aB-Crystallin:ANovelRegulatorofBreastCancerMetastasis to the Brain · Human Cancer Biology aB-Crystallin:ANovelRegulatorofBreastCancerMetastasis to the Brain Dmitry Malin1, Elena

Human Cancer Biology

aB-Crystallin: ANovelRegulatorofBreastCancerMetastasisto the Brain

Dmitry Malin1, Elena Strekalova1, Vladimir Petrovic1, Allison M. Deal3, Abraham Al Ahmad2, Barbara Adamo4,C. Ryan Miller3, Andrey Ugolkov5, Chad Livasy6, Karen Fritchie7, Erika Hamilton8, Kimberly Blackwell8,Joseph Geradts9, Matt Ewend3, Lisa Carey3, Eric V. Shusta2, Carey K. Anders3, and Vincent L. Cryns1

AbstractPurpose: Basal-like breast tumors are typically (ER/PR/HER2) triple-negative and are associated with a

high incidence of brain metastases and poor clinical outcomes. The molecular chaperone aB-crystallin is

predominantly expressed in triple-negative breast cancer (TNBC) and contributes to an aggressive tumor

phenotype in preclinical models. We investigated the potential role of aB-crystallin in brain metastasis in

TNBCs.

Experimental Design: aB-crystallin expression in primary breast carcinomas and brain metastases was

analyzed by immunohistochemistry among patients with breast cancer with brainmetastases. aB-crystallinwas overexpressed or silenced in two different TNBC cell lines. The effects on cell adhesion to human brain

microvascular endothelial cells (HBMEC) or extracellular matrix proteins, transendothelial migration, and

transmigration across a HBMEC/astrocyte coculture blood–brain barrier (BBB) model were examined. In

addition, the effects of overexpressing or silencingaB-crystallin on brainmetastasis in vivowere investigated

using orthotopic TNBC models.

Results: In a cohort of women with breast cancer brain metastasis, aB-crystallin expression in primary

breast carcinomas was associated with poor overall survival and poor survival after brain metastasis, even

among patients with TNBC. Stable overexpression of aB-crystallin in TNBC cells enhanced adhesion to

HBMECs, transendothelial migration, and BBB transmigration in vitro, whereas silencing aB-crystallininhibited these events. aB-crystallin promoted adhesion of TNBC cells to HBMECs, at least in part, through

ana3b1 integrin–dependentmechanism.aB-crystallin overexpression promoted brainmetastasis, whereas

silencing aB-crystallin inhibited brain metastasis in orthotopic TNBC models.

Conclusion: aB-crystallin is a novel regulator of brain metastasis in TNBC and represents a potential

biomarker and drug target for this aggressive disease. Clin Cancer Res; 1–12. �2013 AACR.

IntroductionBrain metastases are a devastating complication in 10%

to 16%of womenwith advanced breast cancer that result inneurologic deficits, including headaches, cognitive pro-

blems, seizures, and/or motor deficits (1). Brain metastasesare often a late occurrence in women with preexistingmetastatic disease in other organs and typically followa rapidly progressive course with a 1-year survival rate lessthan 20%. Unfortunately, the incidence of brainmetastasesappears to be increasing with the advent of improvedsystemic therapies, which are frequently ineffective againstbrain metastases because of their limited ability to cross theblood–brain barrier (BBB), a distinctive permeability bar-rier composed of human brain microvascular endothelialcells (HBMEC) interconnected by tight junctions and rein-forced by astrocytes, pericytes, and a basement membrane(2, 3). The BBB is also a formidable obstacle for circulatingtumor cells to extravasate into the brain, a critical step inbrain metastasis.

Recent studies in animal models have provided newinsights into the mechanisms by which tumor cells crosstheBBBand colonize thebrain.Circulating tumor cells arrestin the brain microvasculature, often at capillary branches,where they adhere to HBMECs and/or the subendothelialbasement membrane, distending the capillary and extrava-sating through gaps in the microvessel wall (2–5). Tumor

Authors' Affiliations: 1Department of Medicine, University of WisconsinCarbone Cancer Center, University of Wisconsin School of Medicine andPublic Health; 2Department of Chemical and Biological Engineering, Uni-versity of Wisconsin, Madison, Wisconsin; 3Lineberger ComprehensiveCancer Center, University of North Carolina at Chapel Hill, Chapel Hill,North Carolina; 4Breast Cancer Unit, Medical Oncology Department, Valld'Hebron Institute of Oncology, Barcelona, Spain; 5Center for Develop-mental Therapeutics, Northwestern University, Evanston, Illinois; 6Depart-ment of Pathology and Lab Medicine, Carolinas Medical Center, Charlotte,North Carolina; 7Department of Laboratory Medicine and Pathology, MayoClinic, Rochester, Minnesota; and 8Department of Medicine and 9Depart-ment ofPathology,DukeUniversityMedicalCenter,Durham,NorthCarolina

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Vincent L. Cryns, Department of Medicine, Uni-versity of Wisconsin School of Medicine and Public Health, MFCB 4144,1685 Highland Avenue, Madison, WI 53705. Phone: 608-262-4786; Fax:608-263-9983; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-1255

�2013 American Association for Cancer Research.

ClinicalCancer

Research

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cells proliferate in the brain in close proximity to the exteriorsurface of capillaries from which they extravasate, a processtermed "vascular cooption," although some metastatictumor cells rely on angiogenesis for perivascular growth(2–6). The metastatic tumor cells initiate a localized inflam-matory response through reciprocal interactions with reac-tive astrocytes andmicroglia that promote tumor growth (2,3, 5). Hence, dynamic interactions between tumor cells anddiverse cell types in the brain contribute to brainmetastasis.

Molecular profiling of breast tumors has provided addi-tional insights into brain metastasis. Brain metastases aremost prevalent in HER2/ErbB2-positive and basal-likebreast tumors; the latter express basal epithelial genes andare often "triple (ER/PR/HER2)-negative" (7–9). Brainmetastases in triple-negative breast cancer (TNBC) are par-ticularly challenging given the lack of targeted agents andpoor clinical outcomes. In one cohort, brain metastaseswere present in 46% of patients with metastatic TNBCs andwere associated with median survival of 4.9 months afterdiagnosis (10). Moreover, patients with early-stage TNBChave a high incidence (4.7%) of brain metastasis as a firstsite of relapse (11). Although a brain metastasis genesignature was recently reported (12), the specific genes thatregulate brain metastasis in TNBC are poorly understood.Identification of these genes is a critical first step in thedevelopment of biomarkers and targeted therapies for brainmetastases.

The molecular chaperone aB-crystallin is predominantlyexpressed inbasal-like breast cancer/TNBCand is associatedwith poor outcomes (13–15). aB-crystallin has been linkedto many biologic characteristics of these aggressive tumors.aB-crystallin promotes apoptosis resistance, at least in part,

by inhibiting caspase-3 activation, thereby enhancing cellsurvival in the setting of oncogenic stress, growth factordepletion, chemotherapy, and other cellular stressors (16–19).aB-crystallin promotes cellmigration and invasion andlocalizes to the infiltrative edge of malignant glioblastomas(13, 20–22). These effects are likely mediated by directinteractions of aB-crystallin with actin and intermediatefilaments, which regulate cytoskeletal stability and dynam-ics (23, 24). In addition, aB-crystallin expression is dra-matically increased in 2 highly metastatic TNBC cell linesidentified by in vivo selection (includingGILM2 cells used inour experiments) compared with the less metastatic paren-tal cells (25). However, the functional role of aB-crystallinin metastasis has not been studied. We postulated that aB-crystallin might contribute to the observed proclivity ofTNBCs to metastasize to the brain.

Here, we report that aB-crystallin is commonly expressedin breast cancer brain metastases and show that its expres-sion in primary breast carcinomas predicts poor survival inpatients with brainmetastasis. Stable overexpression of aB-crystallin in TNBC cells enhanced adhesion to HBMECs,transendothelial migration, and transmigration through aBBB model in vitro, whereas silencing aB-crystallin inhib-ited these events.aB-crystallin promoted adhesion of TNBCcells to HBMECs, at least in part, by an a3b1 integrin–dependent mechanism. Moreover, aB-crystallin promotedbrain metastasis in vivo in orthotopic TNBC models. Ourfindings indicate that aB-crystallin is a novel regulator ofbrain metastasis in TNBC and point to aB-crystallin anda3b1 integrin as potential drug targets for this devastatingdisease.

Materials and MethodsBreast cancer brain metastasis cohort andimmunohistochemical analyses

Patients with a diagnosis of breast cancer and brainmetastases who were treated at the University of NorthCarolina at Chapel Hill (1989–2006) and Duke UniversityMedical Center (1985–2005) with available tumor tissue(breast, brain, or both) and survival data were included.Additional data included age, gender, race, tumor estrogenreceptor (ER)/progesterone receptor (PR)/HER2 status, andtherapies. For cases with sufficient tissue, ER, PR, and HER2status was determined by immunohistochemistry (IHC).Eighty-seven formalin-fixed, paraffin-embedded (FFPE) tis-sues (49 brain metastases and 38 breast tumors, including11 paired tumors) were available from 76 patients. Thestudy was approved by the respective Institutional ReviewBoards.

FFPE tissue sections were incubated in 3% hydrogenperoxide/methanol for 10 minutes, followed by antigenretrieval in steaming citrate buffer for 30 minutes. Sectionswere preincubated in horse serum (Vector Laboratories)and then incubated for 60 minutes with antibodiesagainst ER (1D5, 1:50, Dako), PR (16, 1:70, Vision BioSys-tems), HER2 (CB11, 1:100, BioGenex), or aB-crystallin(1B6.1-3G4, 1:200, Enzo Life Sciences/Stressgen) using aDakoCytomation autostainer. An avidin/biotin complex

Translational RelevanceThe prognosis for patients with breast cancer brain

metastases remains dismal with survival typically mea-sured in months. Unfortunately, the limited availabilityof brain metastases from patients and dearth of animalmodels that recapitulate the entire metastatic cascadehave been major impediments to understanding themolecular pathogenesis of brain metastasis and devel-oping effective therapies. Here, we report that themolec-ular chaperone aB-crystallin, previously linked to thepathogenesis of triple-negative breast cancer (TNBC)and malignant glioblastomas, is commonly expressedin primary breast carcinomas and brain metastasesfrom patients and predicts poor clinical outcomes.aB-crystallin promotes adhesion to brain microvascularendothelial cells, at least in part, by an a3b1 integrin-dependentmechanism, transmigration through amodelof the blood–brain barrier (BBB) in vitro, and brainmetastasis in two orthotopic TNBC models that capturethe entire metastatic cascade. Our findings point to aB-crystallin and a3b1 integrin as potential drug targets forthis deadly disease.

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Clin Cancer Res; 2013 Clinical Cancer Research2




(Vectastain Elite) was applied for 30 minutes followed bydiaminobenzidine (Innovex) and hematoxylin (DakoCy-tomation). ER and PR staining were scored using the Allredsystem (26). HER2 was scored using ASCO/CAP guidelines(27). Breast cancer subtype was assigned by primary tumorIHC as ER/PR or hormone receptor (HR)-positive/HER2-negative (HRþ/HER2�), triple-negative (HR�/HER2�), orHR-positive/negative andHER2-positive (HER2þ).aB-crys-tallin was scored as negative (0%) or positive (>0%) basedon tumor cell expression.Statistical analysis for associations between subtypes and

aB-crystallin expression was conducted using the Fisherexact test. Time-to-event analyses were done for overallsurvival (time from breast tumor diagnosis to death or lastcontact) and overall survival from brain metastasis (timefrom the date of brain metastasis to the date of death orlast contact). The Kaplan–Meier method and log-rank sta-tistics were used to estimate survival and to evaluate asso-ciationswithaB-crystallin expression. Breast cancer subtypewas available for 71 of 76 patients. Date of brain metastasiswas available for 75 patients. Statistical analyses were con-ducted with SAS 9.2 software.

Cell lines and cultureHumanGILM2 andMDA-MB-231 TNBC cells expressing

mCherryfluorescent protein (231-mCherry)were described(28). 231-mCherry cells were grown in DMEM/F12 mediawith 5% FBS, 100 units/mL penicillin/streptomycin, non-essential amino acids, and 1 mmol/L sodium pyruvate(Invitrogen). GILM2 cells were cultured in DMEM/F12mediawith 10%FBS, 100units/mLpenicillin/streptomycinand Insulin/Transferrin/Sodium Selenite mix (Invitrogen).Primary HBMECs and human astrocytes (ScienCell) werecultured according to the manufacturer’s protocol.

Lentiviral and retroviral transductionpLL3.7RSV-mCherry lentivirus was generated in 293T

cells using the ViraPower Lentiviral Expression system (Invi-trogen) and used to stably transduce GILM2 cells. Retro-viruses were produced in Phoenix cells and used to infectbreast cancer cells as described (13, 29). 231-mCherrycells were infected with pLXSN or pLXSN-aB-crystallinretrovirus (13),whereasGILM2-mCherry cellswere infectedwith pSM2, pSM2-CRYABsh1, or pSM2-CRYABsh2 (OpenBiosystems, RHS1764-9393967 and RHS1764-9691062,respectively). Cells were selected for growth in the appro-priate antibiotics. Silent point mutations in aB-crystallin,which rendered it resistant to RNA interference (RNAi) butdid not alter its coding sequence, were made using theQuikChange Site-Directed Mutagenesis kit (Stratagene)with the primer 50-ccgcctcttctttgaccagttctt-30.

Immunocytochemistry of HBMECsHBMECs (passage #4–5) were grown to confluence on

tissue-culture plastic, fixed with 4% paraformaldehyde (or100% ice-cold methanol), and permeabilized with 0.1%Triton-X100. Cells were blocked with 10% goat serum andthen incubated with primary antibodies against b-catenin

(1:100, BD Biosciences), claudin-5 (1:100, Life Technolo-gies), GLUT-1 (1:50, SPM498 clone, Thermo-Fisher),occludin (1:100, Life Technologies), PECAM-1 (1:50,Thermo-Fisher), VE-cadherin (1:50, F8 clone, Santa Cruz),and ZO-1 (1:100, Life Technologies) overnight at 4�C. Anti-mouse and anti-rabbit conjugated Alexa Fluor secondaryantibodies (1:200, Life Technologies) were incubated withthemonolayers for 1 hour. Nuclei were counterstainedwith30 nmol/L 40,6-diamidino-2-phenylindole (DAPI; Sigma).Immunolabeled HBMECs were visualized with an invertedepifluorescence microscope (Olympus) and imagesacquired using a 16-bit SPOT camera (Diagnostic Instru-ments) withMetavue software (Molecular Devices). Imageswere visualized and processed using NIH ImageJ software.

Adhesion to brain endotheliumHBMECs were grown on fibronectin-coated 24-well

plates until confluent. Fifty thousand cancer cells wereadded to each well. After 2 hours (231-mCherry) or 4 hours(GILM2-mCherry), cellswerewashed, and the attached cellswere fixed in 10% formalin and scored per 10� magnifi-cation field (Leica MZ10F stereomicroscope). In someexperiments, cells were preincubated for 1 hour with integ-rin blocking antibodies (Millipore).

Adhesion to extracellular matrix proteinsTwenty-four-well plates were coated with fibronectin,

collagen I, or laminin (20 mg/mL, Invitrogen), washed, andblocked with 2%bovine serum albumin (BSA) for 1 hour at37�C. Fifty thousand cells were added to each well, incu-bated for 20 to 30 minutes at 37�C, washed, and theattached cells were fixed, stained with crystal violet, andscored per 10� field.

Transendothelial migration and BBB transmigrationin vitro

Transendothelial migration was assayed as described(30). Transwell inserts with 8-mm pores (BD Bioscience)were coated with fibronectin (20 mg/mL) overnight. Fiftythousand HBMECs (passages #4–5) were plated on theupper chamber of the inserts, and cells were grown toconfluence. One day after confluence, 50,000 cancer cellswere added to each well. After 24 hours (231-mCherry) or48 hours (GILM2-mCherry), the nonmigrating cancer cellsand HBMECs were removed, and the mCherry-flourescentmigrating cells were fixed in 10% formalin and scored per10� field (Leica MZ10F stereomicroscope). For the BBBassay, primary HBMECs were cocultured with human pri-mary astrocytes on opposite sides of Transwell inserts asdescribed (12, 31). Transwell inserts with 8-mm pores werecoated with fibronectin as described above and placedupside-down in a 15-cm plate. Twenty thousand primaryhuman astrocytes were plated on the membrane surfaceand incubated for 30minutes at 37�C. The inserts were theninverted, HBMECs added as in the transendothelial migra-tion assay, and grown to confluence. One day after conflu-ence, 50,000 cancer cells were plated on the upper chamberof the inserts, and mCherry-fluorescent cancer cells

aB-Crystallin Promotes Brain Metastasis

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migrating through the endothelial and astrocyte layers werescored per 10� field at 48 hours (231-mCherry) or 72 hours(GILM2-mCherry).

ImmunoblottingImmunoblotting was conducted as described (13) with

antibodies for aB-crystallin (Enzo Life Sciences/Stressgen)or b-actin (Sigma).

Cell surface expression of integrinsThe cell surface expression of integrins was determined

using an a/b integrin–mediated Cell Adhesion Array kit(CHEMICON). Absorbance at 570 nmwas measured usinga SpectraMax Plus384 Absorbance MicroPlate Reader(Molecular Devices).

Orthotopic models of breast cancer brain metastasis231-mCherry cells stably expressing vector or aB-crystal-

lin (2.5 � 105) or GILM2-mCherry cells stably expressinga nonsilencing construct or shRNA1 targeting aB-crystallin(1 � 106) were resuspended in 100% Matrigel (BD Biosci-ence) and injected bilaterally into the ducts of the fourthmammary glands of 4- to 5-week-old female NOD scid IL2receptor g chain knockout (NSG) mice (Jackson Laborato-ry). Tumor volume was measured weekly as described (32).For GILM2 xenografts, mammary tumors were resected 9weeks after tumor inoculation to allow sufficient time formetastasis.Micewere euthanized at 7weeks (231-mCherry)or 12 weeks (GILM2-mCherry). Images of isolated wholebrains were obtained (Leica MZ10F fluorescent stereomi-croscope) and analyzed with ImageJ software. All animalexperiments were approved by the institutional AnimalCare and Use Committee.

Statistical methodsStatistical significancewas assessed asdescribed in "Breast

cancer brain metastasis cohort and immunohistochemistryanalyses" or by ANOVA with posttests using GraphPadPrism software.

ResultsaB-crystallin predicts poor survival in patients withbreast cancer brain metastases

We examined aB-crystallin expression by IHC in primarybreast carcinomas and brain metastases in 76 patients withbreast cancerwho developed brainmetastases (Supplemen-tary Table S1). Thirty-seven percent (14 of 38) of primarybreast cancers and 47% (23 of 49) of brain metastasesexpressed aB-crystallin. Representative IHC staining ofpaired breast tumor and brain metastases from 2 patientsis shown (Fig. 1A). Concordance between paired breasttumors and brain metastases was 55% (6 of 11, Supple-mentary Table S2). It was more common for brain metas-tases to gain aB-crystallin expression (36%, 4 of 11) than tolose expression of this protein (9%, 1 of 11). aB-crystallinexpression in primary breast tumors was associated withbreast cancer subtype defined by IHC (P < 0.0001, Supple-mentary Table S3). Triple-negative (HR�/HER2�) breast

cancers were more likely to express aB-crystallin (73%,11 of 15) compared with HRþ/HER2� (10%, 1 of 10) orHER2þ tumors (0%, 0 of 11). These findings indicate thataB-crystallin is commonly expressed in breast cancer brainmetastases and confirm the association between aB-crystal-lin expression and TNBC.

We next examined the relationship betweenaB-crystallinexpression and survival. Themedian follow-up for survivorswas 6.5 years from diagnosis and 1.7 years from time ofbrainmetastases; 79% (60 of 76) of patients had died whenthese analyses were completed.Overall survival was inferioramong patients with aB-crystallin–positive breast tumorscompared with aB-crystallin–negative breast tumors [1.4;95% confidence interval (CI), 0.79–3.01 vs. 4.7; 95% CI,2.79–9.11years; P ¼ 0.0002; Fig. 1B]. Similarly, overallsurvival from the time of brain metastases was inferioramong patients with aB-crystallin–positive breast tumorscompared with aB-crystallin–negative tumors (0.13; 95%CI, 0.01–0.3 vs. 0.91; 95% CI, 0.13–3.37 years; P¼ 0.001).Among patients with TNBC, aB-crystallin expression in theprimary breast tumors was associated with lower overallsurvival rates (1.4; 95%CI, 0.79–3.01 vs. 4.7; 95%CI, 1.43–not estimable years; P¼ 0.01) and survival following brainmetastases (0.14; 95% CI, 0–0.3 vs. 1.5; 95% CI, 0.13–notestimable years; P ¼ 0.02) compared with those with aB-crystallin–negative breast tumors. aB-crystallin expressionin brain metastases did not predict survival in this cohort(data not shown). These results indicate that aB-crystallinexpression in primary breast carcinomas is associated withpoor overall survival and poor survival after brain metas-tasis, even among patients with TNBC.

aB-crystallin promotes adhesionof TNBC cells to brainendothelium, transendothelial migration, and BBBtransmigration in vitro

The extravasation of breast cancer cells into the brain isa critical stepwise process in brain metastasis that beginswith their adhesion to brain microvascular endothelium,followed by migration through the BBB (2, 3). We mod-eled these metastatic steps in vitro by examining the abilityof TNBC cells to adhere to HBMECs, migrate through alayer of HBMECs (transendothelial migration), and passthrough a model of the BBB composed of HBMECs andhuman astrocytes grown on opposite sides of Transwellinserts (12, 31). HBMECs formed a modest restrictivebarrier that was enhanced by coculture with astrocytes(Supplementary Fig. S1). In addition, HBMEC mono-layers expressed the vascular markers PECAM-1 and VE-cadherin, the BBB marker GLUT-1, and the BBB celljunction–associated proteins claudin-5, ZO-1, occludin,and b-catenin (Fig. 2A).

To determine the role of aB-crystallin in these steps,we stably overexpressed vector or aB-crystallin in MDA-MB-231-mCherry TNBC cells (abbreviated 231-mCherry-Vector and 231-mCherry-aB cells, respectively). Immuno-blot analysis confirmed robust expression of aB-crystallinin 231-mCherry-aB cells, whereas 231-mCherry-Vectorcells did not express detectable aB-crystallin protein

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(Fig. 2B). aB-crystallin overexpression increased adhesionto HBMECs (Fig. 2C), transendothelial migration throughHBMECs (Fig. 2D), and BBB transmigration in vitro (Fig.2E). In addition, we stably silenced aB-crystallin in GILM2-mCherry TNBC cells with 2 different shRNAs (sh-aB1 andsh-aB2), which reduced aB-crystallin levels compared withGILM2-mCherry cells stably expressing a nonsilencing (NS)construct (Fig. 3A). To control for potential off-targeteffects, we coexpressed sh-aB1 with an RNAi-resistant

mutant aB-crystallin that restored expression of the wild-type protein (sh1-aBM). Silencing aB-crystallin in GILM2-mCherry cells inhibited adhesion to HBMECs (Fig. 3B),transendothelial migration (Fig. 3C), and BBB transmigra-tion in vitro (Fig. 3D) compared with GILM2-mCherry-NSand GILM2-mCherry-sh1-aBM cells. Notably, altering aB-crystallin levels by overexpression and/or gene silencing didnot affect cell viability under standard cell culture condi-tions (Supplementary Fig. S2A and S2B). Collectively, these

Brain metastasisBreast tumor A

B

PositiveNegative

PositivePositive

Overall survival

Overall survival in TNBC

Survival after BCBM

Survival after BCBM in TNBC

P = 0.0002 P = 0.001

P = 0.01 P = 0.02

ααB-Positive

αB-Negative

Figure 1. aB-crystallin expressionin primary breast tumors isassociated with poor survival inpatients with breast cancer brainmetastases. A, representative IHCstaining of paired breast tumorsand brain metastases from 2patients. B, Kaplan–Meier survivalcurves by aB-crystallin expression(positive in red andnegative in blue)for all patients with breast cancer(top) and for patients with TNBCs(bottom). Overall survival (OS) isplotted in the left and survival afterbreast cancer brain metastasis(BCBM) is shown in the right.






findings indicate that aB-crystallin promotes adhesion ofTNBC cells to HBMECs, transendothelial migration, andBBB transmigration in vitro.

aB-crystallin increases adhesion of TNBC cells toHBMECs, at least in part, through an a3b1 integrin–dependent mechanism and promotes adhesion toextracellular matrix proteins

Because integrins play an important role in the adhesionof cancer cells to endothelial cells in the microvasculature(33–35), we postulated that the observed effects of aB-crystallin in promoting adhesion to HBMECs might bemediated by one or more integrins. Neither overexpressionof aB-crystallin in 231-mCherry cells nor silencing aB-crystallin in GILM2-mCherry cells affected the cell surfaceexpression of several a/b integrin subunits (Fig. 4A). Nev-ertheless, integrin a3 and b1 blocking antibodies (but notseveral other integrin-blocking antibodies) inhibited the

enhanced adhesion of 231-mCherry-aB cells to HBMECs(Fig. 4B, left). Similarly, integrin a3 and b1 blocking anti-bodies attenuated the adhesionofGILM2-mCherry-NS cellsto HBMECs to a level comparable to that observed inGILM2-mCherry-sh1-aB cells (Fig. 4B, right). In contrast,the integrin b1 blocking antibody had no effect on theadhesion of 231-mCherry-Vector or GILM2-sh-aB cells toHBMECs. The integrin a3 blocking antibody inhibitedadhesion of 231-mCherry-Vector cells (albeit to a lesserextent than observed in 231-mCherry-aB cells) but notGILM2-mCherry-sh-aB cells toHBMECs. These results indi-cate that aB-crystallin promotes adhesion of TNBC cells toHBMECs, at least in part, by an a3b1 integrin–dependentmechanism.

We next examined whether aB-crystallin regulatedadhesion of TNBC cells to extracellular matrix (ECM)proteins, which are present in subendothelial microvascu-lature and on the exterior of microvessels in organs (6, 36).

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Figure 2. aB-crystallin overexpression in TNBC cells enhances adhesion to brain endothelium, transendothelial migration, and BBB transmigration in vitro. A,immunocytochemical analysis of confluent primary HBMEC (passages #4–5) monolayers. The tight junction proteins claudin-5, occludin, and ZO-1 wereexpressed at varied levels throughout the monolayer. HBMEC clusters with elevated expression and appropriate localization of these proteins areshown. Negative controls (mouse or rabbit IgG) are also included. Scale bar ¼ 5 mm. B, immunoblot of 231-mCherry breast cancer cells stably expressingvector or aB-crystallin. C, adhesion of 231-mCherry cells stably expressing vector or aB-crystallin (aB) to HBMECs. Cells were seeded onto confluentHBMECs for 2 hours, washed, and attached mCherry-positive cells were scored (mean � SEM, n ¼ 3; ��, P < 0.01). D, transendothelial migration (TEM) of231-mCherry-Vector or 231-mCherry-aB cells at 24 hours (mean � SEM, n ¼ 3; ��, P < 0.01). E, BBB transmigration in vitro. 231-mCherry-Vector or231-mCherry-aB cells that transmigrated through 2 cell layers (HBMECs and astrocytes) were scored at 48 hours (mean � SEM, n ¼ 3; �, P < 0.05).

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231-mCherry-aB cells adhered more robustly to laminin,fibronectin, and collagen than 231-mCherry-Vector cells,whereas GILM2-sh1-aB cells exhibited reduced adhesion tolaminin and collagen compared with control GILM2-mCherry-NS cells (Fig. 4C). These results indicate that aB-crystallin promotes adhesion of TNBC cells tomultiple ECMproteins, including laminin, a key step in both extravasationand perivascular growth of micrometastases.

aB-crystallin overexpression increases brainmetastases in an orthotopic TNBC modelTo explore the potential role of aB-crystallin in brain

metastasis in vivo, we injected 231-mCherry-aB or 231-mCherry-Vector cells intraductally into the fourthmamma-ry glands of NSGmice.aB-crystallin overexpression did notaffect mammary tumor growth in this model (Fig. 5A). aB-crystallin overexpression in mammary tumors was con-firmed by immunoblotting and IHC (Fig. 5B). Mice wereeuthanized 7 weeks after tumor inoculation and mCherry-

fluorescent metastatic lesions were identified at autopsy.Both groups of mice had widespread metastases to manyorgans, including the brain, lungs, liver, lymph nodes, andother tissues (Supplementary Table S4). NSG mice with231-mCherry-aB tumors had a greater number ofmCherry-positive brain metastatic lesions and more extensive tumorburden as determined by the percentage of the surface areaof the brain occupied by metastases compared with micewith 231-mCherry-Vector tumors (Fig. 5C and D). In addi-tion, aB-crystallin overexpression resulted in increasedmetastatic tumor burden in the liver and bone (Supple-mentary Table S4 and Supplementary Fig. S3). aB-crystallinexpression in brainmetastases was observed by IHC inmicewith 231-mCherry-aB tumors but not vector controls (Fig.5B, bottom). Intriguingly, aB-crystallin overexpression didnot affect proliferation as determined by Ki67 IHC orapoptosis as determined by active caspase-3 IHC of mam-mary tumors or brain metastases analyzed at 7 weeks(Supplementary Fig. S5A). These observations indicate that

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Figure 3. Silencing aB-crystallin inTNBC cells inhibits adhesion tobrain endothelium,transendothelial migration, andBBB transmigration in vitro. A,immunoblot of GILM2-mCherrybreast cancer cells stablyexpressing an NS construct,shRNAs targeting different aB-crystallin sequences (sh1-aB andsh2-aB), or sh1-aB and an aB-crystallinmutant that disrupts genesilencing but does not alter itscoding sequence (sh1-aBM). B,adhesion of GILM2-mCherry cellsstably expressing NS, sh1-aB,sh2-aB, or sh1-aBM to HBMECs.Cells were seeded onto confluentHBMECs for 4 hours, washed, andattached mCherry-positive cellswere scored (mean � SEM, n ¼ 3).C, transendothelial migration(TEM) of GILM2-mCherry cellsstably expressing NS, sh1-aB,sh2-aB, or sh1-aBM at 48 hours(mean � SEM, n ¼ 3). D, BBBtransmigration in vitro of GILM2-mCherry cells stably expressingNS, sh1-aB, sh2-aB, or sh1-aBMat 72 h (mean� SEM, n¼ 3). (B–D):�, P < 0.05; ��, P < 0.01;��, P < 0.001 versus NS.






aB-crystallin promotes brain metastases in an orthotopicTNBC model with widespread metastatic dissemination.

Silencing aB-crystallin inhibits brain metastases in anorthotopic TNBC model

We also examined the effect of silencing aB-crystallin inTNBC cells on brain metastases in vivo. GILM2-mCherry-

sh1-aB and GILM2-mCherry-NS cells were injected intra-ductally into the fourth mammary glands of NSG mice.Silencing aB-crystallin did not alter mammary tumorgrowth (Fig. 6A), despite robust reduction of aB-crystallinlevels (Fig. 6B). Mammary tumors were resected at 9 weeksto allow additional time for metastasis; mCherry-positivemetastases were identified at autopsy 3 weeks later. Both

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Figure 4. aB-crystallin promotesadhesion to HBMECs, at least inpart, by an a3b1 integrin–dependent mechanism andenhances adhesion to ECMproteins. A, the cell surfaceexpression of various integrins in231-mCherry breast cancer cellsstably expressing vector or aB-crystallin (top) andGILM2-mCherrycells stably expressing NS or sh-aB (bottom) was determined usingan a/b integrin–mediated celladhesion array.Data are expressedas absorbance at 570 nm (mean �SEM, n ¼ 3). A negative control (c)was included. B, 231-mCherry-Vector, 231-mCherry-aB, GILM2-mCherry-NS, and GILM2-sh-aBcells were preincubated withintegrin-blocking antibodies for1 hour, seeded onto confluentHBMECs for 2 hours (231-mCherrycells) or 4 hours (GILM2-mCherrycells), washed, and attachedmCherry-positive cells werescored (mean � SEM, n ¼ 3). C,adhesion of breast cancer cells todifferent ECM proteins. Cells wereplated on fibronectin-, collagen-, orlaminin-coated 24-well plates for20 minutes (231-mCherry cells) or30 minutes (GILM2-mCherry cells)at 37�C, washed, and attachedcells were counted (mean � SEM,n ¼ 3). (A–C): �, P < 0.05;��, P < 0.01; ��, P < 0.001.

Malin et al.





groups of mice had metastases to many organs, includingthe brain, lungs, liver, lymph nodes, and other organs(Supplementary Table S4). Mice with GILM2-mCherry-sh-aB tumors had fewer brain metastases as determinedby mCherry-fluorescence (Fig. 6C) and hematoxylin andeosin (H&E) staining (Fig. 6D). GILM2-mCherry-sh-aBbrain metastases had reduced expression of aB-crystallincompared with NS controls (Fig. 6B). Moreover, silencingaB-crystallin reduced metastates in other organs, includingthe liver (Supplementary Table S4 and Supplementary Fig.S4). Silencing aB-crystallin did not affect proliferation orapoptosis as determined by IHC of mammary tumors orbrain metastases analyzed at 12 weeks (Supplementary Fig.S5B). These findings provide additional evidence that aB-crystallin promotes brain metastasis in vivo in a secondorthotopic TNBC model.

DiscussionThemolecular pathogenesis of breast cancer brainmetas-

tasis remains poorly understood due to limited access tobrain metastases from patients, a dearth of clinical trials,and the lack of animal models that recapitulate the entiremetastatic cascade (37). The vast majority of animals mod-els reported rely on intracardiac or carotid artery injection ofbreast tumor cells (38).We have shown thataB-crystallin, amolecular chaperone previously linked to an aggressivetumor phenotype in TNBC, glioblastoma multiforme(GBM), and other neoplasms (13, 19, 20, 22, 39), is anovel regulator of breast cancer brain metastasis. Specifi-

cally, we have shown that aB-crystallin is commonlyexpressed in clinical breast cancer brain metastases, includ-ing some brain metastases that developed from breasttumors that did not express aB-crystallin. aB-crystallinexpression in breast carcinomas was associated with tri-ple-negative IHC status and with poor overall survival andpoor survival after brain metastasis in a cohort of breastcancer cases across all subtypes and alsowithin the subset ofTNBC cases. We have also shown a direct causal role for aB-crystallin in promoting breast cancer brainmetastasis in vivoin 2orthotopicmodels inwhichfluorescently labeledTNBCcells metastasize from the mammary gland to the brain inNSG mice. These models recapitulate the entire metastaticcascade and several clinical aspects of breast cancer brainmetastasis, including triple-negative status and aB-crystal-lin expression by tumors, widespread metastatic disease,and late-onset brain metastases. Using overexpression andgene silencing to alter aB-crystallin levels in mammarytumors, we showed that aB-crystallin promotes brainmetastases in vivo without accelerating mammary tumorgrowth. Collectively, our results point to a previously unrec-ognized role for aB-crystallin in breast cancer brain metas-tasis and suggest that aB-crystallin may be a useful bio-marker to identify poor-prognosis patients with breastcancer who might be enrolled in clinical trials for earlydetection or prevention/treatment of brain metastases.Moreover, our observation that silencingaB-crystallin inhi-bits brain metastases suggests that aB-crystallin may be apromising drug target.

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Figure 5. aB-crystallinoverexpression increases brainmetastases in an orthotopic TNBCmodel. 231-mCherry cells stablyexpressing vector or aB-crystallin(aB) were injected bilaterally intothe ducts of the fourth mammaryglands of NSG mice. A, mammarytumor volume expressed as thepercentage original tumor volumeat 2 weeks in mice with 231-mCherry-Vector and 231-mCherry-aB xenografts (n ¼ 10mice per group). B, immunoblot of231-mCherry-Vector and 231-mCherry-aBmammary tumors andaB-crystallin IHC staining ofmammary tumors and brainmetastases in both groups. C,representative fluorescent whole-brain images from mice with231-mCherry xenograftsoverexpressing vector or aB. D,number of mCherry-fluorescentmetastases per brain in vector andaB groups and percentage surfacearea of brain metastases in vectorand aB groups (mean � SEM, n ¼10 mice per group; ��, P < 0.001).






Wehave shown thataB-crystallin promotes several of theearliest steps in the extravasation of circulating tumor cellsacross the BBB, including cell adhesion to HBMECs, trans-endothelial migration, and transmigration across a modelof the BBB in vitro. Of note, our in vitro BBB model mimicssome features of the BBB, but like most in vitro BBBmodels,the barrier is not as tight as that observed in vivo (31).Nevertheless, our observation that aB-crystallin enhancestransmigration through our in vitro BBB model and breastcancer brain metastasis in vivo strongly suggests that aB-crystallin promotes BBB penetration. Moreover, aB-crystal-lin promotes adhesion to several ECM proteins includinglaminin, which are present in the subendothelial vessel walland mediate adhesion to intraluminal circulating tumorcells and support perivascular growth of newly extravasatedtumor cells (6, 36). Notably, the initial adhesion of TNBCcells to HBMECs is dependent, at least in part, on a3b1integrin, which has been broadly implicated in metastasis,including brain metastasis. Specifically, a3b1 integrin hasbeen reported to mediate arrest of circulating tumor cells inthe pulmonary vasculature by engaging its ligand laminin-5in exposed regions of the vessel wall; pulmonary arrest invivowas inhibited by an integrin b1 blocking antibody (36).a3b1 integrin is also robustly expressed in a non–small cell

lung cancer cell line highly metastatic to the brain andmediates adhesion to brain slices and invasion in vitro; ana3 integrin blocking antibody dramatically suppressedbrain metastases when these cells were injected into theleft ventricle of mice (40, 41). In addition, b1 integrin playsa key role in the perivascular growth of early brain micro-metastases upon intracardiac injection of breast and othertumor cells bymediating "vascular cooption," the adhesionof metastatic tumor cells to the exterior surface of thevascular basement membrane of preexisting vessels andsubsequent expansion ofmicrometastses in the perivascularniche (6). Intriguingly, deletionofb1 integrin in theMMTV-activated ErbB2 model did not affect mammary tumorinduction but suppressed lung metastases in this model(42). Although we have yet to determine the nature of theinteraction between aB-crystallin and a3b1 integrin, thesestudies suggest that inhibition of a3b1 integrin might be apromising therapeutic strategy against brain metastasesarising from aB-crystallin–positive TNBCs.

In our orthotopic TNBC models, aB-crystallin increasedbrainmetastases without affectingmammary tumor growth,suggesting that the enhanced adhesion to HBMECs andBBB transmigration may be the principal mechanisms bywhich aB-crystallin promotes brain metastases. However,

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Figure 6. Silencing aB-crystallininhibits brain metastases in anorthotopic TNBC model. GILM2-mCherry cells stably expressing anNS construct or aB-crystallinshRNA (sh1-aB) were injectedbilaterally into the ducts of thefourth mammary glands ofimmunodeficient NSG mice.A, mammary tumor volumeexpressed as the percentageoriginal tumor volume at 2 weeks inmice with GILM2-mCherry-sh-aBand GILM2-mCherry-NSxenografts (n¼ 10mice per group).B, immunoblot of GILM2-mCherry-NS and GILM2-sh-aB mammarytumors and aB-crystallin IHCstaining of mammary tumors andbrain metastases in both groups.C, representative fluorescentwhole brain images and number offluorescent metastases per brainfor NS and sh-aB groups (mean �SEM, n ¼ 10 mice per group;�, P < 0.05). D, representative H&Estaining of brain sections andnumber of metastatic lesions persection for NS and sh-aB groups(mean � SEM; ��, P < 0.001).

Malin et al.





we cannot exclude other potential mechanisms, includingincreased intravasation and survival of circulating tumorcells, increased perivascular growth of micrometastasesand/or diminished tumor dormancy. Although we did notobserve differences in apoptosis or proliferation of mam-mary tumors or brain metastases at the conclusion of theexperiments, aB-crystallin might affect proliferation or apo-ptosis during the initial perivascular expansion of micro-metastases. Furthermore, we have previously reported thatectopic expression of high levels of aB-crystallin in a singleclone of MDA-MB-231 cells promoted mammary tumorgrowth (43). No such mammary tumor growth advantagewas observed in these experiments using a polyclonal pop-ulationofMDA-MB-231cellswithmoremodest levelsofaB-crystallin, suggesting that these effects may be dose-depen-dent. In addition, the prometastatic activity of aB-crystallinis not limited to the brain. In both orthotopic models, aB-crystallin promoted liver metastases, indicating that aB-crystallin may also regulate cell adhesion and/or extrava-sation in the liver microvasculature. Consistent with thisidea, integrin b1 has been implicated in tumor cell extrav-asation and hepatic colonization (44). Although we didnot observe a difference in lungmetastatic tumor burden atautopsy by altering aB-crystallin levels, the lungs in allanimals had extensive tumor burden by the time brainmetastases became apparent, suggesting the need for moredetailed analyses at earlier time points or different tumormodels to determine whether aB-crystallinmay affect lungmetastasis as well.In summary, our results underscore a previously unrec-

ognized role for aB-crystallin in brain metastasis in TNBCand point to aB-crystallin and a3b1 integrin as potentialdrug targets for this devastating disease. We are currentlyusing our orthotopic TNBC models to further delineate theprometastatic mechanisms of aB-crystallin and to evaluatenovel therapies, including neutralizing a3b1 integrin anti-

bodies. Moreover, our findings point to aB-crystallin as apotential biomarker to help identify patients with breastcancer who might benefit from additional diagnostic ortherapeutic interventions.

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

Authors' ContributionsConception and design: D. Malin, E.V. Shusta, C.K. Anders, V.L. CrynsDevelopment of methodology: D. Malin, E. Strekalova, B. Adamo, A.Ugolkov, C.K. Anders, V.L. CrynsAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): V. Petrovic, A. Al Ahmad, B. Adamo, C.R. Miller,C. Livasy, K. Fritchie, E. Hamilton, K. Blackwell, L. Carey, C.K. AndersAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): D. Malin, V. Petrovic, A.M. Deal, A. AlAhmad, B. Adamo, C. Livasy, K. Blackwell, E.V. Shusta, C.K. Anders, V.L.CrynsWriting, review, and/or revision of the manuscript: D. Malin, E. Stre-kalova, V. Petrovic, A.M. Deal, A. Al Ahmad, B. Adamo, C.R. Miller, A.Ugolkov, C. Livasy, E. Hamilton, J. Geradts, M. Ewend, L. Carey, E.V. Shusta,C.K. Anders, V.L. CrynsAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): D. Malin, K. Blackwell, J. Geradts,L. Carey, V.L. CrynsStudy supervision: V.L. Cryns

AcknowledgmentsThe authors thank Drs. Jennifer Koblinski and Janet Price for providing

cell lines.

Grant SupportThe study was supported by Susan G. Komen for the Cure Postdoctoral

Fellowship Award (D. Malin), CALGB Young Investigator Award (C.K.Anders), UNCHematology-OncologyNCI K12 (C.K. Anders), and the BreastCancer Research Foundation (V.L. Cryns).

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

Received May 7, 2013; revised October 9, 2013; accepted October 10,2013; published OnlineFirst October 16, 2013.

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2014;20:56-67. Published OnlineFirst October 16, 2013.Clin Cancer Res Dmitry Malin, Elena Strekalova, Vladimir Petrovic, et al. Brain

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aB-Crystallin:ANovelRegulatorofBreastCancerMetastasis to the Brain · Human Cancer Biology aB-Crystallin:ANovelRegulatorofBreastCancerMetastasis to the Brain Dmitry Malin1, Elena