biii-tubulin regulates breast cancer metastases to the...

Cancer Biology and Signal Transduction

bIII-Tubulin Regulates Breast Cancer Metastasesto the BrainDeepak Kanojia1, Ramin A. Morshed1, Lingjiao Zhang1, Jason M. Miska1, Jian Qiao1,JuliusW. Kim1, Peter Pytel2, IrinaV. Balyasnikova1, Maciej S. Lesniak1, andAtiqueU.Ahmed1

Abstract

Brain metastases occur in about 10% to 30% of breast cancerpatients, which culminates in a poor prognosis. It is, therefore,critical to understand the molecular mechanisms underlyingbrain metastatic processes to identify relevant targets. Wehypothesized that breast cancer cells must express brain-asso-ciated markers that would enable their invasion and survival inthe brain microenvironment. We assessed a panel of brain-predominant markers and found an elevation of several neu-ronal markers (bIII-tubulin, Nestin, and AchE) in brain meta-static breast cancer cells. Among these neuronal predominantmarkers, in silico analysis revealed overexpression of bIII-tubu-lin (TUBB3) in breast cancer brain metastases (BCBM) and its

expression was significantly associated with distant metastases.TUBB3 knockdown studies were conducted in breast cancermodels (MDA-Br, GLIM2, and MDA-MB-468), which revealedsignificant reduction in their invasive capabilities. MDA-Br cellswith suppressed TUBB3 also demonstrated loss of key signalingmolecules such as b3 integrin, pFAK, and pSrc in vitro. Further-more, TUBB3 knockdown in a brain metastatic breast cancercell line compromised its metastatic ability in vivo, and signif-icantly improved survival in a brain metastasis model. Theseresults implicate a critical role of TUBB3 in conferring brainmetastatic potential to breast cancer cells. Mol Cancer Ther; 14(5);1–10. �2015 AACR.

IntroductionBrain metastases occur in about 10% to 30% of breast cancer

patients (1, 2). The median survival of patients with brain metas-tases is extremely poor (<10months), and at present there is a lackof targeted therapy. To identify appropriate treatment regimens, itis critical to understand the biology of breast cancer brain metas-tases (BCBM). To comprehend the brain metastatic process,researchers developed paired models systems of brain metastasis.MDA-MD-231/MDA-MB-231BrM2 and CN34/CN34BrM2 aresuch paired model systems in which MDA-MB-231BrM2/CN34BrM2 cells exhibit a high propensity to metastasize to thebrain after intracardiac administration in athymic nude mice.

Recent literature using this model system combined withBCBM tissues documents that brain metastatic cells overexpressneuronal markers such as ST6GALNAC5 (ST6; ref. 3), Nestin/CD133 (4), GABA receptor (5), and neuroserpin (6). This over-expression of neuronal markers by brain metastatic cells isbelieved to be an adaptation to survive in the brain microenvi-ronment. However, the active role of these predominant brainmarkers in conferring BCBM has not been evaluated. Studies onmolecular mechanisms of brain metastasis have also led to the

identification of hyperactivated signaling pathways, including Src(7), STAT3(8), Notch (9), IGFBP3 (10), and EGFR (11). Theinhibition of these signaling mediators either decreased theformation of brain metastases (9–11) or improved survival inmurine models of preexisting brain metastases (7).

The unique cytoskeletal characteristics of a cancer cell are alsocritical mediators of its invasive behavior. Actin microfilamentsare essential components for conferring metastatic potentialthrough formation of invasive structures (12), whereas the roleof microtubules is still under investigation (12). Microtubules areheterodimers of a and b subunits that are expressed in a tissue-specific manner (13). One such isotype is bIII-tubulin (TUBB3),which is specifically expressed by neurons (14). TUBB3 is absentin normal mammary epithelia (15); however, it is ectopicallyexpressed inbreast, lung, ovarian, and colon cancers, andhasbeenfound to confer paclitaxel resistance (16–19). Moreover, TUBB3overexpression is strongly associated with poor prognosis ofepithelial malignancies (16–20).

In an attempt to understand the role of neuronal predominantmarkers in brain metastasis of breast cancer, we screened a panelof neuronal markers and found ectopic overexpression of TUBB3,Nestin, and AchE in brain metastatic cells as compared withparental cells. Among these neuronal predominant markers, insilico analysis revealed overexpression of TUBB3 in BCBMpatients, and its expression was significantly associated withdistant metastases of breast cancer. Therefore, we focused ourstudy on defining the role of TUBB3 in brainmetastases. Throughin vitro and in vivo analysis, our results uncover a vital role of thecytoskeletal protein bIII-tubulin in conferring brain metastaticpotential to breast cancer cells. Our findings, along with avail-ability of clinically approved antimicrotubule agents, warrantsinvestigation of TUBB3 as a target for prevention of brain meta-stases in patients with breast cancer.

1The Brain Tumor Center, The University of Chicago, Chicago, Illinois.2Department of Pathology, University of Chicago Medical Center,Chicago, Illinois.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Maciej Lesniak, The University of Chicago Hospitals,5841 South Maryland Avenue, Chicago, IL 60637. Phone: 773-834-4757; Fax:773-834-2608; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-14-0950

�2015 American Association for Cancer Research.

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on June 18, 2018. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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Materials and MethodsCell culture and reagents

MDA-MB-231/MDA-MB-231BrM2 (will be referred to asMDA-231/MDA-Br) cells were obtained from Dr. Joan Massague(MSKCC) and were maintained in DMEM (Corning) with 10%FBS (Hyclone). MDA-MB-468 cells (courtesy of Dr. SuzanneConzen, University of Chicago) were maintained in RPMI-1640 (Corning) medium with 10% FBS (Hyclone) whereasGLIM2 cells (obtained from Dr. Vincent Cryns, University ofWisconsin) were maintained in DMEM F/12 medium with10% FBS and insulin/transferrin/sodium selenite mix (Invitro-gen). CN34/CN-34-Br cells were obtained from Dr. Joan Massa-gue (MSKCC) and were maintained in M199 medium supple-mented with 2.5% FBS, 10 mg/mL insulin, 0.5 mg/mL hydrocor-tisone, 20 ng/mL EGF, and 100 ng/mL cholera toxin (Sigma). Nocell line authentication was done by the authors. Penicillin–streptomycin (100 U/mL) was added to all the cell culture media.Cellswere routinely screened and found tobe free ofmycoplasma.For lentiviral transduction, nontarget control shRNA and twodifferent shRNA specific to TUBB3 were procured (Sigma). Lenti-viral particles were generated by transfection of shRNA andpackaging vectors in HEK293 cells (Invitrogen). The primaryantibodies used were bIII-tubulin, phosphor-FAK, Src, phos-phor-Src, STAT3, phosphor-STAT3, and actin (Cell SignalingTechnologies), bIV-tubulin (Sigma), L1CAM (Thermo Fisher sci-entific), Integrin b3 (BioLegend), and Nestin (BD Pharmingen).Densitometric analysis was conducted using Image J software.Flow cytometry was conducted as described before (21).

Patient samples and immunohistochemistryThe tissues were collected in accordance with a protocol

approved by the Institutional Review Board at the University ofChicago. Sections (5mm-thick) of breast cancer and BCBM weredeparaffinized in xylene and then rehydrated. After deparaffiniza-tion and rehydration, tissue sections were treated with antigenretrieval buffer (S1699; DAKO) in a steamer for 20 minutes. bIII-tubulin rabbit monoclonal antibody (Cell Signaling Technology)was applied on tissue sections at a dilution of 1:20 for 1-hour andincubated at room temperature in a humidity chamber. Theantigen–antibody binding was detected by Bond Polymer RefineDetection (DS9800; Leica Microsystems).

Quantitative real-time polymerase chain reaction analysisRNA (1 mg) was isolated from breast cancer cells using the

RNeasy Plus Kit (Qiagen) and was reverse-transcribed using theiScript cDNA Conversion Kit (Bio-Rad) according to the manu-facturer's instructions. qRT-PCR was conducted using the SYBRGreen qPCR Kit (Bio-Rad) using primers indicated in Supple-mentary Table S1. Data analysis was performed using the 2�DDCt

method for relative quantification, and all sample values werenormalized to the GAPDH expression value.

Migration assayCells (1� 105) were seeded in 6-well plates and grown to 95%

confluency. Awoundwas created using a sterile 200mL pipette tip.The cells were fed with fresh medium with 1% serum andobserved under inverted microscope for 28 hours. Images werecaptured at various time points and migration of cells wasmeasured using ImageJ software. The experiments were con-ducted three times in duplicates.

Invasion assayMatrigel invasion assay was conducted using BD Biocoat

Matrigel invasion chambers according to the manufacturer'sinstructions. Briefly, cells were seeded in serum-free medium, inthe top inserts and complete medium was used as a chemoat-tractant at the bottom chamber. After 22 hours, the noninvadingcells were removed and the inserts were fixed and stained withcrystal violet. Five different fields of each chamber were photo-graphed and cells were counted manually. Graph was plotted asrelative invasion to cells to the vector control cells. The experi-ments were repeated twice in triplicates.

Adhesion assaysNinety-six well microplate was coated with laminin (5 mg/mL),

fibronectin (2.5 mg/mL), and collagen (50 mg/mL). Plates wereincubated overnight at 4�C for polymerization of ECM pro-teins. Unpolymerized substrates were washed with PBS and theplates were blocked with 2% BSA for 2 hours at 37�C. Vectorcontrol and TUBB3 knockdown cells were plated (4 � 104) andcell adhesion was monitored for 15 to 60 minutes at 37�C.Following incubation, the nonadherent cells were removed bytwo washes with PBS. The adherent cells were quantified usingMTT assay (Roche) and expressed as a percentage relative of therespective total unwashed cells. The experiments were repeatedtwice in triplicates.

Animal experimentsAll surgical procedures were completed in accordancewithNIH

guidelines on the care and use of laboratory animals for researchpurposed. The protocols were approved by the InstitutionalCommittee on Animal Use at the University of Chicago. Six- to8-week-old athymic nude femalemicewere obtained fromHarlanlaboratories and maintained in a specific pathogen-free facility.Mice were anaesthetized with an i.p. injection of 0.1 mL of astock solution containing ketamine HCl (25 mg/mL), xylazine(2.5 mg/mL), and 14.25% ethyl alcohol (diluted 1:3 IN 0.9%NaCl) and inoculated with 250,000 MDA-Br Vector control andTUBB3 knockdown cells in 100 mL PBS via injection into the leftventricle of the heart. After 28 days or appearance of signs ofmorbidity, mice were sacrificed and whole-brain tissues wereexcised immediately and embedded in OCT freezing agent.Approximately 100 sections with thickness of 10 mm across thehorizontal planewere cut, air dried, and stainedwith hematoxylinand eosin. For orthotopic mammary gland assay, subconfluentMDA-Br Vector control and TUBB3 knockdown cells were sus-pended in plain DMEM medium and mixed with Matrigel (Bec-tonDickinson) at a ratio of 1:1 in a volume of 50 mL. A total of 1�106 cells were injected in third or fourth Mammary gland of HSd:athymic nude female mice, and tumor size was monitored for15 weeks.

Statistical analysisAll statistical analyses were performed using GraphPad Prism 4

(GraphPad Software Inc.). The sample size for each group was�3and numerical data were reported asmean� SEM. The RNA levelsof brain predominant markers were assessed using the one-sam-ple Student t test, and the differences on protein level wereassessed using the two-sample Student t test. Comparisons oncellmigration, adhesion, and invasionwere conducted using one-way or two-way ANOVA with Bonferroni or Dunnett post hoc testas appropriate. For animal survival experiments, Kaplan-Meier

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survival curve was generated and log-rank test was applied tocompare survival distributions. Cox proportional-hazard regres-sion was used to investigate the association between TUBB3 andbreast cancer metastasis on patient data (22), and distant metas-tasis-free survival curvewas generated for patients grouped as highvs. low expression by one SD from mean. All reported P valueswere two-sided and were considered to be statistically significantat �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

ResultsOverexpression of predominant brain markers in BCBM

Given the ectopic overexpression of neuronalmarkers in BCBM(3–6), we hypothesized that theremay be other neuronalmarkersthat are involved in breast cancer metastases to the brain. Toevaluate our hypothesis, we used two different paired modelsystems, MDA-231/MDA-Br and CN34/CN34-Br. MDA-Br/

CN34-Br cells are the brain metastatic derivatives of the parentalMDA-231/CN34 cells (respectively), which were isolated aftertwo roundsof intracardiac injectionwith isolationof transplantedcells from brain tissue (3).

We conducted qRT-PCR to assess the RNA levels of a panel ofbrain predominantmarkers (TUBB3, Nestin, AchE, LMO3, Grin1,Grin2b, GFAP, PIN1, NSE2, NFL, NGFR, andNCAM1) and foundupregulation of TUBB3,Nestin, andAchEby 1.5-, 2-, and1.6-fold,respectively, in MDA-Br cells versus their corresponding parentalcells (Fig. 1A). These results were further confirmed in CN34-Brcells in which TUBB3, Nestin, and AchE were upregulated by 3.6-,1.46-, and 2.1-fold, respectively (Fig. 1B). However, there was nosignificant difference in other brain predominant markers thatwere evaluated (data not shown). We further confirmed thealterations in RNA levels of TUBB3 and Nestin at the proteinlevel using Western blotting (bIII-tubulin; Fig. 1C) and flowcytometry (Nestin; Fig. 1E). Densitometric analysis revealed a

Figure 1.Overexpression of TUBB3 in BCBM. A, quantitative RT-PCR analysis of Nestin, TUBB3, andAchE expression inMDA-Br cells as comparedwith parental MDA-231 cells.B, quantitative RT-PCR analysis of Nestin, TUBB3, and AchE expression in CN34-Br cells as compared with CN34. The primers used for quantitative PCR are listed inSupplementary Table S1. Data are representative of three independent experiments. C,Western blot analysis demonstrating overexpression ofbIII-tubulin inMDA-Brand CN34-Br cells as compared with respective parental cells. D, semiquantitative densitometry analysis was performed using ImageJ software. Data arerepresentative of three independent experiments. E, flow cytometry of MDA-231/MDA-Br and CN34/CN34-Br cells stained with nestin antibody. F, quantitation ofnestin MFI; �� , P < 0.001; �� , P < 0.01; � , P < 0.05.

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2.5/4.9-fold increase of TUBB3 protein inMDA-Br/CN34-Br cells,respectively (Fig. 1D), whereas nestin protein levels were elevatedby 1.4/2-fold in MDA-Br/CN34-Br cells as compared with paren-tal cells, respectively (Fig. 1E and F).

Furthermore, we conducted gene-expression analysis usinggene set enrichment (GSE) datasets, where nestin and AchEoverexpression did not correlate with brain metastases (data notshown). However, we found significant upregulation in TUBB3RNA levels in BCBM samples as comparedwith the primary breasttumors using GSE43837 dataset (Fig. 2A). To evaluate whetherTUBB3 confers metastatic potential to breast cancer cells, weconducted gene-expression analysis on a large dataset containing683 breast cancer patients (22) and found that overexpression ofTUBB3 significantly correlates with distant metastasis (P ¼0.008; Fig. 2B). Moreover using TCGA database, we found thatbreast cancer patients with high TUBB3 expression (22) exhibitedpoor survival as compared with patients with low TUBB3 expres-sion. (P ¼ 0.027 for Fig. 2C; ref. 23) and (P ¼ 0.007 for Fig. 2D;ref. 22).

To substantiate our in vitro and in silico results, we performedimmunohistochemistry on five human primary breast cancersand unmatched brainmetastasis tissues (Fig. 2E). Four samples ofBCBM demonstrated overexpression of bIII-tubulin as compared

with primary breast cancer tissues. Therefore, these results dem-onstrate that bIII-tubulin levels are elevated in brain metastaticbreast cancer cells and associated with poor prognosis.

Knockdown of TUBB3 in breast cancer cells decreases invasionin vitro

Tounderstand the active role ofTUBB3 in breast cancer cells, wesilenced TUBB3 expression via lentiviral transduction using twodifferent shRNA specific to TUBB3 (shRNA1 and shRNA2) inwidely used breast cancer cells, namely MDA-Br, GLIM2, andMDA-MB-468 cells. Densitometric analysis of Western blotsrevealed95%and88%downregulationofbIII-tubulin in shRNA1and shRNA2, respectively, as compared with scrambled vectorcontrol cells (Supplementary Fig. S1A–S1C). TUBB3 knockdowncells exhibited gross morphologic alterations like elongation ofcells and loss of cellular structures (Supplementary Fig. S2A) ascompared with the vector control cells. This observationprompted us to evaluate whether there are alterations in migra-tion of TUBB3 suppressed MDA-Br cells by a scratch woundmigration assay. MDA-Br cells with downregulated TUBB3migrated at significantly slower rate (n ¼ 6; P < 0.05) as shownby the images captured after 18 hours (Fig. 3A) in comparisonwith vector control cells. In addition, when complete closure of

Figure 2.Overexpression of TUBB3 in brain metastasis and distant metastasis patients. A, analysis of GSE43837 dataset demonstrating overexpression of TUBB3 inbrain metastasis as compared with primary breast cancer (n ¼ 38). B, analysis of the microarray dataset, demonstrating a significant correlation of TUBB3expression with distant metastasis of breast cancer (n ¼ 682; ref. 22), P ¼ 0.008. C and D, distant metastasis-free survival analysis of breast cancer patientsexhibiting high or low levels of TUBB3 (n ¼ 171 and n ¼ 198, respectively). E, immunohistochemical analysis of bIII-tubulin in breast cancer and BCBM. In brainmetastasis, bIII-tubulin shows strong staining in brain metastatic cells as compared with the breast cancer cells (n ¼ 5); scale bar, 50 mm. There is someweaker staining in the adjacent brain parenchyma and in particular in gemistocytic astrocytes at the edge of the lesion. Arrow indicates BCBM and arrowheadindicates brain tissue; scale bar, 100 mm. �� , P < 0.01; � , P < 0.05.

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the wound was noted in vector control cells, the knockdowncells were unable to close the wound even at the end of 28 hours(Fig. 3A). These results indicate reduced migratory potential ofTUBB3 knockdown MDA-Br cells.

It is well known that augmented invasion is essential formetastasis of cancer cells. Hence, to evaluate whether TUBB3knockdown cells possess the ability to invade the basementmembrane components, an invasion assay was performed usingMatrigel invasion chambers. MDA-Br cells with suppressedTUBB3 demonstrated 2.7- and 2.3-fold reduction in invasion forshRNA1 and shRNA2, respectively (Fig. 3B), as compared withvector control cells. To substantiate the role of TUBB3 in invasion,we conducted invasion assay in GLIM2 cells, which were previ-ously shown to have intrinsic brainmetastasis capability (24).Wenoted 8-(shRNA1) and 2.1 (shRNA2)-fold reduction in invasionofTUBB3-suppressedMDA-MB-468 cells as comparedwith vectorcontrol cells (Fig. 3C). Moreover, TUBB3-suppressed GLIM2 cellsdemonstrated 9.8-(shRNA1) and 4.5 (shRNA2)-fold decrease ininvasion (Fig. 3D). These results demonstrate that ectopic expres-

sion of TUBB3 regulates invasive migration of breast cancer andbrain metastatic cells.

TUBB3 knockdown in brain metastatic cells decreases celladhesion to ECM accompanied by decreased levels of b3integrins

Adhesion to extracellular matrix (ECM) components assists thecancer cells in the metastatic cascade. Laminin, fibronectin, andcollagen are abundant ECM proteins in the brain endothelial cells.Hence, we conducted adhesion assays using laminin-,fibronectin-,and collagen-coated dishes. TUBB3 knockdown cells exhibited asignificantly decreased ability to attach to laminin and fibronectin(Fig. 4A–C). These results depict that bIII-tubulin modulates theadhesion of cells to the ECM components.

Integrins are cell-surface proteins that mediate the process celladhesion with ECM proteins. Previous reports documented ele-vated levels of aVb3, aVb6, and aVb8 integrins in human BCBM(25). Therefore, we evaluated the transcript levels of these integ-rins in our brain metastasis model. Using the parental and brain

Figure 3.TUBB3 suppression decreases migration and invasion of breast cancer cells. A, scratch wound assay for migration. Images demonstrating wound closure ofvector control MDA-Br and TUBB3 knockdown cells at 0 and 28 hours time points. Right, statistical analysis of migration using ImageJ software. Images showinginvasion of vector control and TUBB3 knockdown cells. MDA-Br (B), MDA-MB-468 (C), andGLIM2 (D) cell lines, respectively. Left to right, vector control, shRNA1, andshRNA2. Graph indicates the percentage of invasion as compared with vector control cells. The results represent means � SEM for triplicate experiments; �� , P <0.001; ��, P < 0.01; � , P < 0.05.






metastatic derivative of MDA- 231 cells, qRT-PCR analysisrevealed a 3-fold increase in integrin b3 transcripts in brainmetastatic cells (MDA-Br) as compared with parental cells(MDA-231; Fig. 4D). This prompted us to evaluate whether thereare any alterations in integrin b3 levels when TUBB3 was sup-pressed in brain metastatic cells. Flow-cytometry analysis ofTUBB3 knockdown cells demonstrated 2.2- and 3.2-fold down-regulation of cell surface b3 integrin levels in shRNA1 andshRNA2, respectively (Fig. 4E), as compared with the vectorcontrol cells. These results demonstrate that bIII-tubulin regulatesadhesion ability of brainmetastatic cells possibly through integrinb3. Integrin downstream signaling is known to promote metas-tasis in a number of epithelial malignancies; therefore, it was ofinterest to evaluate whether the alteration in the integrin b3surface expression in knockdown cells was accompanied byalterations in integrin downstream signaling.

Knockdown of TUBB3 alters integrin-Src and STAT3 signalingIntegrins mediate signaling through phosphorylation of focal

adhesion kinase (FAK), which is activated when autophosphory-lated at Y397 residues. To ascertain whether decreased integrin b3in TUBB3 knockdown cells leads to decrease in FAK phosphor-ylation, we performed Western blotting in vector control andTUBB3 knockdown cells, which revealed reduced FAK phosphor-ylation (Fig. 5A). Furthermore, reduced FAK activation alsoresulted in blunted phosphorylation of Src at Y416 residues(Fig. 5A).

Integrin-mediated adhesion and signaling regulates activationof STAT3 (26). To evaluate whether abrogated integrin–FAK–Srcsignaling leads to alterations in STAT3 signaling, we conductedWestern blotting in MDA-Br vector control and TUBB3 knock-down cells.We foundattenuated STAT3phosphorylation at Y705,which is responsible for its transcriptional activation (Fig. 5A).These results demonstrate an important role of TUBB3 in main-

taining the integrin–Src–STAT3 axis in the brain metastatic cells(Fig. 5A).

Recently, it was shown that L1 cell adhesion molecule(L1CAM) is important for the colonization of metastatic cellsin the brain (6). To address the role of TUBB3 in regulation ofL1CAM levels, we conducted Western blotting using MDA-Brvector control and TUBB3 knockdown cells. We found dramaticreduction in L1CAM protein levels in of L1CAM in TUBB3knockdown cells (Fig. 5B). These results underscore the role ofbIII-tubulin in regulating the key signaling molecules that areinvolved in the process of BCBM.

Knockdown of TUBB3 decreases the brain metastaticcapabilities of breast cancer cells and increases survival of micewithout altering primary tumor growth

TUBB3 knockdown alters in vitromigratory and invasive prop-erties. However, our in vitro results demonstrated a marginalstatistical difference in the proliferative ability of TUBB3 knock-down cells (Supplementary Fig. 2SB–S2D). Therefore, it was ofinterest to understand whether there are any differences in theability of vector control MDA-Br and TUBB3 knockdown cells togrow in orthotopic mammary gland. Vector control MDA-Br,TUBB3 shRNA1, and TUBB3 shRNA2 cells were injected in thethird and fourthmammary glandof nudemice and tumor volumewasmonitored for a period of 15weeks. There were no significantdifferences in the growthof vector control andTUBB3 knockdowncells (Fig. 6A).

To evaluate the in vivo functional consequences of TUBB3knockdown on metastatic behavior of cells, a brain metastasismodel was used. Vector control and TUBB3 knockdown cells wereinjected through an intracardiac route, and after 28 days, whole-brain tissues were sectioned to determine the number of meta-static lesions. One hundred sections (10 mm) were isolated fromeach mouse followed by hematoxylin and eosin staining. Ten

Figure 4.Decreased adhesion of MDA-BrTUBB3 knockdown cells to laminin,fibronectin, and collagen. Adhesionassay was performed by seedingequal amount of cells of differentgroups in 96-well tissue culture platescoated with laminin (A), fibronectin(B), and collagen (C). The resultsrepresent means � SEM for triplicateexperiments. D, quantitative RT-PCRanalysis of the expression of Integringenes in MDA-Br cells as comparedwith parental MDA-MB-231 cells. E,flow cytometric analysis of b3 integrinshowing TUBB3 shRNA cellsdemonstrating reduction in b3integrin levels; ��, P < 0.01 and�� , P < 0.001.

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sections of each brainwere analyzed for the presence of number ofmicrometastasis. Within each group an average of 9 mice wereconsidered for calculating the number of micrometastasis. Miceinjected with TUBB3 knockdown cells exhibited significant reduc-tion in the formation of micrometastasis (Fig. 6B and C).

To understand the functional consequences of TUBB3 knock-down in ametastaticmodel on the survival ofmice, we conductedsurvival experiments. Here, the vector control and stable TUBB3knockdown cells were injected via an intracardiac route in the leftventricle of nude mice, and survival outcomes were determined.The vector control group exhibited a median survival of 34 dayswhereas shRNA1 and shRNA2 demonstrated median survival of45 (P¼ 0.0053) and 50 (P¼ 0.0011) days, respectively (Fig. 6D).These results demonstrate that TUBB3 knockdown increases sur-vival of mice via decreasing brain metastatic ability of breastcancer cells.

DiscussionThere is an urgent need to identify rationalmolecular targets for

the treatment of brain metastases. In this report, we present thefirst evidence that a microtubule family member, bIII-tubulin,confers brain metastatic potential to breast cancer cells by regu-lating invasion and Integrin-Src signaling.Our studies support theconcept that (i) TUBB3 overexpression is a malignant adaptationof breast cancer cells for invasion in the brain and (ii) suppressionof TUBB3 function holds the potential for improving the survivalin brain metastasis patients.

TUBB3 plays an important role in axonal guidance of neuronsand perturbations in TUBB3 function lead to ocular motilitydisorders (14). Within the brain only a specific subset of tubulins(TUBB3, TUBB2C, TUBB, TUBB2A, and TUBB2B) are expressed(15), whereas in normal breast epithelium (which expressTUBB2C, TUBB1, TUBB2A, and TUBB6) TUBB3 is virtually absent(15). Overexpression of TUBB3 is associated with poor prognosisof multiple epithelial malignancies, including lung, colorectal,and ovarian cancers (16–20). Moreover, 85% of small-cell lungcancer patients exhibit TUBB3 overexpression (27), a cohort ofwhich shows the highest incidence of CNS metastasis (28). Thesereports support the notion that TUBB3 overexpression impartsmetastatic potential to cancer cells. However, there are no studiesdemonstrating a direct correlation of TUBB3 overexpression andbrain metastases. We found overexpression of TUBB3 in humanBCBM tissues in silico, which is in agreement with the report thatTUBB3overexpression in primary breast cancers is associatedwithpoor prognosis (29). Although a clear relation between over-expression of TUBB3 and prognosis exist in the literature, its rolein invasion and metastasis has not been evaluated.

Invasion of cancer cells holds a central role in the process ofmetastasis. It is a combined interplay of various cancer hallmarksthat are governed by both interactions of cancer cell with the ECMcomponents and cell signaling through surface receptors. In thiscontext, we have evaluated the functional role of TUBB3 ininvasion by generating TUBB3 knockdown cells in different breastcancer cell lines. In all these models, knockdown of TUBB3 levelssignificantly decreased the invasion of cells. Moreover, we found

Figure 5.TUBB3 knockdown in brain-seeking MDA-Br cells attenuates FAK, Src, and STAT3 signaling. A, Western blotting demonstrating decrease in phosphorylationof FAK/Src/STAT3 and decreased levels of L1CAM in TUBB3 knockdown cells as compared with vector control MDA-Br cells. B, densitometric analysis ofpFAK, pSrc, pSTAT3, and L1CAM. The experiments were repeated three times. �� , P < 0.001; �� , P < 0.01; � , P < 0.05.






significant reduction in the adhesion ability of TUBB3-suppressedbrain metastatic cells to ECM components (Laminin, fibronectinand collagen). This decrease in adhesion was also associated withcompromised levels of L1CAM, a critical protein involved in theprocess of BCBM (6).

L1CAM binds to integrin b1, b2, and b3, and hetrodimerzationof L1CAM to integrins results in recruitment and auto phosphor-ylation of FAK. Phosphorylation of FAK recruits Src, which is thenautophosphorylated, leading to activation of MAPK (30). Thisadhesion-mediated cascade of signaling then promotes invasionand colonizationof cancer cells (6, 30). Integrins areoverexpressedin malignant cells, and hence promote invasion and metastasis(31). Integrin aVb3, aVb6 and aVb8 are overexpressed in humanBCBM tissues (25). Given the critical role of integrin signaling ininvasion and metastasis, we investigated the levels of integrin andfound downregulation of integrin b3 levels in TUBB3-suppressedcells. These results are in concordance with decrease in invasion inMDA-Br TUBB3 knockdown cells. Taken together, our resultssuggest that TUBB3 regulates L1CAM and Integrin b3 levels toregulate the invasive nature of brain metastatic cells.

Moreover, to evaluate the consequences of TUBB3 knock-down on metastatic burden, we conducted an in vivo brain

metastasis assay, which demonstrated a significant decrease inthe number of spontaneous micrometastasis in mice injectedwith TUBB3-suppressed cells. Decreased metastatic loadalso correlated with improved survival in the preclinical mod-el, which establishes TUBB3 as a critical mediator of brainmetastasis.

Our results on proliferation of TUBB3-suppressed cells in vitrodemonstrated marginal difference in proliferation (Supplemen-tary Fig. S2B–S2D). However, tumor volume analysis of TUBB3-suppressed metastatic MDA-Br cells showed no difference. Theseresults are in concordance with a very recent report on non–smallcell lung cancer model, in which TUBB3 regulates lung cancermetastasis without altering growth of tumor cells in vivo in ananchorage-dependent manner (32). Our results are further sup-ported by the fact that parental (MDA-231) and brain metastaticcells (MDA-Br) do not exhibit differences in proliferation (3),although MDA-Br demonstrated a 2.5-fold increase in bIII-tubu-lin protein levels. We therefore believe that TUBB3 in brainmetastatic cells predominantly regulate the molecular players ofinvasion and metastasis.

Pharmacologic inhibition of invasion is believed to be aplausible approach for successful intervention of cancer

Figure 6.In vivo brain metastatic capability of TUBB3 knockdown cells. A, orthotopic mammary gland injections of vector control and TUBB3 knockdown cells. A total of1 � 106 cells were injected in mammary fat pad and tumor growth was monitored for 15 weeks. B, in vivo brain metastasis assay. A total of 2.5 � 105 cellswere injected in nude mice via the intracardiac route. Mice were sacrificed 28 days and later the brains were removed for histologic analysis. Whole-brainhistology image of mice injected with MDA-Br vector control, TUBB3 knockdown cells. C, evaluation of micrometastatic lesions. Average values from 10 sectionsper mouse brain (mean value and range; n ¼ 9). D, Kaplan–Meier survival curve of vector control and TUBB3 knockdown cells. The log-rank test was appliedto compare mice survival; (n ¼ 9); �� , P < 0.01.

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progression. For example, inhibition of cathepsin B (via VBY-999;ref. 33) and COX2 (via cetuximab; ref. 3) decrease the occurrenceofmetastasis. These reports provide a clear evidence of preventingbrain metastases before they arise. bIII-tubulin protein is knownto be sensitive to vinorelbine-mediated drug therapy. In systemiclung cancer patients with TUBB3 overexpression, it has beenshown that these patients benefit from vinorelbine-mediatedchemotherapy with improvements seen in progression-free sur-vival (34). Hence, it will be of interest to evaluate vinorelbinemediated prevention studies for inhibiting the occurrence ofBCBM. In this context, our analysis of TCGA datasets (22, 23)implicates that, over time, the presence of high levels of TUBB3 inprimary breast cancers predicts distant metastasis. Furthermore,our results in a brainmetastasis model reveal that the suppressionof TUBB3 in these cells decreases the incidence of BCBM. Takentogether, TUBB3 in the primary/disseminated breast cancer cellsregulates brain metastases of breast cancer and TUBB3 is apotential target for prevention of BCBM. Moreover, given thatoverexpression of TUBB3 predicts poor prognosis of lung (34, 35)and ovarian (19) cancer, this finding supports a broad role ofTUBB3 in metastasis of different malignancies, which are knowntometastasize to the brain (36–39). Hence, research on the role ofTUBB3 in brainmetastasis of other malignancies warrants furtherinvestigation.

In conclusion, ourwork suggests for thefirst time a novel role ofTUBB3/bIII-tubulin in regulation of metastases and integrin-Srcsignaling. TUBB3/bIII-tubulin is, therefore, a possible marker forhigh-risk patients for development of brainmetastases. Our workalso demonstrates that TUBB3/bIII-tubulin regulates key media-tors of brain metastases (Src and STAT3), and thus TUBB3/bIII-tubulin should be targeted to improve the prognosis of patientswith metastatic brain cancer.

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

Authors' ContributionsConception and design: D. Kanojia, M.S. Lesniak, A.U. AhmedDevelopment of methodology: D. Kanojia, R.A. Morshed, M.S. Lesniak,A.U. AhmedAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Kanojia, R.A. Morshed, J.M. MiskaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D. Kanojia, R.A. Morshed, L. Zhang, J.M. Miska,P. Pytel, M.S. LesniakWriting, review, and/or revision of the manuscript:D. Kanojia, R.A. Morshed,J.M. Miska, J. Qiao, J.W. Kim, I.V. Balyasnikova, M.S. Lesniak, A.U. AhmedAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D. Kanojia, J.W. Kim, M.S. LesniakStudy supervision: M.S. Lesniak, A.U. Ahmed

AcknowledgmentsThe authors thank Dr. Maria Kavallaris for helpful discussions during the

preparation of this article. The authors thank Alan Chang for help with flow-cytometry analysis. The authors thank University of Chicago histology corefacility for conducting immunohistochemistry. MDA-MB-468 cells were a kindgift from Dr. Suzanne Conzen (University of Chicago) and GLIM2 cells were akind gift fromProf. Vincent Cryns (University ofWisconsin) andProf. Janet Price.

Grant SupportThisworkwas supported by theNIH (R01CA138587 and R01NS077388) and

the National Institute of Neurological Disorders and Stroke (U01NS069997)awarded to M.S. Lesniak

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedNovember 4, 2014; revised February 14, 2015; accepted February 18,2015; published OnlineFirst February 27, 2015.

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Published OnlineFirst February 27, 2015.Mol Cancer Ther Deepak Kanojia, Ramin A. Morshed, Lingjiao Zhang, et al. III-Tubulin Regulates Breast Cancer Metastases to the Brainβ

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