Breast Cancer Resistance to Antiestrogens Is …...Therapeutics, Targets, and Chemical Biology Breast Cancer Resistance to Antiestrogens Is Enhanced by Increased ER Degradation and

Therapeutics, Targets, and Chemical Biology

Breast Cancer Resistance to Antiestrogens IsEnhanced by Increased ER Degradation andERBB2 ExpressionTomohiro 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

Abstract

Endocrine therapies effectively improve the outcomes ofpatients with estrogen receptor (ER)-positive breast cancer.However, the emergence of drug-resistant tumors creates a coreclinical challenge. In breast cancer cells rendered resistant to theantiestrogen fulvestrant, we defined causative mechanistic rolesfor the transcription factor YBX1 and the levels of ER and theERBB2 receptor. Enforced expression of YBX1 in parental cellsconferred resistance against tamoxifen and fulvestrant in vitroand in vivo. Furthermore, YBX1 overexpression was associatedwith decreased and increased levels of ER and ERBB2 expres-

sion, respectively. In antiestrogen-resistant cells, increasedYBX1 phosphorylation was associated with a 4-fold higherdegradation rate of ER. Notably, YBX1 bound the ER, leadingto its accelerated proteasomal degradation, and induced thetranscriptional activation of ERBB2. In parallel fashion, tamox-ifen treatment also augmented YBX1 binding to the ERBB2promoter to induce increased ERBB2 expression. Together,these findings define a mechanism of drug resistance throughwhich YBX1 contributes to antiestrogen bypass in breast cancercells. Cancer Res; 77(2); 545–56. �2016 AACR.

IntroductionThe detection of estrogen receptor (ER) in 70% of invasive

breast cancers led to the identification of ER expression as themost significant risk factor for breast cancer (1). ER is activatedthrough interaction with its ligand estradiol (E2), and thegrowth and survival of ERþ breast cancers mainly dependsupon activation of the ER signal transduction pathway (2).Selective ER modulators and third-generation aromatase inhi-bitors are widely used to treat patients with ERþ breast cancer

(3). Antiestrogens provide significant benefits when used asadjuvants as well as to treat recurrent or metastatic breast cancer(3, 4). Furthermore, the selective ER downregulator fulvestrantimproves the prognosis of postmenopausal women withadvanced breast cancer who experience tumor progression afterendocrine therapy (5).

The EGFR family member erb-b2 receptor tyrosine kinase 2(ERBB2) is a driver of breast cancer (6). Gene amplification andoverexpression of ERBB2 occur in 20% to 30% of invasive breastcancers (7). An antibody against ERBB2 (trastuzumab) signifi-cantly improves outcomes (8, 9) and reduces the rate of recurrenceby greater than 50% in patients with early-stage ERBB2þ breastcancer (10). Treatment with lapatinib, a dual inhibitor of EGFRand ERBB2 tyrosine kinases, improves therapeutic efficacy whencombined with capecitabine to treat ERBB2þ breast cancer (11).Moreover, the combination of the anti-ERBB2 mAb pertuzumabwith trastuzumab plus docetaxel significantly improves medianoverall survival from 40.8 months, achieved using trastuzumabplus docetaxel, to 56.5 months of patients with ERBB2þ meta-static breast cancer (12, 13).

The Y-box binding protein-1 (YBX1) mediates the acquisitionof global resistance to anticancer drugs (14). YBX1-knockin miceprovoke breast cancers with diverse histologic characteristics,implicating YBX1 as an oncoprotein (15). Furthermore, YBX1knockdown inhibits the proliferation of humanbreast cancer cellsand inhibits the expression of ERBB2 and genes that mediate thecell cycle (16–20). YBX1 transforms human mammary epithelialcells into aggressive breast cancer cells through chromatin remo-deling (21). Together, these studies strongly suggest the associa-tion of YBX1 with the oncogenic potential of breast cancer cells.Furthermore, in breast cancer, expression of YBX1 in the nucleus is

1Department of Pharmaceutical Oncology, Graduate School of PharmaceuticalSciences, Kyushu University, Fukuoka, Japan. 2Department of OccupationalPneumology, Institute of Industrial Ecological Sciences, University of Occupa-tional and Environmental Health, Kitakyushu, Japan. 3Department of DiagnosticPathology, 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 CareCenter, Kyushu Medical Center, Fukuoka, Japan. 7Department of Surgery,Kurume University School of Medicine, Kurume, Japan. 8Division of Breast andEndocrine Surgery, Department of Surgery, Shinshu University School of Med-icine, Matsumoto, Japan. 9Department of Pharmaceutics, Graduate School ofPharmaceutical Sciences, Kyushu University, Fukuoka, Japan. 10Kurume GeneralHospital, Japan Community Health Care Organization (JCHO), Kurume, Japan.

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

Corresponding Author:Mayumi Ono, Department of Pharmaceutical Oncology,Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi,Higashi-ku, Fukuoka 812-8582, Japan. Phone/Fax: 81-92-642-6296; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-16-1593

�2016 American Association for Cancer Research.

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an independent prognostic factor for overall and progression-freesurvival (18), and YBX1 expression predicts relapse and disease-specific survival (22, 23). Moreover, biostatistical modeling indi-cates that nuclear localization of YBX1 positively and negativelycorrelates with ERBB2 and ER expression, respectively, in patientswith breast cancer (18).

ERþ breast cancers are sensitive to endocrine therapeuticdrugs (3). However, tumors develop drug resistance, leadingto relapse and progression (24, 25). Loss of ER expression,E2-independent ER activation, and ER-inactivating mutationsincrease resistance to antiestrogens (24, 26–28). Moreover,activation of bypass pathways induces resistance to tamoxifen,fulvestrant, and letrozole (29–33). The mTOR inhibitor ever-olimus or the cyclin-dependent kinase-4 and -6 inhibitor pal-bociclib, in combination with antiestrogens, are effective fortreating patients with ERþ breast cancer (34, 35). Moreover,ERBB2 is frequently associated with antiestrogen-resistantbreast cancers (29).

Here, we established breast cancer cells resistant to fulvestrantand used them to show that YBX1 was specifically activated inthese cells, leading us to ask whether YBX1 contributes to theantiestrogen resistance of breast cancer cells in association withER and ERBB2.

Materials and MethodsCell lines and chemicals

The human breast cancer cell lines MCF-7, T-47D, SKBr-3,MDA-MB231, and MDA-MB453 were purchased from the ATCC.KPL-1 was purchased from Health Science Research ResourcesBank. All cell lines were obtained between 2005 to 2010. All celllines were cultured at 37�C in DMEM supplemented with 10%FBS in a humidified atmosphere containing 5%CO2. All cell lineswere passaged for �6 months and were not further tested orauthenticated by the authors. We generated an antibody againstYBX1 designated (st1968) by immunizing New Zealand whiterabbits with a synthetic peptide representing YBX1 C-terminalamino acid residues 299–313 (19). This antibody detects cyto-plasmic and nuclear YBX1 in IHC. An antibody against YBX1(EP2708Y, ab76149; Abcam) was used for chromatin immuno-precipitation (ChIP) and coimmunoprecipitation (Co-IP) assays.

Establishment of fulvestrant-resistant cell linesWe established two fulvestrant-resistant cell lines designated

T-47D/FR-1 and T-47D/FR-2 from T-47D cells by exposing themcontinuously for approximately 6 months to step-wise increasesin fulvestrant concentrations up to 1 mmol/L. T-47D/FR-1 andT-47D/FR-2 were established from different flasks and were notcloned.

Cell proliferation assayCells (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� SD of triplicate wells.

YBX1/Tet-On plasmid constructionpEB-Tet-On YBX1 was generated by inserting the genes encod-

ing resistance to ampicillin and hygromycin from pcDNA3(Clontech Laboratories, Inc.), into the pEB-multi vector (Clon-tech). The TRE-Tight, rtTA, and YBX1-3�NLS (AGATCCAAAAA-

AGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAGAT-CCAAAAAAGAAGAGAAAGGTAGATACGGCC)-3�FLAG wereinserted into the BamHI–EcoRV sites of the pEB-multi vector.

Pull-down assayDeletion constructs of YBX1 and other aspects of this pull-

down assay were performed as described previously (36). Briefly,GST fusion proteins were dialyzed against X-buffer (50 mmol/LTris-HCl, pH 8.0; 1 mmol/L EDTA; 120 mmol/L NaCl; 0.5% NP-40 10% glycerol; and 1mmol/L PMSF). Immobilized GST or GSTfusion proteins were incubated with Glutathione Sepharose 4B(GEHealthcare) for 4 hours at 4�C. After fivewasheswith X-buffer(1 mL), GST fusion proteins were incubated with ER in thepresence or absence of tamoxifen (20 nmol/L) overnight at 4�C.The complex was washed five times with X-buffer and thensubjected to SDS-PAGE.

MiceThe Ethics of Animal Experiments Committee of Kyushu Uni-

versity Graduate School of Medical Sciences (Fukuoka, Japan)approved the animal experiments, which were conducted accord-ing to the recommendations of the United States Public HealthService Policy on Humane Care and Use of Laboratory Animals(Office of LaboratoryAnimalWelfare,NIH,Department ofHealthand Human Services, Bethesda, MD). Female BALB/c nu/nuathymic nude mice (6–7 weeks old) were purchased from CLEAand housed in microisolator cages under a 12-hour light/darkcycle. Water and food were supplied ad libitum. Animals wereobserved for tumor growth, activity, feeding, and pain accordingto the guidelines of the Harvard Medical Area Standing Commit-tee on Animals.

Xenograft studiesApproximately 5.0� 106 T-47D/mock or T-47D/Tet YBX1 cells

in 200 mL of 50%Matrigel were implanted into the subcutaneoustissue of the right abdominal wall of the mice that were admin-istered 0.75 mg 60-day release estrogen pellets (InnovativeResearch). Tumor sizes were measured, and tumor volumes(mm3) were calculated as follows: length � width2 � 0.5. Whentumors reached 100 to 200 mm3, 6 mice each were randomlyallocated into groups (n ¼ 6/group) administered doxycycline(1 mg per mouse, daily, orally) or doxycycline plus tamoxifencitrate (500 mg per mouse in peanut oil, daily, s.c.). The tumorswere harvested after 2 weeks, stored at �80�C, or fixed immedi-ately in 10% paraformaldehyde overnight at 4�C.

Patient selectionWe screened 116 premenopausal and 114 postmenopausal

patients with breast cancer who were treated between 2007 and2013 at Kurume University Hospital or Kurume General Hospital(Kurume, Japan), and who underwent percutaneous biopsy withno prior treatment, such as endocrine therapy. Histologic typesand numbers of carcinomas were 190 invasive ductal, 14 nonin-vasive ductal, 5mucinous, 4 neuroendocrine, 5 invasive lobular, 4invasive micropapillary, 3 apocrine, 3 metaplastic, 1 tubular, and1 medullary. The average ages of 116 premenopausal and 114postmenopausal women were 45.1 (range, 28–55) and 66.3(range, 50–102), respectively. This study conforms to the princi-ples of the Declaration of Helsinki and was approved by theInstitutional Review Board of Kurume University Hospital (Kur-ume, Japan).

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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 proteinexpression 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 showingER and ERBB2 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 ofcycloheximide (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 hour.E, Western blots showing YBX1, ER, and ERBB2 expression after treatment with YBX1 siRNA (100 nmol/L) for the indicated times in T-47D and T-47D/FR-1cells. F, T-47D and T-47D/FR-1 cells were treated with YBX1 siRNA (100 nmol/L) for 24 hours, exposed to fulvestrant or lapatinib for 72 hours, andsubjected to cell proliferation assays. Data represent the mean � SD of triplicate dishes. � , P < 0.05; �� , P < 0.01, two-sided Student t test. Values areexpressed as the percentage of the value in the absence of drugs.

YBX1 Mediates Antiestrogen Resistance

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Effects of YBX1 on sensitivity to antiestrogens and ERBB2-targeted drugs. A, Western blots showing FLAG-YBX1, ER, ERBB2, and CDC6 expression withor without doxycycline in six breast cancer cell lines transfected with the YBX1/Tet-On expression vector. B, Effect of YBX1 overexpression on ER andERBB2 expression. After 24 and 120 hours of doxycycline (Dox) treatment of T-47D/mock and T-47D/Tet YBX1 cells, the expression levels of ER andERBB2 were determined using Western blotting. C, T-47D/mock and T-47D/Tet YBX1 cells were treated with doxycycline for 24 hours, followed byexposure to doxycycline and fulvestrant or lapatinib for 96 hours and subjected to cell proliferation assays. (Continued on the following page.)

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In vitro assays and statistical analysisWestern blot analysis, preparation of charcoal-stripped serum,

Co-IP assays, and statistical analysis are described in Supplemen-tary Materials and Methods.

ResultsIncreased activation of YBX1 in fulvestrant-resistant cells isassociated with respective decreases and increases of ER andERBB2 expression

The T-47D/FR-1 and T-47D/FR-2 cell lines were >100-foldmore resistant to fulvestrant and tamoxifen compared with theparental cells (Fig. 1A). Furthermore, increased phosphoryla-tion of YBX1-Ser102 was accompanied by markedly decreasedand increased expression of ER and ERBB2, respectively, com-pared with their parental cells (Fig. 1B). Quantitative RT-PCRanalysis detected approximately 30% to 40% decreases and3- to 4-fold increases in ER and ERBB2 mRNA levels, respec-tively, 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-1cells were approximately 8 and 2 hours, respectively. Treatmentwith the proteasome inhibitor MG-132 increased the half-lifeof ER in T-47D/FR-1 cells (Fig. 1D). Treatment with YBX1-siRNA enhanced ER expression and suppressed ERBB2 expres-sion in T-47D/FR-1 cells (Fig. 1E), associated with significantlyincreased and decreased sensitivities to fulvestrant and lapati-nib, respectively (Fig. 1F).

YBX1 induces resistance to antiestrogens in ERþ cellsTo determine whether YBX1 directly influenced sensitivity to

antiestrogens and ER and ERBB2 expression, we analyzed breastcancer cells transfected with the doxycycline-inducible constructYBX1/Tet-On. The levels of ER and ERBB2 decreased andincreased, respectively, in the three ERþ cell lines KPL-1, MCF-7, and T-47D, following doxycycline induction (Fig. 2A),although their levels were unchanged in the ER� cell linesSKBr-3, MDA-MB231, and MDA-MB453. Consistent with previ-ous research (19), the expression of CDC6 increased in the six celllines when YBX1 was expressed (Fig. 2A).

We determined whether YBX1 overexpression affected cellularsensitivities to the antiestrogen fulvestrant and the ERBB2-tar-geted drug lapatinib. T-47D/Tet YBX1 cells expressed decreasedand increased levels of ER and ERBB2, respectively, (Fig. 2B),which were associated with respective decreased and increasedsensitivities to fulvestrant and lapatinib compared with those ofT-47D/mock cells (Fig. 2C).

Furthermore, we determined whether YBX1 overexpressionaffected the sensitivity of tumor xenografts to tamoxifen in vivo.The growth of tumors formed by T-47D/mock cells, but notthose formed by T-47D/Tet YBX1 cells, was significantly inhib-

ited by tamoxifen (Fig. 2D). YBX1 overexpression significantlyincreased the levels of ERBB2mRNA and ERBB2 (Fig. 2E and F)compared with those of T-47D/mock-induced tumors. Further-more, tamoxifen increased ERBB2 mRNA and ERBB2 levelsin tumors induced by T-47D/mock and T-47D/Tet YBX1 cellscompared with those induced by T-47D/mock cells in untreat-ed mice. Immunohistochemical analysis demonstrated anincreased number of engrafted tumor cells that expressedYBX1 in the nucleus as well as increased and decreased levelsof ERBB2 and ER, respectively, in tumors formed by T-47D/TetYBX1 cells compared with tumors induced by T-47D/mockcells (Fig. 2G). Tamoxifen treatment was associated withincreased levels of ERBB2 in tumors formed by T-47D/mockand T-47D/Tet YBX1 cells compared with T-47D/mock cells inuntreated mice (Fig. 2G).

YBX1 promotes proteasomal ER degradationWe asked whether the half-life of ER was influenced by

YBX1 overexpression. YBX1 was highly expressed in the cyto-plasm and nucleus of YBX1/Tet-On transfectants comparedwith the control (Fig. 3A). Furthermore, ER levels decreasedafter YBX1 expression was induced (Fig. 3B and C), whereasER mRNA levels remained unchanged (Fig. 3D). E2–ER bind-ing induces the degradation of ER through the proteasomalpathway (37, 38). Similarly, the half-life of ER decreased fromapproximately 10 to 4 hours when YBX1 expression wasinduced in exponentially growing T-47D cells (Fig. 3E), andtreatment with the proteasome inhibitor MG-132 increasedthe half-life of ER after YBX1 induction (Fig. 3E). Furthermore,ubiquitination of ER increased when YBX1 expression wasinduced (Fig. 3F).

ER degradation increases through interaction with YBX1Co-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 withER, we individually deleted the N-terminal, cold shock domain(CSD), and C-terminal regions of YBX1 (Fig. 4C and Supple-mentary Fig. S1A; ref. 36). ER did not bind the CSD deletionmutant GST-YBX1 D3 (Fig. 4C). Furthermore, pull-down assaysrevealed that tamoxifen directly inhibited YBX1–ER binding(Fig. 4D).

To identify the YBX1 interaction domain of ER, we gener-ated ER deletion mutants (Fig. 4E) and found that YBX1bound only the mutant (FLAG-ER D2) with an intactligand-binding domain (Fig. 4E). Furthermore, YBX1-induceddegradation of ER was accelerated by E2 (Fig. 4F). Moreover,when we transiently overexpressed FLAG-YBX1 and theYBX1 mutants, the former reduced the cellular levels of ER(Supplementary Fig. S1B). In contrast, ER expression was

(Continued.) Data represent the mean � SD of triplicate dishes. �� , P < 0.01, two-sided Student t test. Values are expressed as the percentage of the valuein the absence of drugs. D, Tumor growth of T-47D/mock (left; n ¼ 5) and T-47D/Tet YBX1 (right; n ¼ 6) cells during treatment with doxycycline alone(black line) or doxycycline and tamoxifen (Tam; red line) in a mouse xenograft experiment for 14 days. Administration of doxycycline (1 mg per mouse,daily, orally) with or without tamoxifen was started when the tumors reached 100 to 200 mm3. The tumor growth rates (fold changes) are indicatedcompared with day 1 of doxycycline administration. The graphs represent individual tumor sizes. E, Quantitative RT-PCR showing ERBB2 mRNA levels inT-47D/mock (n ¼ 4) and T-47D/Tet YBX1 (n ¼ 5) tumors. The ERBB2 mRNA levels (fold changes) compared with T-47D/mock tumors treated withdoxycycline 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/TetYBX1 tumors after 14 days of treatment with doxycycline, tamoxifen, or both. G, Effect of YBX1 with or without tamoxifen on ERBB2 and ER expression inT-47D/mock and T-47D/Tet YBX1 tumors. After doxycycline treatment for 14 days with or without tamoxifen, tumors were analyzed using IHC usingantibodies specific for YBX1, ERBB2, and ER (original magnification, �200). A representative tumor sample of each group is shown.






unaffected by FLAG-YBX1 D3 overexpression (SupplementaryFig. S1C).

When we overexpressed FLAG-YBX1 or FLAG-YBX1 D3, ERdegradation was significantly lower in cells transfected with thelatter that did not bind ER (Fig. 4G). MG-132 treatmentmarkedly increased the half-life of ER in cells transfected withFLAG-YBX1 or FLAG-YBX1 D3 (Supplementary Fig. S1D). Thelevels of ubiquitinated ER were much higher in cells over-expressing FLAG-YBX1 versus FLAG-YBX1 D3 (Fig. 4H).

Transcriptional activation of the ERBB2 by YBX1 isinhibited by E2 or ER

We asked whether YBX1 expression or antiestrogens influ-enced ERBB2 expression in T-47D cells. We found that ERBB2levels and ERBB2 mRNA levels increased significantly indoxycycline-treated T-47D/Tet YBX1 cells but not in T-47D/mock cells (Fig. 5A and B). Tamoxifen markedly increasedERBB2 expression in the ERþ cell lines KPL-1 and T-47D, butnot in ER� cell lines SKBr-3 and MDA-MB231. Fulvestrantincreased ERBB2 levels in T-47D cells (Fig. 5C), and tamoxifenor fulvestrant increased ERBB2 mRNA levels (Fig. 5D).

We performed ChIP assays to determine whether the ab-sence or presence of activated ER affected YBX1 binding to theputative Y-box sites #1 and #2 in ERBB2 (Fig. 5E). The Y-box–like elements are located in the 50-promoter region of ERBB2(17, 39, 40); ERBB2 comprises two transcriptional variants,and two promoters initiate at two different transcription startsites, respectively (NM_001005862 and NM_004448; ref. 39).Treatment with tamoxifen stimulated YBX1 binding to sites #1and #2 in T-47D cells (Fig. 5F).

Exogenous E2 inhibited the expression of ERBB2 mRNA andERBB2, while treatment with tamoxifen or fulvestrant abrogatedE2-induced inhibition of ERBB2 expression (Fig. 5G and H). E2inhibited YBX1 binding to ERBB2 site #2, whichwas abrogated bytamoxifen or fulvestrant in ERþ T-47D cells but not in ER� SKBr-3cells (Fig. 5I).

We generated the stably ER-transfected cell lines MDA-MB231/ER-1 and MDA-MB231/ER-2 from ER� MDA-MB231cells. Enforced expression of ER inhibited YBX1 binding toERBB2 sites #1 and #2 in MDA-MB231-ER-1 cells, but notcontrol cells (Fig. 5J). In contrast, ER knockdown by its cognatesiRNAs increase the levels of ERBB2mRNA and ERBB2 in T-47Dcells (Supplementary Fig. S2A and S2B).

Expression of YBX1, ERBB2, and ER in patients with breastcancer

We used IHC and dual in situ hybridization (DISH) toanalyze the YBX1, ERBB2, and ER expression in breast cancertissues representing the subtypes luminal A, luminal B(ERBB2�), luminal B (ERBB2þ), ERBB2 disease, and triplenegative (Fig. 6A; ref. 41). The intracellular locations of YBX1,ERBB2, ER, and progesterone receptor (PGR) in cancer cellsfrom serial sections of luminal A and ERBB2 disease specimensare shown in Fig. 6B. ER and PGR were expressed at higherlevels in luminal A tissue, and ERBB2 or nuclear YBX1 was notdetected. In contrast, ERBB2 and nuclear YBX1 were expressedat higher levels without detectable ER or PGR expression inERBB2 disease tissue. ER and ERBB2 expression negatively andpositively 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 BERBB2þ tissue.

DiscussionThis study demonstrates that resistance to antiestrogens was

associated with increased activation of YBX1 and respectivedecreases and increases of ER and ERBB2 expression in breastcancer cell lines. Furthermore, YBX1 knockdown increasedsensitivity to fulvestrant and resistance to lapatinib, anERBB2/EGFR-targeted drug, in fulvestrant-resistant cells. More-over, ectopic expression of YBX1 conferred resistance of breastcancer cells to fulvestrant or tamoxifen in vitro and in vivo,associated with decreased and increased expression of ER andERBB2, respectively. Together, these findings support the con-clusion that YBX1 contributes to antiestrogen resistancethrough regulation of ER and ERBB2 expression in breast cancercells (Fig. 7).

We further show that YBX1 decreased the levels of ER throughposttranslational control. ER is degraded via the proteasomalpathway, with half-lives ranging from 8 to 10 hours in ERþ breastcancer cells (37). Similarly, here the half-lives of ER were approx-imately 8 hours in T-47D cells and approximately 2 to 3 hours infulvestrant-resistant T-47D cells. Furthermore, YBX1 directlybound ER in association with accelerated proteasomal degrada-tion of ER. In contrast, YBX1mutants that did not bind ER did notaccelerate degradation, indicating that YBX1 inhibited ER expres-sion through specific binding to ER to confer resistance to anti-estrogens upon T-47D cells.

Here, YBX1 increased ERBB2 mRNA expression at the tran-scriptional level and that E2 inhibited YBX1-dependent ERBB2expression by preventing YBX1 binding to the ERBB2 promot-er. Furthermore, treatment with tamoxifen or fulvestrant withE2 markedly restored ERBB2 expression, indicating that acti-vated ER inhibited YBX1-dependent transcriptional activationof ERBB2 in breast cancer cells. We found that localizationof YBX1 to the nucleus positively and negatively associatedwith ERBB2 and ER expression, respectively, in patients withbreast cancer and that higher levels of YBX1 were present in thenucleus of tumor cells with the ERBB2 disease subtype com-pared with the luminal B ERBB2þ subtype (Fig. 6C). TheERBB2 disease subtype is mainly characterized by ERBB2amplification or ERBB2 overexpression without amplification(42). Furthermore, YBX1 overexpression induces low levels ofERBB2 amplification in 20% of human mammary epithelialcells in vitro when YBX1 is ectopically expressed (43). Here, weassessed whether nuclear YBX1 activation induced ERBB2 geneamplification in vitro and in vivo. However, ERBB2 amplifica-tion was undetectable when YBX1 was overexpressed (datanot shown).

These findings indicate that YBX1 transcriptionally activatedERBB2 in vitro and that YBX1 expression was not associated withERBB2 amplification. In contrast, long-term adjuvant therapywith tamoxifen or aromatase inhibitors is associated with 5% to30% incidence of ERBB2þ breast cancers (44, 45), and thelocalization of YBX1 to the nucleus may contribute. Periodicanalysis of YBX1 expression in the nucleus during long-termtreatment with antiestrogens might contribute to the detectionof ERBB2þ cancer cells.

In conclusion, we show that antiestrogen resistance of breastcancer cells involved activation of YBX1 associated with increased

Shibata et al.





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Figure 3.

Effect of YBX1 on ER protein stability. A, Western blots showing YBX1 induction by doxycycline in the cytoplasm and nucleus of T-47D/Tet YBX1 cells. B,Western blots showing ER expression 6 to 72 hours after YBX1 induction of the YBX1/Tet-On expression vector in T-47D cells. C, Relative ER levels shown in Bat various times after YBX1 induction normalized to ER expression in the absence of doxycycline (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 inthe presence of cycloheximide (CHX) after treatment without or with doxycycline for 12 hours (top) and Western blots showing ER protein stability inthe presence of cycloheximide and MG-132 after treatment without or with doxycycline for 12 hours (bottom); right, levels of ER, with or without MG-132normalized to ER levels at 0 hour. F, ER ubiquitination with or without MG-132 after treatment without or with doxycycline for 12 hours in T-47D/TetYBX1 cells. Left, Co-IP using an anti-ER antibody and immunoblotting (IB) with an anti-ubiquitin (Ub) antibody; right, Co-IP using an anti-ubiquitinantibody and immunoblotting with an anti-ER antibody.






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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 andimmunoblotting 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 hours. C, Left, YBX1 deletion mutants;right, Western blots showing ER binding to YBX1 deletion mutants. Immobilized tagged proteins were incubated with ER, and the bound proteins wereanalyzed usingWestern blottingwith an anti-ER antibody.D,Pull-downassays showingYBX1 binding to ERwith orwithout tamoxifen for 2 hours.E, Left, ER deletionmutants; right, Western blots showing ER mutants binding to YBX1. Immobilized tagged YBX1 proteins were incubated with various ER deletion mutants, andthe bound proteins were analyzed using Western blotting with an anti-FLAG antibody. F, Left, Western blots showing ER protein levels in the absence orpresence of E2 after treatment without or with doxycycline (Dox) for 12 hours in DMEM supplemented with 10% charcoal-stripped FBS; right, degradationcurves for ER with or without E2, normalized to ER levels at 0 hour. G, Left, Western blots showing ER levels in the presence of cycloheximide 24 hours aftertransfection of FLAG-YBX1 or FLAG-YBX1 D3; right, ER levels normalized to those of ER at 0 hour. H, ER ubiquitination 24 hours after transfection of FLAG-YBX1 orFLAG-YBX1 D3. Co-IP assays involving immunoprecipitation using an anti-ER antibody and immunoblotting using an anti-ubiquitin antibody.

Shibata et al.





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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 ERexpression with or without doxycycline (Dox) in T-47D/mock and T-47D/Tet YBX1 cells. B, Quantitative RT-PCR analysis showing ERBB2 mRNAexpression with or without doxycycline 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 hours; right,Western blots showing ERBB2 expression in T-47D cells incubated with fulvestrant for 72 hours. D, Quantitative RT-PCR showing ERBB2 mRNAexpression in T-47D cells incubated with tamoxifen or fulvestrant for 48 hours. E, Potential YBX1-binding sites in the ERBB2 promoter region and primerlocations for ChIP assays. F, ChIP assays with or without tamoxifen for 24 hours in DMEM supplemented with 10% FBS in T-47D cells. G and H,Western (G) andquantitative RT-PCR analyses (H) showing ERBB2 expression in T-47D cells incubated with E2 in the presence or absence of tamoxifen or fulvestrant for72 hours. I, ChIP assays with or without E2 in the presence or absence of tamoxifen or fulvestrant for 24 hours in DMEM supplemented with 10% charcoal-stripped FBS in ERþ T-47D cells and ER� SKBr-3 cells. J, Left, ER cDNA-transfected cell lines (MDA-MB231-ER-1 and MDA-MB231-ER-2) and mock-transfectedcell line (MDA-MB231-mock); right, ChIP assays of MDA-MB231-mock and MDA-MB231-ER-1 cells after 48 hours in DMEM supplemented with 10% FBS.






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C

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 and f), ER (i and j), PGR (k and l), and Ki67 (m andn) in breast cancer specimens (original magnification, �400). DISH analyses showing amplified (g) and unamplified (h) ERBB2 (original magnification, �400).Scale bar, 10 mm. B, Immunohistochemical analysis of serial sections (magnification, �200) for nuclear YBX1 (inset, magnification, �400), ERBB2 (inset,ERBB2 DISH; original magnification, �400), ER, and PGR. Scale bar, 20 mm. C, Scatter plots of nuclear YBX1 versus ER and ERBB2 expression. Nuclear YBX1expression 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 ERBB2þ, green; ERBB2 disease, red; triple negative, black.

Shibata et al.





ERBB2 expression and decreased ER expression. Our findingsstrongly suggest that YBX1 will serve as a target to treat anties-trogen-resistant breast cancer.

Disclosure of Potential Conflicts of InterestS. Hattori is a consultant/advisory board member for Chugai Pharma-

ceuticals. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: T. Shibata, H. Izumi, S. Ohdo,M. Tanaka, M. Kuwano,M. OnoDevelopment of methodology: T. Shibata, H. Izumi, M. Tanaka, M. Kuwano,M. OnoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Shibata, K. Watari, Y. Murakami, R. Takahashi,M. TanakaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Hattori, M. TanakaWriting, review, and/or revisionof themanuscript:T. Shibata, K.Watari, K. Ito,M. Tanaka, M. Kuwano, M. Ono

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. Izumi, A. Kawahara, C. Fukumitsu,R. Takahashi, U. Toh, M. TanakaStudy supervision: U. Toh, M. Tanaka, M. Kage, M. Kuwano

AcknowledgmentsWe thank Kimitoshi Kohno (University of Occupational and Environ-

mental Health, Kitakyushu, Japan) for fruitful discussions.

Grant SupportThis work is supported by JSPS KAKENHI grant number 15J03033

(T. Shibata), the Fukuoka Foundation for Sound Health Cancer Research Fund(T. Shibata), the Life Science Foundation of Japan (M. Ono), and St. Mary'sInstitute of Health Sciences (K. Watari, M. Kuwano, and M. Ono).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 7, 2016; revised September 26, 2016; accepted November 4,2016; published OnlineFirst November 22, 2016.

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Antiestrogen (D)

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Figure 7.

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2017;77:545-556. 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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