ERRα cancer....Nov 05, 2015 · (EREs), while ERRα binds to a distinct genomic motif termed the ERRE, and only competes with ERα on EREs that also contain an embedded ERRE (11)

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ERRα is a marker of tamoxifen response and survival in triple-negative breast cancer.

Running Title:

ERRα is a marker of tamoxifen response in TNBC

Authors

Subrata Manna1, Josefine Bostner2, Yang Sun3, Lance D. Miller4, Anya Alayev1, Naomi S. Schwartz1, Elin Lager2, Tommy Fornander5, Bo Nordenskjöld2, Jane J. Yu3, Olle Stål2, Marina K. Holz1,6,

1. Department of Biology; Stern College for Women of Yeshiva University; New York, NY USA;

2. Department of Clinical and Experimental Medicine, and Department of Oncology, Linköping University, Linköping, SE-58185, Sweden;

3. Division of Pulmonary and Critical Care, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School; Boston, MA USA;

4. Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC USA ; The Comprehensive Cancer Center of Wake Forest University, Winston Salem, NC USA;

5. Department of Oncology, Karolinska University Hospital, Stockholm South General Hospital, Karolinska Institute, Stockholm, SE-11883, Sweden;

6. Department of Molecular Pharmacology; Albert Einstein College of Medicine; New York, NY USA; Albert Einstein Cancer Center; Albert Einstein College of Medicine; Bronx, NY USA.

Corresponding author: Marina K. Holz, Yeshiva University, 245 Lexington Avenue, New York, NY 10016; Phone: 212-30-7838; [email protected]

Grant support This work was supported by grants to MKH from the NIH (CA151112), Atol Charitable Trust, American Cancer Society (RSG-13-287-01-TBE), NIH grant to JJY (HL098216), fellowship support to AA from the National Cancer Center, and funding from Yeshiva University and the Swedish Cancer Society.

The authors disclose no potential conflicts of interest

Research. on January 22, 2021. © 2015 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 5, 2015; DOI: 10.1158/1078-0432.CCR-15-0857

http://clincancerres.aacrjournals.org/

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Statement of Translational Relevance

While many targeted and hormonal treatment options exist for hormone-positive breast cancer,

only standard chemotherapy is available for the treatment of triple-negative breast cancer

(TNBC). Hereby, we identified a subgroup within the TNBC population that could potentially gain

from endocrine treatment. The clinical value of Estrogen-Related Receptor alpha (ERRα) was

explored in randomized cohorts of tamoxifen-treated and adjuvant-untreated patients. Analysis

of a large multi-institutional cohort revealed that gene expression of ERRα predicts tamoxifen

response and improved distal metastasis-free survival (DMFS) of patients with basal-like breast

cancer. Protein analysis of ERRα expression from a large trial of adjuvant tamoxifen-untreated

patients revealed a worse prognosis for TNBC patients harboring tumors with high nuclear

expression of ERRα. Surprisingly, analysis of patient outcome in the tamoxifen-treated arm of

this trial revealed that high ERRα expression is a good prognostic factor for tamoxifen-treated

TNBC patients. This finding may have a great clinical value.




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Abstract

Purpose: Estrogen-related receptor alpha (ERRα) signaling has recently been implicated in

breast cancer. We investigated the clinical value of ERRα in randomized cohorts of tamoxifen-

treated and adjuvant-untreated patients.

Experimental design: Cox proportional hazards regression was used to evaluate the

significance of associations between ERRα gene expression levels and patient DMFS in a

previously published microarray dataset representing two thousand breast tumor cases derived

from multiple medical centers worldwide. The 912 tumors used for immunostaining were from a

tamoxifen-randomized primary breast cancer trial conducted in Stockholm, Sweden, during

1976-1990. Mouse model was used to study the effect of tamoxifen treatment on lung

colonization of MDA-MB-231 control cells and MDA-MB-231 cells with stable knockdown of

ERRα. The phenotypic effects associated with ERRα modulation were studied using

immunoblotting analyses and wound healing assay.

Results: We found that in ER-negative and triple-negative breast cancer (TNBC) adjuvant-

untreated patients, ERRα expression indicated worse prognosis and correlated with poor

outcome predictors. However, in tamoxifen-treated patients, an improved outcome was

observed with high ERRα gene and protein expression. Reduced ERRα expression was

oncogenic in the presence of tamoxifen, measured by in vitro proliferation and migration assays

and in vivo metastasis studies.

Conclusion: Taken together, these data show that ERRα expression predicts response to

tamoxifen treatment, and ERRα could be a biomarker of tamoxifen sensitivity and a prognostic

factor in TNBC.




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Introduction

Triple-negative breast cancer (TNBC; estrogen receptor negative, progesterone receptor

negative, human epidermal growth factor receptor 2 (HER2) negative) is a subtype of cancer

that is particularly challenging to treat. TNBC accounts for 15-20 % of newly diagnosed breast

cancer cases. TNBC incidence rates are higher in younger women, African-Americans and in

patients with mutations in the BRCA1 gene (1). These tumors respond to conventional

chemotherapy but have a significantly higher probability of relapse with visceral and cerebral

metastasis and poorer overall survival in the first few years after diagnosis compared with other

breast cancer subtypes (1). Unlike other breast cancer subtypes, targeted therapies for TNBC

have yet to progress past clinical trial stage to approval. In the absence of a well-defined

therapeutic target, chemotherapy is the only effective treatment option. Retrospective correlative

studies focusing on identification of the subgroup of TNBC that may benefit from standard drugs

have become an important approach (2).

Tamoxifen was the first selective estrogen receptor modulator (SERM) approved for prevention

and long-term adjuvant therapy of breast cancer. Although tamoxifen has been found to be an

effective therapeutic strategy for ER-positive breast cancer, it was generally found ineffective in

ER-negative breast cancer (3). Nonetheless, stratification of TNBC based on molecular

expression signatures revealed that ERβ may serve as a predictive factor of tamoxifen response

in ER-negative breast cancer (4, 5). In addition, it was shown that co-targeting Akt may sensitize

ER-negative cells to tamoxifen (6, 7). These data underscore the importance of considering the

role of oncogenes and members of the steroid hormone receptor family as predictors of

tamoxifen sensitivity in TNBC.

Estrogen-related receptor alpha (ERRα) is an orphan nuclear receptor and an important

component of signaling networks in breast cancer cells (8). Although its ligand has not yet been

identified, ERRα activity depends on the expression level and/or activity of its co-regulators,




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peroxisome proliferator activated receptor gamma coactivator-1 alpha and beta (PGC-1α and

PGC-1β) (9). ERRα regulates transcription of various enzymes involved in glycolysis,

tricarboxylic acid cycle (TCA), lipid, amino- and nucleic acid metabolism thus accommodating

energy demands of proliferating cancer cells (9-12). Notably, there exists a distinct pattern of

genomic recruitment for ERα and ERRα, whereby ERα binds to Estrogen Response Elements

(EREs), while ERRα binds to a distinct genomic motif termed the ERRE, and only competes

with ERα on EREs that also contain an embedded ERRE (11). Moreover, the two transcription

factors regulate specialized cohorts of target genes in breast cancer cells. While ERα directs

gene expression in development and proliferation, it was determined that ERRα target genes

show enrichment for metabolic-related functions and genes associated with breast cancer

biology (11).

High ERRα expression was shown to associate with poor prognosis in breast and ovarian

cancer patients (10, 13, 14). Moreover, studies have shown that downregulation of ERRα

activity by pharmacological antagonists decreased cell proliferation and tumorigenicity in both

ER-positive and ER-negative breast cancer (15-17). These findings have triggered a significant

interest in targeting ERRα for the treatment of TNBC.

Because of the distinct genomic and physiological activities of ERRα and ERα (11), and the

association between high ERRα expression and a worse prognosis in breast cancer patients

(13), we investigated the correlation between ERRα levels and sensitivity to tamoxifen in breast

cancer cell lines and in tumor specimens derived from clinical trials of adjuvant tamoxifen. We

found ERRα to modulate tamoxifen sensitivity. Interestingly, ERRα expression was shown to

potentially predict tamoxifen sensitivity in ER-negative breast cancer and TNBC, emerging as a

novel biomarker of tamoxifen response with a potential for personalized treatment strategy for

patients with ER-negative and triple-negative breast cancer.




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Materials and Methods

Cell Culture

MDA-MB-231 were obtained from Dr. Juan Massague (Memorial Sloan Kettering Cancer

Center), MDA-MB-436 and MDA-MB-468 cells were obtained from ATCC. All cells were grown

in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1%

Penicillin-Streptomycin in 37oC incubator with humidified 5% CO2 atmosphere. Stable ERRα

knockdown and control MDA-MB-231 luciferase-expressing cells were a generous gift from

Donald P. McDonnell (Duke University). (No independent authentication of cell lines was done).

Immunoblotting

Cells were lysed, resolved using sodium dodecyl sulphate polyacrylamide gel electrophoresis

(SDS-PAGE) and transferred onto nitrocellulose membrane for immunoblotting and near-

infrared detection using IRDye-conjugated secondary antibodies (LI-COR) as previously

described (18). Anti-ERRα and anti-actin antibodies were purchased from Millipore and Santa

Cruz Biotechnology, respectively; Survivin, E-cadherin and Fibronectin antibodies were from

Sigma. Quantification of protein levels was performed using Odyssey Image Studio Version 5.2.

Cell Proliferation Assays

Cells were seeded at a density of 5000 cells/well in 96 well plates and allowed to attach

overnight. Next day, cells were placed in media containing 1% FBS with or without 100 nM 4-

hydroxytamoxifen. Cell proliferation was assayed using the neutral red (NR) assay as described

(19, 20).

Animal Studies

All animal work was performed in accordance with protocols approved by the Institutional

Animal Care and Use Committee-Children Hospital Boston. Female CB17 SCID mice were

purchased from Taconic. MDA-MB-231 cells-LUC control cells or MDA-MB-231 cells-LUC cells




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with stable ERRα knockdown were harvested, suspended in 100 μL PBS (5×106/mL), and

injected intravenously into mice (n=5/group). Lung colonization was monitored using

bioluminescent live imaging at 1, 6 and 24 h post cell injection. Ten minutes prior to imaging,

animals were injected with D-luciferin (Perkin Elmer, 120 mg/kg, i.p.). Bioluminescent signals

were recorded using the Xenogen IVIS 200 System. Total photon flux of chest area was

analyzed. Mice were administrated with vehicle or tamoxifen (80mg/kg, p.o.) after imaging at

one-hour post cell inoculation. The photon flux at the chest regions was evaluated.

Wound Healing Assay

Cells were seeded in 6-well plates in complete DMEM media and grown to confluency in

monolayer overnight. Wound/scratch was created along the diameter of each well using a 200μl

pipette tip. Cell debris were removed by washing once with PBS, followed by addition of fresh

media containing 100 nM 4-hydroxytamoxifen. Wound healing was measured as described (21).

ESRRA Expression Survival analysis

Cox proportional hazards regression was used to evaluate the significance of associations

between ESSRA (the gene encoding for ERRα) expression levels and patient DMFS in a

previously published microarray dataset representing two thousand breast tumor cases derived

from multiple medical centers worldwide (22). The analysis focused on previously classified

basal-like and luminal (A and B) breast cancers corresponding to patients receiving tamoxifen

(hormone therapy) and chemotherapy (taxane and/or anthracycline-based therapy, or

combination of both). ESRRA gene expression was quantified by averaging the MAS5.0-

normalized log2 signal intensities corresponding to Affymetrix probe set IDs 203193_at and

1487_at, both of which were designed against the ESRRA gene. The decision to average these

two probe sets was based on an observed highly statistically significant positive correlation that




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suggested both probe sets reflect ESRRA expression. Independent analysis of the two probe

sets did not yield substantially different results (data not shown).

Patients and tumors for protein detection analysis

The 912 tumors used for immunostaining were from a tamoxifen-randomized primary breast

cancer trial conducted in Stockholm, Sweden, during 1976-1990 (23). Women were

postmenopausal at the time of diagnosis. The present study was designed and presented with

regard to the reporting recommendations for tumor marker prognostic studies (REMARK)

guidelines (24). Ethical approval was from the Karolinska Institute Ethics Council. Demographic

data and detailed information on the cohort was previously described (25). Hormone receptor

and HER2 status was determined retrospectively, and previously described (26). ER status was

not a determinant for tamoxifen treatment.

Immunohistochemistry

Tissue micro arrays (TMA) with 4µm sections of three individual cores per tumor were

constructed from formalin-fixed paraffin-embedded tumors. The PT-link system (Dako,

Denmark; PT10126) with the Envision FLEX Target Retrieval Solution Low pH was used for

deparaffinization, rehydration, and epitope retrieval. Slides were washed in PBS/0.05% tween

20, subjected to endogenous peroxidase inactivation in 3% hydrogen peroxide, blocked for 10

min in serum-free protein block (Spring Bioscience, Freemont, CA), and incubated with

ERRα primary antibody (Abcam, UK, H5844; ab41868, 1:100 dilution) in a moisturized chamber

at 4°C overnight. On day 2, slides were incubated with secondary antibody (Dako Cytomation

Envision+ HRP system) at room temperature for 30 min, developed in DAB (3.3-

diaminobenzidine hydrochloride) for 8 min, counterstained with haematoxylin for 1 min,

dehydrated in ethanol series and mounted with Pertex (HistoLab, Sweden). Washing steps were

conducted in PBS/0.5% BSA. Images at 20x and 40x magnification were produced with the




9

Olympus BX21 microscope/Olympus DP70 camera, and whole slide images were generated

using the ScanScope AT at 200x magnification (Aperio, Vista, CA).

ERRα protein scoring

Staining was evaluated in 808 tumors, giving a success rate of 89%. Tumors were evaluated for

intensity levels (negative=0, weak=1, medium=2 and strong=3) and location (nucleus or

cytoplasm). In addition, the percentage of positive nuclei was determined (0%=0, 1-25%=1, 26-

75%=2, and >75%=3). Scoring was thereafter conducted by two individual observers (J.B and

E.L), blinded to clinical data. A consensus on the score of each tumor was reached after

individual scoring. For cytoplasmic staining, the cutoff for high expression was set at >1.

Nuclear staining intensity and frequency score was added to range from 0-6, where cutoff for

high expression was set at >4.

Statistics

The Pearson χ2-test and the Spearman rank order correlation were performed to compare

protein expression levels with clinicopathological data and other previously defined protein

expression levels in the tumor set. R-values were produced within the Spearman analysis. The

end-point used to create Kaplan-Meier curves and log-rank p-values was recurrence-free

survival (RFS). In addition to RFS, hazard ratios (HR) with 95% confidence interval (CI) for

metastasis-free survival (MFS) and breast-cancer survival (BCS) were generated with the Cox

proportional hazards model. Endocrine-treated patients were excluded in the prognostic

analyses, and ER-negative patients were excluded in the treatment prediction analyses, if not

otherwise specified. P<0.01 was considered significant to adjust for the effect of multiple

comparisons in tables 2 and 3. All statistical analyses of the patient cohort were conducted in

the Statistica 10 software.




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Results

Gene expression analysis reveals that expression of ESSRA, the gene encoding for

ERRα, may be predictive of tamoxifen response in patients with basal-like breast cancer.

Using a previously described (22) multi-institutional breast tumor cohort profiled for gene

expression on Affymetrix GeneChip microarrays, we examined the association between ESRRA

expression levels in tumors and patient distant metastasis-free survival (DMFS). In this cohort,

814 patients received tamoxifen and 152 received chemotherapy, of those, 757 ER-positive and

54 ER-negative patients received tamoxifen. First, we analyzed the expression distributions of

ESRRA in breast tumor subtypes and we observed significant differences among subtypes (Fig.

1A). Specifically, ESRRA was overexpressed in ER-negative compared to ER-positive breast

cancer cohort (p<0.001), and ESRRA was overexpressed in basal breast cancer molecular

subtypes compared to luminal (p<0.001).

With regard to disease outcome in tamoxifen-treated patients (Fig. 1B), while there was no

correlation between ESRRA expression and outcome (HR=1.04) for ER-positive patients,

ESRRA expression trended towards a good outcome association in ER-negative patients

(HR=0.63), but did not reach significance (p=0.22), which may result from the small sample size

as ER-negative patients typically did not receive tamoxifen in these cohorts (as expected).

Since data on clinical estrogen receptor status was incomplete, we examined the association

between ESRRA and DMFS in the luminal (ER-positive like) and basal-like (ER-negative like)

breast cancer molecular subtypes (27) by Cox proportional hazards regression (Fig. 1B). In

luminal breast tumors, ESRRA expression was not found to be significantly associated with

DMFS in either of tamoxifen or chemotherapy-treated patients (427 and 46 patients,

respectively). However, for patients with basal-like breast tumors treated with tamoxifen (102

individuals), ESRRA was found to be marginally associated with prolonged DMFS (HR=0.51,

p=0.067). By contrast, in those with basal-like tumors treated only with chemotherapy (53




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patients), ESRRA was significantly associated with a shorter interval to metastasis (HR=2.61,

p=0.021). These findings support the notion that ESSRA expression may predict response to

tamoxifen treatment to enhance DMFS of patients with basal-like breast cancer. Together, our

data indicate that patients with ER-negative tumors characterized by high ESRRA expression

may be adversely affected by chemotherapy, but may derive clinical benefit from tamoxifen.

Cytoplasmic ERRα is mostly expressed in ER- and PR-negative, high grade, and large

tumors. We next analyzed ERRα protein expression using TMA constructed using material

from a large trial of adjuvant tamoxifen in breast cancer (23). Association analysis of ERRα

protein levels with clinicopathological data showed strong correlations of cytoplasmic ERRα with

ER-negative, PR-negative, high grade and tumors larger than 20 mm at surgery (Table 1). A

ranked correlation with all ERRα-expression levels without regard to the set cut-offs showed

stable results (data not shown). This was not evident for nuclear ERRα, although trends were in

the same direction. In the ER-positive group we found a close association between cytoplasmic

ERRα and the 40S ribosomal S6 kinase 1 (S6K1), the best-characterized kinase downstream of

the mechanistic target of rapamycin (mTOR) (28, 29), which we recently found to reduce

proliferation and response to tamoxifen (30), but the association was not evident when ERRα

was localized to the nucleus (Supplementary Table S1). Nuclear ERRα was frequently

overexpressed in tumors with an active AKT/mTOR-pathway, a signature we found to predict

tamoxifen benefit (25). Also worth mentioning is the strong correlation of nuclear ERRα with

HER2-positivity and large tumor size seen in the ER-negative subgroup.

ERRα protein expression has prognostic value in triple-negative breast cancer. The

antibody specificity was tested in formalin-fixed paraffin-embedded MCF7 cells (Supplementary

Fig. S1). Protein expression and location was determined with cutoffs dividing the subgroups in

similarly large groups. To approach the true prognostic value of ERRα, the adjuvant tamoxifen-




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treated patients were omitted in the analysis. We observed a worse prognosis for ER-negative

(Supplementary Fig. S2A-B) and TNBC patients (Fig. 2A-B) harboring tumors with high nuclear

expression of ERRα. This was also true in a Cox-proportional hazard multivariate analysis

adjusting for tumor size (ER-negative breast cancer (p=0.025) and TNBC (p=0.0057)).

However, no prognostic value was evident for cytoplasmic ERRα-protein levels regardless of

subgroup (Fig. 2A-C); also no prognostic significance was observed for nuclear ERRα in ER-

positive tumors (Supplementary Fig. 3A-B).

High ERRα expression is a good prognostic factor in tamoxifen-treated TNBC patients.

To compare ERRα gene-expression data with ERRα protein-expression results the prognostic

value was analyzed in tamoxifen-treated patients (Fig. 2C-D, and Supplementary Fig. S3C-D).

In ER-positive patients, the levels or subcellular localization of ERRα had no consequence for

the prognosis, irrespective of the end-points used - breast-cancer survival (BCS), recurrence-

free survival (RFS), or metastasis-free survival (MFS) (Supplementary Fig. S3C-D). In contrast,

in TNBC patients treated with tamoxifen, high protein expression of ERRα indicated an

improved prognosis, irrespective of subcellular localization (Fig. 2C-D). This was most evident

for cytoplasmic ERRα with the end-point RFS, although all end-points and nuclear location

trended in the same direction.

Patients with ER-negative tumors and high nuclear ERRα benefit from tamoxifen. The

cohort used in this study was randomized to tamoxifen or no tamoxifen, regardless of ER-status.

This made it possible to evaluate the predictive role of ERRα with regard to tamoxifen

treatment. For ER-positive breast cancer patients, ERRα did not associate with tamoxifen

response (Fig. 3A-B). Although ER-positive breast cancer patients with high ERRα nuclear

expression did better with tamoxifen treatment, that effect could be due to the fact that ER-

positive breast cancers respond well to tamoxifen treatment in general. Interestingly, however,




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ERRα expression significantly improved the effect of tamoxifen on recurrence-free survival in

the ER-negative subgroup when nuclear ERRα was tested for, p (interaction)=0.0052 (Fig. 3C-

D). This is an important finding because previous analyses indicated no benefit from tamoxifen

in ER-negative patients (3).

ERRα affects expression of mesenchymal markers and proliferation upon tamoxifen

treatment. We next examined whether tamoxifen may act via ERRα to affect phenotypic

changes, such as Epithelial-Mesenchymal Transition (EMT), proliferation, migration and

metastasis.

Over-expression of ERRα increased the levels of fibronectin in ER-negative cells, an effect most

pronounced in MDA-MB-468 and MDA-MB-231 cells (Fig. 4A and 4B). We observed that while

MDA-MB-231 cells did not express E-cadherin, as previously described (31), ERRα

overexpression also resulted in loss of E-cadherin expression (Fig. 4A and 4C) in MDA-MB-436

and MDA-MB-468 cells. The overexpression of fibronectin and loss of E-cadherin suggested

that ERRα reinforces the mesenchymal phenotype of ER-negative cells, and consistent with the

finding that high ERRα expression is a negative prognostic marker in patients (Fig. 2B).

Treatment with tamoxifen caused reduction in expression of survivin, an anti-apoptotic protein,

underscoring the cell death-inducing potential of tamoxifen in vitro (Fig. 4D). To examine the

effect of ERRα modulation on cell proliferation, we obtained MDA-MB-231 cells with stable

reduction in ERRα expression (10). After five days in culture, MDA-MB-231-ERRα shRNA cells

grew faster than the control cells over a five-day period (Fig. 4E). This finding is not consistent

with published data indicating that these cells exhibit no change in proliferation rates in vitro,

and could stem from different culture media conditions allowing us to detect those differences

(10). Surprisingly, when analyzing the effects of tamoxifen in MDA-MB-231-ERRα shRNA, we

observed that tamoxifen potentiated cell proliferation, indicating a relationship between




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ERRα expression and tamoxifen sensitivity (Fig. 4E). Finally, we examined migration of MDA-

MB-231 cells, as measured by the wound healing assay. As shown in Fig. 4F and 4G, ERRα

knockdown increased the migration of MDA-MB-231 cells. Interestingly, tamoxifen potentiated

the migration of MDA-MB-231 cells, consistent with the proliferation effects observed in Fig. 4E.

While in the knockdown cells migration was slightly increased by tamoxifen, it was not

statistically significant when compared to control cells, although when compared to untreated

knockdown cells, there was a statistical difference. Although tamoxifen treatment of ERRα-

positive cell did not lead to changes in proliferation and migration to the same extent as

observed by clinical data, this could be due to several factors. In vitro treatment of cells does not

wholly replicate in vivo conditions, such as the effect of the tumor microenvironment. Moreover,

the cells were treated with tamoxifen for a relatively short period of time as opposed to years-

long treatment in patients.

Tamoxifen increases metastasis of MDA-MB-231-ERRα shRNA cells in vivo. To determine

the impact of ERRα on the sensitivity to tamoxifen in a lung colonization model of breast cancer

cells, we inoculated immunodeficient mice with luciferase-expressing MDA-MB-231-ERRα

shRNA cells or MDA-MB-231-control shRNA cells (Fig. 5A). The level of bioluminescence was

measured using the Xenogen IVIS System. Within one hour post-cell inoculation, similar levels

of bioluminescence were observed in the chest regions of all mice (Fig. 5B). At 24 h,

chemiluminescence was greater in the mice inoculated with MDA-MB-231-ERRα shRNA cells

compared to control shRNA cells. Moreover, tamoxifen treatment enhanced the levels of

bioluminescence in the chest regions of mice inoculated with both MDA-MB-231-ERRα shRNA

cells and control shRNA cells, suggesting that tamoxifen potentiates metastatic potential of ER-

negative cells, consistent with proliferation and migration data presented in Fig. 4.




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Discussion

In this study we present new functional and clinical data describing the transcription factor

ERRα in breast cancer. A novel conclusion we present here is that ERRα expression plays an

important role in the tamoxifen sensitivity of postmenopausal women with TNBC, supported by

in vitro and in vivo data, as well as gene and protein expression analyses from patient studies.

This is an important and exciting finding because it was previously thought that only ER-positive

patients derive benefit from tamoxifen (32).

The inverse correlation between ERRα and ERα in vitro was determined in breast cancer

patients at the gene and protein expression level. ERRα expression was higher in basal-like and

ER-negative tumors than in luminal and ER-positive tumors, respectively. These findings led us

to investigate the prognostic and treatment predictive role of ERRα in subgroups based on ER-

status. The cohort tested for immunoreactivity of the ERRα protein was adjuvant-untreated after

surgical removal of the tumor. A high nuclear expression of ERRα predicted worse prognosis of

ER-negative and TNBC cohorts. ERRα was previously suggested to be oncogenic, with higher

expression in tumors than in normal tissue, and high levels were associated with increased risk

of recurrence in breast cancer (13, 33). Taken together, these data suggest ERRα as a target

for inhibition in ER-negative tumors.

ERRα expression did not affect RFS of tamoxifen-treated and endocrine-untreated patients

diagnosed with ER-positive tumors. In contrast, testing the treatment-predictive role of ERRα in

ER-negative tumors, commonly not responsive to endocrine treatment, we observed improved

outcome with tamoxifen treatment compared with untreated controls with high ERRα nuclear

levels. Interestingly, with low ERRα levels in the tumor, patients tended to have shorter time to

recurrence and significantly shorter time to breast cancer-related death. This result is in line with




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our in vitro data showing a protective role of ERRα, as tamoxifen increased proliferation and

migration when ERRα was reduced in TNBC cells. Hence, in the ER-negative, tamoxifen-

treated setting, ERRα seems to act as a tumor suppressor. To follow up these results, the

prognostic value of the ERRα in tamoxifen-treated patients was analyzed in two cohorts. First,

high mRNA levels of ERRα showed a marginally improved prognosis for basal-like tumors, and

second, a high ERRα-protein expression showed improved prognosis for TNBC.

Tumor metastasis or transition of in situ tumors to invasive tumors is a complex process

associated with loss of cell-cell adhesion, migration, invasion, cytoskeletal reorganization,

morphological transition and proliferation of cancer cells receiving signals and interacting with

the extracellular matrix, neighboring cells and growth factors (34-36). Decreased E-cadherin

and increased expression of fibronectin characterize EMT, one of the major mechanisms

associated with breast cancer metastasis (31, 37). Fibronectin expression is also inversely

correlated with survival and clinical outcome of the breast cancer patients as fibronectin

upregulation leads to increased invasion and metastasis of breast cancer (34-36, 38). In

agreement with the above-described studies, we observed increased fibronectin expression in

cells overexpressing ERRα.

According to previous studies, tamoxifen lacks selectivity and showed pro-apoptotic effects by

modulation of various cell signaling proteins in an ER-independent manner in ER-negative

breast cancer cells (39-41). In this study, we found that tamoxifen decreased the expression of

survivin, an anti-apoptotic protein, which may lead cell death after long-term treatment with

tamoxifen. Tamoxifen has been shown not to bind ERRα (42), hence its role in this setting is

more complex allowing for tamoxifen off-target effects to be manifested as a function of ERRα

expression. The activity of ERRα on gene promoters is regulated by miRs (eg 125a and 137)

(43, 44), coactivators and corepressors (9). Its ability to control transcription through activation




17

and repression at specific gene promoters may be regulated strongly by the cellular setting,

including proliferative pathways such as the AKT/mTOR/S6K1 and the ERα signaling pathways.

In addition, we cannot rule out the possibility that the effects of tamoxifen in TNBC are not cell-

autonomous, and instead result from complex interaction between downstream effectors of the

ERRα−regulated gene program and the tumor stroma and microenvironment.

Taken together, our data support the role of ERRα as a prognostic marker in TNBC and a

predictor of response to tamoxifen. It will be important to confirm our results in other larger

cohorts of patients with TNBC, randomized based on expression of ERRα, and uniformly treated

with tamoxifen. In addition, it would be important to determine whether the predictive value of

ERRα exists for other forms of endocrine therapy, such as aromatase inhibitors. In addition, our

results may motivate further investigation into the molecular and biochemical relationship

between ERRα and tamoxifen. Because tamoxifen is a well-tolerated globally-available generic

drug, it may be an attractive alternative for some patients in lieu of cytotoxic therapies.

Acknowledgements We thank Donald P. McDonnell for generous providing the luciferase-expressing MDA-MB-231 cells. We thank the following individuals: Dennis Sgroi (grade), Birgitta Holmlund (TMA), Lambert Skoog (ER, PgR, HER2, pathological evaluation), Elin Karlsson (pAkt, S6K1).

References

1. Mayer IA, Abramson VG, Lehmann BD, Pietenpol JA. New strategies for triple-negative breast cancer--deciphering the heterogeneity. Clinical cancer research : an official journal of the American Association for Cancer Research. 2014;20:782-90. 2. Schneider BP, Winer EP, Foulkes WD, Garber J, Perou CM, Richardson A, et al. Triple-negative breast cancer: risk factors to potential targets. Clinical cancer research : an official journal of the American Association for Cancer Research. 2008;14:8010-8. 3. Early Breast Cancer Trialists' Collaborative G, Clarke M, Coates AS, Darby SC, Davies C, Gelber RD, et al. Adjuvant chemotherapy in oestrogen-receptor-poor breast cancer: patient-level meta-analysis of randomised trials. Lancet. 2008;371:29-40. 4. Gruvberger-Saal SK, Bendahl PO, Saal LH, Laakso M, Hegardt C, Eden P, et al. Estrogen receptor beta expression is associated with tamoxifen response in ERalpha-negative breast carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research. 2007;13:1987-94.




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5. Yan Y, Li X, Blanchard A, Bramwell VH, Pritchard KI, Tu D, et al. Expression of both estrogen receptor-beta 1 (ER-beta1) and its co-regulator steroid receptor RNA activator protein (SRAP) are predictive for benefit from tamoxifen therapy in patients with estrogen receptor-alpha (ER-alpha)-negative early breast cancer (EBC). Annals of oncology : official journal of the European Society for Medical Oncology / ESMO. 2013;24:1986-93. 6. Liu CY, Hung MH, Wang DS, Chu PY, Su JC, Teng TH, et al. Tamoxifen induces apoptosis through cancerous inhibitor of protein phosphatase 2A-dependent phospho-Akt inactivation in estrogen receptor-negative human breast cancer cells. Breast cancer research : BCR. 2014;16:431. 7. Weng SC, Kashida Y, Kulp SK, Wang D, Brueggemeier RW, Shapiro CL, et al. Sensitizing estrogen receptor-negative breast cancer cells to tamoxifen with OSU-03012, a novel celecoxib-derived phosphoinositide-dependent protein kinase-1/Akt signaling inhibitor. Molecular cancer therapeutics. 2008;7:800-8. 8. Jarzabek K, Koda M, Kozlowski L, Sulkowski S, Kottler ML, Wolczynski S. The significance of the expression of ERRalpha as a potential biomarker in breast cancer. The Journal of steroid biochemistry and molecular biology. 2009;113:127-33. 9. Deblois G, Giguere V. Oestrogen-related receptors in breast cancer: control of cellular metabolism and beyond. Nature reviews Cancer. 2013;13:27-36. 10. Stein RA, Chang CY, Kazmin DA, Way J, Schroeder T, Wergin M, et al. Estrogen-related receptor alpha is critical for the growth of estrogen receptor-negative breast cancer. Cancer Res. 2008;68:8805-12. 11. Deblois G, Hall JA, Perry MC, Laganiere J, Ghahremani M, Park M, et al. Genome-wide identification of direct target genes implicates estrogen-related receptor alpha as a determinant of breast cancer heterogeneity. Cancer research. 2009;69:6149-57. 12. Deblois G, St-Pierre J, Giguere V. The PGC-1/ERR signaling axis in cancer. Oncogene. 2013;32:3483-90. 13. Suzuki T, Miki Y, Moriya T, Shimada N, Ishida T, Hirakawa H, et al. Estrogen-related receptor alpha in human breast carcinoma as a potent prognostic factor. Cancer research. 2004;64:4670-6. 14. Fujimoto J, Alam SM, Jahan I, Sato E, Sakaguchi H, Tamaya T. Clinical implication of estrogen-related receptor (ERR) expression in ovarian cancers. The Journal of steroid biochemistry and molecular biology. 2007;104:301-4. 15. Chisamore MJ, Wilkinson HA, Flores O, Chen JD. Estrogen-related receptor-alpha antagonist inhibits both estrogen receptor-positive and estrogen receptor-negative breast tumor growth in mouse xenografts. Molecular cancer therapeutics. 2009;8:672-81. 16. Chang CY, Kazmin D, Jasper JS, Kunder R, Zuercher WJ, McDonnell DP. The metabolic regulator ERRalpha, a downstream target of HER2/IGF-1R, as a therapeutic target in breast cancer. Cancer cell. 2011;20:500-10. 17. Bianco S, Lanvin O, Tribollet V, Macari C, North S, Vanacker JM. Modulating estrogen receptor-related receptor-alpha activity inhibits cell proliferation. The Journal of biological chemistry. 2009;284:23286-92. 18. Alayev A, Sun Y, Snyder RB, Berger SM, Yu JJ, Holz MK. Resveratrol prevents rapamycin-induced upregulation of autophagy and selectively induces apoptosis in TSC2-deficient cells. Cell cycle. 2014;13:371-82. 19. Yamnik RL, Digilova A, Davis DC, Brodt ZN, Murphy CJ, Holz MK. S6 kinase 1 regulates estrogen receptor alpha in control of breast cancer cell proliferation. The Journal of biological chemistry. 2009;284:6361-9. 20. Yamnik RL, Holz MK. mTOR/S6K1 and MAPK/RSK signaling pathways coordinately regulate estrogen receptor alpha serine 167 phosphorylation. FEBS letters. 2010;584:124-8. 21. Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nature protocols. 2007;2:329-33.




19

22. Nagalla S, Chou JW, Willingham MC, Ruiz J, Vaughn JP, Dubey P, et al. Interactions between immunity, proliferation and molecular subtype in breast cancer prognosis. Genome biology. 2013;14:R34. 23. Rutqvist LE, Johansson H, Stockholm Breast Cancer Study G. Long-term follow-up of the randomized Stockholm trial on adjuvant tamoxifen among postmenopausal patients with early stage breast cancer. Acta oncologica. 2007;46:133-45. 24. McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM, et al. REporting recommendations for tumor MARKer prognostic studies (REMARK). Breast cancer research and treatment. 2006;100:229-35. 25. Bostner J, Karlsson E, Pandiyan MJ, Westman H, Skoog L, Fornander T, et al. Activation of Akt, mTOR, and the estrogen receptor as a signature to predict tamoxifen treatment benefit. Breast cancer research and treatment. 2013;137:397-406. 26. Jerevall PL, Jansson A, Fornander T, Skoog L, Nordenskjold B, Stal O. Predictive relevance of HOXB13 protein expression for tamoxifen benefit in breast cancer. Breast cancer research : BCR. 2010;12:R53. 27. Fan C, Oh DS, Wessels L, Weigelt B, Nuyten DS, Nobel AB, et al. Concordance among gene-expression-based predictors for breast cancer. The New England journal of medicine. 2006;355:560-9. 28. Holz MK. The role of S6K1 in ER-positive breast cancer. Cell cycle. 2012;11:3159-65. 29. Maruani DM, Spiegel TN, Harris EN, Shachter AS, Unger HA, Herrero-Gonzalez S, et al. Estrogenic regulation of S6K1 expression creates a positive regulatory loop in control of breast cancer cell proliferation. Oncogene. 2012;31:5073-80. 30. Bostner J, Karlsson E, Eding CB, Perez-Tenorio G, Franzen H, Konstantinell A, et al. S6 kinase signaling: tamoxifen response and prognostic indication in two breast cancer cohorts. Endocrine-related cancer. 2015;22:331-43. 31. Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, et al. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature. 2007;449:557-63. 32. Rutqvist LE, Cedermark B, Glas U, Johansson H, Nordenskjold B, Skoog L, et al. The Stockholm trial on adjuvant tamoxifen in early breast cancer. Correlation between estrogen receptor level and treatment effect. Breast cancer research and treatment. 1987;10:255-66. 33. Ariazi EA, Clark GM, Mertz JE. Estrogen-related receptor alpha and estrogen-related receptor gamma associate with unfavorable and favorable biomarkers, respectively, in human breast cancer. Cancer research. 2002;62:6510-8. 34. Hong H, Zhou T, Fang S, Jia M, Xu Z, Dai Z, et al. Pigment epithelium-derived factor (PEDF) inhibits breast cancer metastasis by down-regulating fibronectin. Breast cancer research and treatment. 2014;148:61-72. 35. Zabouo G, Imbert AM, Jacquemier J, Finetti P, Moreau T, Esterni B, et al. CD146 expression is associated with a poor prognosis in human breast tumors and with enhanced motility in breast cancer cell lines. Breast cancer research : BCR. 2009;11:R1. 36. Babina IS, McSherry EA, Donatello S, Hill AD, Hopkins AM. A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44. Breast cancer research : BCR. 2014;16:R19. 37. Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nature reviews Cancer. 2009;9:265-73. 38. Bae YK, Kim A, Kim MK, Choi JE, Kang SH, Lee SJ. Fibronectin expression in carcinoma cells correlates with tumor aggressiveness and poor clinical outcome in patients with invasive breast cancer. Human pathology. 2013;44:2028-37. 39. Fattman CL, An B, Sussman L, Dou QP. p53-independent dephosphorylation and cleavage of retinoblastoma protein during tamoxifen-induced apoptosis in human breast carcinoma cells. Cancer letters. 1998;130:103-13.




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40. Ferlini C, Scambia G, Distefano M, Filippini P, Isola G, Riva A, et al. Synergistic antiproliferative activity of tamoxifen and docetaxel on three oestrogen receptor-negative cancer cell lines is mediated by the induction of apoptosis. British journal of cancer. 1997;75:884-91. 41. Mints M, Souchelnytskyi S. Impact of combinations of EGF, TGFbeta, 17beta-oestradiol, and inhibitors of corresponding pathways on proliferation of breast cancer cell lines. Experimental oncology. 2014;36:67-71. 42. Coward P, Lee D, Hull MV, Lehmann JM. 4-Hydroxytamoxifen binds to and deactivates the estrogen-related receptor gamma. Proceedings of the National Academy of Sciences of the United States of America. 2001;98:8880-4. 43. Tiwari A, Shivananda S, Gopinath KS, Kumar A. MicroRNA-125a Reduces Proliferation and Invasion of Oral Squamous Cell Carcinoma Cells by Targeting Estrogen-related Receptor alpha: IMPLICATIONS FOR CANCER THERAPEUTICS. The Journal of biological chemistry. 2014;289:32276-90. 44. Zhao Y, Li Y, Lou G, Zhao L, Xu Z, Zhang Y, et al. MiR-137 targets estrogen-related receptor alpha and impairs the proliferative and migratory capacity of breast cancer cells. PloS one. 2012;7:e39102.




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Figure Legends:

Figure 1: ESRRA expression distribution varies among Basal-like/Luminal and ER+/ER- breast tumor and association with DMFS. (A) Box and whisker plots of ESRRA expression distributions are shown as a function of breast tumor type. Box upper and lower boundaries demarcate the 75th and 25th percentiles, respectively. Horizontal line marks the median. Whiskers (error bars) denote the 95th (upper) and 5th (lower) percentiles. Circles represent outlier values. P-values reflect the significance of the difference between the means of the distributions. (B) Cox regression analysis of associations between ESRRA and DMFS by treatment and tumor subtype. ESSRA expression may synergize with tamoxifen treatment to enhance DMFS of patients with basal-like breast cancer. Higher hazard ratio (HR) indicates poor prognosis.

Table 1: Two-times-two table of ERRα protein expression in relation to clinocopathological variables using Pearson chi2 correlation. Strong positive correlations of cytoplasmic ERRa with ER-negative, PR-negative, high grade, high mitosis and tumors larger than 20 mm at surgery. All p-values are Pearson chi2 for 2x2 tables, except grade, which is a Spearman rank order correlation.

Figure 2: The prognostic value of ERRα protein expression and subcellular location in adjuvant-untreated and tamoxifen-treated TNBC patients. The prognostic value of ERRα levels evaluated according to hormone receptor status in adjuvant untreated patients (A-B), and in tamoxifen-treated patients (C, D). Breast cancer survival (BCS), Recurrence-free survival (RFS), Metastasis-free survival (MFS), Hazard ratio (HR); (95% confidence interval). Figure 3: Tamoxifen treatment response and ERRα expression. Patients with ERα-positive breast tumors with low ERRα expression did not show an obvious gain from tamoxifen, (A, B). In ER-negative tumors, a treatment predictive value of ERRα was found. A high expression of nuclear ERRα was associated with a significant benefit from tamoxifen treatment (C, D).

Figure 4: ERRα affects expression of mesenchymal markers, and proliferation upon tamoxifen treatment of ER-negative cells. (A) MDA-MB-231, MDA-MB-436 and MDA-MB-468 cells were transfected with FLAG-ERRα, lysed and analyzed for expression by immunoblotting using the indicated antibodies. (B) Quantification of firbronectin protein levels normalized to actin from (A). (C) Quantification of E-cadherin protein levels normalized to actin from (A). (D). Cells were treated with tamoxifen for 24 h, lysed an analyzed for expression of the indicated proteins compared to vehicle-treated controls. (E) MDA-MB-231 control cells or cells with stable ERRα knockdown were plated in media with 1% FBS with 100 nM tamoxifen on Day 0. Cell proliferation was measured on day 5. Data was graphed using Excel. * p<0.05, Student’s t-test. (F) MDA-MD-231 cells with stable ERRα knockdown or control cells were seeded in 6-well plate in complete DMEM media and grown to confluency in monolayer overnight. Wound/scratch was created and cells were treated with 100 nM tamoxifen for 22h, as indicated. (G) Quantification of the wound healing assay from (F) was performed using Excel. ** p<0.01, Student’s t-test.




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Figure 5: Tamoxifen effects on metastasis of MDA-MB-231-ERRα cells in vivo. (A) Representative bioluminescent images of MDA-MB-231 cells. The total flux (photons/second) of cells is illustrated. (B). MDA-MB-231-luciferase expressing cells were inoculated intravenously. Representative bioluminescent images of lung colonization at 1, 6, and 24 h post cell injection are shown. The levels of bioluminescent intensity (total photon flux/second) present in the chest regions were quantified and compared among treatment groups. Mice were administrated with vehicle or tamoxifen (80 mg/kg, p.o.) one hour post-cell inoculation.* p<0.05, ** <0.01, Student’s t-test.






















Published OnlineFirst November 5, 2015.Clin Cancer Res Subrata Manna, Josefine Bostner, Yang Sun, et al. triple-negative breast cancer.

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