCCTam cells, we examined the activity of ERE-, SF1RE-, and AP1-driven luciferase promoter-reporter constructs transiently expressed in SUM44 and LCCTam cells (Figure 5A). Luciferase expression controlled by the ERE and SF1RE response elements is significantly increased by 5- and 3-fold, respectively, in LCCTam cells as compared to SUM44 cells (p<0.005). When LCCTam cells are cultured in 4HT (“LCCTam+4HT”), ERE-luciferase activity is somewhat reduced but still shows a nearly 2-fold increase relative to SUM44 (black bars, p<0.005), while SF1RE-luciferase activity remains high (3-fold above the levels in SUM44 cells, white bars, p<0.005). In contrast, AP1-luciferase activity increases up to 8-fold that observed in SUM44 cells in the presence of 4HT (hatched bars, p<0.005).

Fig 5
ERRγ-associated transcriptional activity is increased in resistant LCCTam cells, and inhibition of AP1 restores TAM sensitivity

ERRγ/AP1 activity appears to drive Tamoxifen resistance in LCCTam cells

To test whether the observed robust AP1 activity plays a functional role in the TAM resistant phenotype, we used a cell-permeable peptide fragment of c-JUN that blocks its interaction with the JUN N-terminal protein kinase (JNK), resulting in strong AP1 inhibition (41). This c-JUN peptide has virtually no effect on SF1RE-luciferase activity (Figure 5B, N.S.), but can inhibit AP1-luciferase activity by more than 2-fold (Figure 5C, p=0.04). Importantly, this level of AP1 inhibition restores 4HT-mediated growth inhibition to LCCTam cells (Figure 5D, p=0.001) and enhances the sensitivity of the parental SUM44 cells to the growth-inhibitory effects of 4HT (p=0.002).

Our functional data suggest that in LCCTam cells, increased ERRγ-driven AP1 transcriptional activity is most strongly associated with TAM resistance. However, endogenous ERRγ/AP1 target genes have yet to be identified; ERRγ-dependent AP1 activity has previously been reported only on heterologous promoter constructs (42). We therefore used the TRANSFAC Professional 11.1 database (43) to search the proximal promoter regions of genes upregulated ≥2-fold in LCCTam cells for AP1 consensus sites (V$AP1) within 5000 base pairs of the start site. The MatchTM algorithm (44) was used to analyze the DNA sequences and search for potential AP1 binding sites, using Position Weight Matrices (PWMs) to minimize false positives. Several genes had multiple AP1 response elements in their promoter regions (Figure 6A). Western blot analysis was then used to confirm the overexpression of two of these genes, HMGCS2 and FASN (Figure 6B). HMGCS2 is a nuclear-encoded mitochondrial matrix gene that can regulate ketogenesis and cholesterol synthesis (45;46) and FASN is the final enzyme of the fatty acid biosynthetic pathway (47). Components of all three processes have been implicated in the etiology or progression of breast cancer, and FASN activity can affect hormonal sensitivity in breast and endometrial cancer cells (48-50). Therefore we suggest that HMGCS2 and FASN may be two novel ERRγ/AP1 targets in TAM resistant breast cancer.

Fig 6
Genes overexpressed in LCCTam cells contain multiple AP1 response elements in their promoter regions


In this study we report the development of the first model of endocrine-resistant breast cancer in a cell line derived from an invasive lobular breast carcinoma, and show that the orphan nuclear receptor ERRγ and its ability to drive AP1 transcriptional activity are central to the TAM resistance phenotype.

Selection of SUM44 cells against 4HT led to the establishment of the LCCTam cell line, which is stably resistant to TAM. In the resistant LCCTam cells we observe a significant downregulation of ERα (though they remain ER+), accompanied by a significant increase in the expression of ERRγ. Resistance to antiestrogens has been hypothesized to take place through several diverse mechanisms (10;12). One is loss or mutation of ERα, while others include alterations in the profile of hormone receptor co-activators and co-repressors expressed by the tumor, differential metabolism of antiestrogens, and changes in the expression of additional genes that control cell proliferation and/or apoptosis (13). One or more of these mechanisms is likely contributing to the TAM-resistance phenotype of LCCTam cells. Relative to SUM44, the resistant LCCTam cells express 3-fold less ERα. However, SUM44 cells express high basal levels of ERα (D. Zajchowski and S. Ethier, unpublished data). Consequently, the reduced level of ERα expression in LCCTam is comparable to that observed in MCF-7 breast cancer cells (∼73% of basal MCF-7 ERα levels by qPCR, data not shown). Since ERα levels in MCF-7 cells are clearly sufficient to confer antiestrogen sensitivity, it is unlikely that ERα downregulation in LCCTam is the major determinant of resistance in this model.

Our siRNA knock-down and cDNA overexpression studies are the first to show that ERRγ is an essential regulator of TAM responsiveness in lobular breast cancer cells. Until now, the role of ERRs (and specifically ERRγ) in breast cancer therapeutic response has not been well-understood. In 2002 Ariazi et al. published a study of ERR family expression in 38 breast tumors as compared to normal mammary epithelial cells (MECs) (36). ERRγ mRNA expression is nearly 4-fold higher in breast tumors than in MECs and is positively associated with ER and PR expression. These authors conclude that the correlation of ERRγ with ER and PR is indicative of a better prognosis (36). While this is certainly plausible, the presence of ER and PR do not always indicate hormone sensitivity in breast cancer. As discussed above, TAM therapy is ineffective in approximately 30% of patients with ER+/PR+ breast tumors, and the majority of initial responders who acquire resistance to TAM and other endocrine agents do so without losing detectable ER expression (10). 4HT-bound ERRγ is also known to activate transcription at AP1 sites, and elevated AP1 activity has previously been linked to TAM resistance in vitro (51;52) and in vivo (53;54). This is consistent with our findings that AP1 activity is robustly increased in the resistant LCCTam cells in the presence of 4HT, and that AP1 inhibition reverses the TAM-resistant phenotype of LCCTam cells while increasing the sensitivity of SUM44 cells to growth inhibition by this antiestrogen. To our knowledge, this is the first functional consequence of ERRγ-driven AP1 transcriptional activity that has been reported.

No endogenous ERRγ/AP1 target genes have yet been identified. The genes in Figure 6A are strong candidates as ERRγ/AP1 targets in breast cancer. We confirmed the differential regulation of the endogenous HMGCS2 and FASN, and we propose that HMGCS2 and FASN are putative downstream targets of ERRγ in the resistant LCCTam cell line. Further assessment of their direct regulation by ERRγ/AP1 is in progress. HMGCS2 is a nuclear-encoded mitochondrial matrix gene that can regulate ketogenesis and cholesterol synthesis (45;46) and FASN is the final enzyme of the fatty acid biosynthetic pathway (47). Components of all three processes have been implicated in the etiology or progression of breast cancer, and FASN activity can affect hormonal sensitivity in breast and endometrial cancer cells (48-50). Moreover, ERRγ has been shown to control the switch from fetal utilization of carbohydrates to lipid-dependent oxidative metabolism in the adult mouse heart by regulating a series of genes that drive fatty acid oxidation, oxidative phosphorylation, and mitochondrial electron transport (55). That ERRγ might also affect these metabolic processes in the context of breast cancer and TAM resistance is intriguing and will be the focus of future studies. Notably, this possibility is supported by a very recent publication by Montero et al., which reports that increased mitochondrial cholesterol content promotes resistance to doxorubicin in hepatocellular carcinoma (56). Other genes in Figure 6A also are of interest. High LRRC15 expression has been previously linked to invasive and aggressive behavior in breast and prostate cancer (57;58), and MSN is a marker of basal-like breast cancers (59); our future studies will also pursue the role(s) of these genes in endocrine-resistant breast cancer and their regulation by ERRγ/AP1.

Supplementary Material

figures S1_S2

table S1


We would like to thank past and current members of the laboratory for their critical comments and insightful discussions, as well as the staff of the Lombardi Comprehensive Cancer Center Flow Cytometry, Macromolecular Analysis, Microscopy and Imaging, and Tissue Culture Shared Resources for technical assistance.

Research Support: This work was generously supported by funding from the Ladies Auxiliary to the VFW, the Susan G. Komen Foundation (PDF0503551), and the Department of Defense Breast Cancer Research Program (BC051851) to RBR, and Public Health Service award CA096483-01A1 from the National Cancer Institute, and BC030280 from the Department of Defense Breast Cancer Research Program to RC. Technical services were provided by the Flow Cytometry, Macromolecular Analysis, Microscopy & Imaging, and Tissue Culture Shared Resources, which are supported by Public Health Service award 1P30-CA-51008-16 (Cancer Center Support Grant to the Lombardi Comprehensive Cancer Center).


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