Breast cancer resistance to antiestrogens is enhanced by ......creates a core clinical challenge. In breast cancer cells rendered resistant to the ... signal transduction pathway (2)

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Breast cancer resistance to antiestrogens is enhanced by increased ER degradation and

ERBB2 expression

Tomohiro Shibata1, Kosuke Watari1, Hiroto Izumi2, Akihiko Kawahara3, Satoshi

Hattori4, Chihiro Fukumitsu3, Yuichi Murakami1,5, Ryuji Takahashi6, Uhi Toh7,

Ken-ichi Ito8, Shigehiro Ohdo9, Maki Tanaka10, Masayoshi Kage3, Michihiko

Kuwano5 and Mayumi Ono1*

1Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences,

Kyushu University, Fukuoka, Japan

2Department of Occupational Pneumology, Institute of Industrial Ecological Sciences,

University of Occupational and Environmental Health, Kitakyushu, Japan

3Department of Diagnostic Pathology, Kurume University Hospital, Kurume, Japan

4Biostatistics Center, Kurume University, Kurume, Japan

5Cancer Translational Research Center, St. Mary's Institute of Health Sciences, Kurume,

Japan

6Department of Breast Care Center, Kyushu Medical Center, Fukuoka, Japan

7Department of Surgery, Kurume University School of Medicine, Kurume, Japan

8Division of Breast and Endocrine Surgery, Department of Surgery, Shinshu University

School of Medicine, Matsumoto, Japan

9Department of Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kyushu

University, Fukuoka, Japan

10Kurume General Hospital, Japan Community Health Care Organization (JCHO),

Kurume, Japan

on March 13, 2020. © 2016 American Association for Cancer Research.cancerres.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 22, 2016; DOI: 10.1158/0008-5472.CAN-16-1593

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Running title: YBX1 mediates antiestrogen resistance

Keywords: YBX1, ESR, Antiestrogen resistance

Financial Support: This work is supported by JSPS KAKENHI grant number

15J03033 (T.S.), the Fukuoka Foundation for Sound Health Cancer Research Fund

(T.S.), the Life Science Foundation of Japan (M.O.), and St. Mary’s Institute of Health

Sciences (K.W., M.Ku., M.O).

Disclosure of Potential Conflicts of Interest

All the authors declare no potential conflicts of interest

*Corresponding author:

Mayumi Ono, PhD.,

Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences,

Kyushu University

3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582,

Japan

Phone and Fax: +81-92-642-6296

E-mail: [email protected]

Word count: 4593 words

Total number of figures: 7

Total number of supplementary figures: 2




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Abstract

Endocrine therapies effectively improve the outcomes of patients with estrogen

receptor (ER)-positive breast cancer. However, the emergence of drug-resistant tumors

creates a core clinical challenge. In breast cancer cells rendered resistant to the

antiestrogen fulvestrant, we defined causative mechanistic roles for the transcription

factor YBX1 and the levels of ER and the ERBB2 receptor. Enforced expression of

YBX1 in parental cells conferred resistance against tamoxifen and fulvestrant in vitro

and in vivo. Further, YBX1 overexpression was associated with decreased and increased

levels of ER and ERBB2 expression, respectively. In antiestrogen-resistant cells,

increased YBX1 phosphorylation was associated with a 4-fold higher degradation rate

of ER. Notably, YBX1 bound the ER, leading to its accelerated proteasomal degradation,

and induced the transcriptional activation of ERBB2. In parallel fashion, tamoxifen

treatment also augmented YBX1 binding to the ERBB2 promoter to induce increased

ERBB2 expression. Together, these findings define a mechanism of drug resistance

through which YBX1 contributes to antiestrogen bypass in breast cancer cells.




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Introduction

The detection of estrogen receptor (ER) in 70% of invasive breast cancers led to

the identification of ER expression as the most significant risk factor for breast cancer

(1). ER is activated through interaction with its ligand estradiol (E2), and the growth

and survival of ER-positive breast cancers mainly depends upon activation of the ER

signal transduction pathway (2). Selective estrogen receptor modulators and

third-generation aromatase inhibitors are widely used to treat patients with ER-positive

breast cancer (3). Antiestrogens provide significant benefits when used as adjuvants as

well as to treat recurrent or metastatic breast cancer (3, 4). Further, the selective

estrogen receptor down-regulator fulvestrant improves the prognosis of postmenopausal

women with advanced breast cancer who experience tumor progression after endocrine

therapy (5).

The epidermal growth factor receptor (EGFR) family member erb-b2 receptor

tyrosine kinase 2 (ERBB2) is a driver of breast cancer (6). Gene amplification and

overexpression of ERBB2 occur in 20%–30% of invasive breast cancers (7). An

antibody against ERBB2 (trastuzumab) significantly improves outcomes (8, 9) and

reduces the rate of recurrence by greater than 50% in patients with early-stage ERBB2–

positive breast cancer (10). Treatment with lapatinib, a dual inhibitor of EGFR and

ERBB2 tyrosine kinases, improves therapeutic efficacy when combined with

capecitabine to treat ERBB2-positive breast cancer (11). Moreover, the combination of

the anti-ERBB2 monoclonal antibody pertuzumab with trastuzumab plus docetaxel

significantly improves median overall survival from 40.8 months, achieved using

trastuzumab plus docetaxel, to 56.5 months of patients with ERBB2-positive metastatic

breast cancer (12, 13).




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The Y-box binding protein-1 (YBX1) mediates the acquisition of global resistance to

anticancer drugs (14). YBX1 knockin mice provokes breast cancers with diverse

histological characteristics, implicating YBX1 as an oncoprotein (15). Further, YBX1

knockdown inhibits the proliferation of human breast cancer cells and inhibits the

expression of ERBB2 and genes that mediate the cell cycle (16–20). YBX1 transforms

human mammary epithelial cells into aggressive breast cancer cells through chromatin

remodeling (21). Together, these studies strongly suggest the association of YBX1 with

the oncogenic potential of breast cancer cells. Further, in breast cancer, expression of

YBX1 in the nucleus is an independent prognostic factor for overall and

progression-free survival (18), and YBX1 expression predicts relapse and

disease-specific survival (22, 23). Moreover, biostatistical modeling indicates that

nuclear localization of YBX1 positively and negatively correlates with ERBB2 and ER

expression, respectively, in patients with breast cancer (18).

ER-positive breast cancers are sensitive to endocrine-therapeutic drugs (3). However,

tumors develop drug-resistance, leading to relapse and progression (24, 25). Loss of ER

expression, E2-independent ER activation, and ER-inactivating mutations increase

resistance to antiestrogens (24, 26–28). Moreover, activation of bypass pathways

induces resistance to tamoxifen, fulvestrant, and letrozole (29–33). The mTOR inhibitor

everolimus or the cyclin-dependent kinase-4 and -6 inhibitor palbociclib, in

combination with antiestrogens, are effective for treating patients with ER-positive

breast cancer (34, 35). Moreover, ERBB2 is frequently associated with

antiestrogen-resistant breast cancers (29).

Here we established breast cancer cells resistant to fulvestrant and used them to show

that YBX1 was specifically activated in these cells, leading us to ask whether YBX1




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contributes to the antiestrogen-resistance of breast cancer cells in association with ER

and ERBB2.

Material and Methods

Cell lines and chemicals

The human breast cancer cell lines MCF-7, T-47D, SKBr-3, MDA-MB231, and

MDA-MB453 were purchased from the American Type Culture Collection (Manassas,

VA). KPL-1 was purchased from Health Science Research Resources Bank (Osaka,

Japan). All cell lines were obtained between 2005 to 2010. All cell lines were cultured at

37 °C in DMEM supplemented with 10% fetal bovine serum (FBS) in a humidified

atmosphere containing 5% CO2. All cell lines were passaged for ≤6 months and were

not further tested or authenticated by the authors. We generated an antibody against

YBX1 designated (st1968) by immunizing New Zealand white rabbits with a synthetic

peptide representing YBX1 C-terminal amino acid residues 299–313 (19). This antibody

detects cytoplasmic and nuclear YBX1 in immunohistochemistry (IHC). An antibody

against YBX1 (EP2708Y, ab76149) (Abcam; Cambridge, UK) was used for chromatin

immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) assays.

Establishment of fulvestrant-resistant cell lines

We established two fulvestrant-resistant cell lines designated T-47D/FR-1 and

T-47D/FR-2 from T-47D cells by exposing them continuously for approximately 6

months to step-wise increases in fulvestrant concentrations up to 1 μM. T-47D/FR-1 and

T-47D/FR-2 were established from different flasks and were not cloned.




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Cell proliferation assay

Cells (5 × 103) were seeded in 24-well plates and counted using a Z2 Coulter Particle

Count and Size Analyzer (Beckman Coulter) 5 days after siRNA transfection. Results

are expressed as the mean ± standard deviation (SD) of triplicate wells.

YBX1/Tet-On plasmid construction

pEB-Tet-On YBX1 was generated by inserting the genes encoding resistance to

ampicillin and hygromycin from pcDNA3 (Clontech Laboratories, Inc., Mountain View,

CA), into the pEB-multi vector (Clontech). The TRE-Tight, rtTA, and YBX1-3×NLS

(AGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAG

ATCCAAAAAAGAAGAGAAAGGTAGATACGGCC)-3×FLAG were inserted into the

BamHI–EcoRV sites of the pEB-multi vector.

Pull-down assay

Deletion constructs of YBX1 and other aspects of this pull-down assay were

performed as previously described (36). Briefly, GST fusion proteins were dialyzed

against X-buffer (50 mM Tris-HCl, pH 8.0; 1 mM EDTA; 120 mM NaCl; 0.5% NP-40

10% glycerol; and 1 mM PMSF). Immobilized GST or GST fusion proteins were

incubated with Glutathione Sepharose 4B (GE Healthcare) for 4 h at 4° C. After five

washes with X-buffer (1 mL), GST fusion proteins were incubated with ER in the

presence or absence of tamoxifen (20 nM) overnight at 4°C. The complex was washed

five times with X-buffer and then subjected to SDS-PAGE.




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Mice

The Ethics of Animal Experiments Committee of Kyushu University Graduate

School of Medical Sciences approved the animal experiments, which were conducted

according to the recommendations of the United States Public Health Service Policy on

Humane Care and Use of Laboratory Animals (Office of Laboratory Animal Welfare,

National Institutes of Health, Department of Health and Human Services, RKLI, Suite

360, MSC 7982, 6705 Rockledge Drive, Bethesda, MD 20892–7982). Female BALB/c

nu/nu athymic nude mice (6–7 weeks old) were purchased from CLEA (Saga, Japan),

and housed in micro-isolator cages under a 12 h light/dark cycle. Water and food were

supplied ad libitum. Animals were observed for tumor growth, activity, feeding, and

pain according to the guidelines of the Harvard Medical Area Standing Committee on

Animals.

Xenograft studies

Approximately 5.0 × 106 T-47D/mock or T-47D/Tet YBX1 cells in 200 μL of 50%

Matrigel were implanted into the subcutaneous tissue of the right abdominal wall of the

mice that were administered 0.75 mg 60-day release estrogen pellets (Innovative

Research). Tumor sizes were measured, and tumor volumes (mm3) were calculated as

follows: length × width2 × 0.5. When tumors reached 100–200 mm3, six mice each were

randomly allocated into groups (n = 6 per group) administered doxycycline (Dox) (1 mg

per mouse, daily, p.o.) or Dox plus tamoxifen citrate (500 mg per mouse in peanut oil,

daily, s.c.), The tumors were harvested after 2 weeks, stored at –80°C, or fixed

immediately in 10% paraformaldehyde overnight at 4 °C.




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Patient selection

We screened 116 premenopausal and 114 postmenopausal patients breast cancer who

were treated between 2007 and 2013 at Kurume University Hospital or Kurume General

Hospital, underwent percutaneous biopsy with no prior treatment such as endocrine

therapy. Histological types and numbers of carcinomas were 190 invasive ductal, 14

noninvasive ductal, 5 mucinous, 4 neuroendocrine, 5 invasive lobular, 4 invasive

micropapillary, 3 apocrine, 3 metaplastic, 1 tubular, and 1 medullary. The average ages

of 116 premenopausal and 114 postmenopausal women were 45.1 (range, 28–55) and

66.3 (range, 50–102), respectively. The present study conforms to the principles of the

Declaration of Helsinki and was approved by the Institutional Review Board of Kurume

University Hospital.

In vitro assays and statistical analysis

Western blot analysis, preparation of charcoal-stripped serum (CSS), Co-IP assays, and

statistical analysis are described in Supplementary Materials and Methods.

RESULTS

Increased activation of YBX1 in fulvestrant-resistant cells is associated with respective

decreases and increases of ER and ERBB2 expression

The T-47D/FR-1 and T-47D/FR-2 cell lines were >100-fold more resistant to

fulvestrant and tamoxifen compared with the parental cells (Fig. 1A). Further, increased

phosphorylation of YBX1-Ser102 was accompanied by markedly decreased and

increased expression of ER and ERBB2, respectively, compared with their parental cells




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(Fig. 1B). Quantitative RT-PCR analysis detected approximately 30%–40% decreases

and 3–4-fold increases in ER and ERBB2 mRNA levels, respectively, in T-47D/FR-1

and T-47D/FR-2 cells (Fig. 1C). The half-lives of ER in exponentially growing T-47D

and T-47D/FR-1 cells were approximately 8 h and 2 h, respectively. Treatment with the

proteasome inhibitor MG-132 increased the half-life of ER in T-47D/FR-1 cells (Fig.

1D). Treatment with YBX1-siRNA enhanced ER expression and suppressed ERBB2

expression in T-47D/FR-1 cells (Fig. 1E), associated with significantly increased and

decreased sensitivities to fulvestrant and lapatinib, respectively (Fig. 1F).

YBX1 induces resistance to antiestrogens in ER-positive cells

To determine whether YBX1 directly influenced sensitivity to antiestrogens and ER

and ERBB2 expression, we analyzed breast cancer cells transfected with the

Dox-inducible construct YBX1/Tet-On. The levels of ER and ERBB2 decreased and

increased, respectively, in the three ER-positive cell lines KPL-1, MCF-7, and T-47D,

following Dox induction (Fig. 2A), although their levels were unchanged in the

ER-negative cell lines SKBr-3, MDA-MB231, and MDA-MB453. Consistent with

previous research (19), the expression of CDC6 increased in the six cell lines when

YBX1 was expressed (Fig. 2A).

We determined whether YBX1 overexpression affected cellular sensitivities to the

antiestrogen fulvestrant and the ERBB2-targeted drug lapatinib. T-47D/Tet YBX1 cells

expressed decreased and increased levels of ER and ERBB2, respectively, (Fig. 2B),

which were associated with respective decreased and increased sensitivities to

fulvestrant and lapatinib compared with those of T-47D/mock cells (Fig. 2C).




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Further, we determined whether YBX1 overexpression affected the sensitivity of

tumor xenografts to tamoxifen in vivo. The growth of tumors formed by T-47D/mock

cells, but not those formed by T-47D/Tet YBX1 cells, was significantly inhibited by

tamoxifen (Fig. 2D). YBX1 overexpression significantly increased the levels of ERBB2

mRNA and ERBB2 (Fig. 2E and F) compared with those of T-47D/mock-induced

tumors. Further, tamoxifen increased ERBB2 mRNA and ERBB2 levels in tumors

induced by T-47D/mock and T-47D/Tet YBX1 cells compared with those induced by

T-47D/mock cells in untreated mice. IHC analysis demonstrated an increased number of

engrafted tumor cells that expressed YBX1 in the nucleus as well as increased and

decreased levels of ERBB2 and ER, respectively, in tumors formed by T-47D/Tet YBX1

cells compared with tumors induced by T-47D/mock cells (Fig. 2G). Tamoxifen

treatment was associated with increased levels of ERBB2 in tumors formed by

T-47D/mock and T-47D/Tet YBX1 cells compared with T-47D/mock cells in untreated

mice (Fig. 2G).

YBX1 promotes proteasomal ER degradation

We asked whether the half-life of ER was influenced by YBX1 overexpression.

YBX1 was highly expressed in the cytoplasm and nucleus of YBX1/Tet-On

transfectants compared with the control (Fig. 3A). Further, ER levels decreased after

YBX1 expression was induced (Fig. 3B and C), while ER mRNA levels remained

unchanged (Fig. 3D). E2–ER binding induces the degradation of ER through the

proteasomal pathway (37, 38). Similarly, the half-life of ER decreased from

approximately 10 h to 4 h when YBX1 expression was induced in exponentially

growing T-47D cells (Fig. 3E), and treatment with the proteasome inhibitor MG-132




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increased the half-life of ER after YBX1 induction (Fig. 3E). Further, ubiquitination of

ER increased when YBX1 expression was induced (Fig. 3 F).

ER degradation increases through interaction with YBX1

Co-IP assays revealed that YBX1 bound ER (Fig. 4A), which was further increased

by E2 and inhibited by tamoxifen (Fig. 4B). To determine the YBX1 domain for

interaction with ER, we individually deleted the N-terminal, cold shock domain (CSD),

and C-terminal regions of YBX1 (Fig. 4C and Fig. S1A) (36). ER did not bind the CSD

deletion mutant GST-YBX1 Δ3 (Fig. 4C). Further, pull-down assays revealed that

tamoxifen directly inhibited YBX1–ER binding (Fig. 4D).

To identify the YBX1-interaction domain of ER, we generated ER deletion mutants

(Fig. 4E) and found that YBX1 bound only the mutant (FLAG-ER Δ2) with an intact

ligand-binding domain (Fig. 4E). Further, YBX1-induced degradation of ER was

accelerated by E2 (Fig. 4F). Moreover, when we transiently overexpressed

FLAG-YBX1 and the YBX1 mutants, the former reduced the cellular levels of ER (Fig.

S1B). In contrast, ER expression was unaffected by FLAG-YBX1 Δ3 overexpression

(Fig. S1C).

When we overexpressed FLAG-YBX1 or FLAG-YBX1 Δ3, ER degradation was

significantly lower in cells transfected with the latter that did not bind ER (Fig. 4G).

MG-132 treatment markedly increased the half-life of ER in cells transfected with

FLAG-YBX1 or FLAG-YBX1 Δ3 (Fig. S1D). The levels of ubiquitinated ER were

much higher in cells overexpressing FLAG-YBX1 vs FLAG-YBX1 Δ3 (Fig. 4H).

Transcriptional activation of the ERBB2 by YBX1 is inhibited by E2 or ER




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We asked whether YBX1 expression or antiestrogens influenced ERBB2 expression

in T-47D cells. We found that ERBB2 levels and ERBB2 mRNA levels increased

significantly in Dox-treated T-47D/Tet YBX1 cells but not in T-47D/mock cells (Fig. 5A

and B). Tamoxifen markedly increased ERBB2 expression in the ER-positive cell lines

KPL-1 and T-47D, but not in ER-negative cell lines SKBr-3 and MDA-MB231.

Fulvestrant increased ERBB2 levels in T-47D cells (Fig. 5C), and tamoxifen or

fulvestrant increased ERBB2 mRNA levels (Fig. 5D).

We performed ChIP assays to determine whether the absence or presence of activated

ER affected YBX1 binding to the putative Y-box sites #1 and #2 in ERBB2 (Fig. 5E).

The Y-box-like elements are located in the 5′-promoter region of ERBB2 (17, 39, 40),

ERBB2 comprise two transcriptional variants, and two promoters initiate at two

different transcription start sites, respectively (NM_001005862 and NM_004448) (39).

Treatment with tamoxifen stimulated YBX1 binding to sites #1 and #2 in T-47D cells

(Fig. 5F).

Exogenous E2 inhibited the expression of ERBB2 mRNA and ERBB2, while

treatment with tamoxifen or fulvestrant abrogated E2-induced inhibition of ERBB2

expression (Fig. 5G and H). E2 inhibited YBX1 binding to ERBB2 site #2, which was

abrogated by tamoxifen or fulvestrant in ER-positive T-47D cells but not in ER-negative

SKBr-3 cells (Fig. 5I).

We generated the stably ER-transfected cell lines MDA-MB231/ER-1 and

MDA-MB231/ER-2 from ER-negative MDA-MB231 cells. Enforced expression of ER

inhibited YBX1 binding to ERBB2 sites #1 and #2 in MDA-MB231-ER-1 cells, but not

control cells (Fig. 5J). In contrast, ER knockdown by its cognate siRNAs increase the

levels of ERBB2 mRNA and ERBB2 in T-47D cells (Fig. S2A and B).




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Expression of YBX1, ERBB2, and ER in patients with breast cancer

We used IHC and dual in situ hybridization (DISH) to analyze the YBX1, ERBB2,

and ER expression in breast cancer tissues representing the subtypes Luminal A,

Luminal B (ERBB2−), Luminal B (ERBB2+), ERBB2-disease, and Triple-negative (41)

(Fig. 6A). The intracellular locations of YBX1, ERBB2, ER, and progesterone receptor

(PGR) in cancer cells from serial sections of Luminal A and ERBB2-disease specimens

are shown in Fig. 6B. ER and PGR were expressed at higher levels in Lumina A tissue,

and ERBB2 or nuclear YBX1 was not detected. In contrast, ERBB2 and nuclear YBX1

were expressed at higher levels without detectable ER or PGR expression in

ERBB2-disease tissue. ER and ERBB2 expression negatively and positively correlated

with nuclear YBX1 expression, respectively (Fig. 6C). Expression of YBX1 in the

nucleus was significantly higher in the ERBB2 disease tissue compared with Luminal B

ERBB2-positive tissue.

DISCUSSION

The present study demonstrates that resistance to antiestrogens was associated with

increased activation of YBX1 and respective decreases and increases of ER and ERBB2

expression in breast cancer cell lines. Further, YBX1 knockdown increased sensitivity

to fulvestrant and resistance to lapatinib, an ERBB2/EGFR targeted drug, in

fulvestrant-resistant cells. Moreover, ectopic expression of YBX1 conferred resistance

of breast cancer cells to fulvestrant or tamoxifen in vitro and in vivo, associated with

decreased and increased expression of ER and ERBB2, respectively. Together, these




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findings support the conclusion that YBX1 contributes to antiestrogen resistance

through regulation of ER and ERBB2 expression in breast cancer cells (Fig. 7).

We further show that YBX1 decreased the levels of ER through posttranslational

control. ER is degraded via the proteasomal pathway with half-lives ranging from 8–10

h in ER-positive breast cancer cells (37). Similarly, here the half-lives of ER were

approximately 8 h in T-47D cells and approximately 2–3 h in fulvestrant-resistant

T-47D cells. Further, YBX1 directly bound ER in association with accelerated

proteasomal degradation of ER. In contrast, YBX1 mutants that did not bind ER did not

accelerate degradation, indicating that YBX1 inhibited ER expression through specific

binding to ER to confer resistance to antiestrogens upon T-47D cells.

Here, YBX1 increased ERBB2 mRNA expression at the transcriptional level and that

E2 inhibited YBX1-dependent ERBB2 expression by preventing YBX1 binding to the

ERBB2 promoter. Further, treatment with tamoxifen or fulvestrant with E2 markedly

restored ERBB2 expression, indicating that activated ER inhibited YBX1-dependent

transcriptional activation of ERBB2 in a breast cancer cells. We found that localization

of YBX1 to the nucleus positively and negatively associated with ERBB2 and ER

expression, respectively, in patients with breast cancer and that higher levels of YBX1

were present in the nucleus of tumor cells with the ERBB2-disease subtype compared

with the Luminal-B ERBB2+ subtype (Fig. 6C). The ERBB2-disease subtype is mainly

characterized by ERBB2 amplification or ERBB2 overexpression without amplification

(42). Further, YBX1 overexpression induces low levels of ERBB2 amplification in 20%

of human mammary epithelial cells in vitro when YBX1 is ectopically expressed (43).

Here we assessed whether nuclear YBX1 activation induced ERBB2 gene amplification




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in vitro and in vivo. However, ERBB2 amplification was undetectable when YBX1 was

overexpressed (data not shown).

The present findings indicate that YBX1 transcriptionally activated ERBB2 in vitro

and that YBX1 expression was not associated with ERBB2 amplification. In contrast,

long-term adjuvant therapy with tamoxifen or aromatase inhibitors is associated with

5%–30% incidence of ERBB2-positive breast cancers (44, 45), and the localization of

YBX1 to the nucleus may contribute. Periodic analysis of YBX1 expression in the

nucleus during long-term treatment with antiestrogens might contribute to the detection

of ERBB2-positive cancer cells.

In conclusion, we show that antiestrogen resistance of breast cancer cells involved

activation of YBX1 associated with increased ERBB2 expression and decreased ER

expression. Our findings strongly suggest that YBX1 will serve as a target to treat

antiestrogen-resistant breast cancer.

Acknowledgments

This work was supported by JSPS KAKENHI grant number 15J03033 (T.S.), the

Fukuoka Foundation for Sound Health Cancer Research Fund (T.S.), the Life Science

Foundation of Japan (M.O.), and St. Mary’s Institute of Health Sciences (K.W., M.

Kuwano., M.O). We thank Kimitoshi Kohno (University of Occupational and

Environmental Health, Kitakyushu, Japan) for fruitful discussions.

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104.




26

FIGURE LEGENDS

Figure 1.

Generation of fulvestrant-resistant breast cancer cells. A, The sensitivity to fulvestrant

and tamoxifen was assessed using WST assays for 3 days. B, Left: Western blots

showing p-YBX1 (Ser102), YBX1, ER, ERBB2, and EGFR expression in T-47D,

T-47D/FR-1, and T-47D/FR-2 cells. Right: Relative protein expression levels of ER and

ERBB2 in T-47D, T-47D/FR-1, and T-47D/FR-2 cells normalized to GAPDH

expression. C, Quantitative RT-PCR showing ERBB2 and ER mRNA expression in

T-47D, T-47D/FR-1, and T-47D/FR-2 cells. D, Left: Western blots showing ER protein

stability in the presence of cycloheximide (CHX) with or without MG-132 in T-47D and

T-47D/FR-1 cells. Right: Degradation curves for ER normalized to ER expression levels

at 0 h. E, Western blots showing YBX1, ER, and ERBB2 expression after treatment

with YBX1 siRNA (100 nM) for the indicated times in T-47D and T-47D/FR-1 cells. F,

T-47D and T-47D/FR-1 cells were treated with YBX1 siRNA (100 nM) for 24 h,

exposed to fulvestrant or lapatinib for 72 h, and subjected to cell proliferation assays.

Data represent the mean ± SD of triplicate dishes. *P < 0.05, **P < 0.01, two-sided

Student t test. Values are expressed as the percentage of the value in the absence of

drugs.

Figure 2.

Effects of YBX1 on sensitivity to antiestrogens and ERBB2-targeted drugs. A, Western

blots showing FLAG-YBX1, ER, ERBB2, and CDC6 expression with or without Dox

in six breast cancer cell lines transfected with the YBX1/Tet-On expression vector. B,




27

Effect of YBX1 overexpression on ER and ERBB2 expression. After 24 h and 120 h of

Dox treatment of T-47D/mock and T-47D/Tet YBX1 cells, the expression levels of ER

and ERBB2 were determined using western blotting. C, T-47D/mock and T-47D/Tet

YBX1 cells were treated with Dox for 24 h, followed by exposure to Dox and

fulvestrant or lapatinib for 96 h and subjected to cell proliferation assays. Data represent

the mean ± SD of triplicate dishes. **P < 0.01, two-sided Student t test. Values are

expressed as the percentage of the value in the absence of drugs. D, Tumor growth of

T-47D/mock (left) (n = 5) and T-47D/Tet YBX1 (right) (n = 6) cells during treatment

with Dox alone (black line) or Dox and tamoxifen (Tam) (red line) in a mouse xenograft

experiment for 14 days. Administration of Dox (1 mg per mouse, daily, p.o.) with or

without tamoxifen was started when the tumors reached 100–200 mm3. The tumor

growth rates (fold-changes) are indicated compared with day 1 of Dox administration.

The graphs represent individual tumor sizes. E, Quantitative RT-PCR showing ERBB2

mRNA levels in T-47D/mock (n = 4) and T-47D/Tet YBX1 (n = 5) tumors. The ERBB2

mRNA levels (fold-changes) compared with T-47D/mock tumors treated with Dox

alone are indicated. *P < 0.05, **P < 0.01, two-sided Student t test. F, Western blots

showing ERBB2 expression in T-47D/mock and T-47D/Tet YBX1 tumors after 14 days

of treatment with Dox, tamoxifen, or both. G, Effect of YBX1 with or without

tamoxifen on ERBB2 and ER expression in T-47D/mock and T-47D/Tet YBX1 tumors.

After Dox treatment for 14 days with or without tamoxifen, tumors were analyzed using

IHC using antibodies specific for YBX1, ERBB2, and ER (×200 original magnification).

A representative tumor sample of each group is shown.

Figure 3.




28

Effect of YBX1 on ER protein stability. A, Western blots showing YBX1 induction by

Dox in the cytoplasm and nucleus of T-47D/Tet YBX1 cells. B, Western blots showing

ER expression 6–72 h after YBX1 induction of the YBX1/Tet-On expression vector in

T-47D cells. C, Relative ER levels shown in (B) at various times after YBX1 induction

normalized to ER expression in the absence of Dox in T-47D/mock and T-47D/Tet

YBX1 cells. D, Quantitative RT-PCR showing ER mRNA levels under the same

experimental conditions used for (B). E, Left: Western blots showing ER protein

stability in the presence of cycloheximide (CHX) after treatment without or with Dox

for 12 h (top) and western blots showing ER protein stability in the presence of CHX

and MG-132 after treatment without or with Dox for 12 h (bottom). Right: Levels of ER,

with or without MG-132 normalized to ER levels at 0 h. F, ER ubiquitination with or

without MG-132 after treatment without or with Dox for 12 h in T-47D/Tet YBX1 cells.

Left: Co-immunoprecipitation (Co-IP) using an anti-ER antibody and immunoblotting

with an anti-ubiquitin antibody. Right: Co-IP using an anti-ubiquitin antibody and

immunoblotting with an anti-ER antibody.

Figure 4.

Effect of YBX1 binding to ER on ER stability. A, Co-IP assays showing YBX1 binding

to ER in T-47D cells, by immunoprecipitation using an anti-ER antibody and

immunoblotting with an anti-YBX1 antibody (top), and by immunoprecipitation using

an anti-YBX1 antibody and immunoblotting with an anti-ER antibody (bottom). B,

Co-IP assays showing YBX1 binding to ER in T-47D cells incubated with E2 (left) or

tamoxifen (Tam) (right) for 24 h. C, Left YBX1 deletion mutants. Right: Western blots

showing ER binding to YBX1 deletion mutants. Immobilized tagged proteins were




29

incubated with ER, and the bound proteins were analyzed using western blotting with

an anti-ER antibody. D, Pull-down assays showing YBX1 binding to ER with or without

tamoxifen for 2 h. E, Left: ER deletion mutants. Right: Western blots showing ER

mutants binding to YBX1. Immobilized tagged YBX1 proteins were incubated with

various ER deletion mutants, and the bound proteins were analyzed using western

blotting with an anti-FLAG antibody. F, Left: Western blots showing ER protein levels

in the absence or presence of E2 after treatment without or with Dox for 12 h in DMEM

supplemented with 10% charcoal-stripped FBS. Right: Degradation curves for ER with

or without E2, normalized to ER levels at 0 h. G, Left: Western blots showing ER levels

in the presence of CHX 24 h after transfection of FLAG-YBX1 or FLAG-YBX1 Δ3.

Right: ER levels normalized to those of ER at 0 ht. H, ER ubiquitination 24 h after

transfection of FLAG-YBX1 or FLAG-YBX1 Δ3. Co-IP assays involving

immunoprecipitation using an anti-ER antibody and immunoblotting using an

anti-ubiquitin antibody.

Figure 5.

Effect of YBX1 on ERBB2 expression and effect of tamoxifen or fulvestrant on YBX1

binding to the ERBB2 promoter. A, Western blots showing ERBB2 and ER expression

with or without Dox in T-47D/mock and T-47D/Tet YBX1 cells. B, Quantitative

RT-PCR analysis showing ERBB2 mRNA expression with or without Dox in

T-47D/mock and T-47D/Tet YBX1 cells. Data represent the mean ± SD of three

independent experiments. **P < 0.01, two-sided Student t test. C, Left: Western blots

showing ERBB2 expression in four cell lines incubated with tamoxifen (Tam) for 72 h.

Right: Western blots showing ERBB2 expression in T-47D cells incubated with




30

fulvestrant for 72 h. D, Quantitative RT-PCR showing ERBB2 mRNA expression in

T-47D cells incubated with tamoxifen or fulvestrant for 48 h. E, Potential

YBX1-binding sites in the ERBB2 promoter region and primer locations for ChIP

assays. F, ChIP assays with or without tamoxifen for 24 h in DMEM supplemented with

10% FBS in T-47D cells. G and H, Western (G) and quantitative RT-PCR analyses (H)

showing ERBB2 expression in T-47D cells incubated with E2 in the presence or

absence of tamoxifen or fulvestrant for 72 h. I, ChIP assays with or without E2 in the

presence or absence of tamoxifen or fulvestrant for 24 h in DMEM supplemented with

10% charcoal-stripped FBS in ER-positive T-47D cells and ER-negative SKBr-3 cells. J,

Left: ER cDNA-transfected cell lines (MDA-MB231-ER-1 and MDA-MB231-ER-2)

and mock-transfected cell line (MDA-MB231-mock). Right: ChIP assays of

MDA-MB231-mock and MDA-MB231-ER-1 cells after 48 h in DMEM supplemented

with 10% FBS.

Figure 6.

Nuclear YBX1, ERBB2, and ER expression in specimens from patients with breast

cancer. A, IHC images of nuclear YBX1-positive (a) and nuclear YBX1-negative (b)

samples, tumors with high (c) and low (d) YBX1 expression in the nucleus and

cytoplasm, and ERBB2 (e, f), ER (i, j), PGR (k, l), and Ki67 (m, n) in breast cancer

specimens (×400 original magnification). (g, h) Dish analyses showing amplified (g)

and unamplified (h) ERBB2 (×400 original magnification). Scale bar, 10 μm. B, IHC

analysis of serial sections (×200 magnification) for nuclear YBX1 (insert: ×400

magnification), ERBB2 (insert: ERBB2 DISH; ×400 original magnification), ER, and

PGR. Scale bar, 20 μm. C, Scatter plots of nuclear YBX1 vs ER and ERBB2 expression.




31

Nuclear YBX1 expression positively and negatively correlated with ERBB2 and ER

levels, respectively. The five breast cancer subtypes are indicated as follows: Luminal A,

yellow; Luminal B ERBB2−, blue; Luminal B ERBB2+, green; ERBB2 disease, red;

Triple-negative, black.

Figure 7.

Model depicting YBX1-mediated resistance to antiestrogens of breast cancer cells. In

estrogen-dependent ER-positive breast cancer cells, YBX1-induced ERBB2 expression

is inhibited by YBX1 binding to active ER. Treatment with antiestrogens interferes with

binding, and free, active YBX1 promotes ERBB2 expression.




Figure 1

A

0

20

40

60

80

100

120

0 0.3 1 3 10 30

Fulvestrant (nM)

% o

f co

ntr

ol

0

20

40

60

80

100

120

0

0.0

1

0.0

3

0.1

0.3 1 3

10

T-47D

T-47D/FR-1

T-47D/FR-2

ESR

ERBB2

EGFR

p-YBX1 (Ser102)

YBX1

GAPDH

B

E

C

Rela

tive e

xpre

ssio

n

0

0.002

0.004

0.006

0.008

0.01

ERBB2 mRNA

0

0.02

0.04

0.06

0.08

0.1

ESR mRNA

D

ERBB2

YBX1

GAPDH

T-47D T-47D

/FR-1

24 h

T-47D T-47D

/FR-1

96 h

ESR

Long exposure

ESR

Short exposure

ERBB2

p-IκBα

GAPDH

T-47D

CHX CHX

+MG-132

4 8 12 4 8 12 0

T-47D/FR-1

CHX CHX

+MG-132

4 8 12 4 8 12 0

0 4 8 12

Time after treatment (h)

Tamoxifen (μM)

0.001

0.01

0.1

1

10

T-47D CHX

T-47D CHX+MG-132

T-47D/FR-1 CHX

T-47D/FR-1 CHX+MG132

0

1

2

3

ESR protein

0

0.5

1

1.5

2

ERBB2 protein

Rela

tive e

xpre

ssio

n

(h)

ESR

0

20

40

60

80

100

120

140

0 1

Fulvestrant (nM)

0

20

40

60

80

100

120

0 10

Lapatinib (μM)

T-47D siControl

T-47D siYB-1

T-47D/FR-1 siControl

T-47D/FR-1 siYB-1

Cell

via

bil

ity (

%)

F

**

*

**

**

**

**

ES

R p

rote

in

siYBX1

siYBX1




Figure 2

0

50

100

150

200

250

1 4 7 11 14Days after administration

0

50

100

150

200

250

1 4 7 11 14Days after administration

Dox±Tam

D T-47D/Tet YBX1

E

T-47D/mock

Dox±Tam Dox

Dox+Tam

Rela

tive t

um

or

volu

me (

%)

F

T-47D/mock

Tumor # 1 2 3

FLAG-YBX1

ERBB2

GAPDH

Dox Dox

1 2 3 4

T-47D/Tet YBX1

Rela

tive t

um

or

volu

me (

%)

G

Dox+Tam

mock Tet YBX1

Dox

YBX1

ERBB2

ESR

mock Tet YBX1

T-47D/mock

Dox Dox+Tam Dox Dox+Tam

T-47D/Tet YBX1

* **

*

ER

BB

2 m

RN

A

0

1

2

3

4

Dox+Tam

1 2 3 4

Dox+Tam

1 2 3

B

T-47D

/Tet YBX1

T-47D

/mock

Dox - + - +

ERBB2

ESR

GAPDH

24 h 120 h

FLAG-YBX1

T-47D

/Tet YBX1

T-47D

/mock

- + - +

0

20

40

60

80

100

120

0 0.3 3

cell

via

bili

ty (

%)

Lapatinib (μM)

T-47D/mock (Dox+)

T-47D/Tet-YB-1 (Dox+)

0

20

40

60

80

100

120

0 0.1 10cell

via

bili

ty (

%)

Fulvestrant (nM)

**

C

**

**

**

A KPL-1

/Tet YBX1

MCF-7

/Tet YBX1

T-47D

/Tet YBX1

FLAG-YBX1

Dox - +

ESR

GAPDH

CDC6

MDA-MB231

/Tet YBX1

- + - + - +

SKBr-3

/Tet YBX1

- +

MDA-MB453

/Tet YBX1

- +

ERBB2

YBX1




Figure 3

0.1

1

Dox(-)Dox(+)Dox(-)+MG-132Dox(+)+MG-132

0.5

0 2 4 6 8 10 12

CHX (h)

ESR

GAPDH

FLAG-YBX1

CHX+

MG-132 (hr)

p-IκBα

ESR

GAPDH

FLAG-YBX1

CHX (h)

Dox(-)

0 4 6 8 12 24

Dox(+)

0 4 6 8 12 24

ES

R p

rote

in

Dox(-)

0 4 6 8 12 24

Dox(+)

0 4 6 8 12 24

E

6 h 12 h 24 h 48 h 72 h

Dox - + - +

mock Tet YBX1

ESR

GAPDH

FLAG-YBX1

- + - +

mock Tet YBX1

- + - +

mock Tet YBX1

- + - +

mock Tet YBX1

- + - +

mock Tet YBX1

B

ES

R m

RN

A

00.20.40.60.81

1.21.4

6 h 24h

48h

72h

6 h 24h

48h

72h

mock Tet YB-1

Dox(-)

Dox(+)

00.20.40.60.81

1.2

6 h 12h

24h

48h

72h

6 h 12h

24h

48h

72h

mock Tet YB-1

Dox(-)Dox(+)

ES

R p

rote

in

C D

Ub

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R

76

225

102

150

IgG Ub

IP

MG-132

Dox

- -

- +

+ -

+ +

- -

- +

+ -

+ +

52

(kDa)

IB:ESR

ESR

(66kDa)

Ub

-ES

R

IgG ESR

IP

MG-132 Dox

76

52

225

102 150

(kDa) IB:Ub

FLAG-YBX1

α-tubulin

CREB

Dox

Cytoplasm

- + - +

T-47D/Tet YBX1

Nucleus

A

F

- -

- +

+ -

+ +

- -

- +

+ -

+ +

YBX1 YBX1




Figure 4

C

Input

(ESR) 76kDa

52kDa

ESR

B

ESR

A

Whole cell

YBX1

IgG

IP

IB ESR

Whole cell

YBX1

IgG YBX1

IP

IB ESR

D

Tam (20nM) - +

GST GST-YBX1

-

Input

(ESR)

- 76kDa

52kDa

ESR

IP

Whole cell IgG ESR

- + Tam (1μM) - + - +

YBX1 IB

ESR

E2(10nM)

IP

Whole cell IgG ESR

- + - + - +

YBX1 IB

ESR

E

38kDa

31kDa

24kDa

52kDa

Input

FLAG-

ESR Δ1 Δ2 Δ3

GST

FLAG-

ESR Δ1 Δ2 Δ3

GST-YBX1

FLAG-

ESR Δ1 Δ2 Δ3

24

ESR

α-tubulin

FLAG-YBX1

CHX (h)

Dox(-)

0 4 6 8 12 24

Dox(+)

0 4 6 8 12 24

Dox(-)

0 4 6 8 12 24

Dox(+)

0 4 6 8 12

F E2(-) E2(+)

0.1

1

E2(-) Dox(-)

E2(-) Dox(+)

E2(+) Dox(-)

E2(+) Dox(+)

0 2 4 6 8

0.5

ES

R p

rote

in

CHX (h)

FLAG

ESR

α-tubulin

pcDNA3 FLAG-YBX1 FLAG-YBX1 Δ3

CHX (h) 0 4 6 8 12 24 0 4 6 8 12 24 0 4 6 8 12 24

G H

Ub-E

SR

IgG ESR

IP

IB:Ub

76

225

102

150

52

(kDa)

ESR

(66kDa)

0.1

1

pcDNA3

FLAG-YB-1

FLAG-YB-1 Δ3

0 2 4 6 8 10 12

CHX (h)

ES

R p

rote

in

0.5

YBX1

YBX1 Δ3




Figure 5

D

0

0.2

0.4

0.6

0.8

0 0.1 1Tam (μM)

**

ER

BB

2 m

RN

A

0

0.5

1

1.5

2

0 0.01 0.1Fulvestrant (μM)

**

**

ER

BB

2 m

RN

A

C

Fulvestrant (μM)

T-47D

0 0.01 0.1

ERBB2

ESR

GAPDH

YBX1

KPL-1 T-47D

Tam (μM) 0 0.1 1

ERBB2

ESR

GAPDH

YBX1

SKBr-3 MDA-MB231

0 0.1 1 0 0.1 1 0 0.1 1

Tam (μM)

ERBB2

GAPDH

0 0.1 1

E2 (10nM) -

0

+ + +

ERBB2

GAPDH

Fulvestrant (μM) 0 0.01 0.1

E2 (10nM) -

0

+ + +

G

ER

BB

2 m

RN

A

0

0.001

0.002

0.003

0.004

0.005

0.006 **

Tam (μM) 0 0.1 1

E2 (10nM) -

0

+ + +

**

0

0.001

0.002

0.003

0.004

0.005

0.006

Fulvestrant (μM) 0 0.01 0.1

E2 (10nM) -

0

+ + +

** ** E

RB

B2

mR

NA

H

#1

#2

Input IgG YBX1

- + Tam (1μM) - + - +

T-47D

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

T-47D

/Tet YBX1

T-47D

/mock

Dox - + - +

**

ER

BB

2 m

RN

A

B

F

Input

- +

Tam (1μM)

Fulvestrant (0.1μM)

E2 (10nM) + +

- - + -

- - - +

IgG YBX1

- + + +

- - + -

- - - +

- + + +

- - + -

- - - +

T-47D (ESR+) / #2

SKBr-3 (ESR-) / #2

I

ERBB2

ESR

GAPDH

YBX1 #1

#2

Input IgG YBX1

MDA-MB231

J

T-47D

/Tet YBX1

T-47D

/mock

A

ERBB2

Dox

ESR

GAPDH

- + - +

: ATTGG

#1 #2

Start point of

transcription 2

Start point of

transcription 1

Promoter 1

Promoter 2

: ATTG

: CAAT

ERBB2 gene

E




A

a b

c d

e f

g h

j

k l

m n

i

ERBB2 disease Luminal A

H&E

Nuclear

YBX1

ERBB2

ESR

PGR

B

ESR

YB

X1

0

0.2

0

.4

0.6

0

.8

0 0.2 0.4 0.6 0.8 1.0

=-0.26 (P<0.001)

1.0

YB

X1

0

0.2

0

.4

0.6

0

.8

0 2000 4000 6000 8000 10000 12000

=0.23(P<0.001)

1.0

ERBB2

● Luminal A

● Luminal B ERBB2-

● Luminal B ERBB2+

● ERBB2 disease

● Triple negative

C

Figure 6




Figure 7

ESR-positive breast

cancer cell

Antiestrogen (D)

Resistance to

antiestrogen




Published OnlineFirst November 22, 2016.Cancer Res Tomohiro Shibata, Kosuke Watari, Hiroto Izumi, et al. increased ER degradation and ERBB2 expressionBreast cancer resistance to antiestrogens is enhanced by

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