Anxa2 Plays a Critical Role in Enhanced Invasiveness of theMultidrug Resistant Human Breast Cancer Cells

Fei Zhang, Lin Zhang, Bin Zhang, Xiyin Wei, Yi Yang, Robert Z. Qi, Guoguang Ying,

Ning Zhang,*, and Ruifang Niu*,

Key Laboratory of Breast Cancer Prevention and Treatment, Ministry of Education, Tianjin Medical UniversityCancer Institute and Hospital, Tianjin 300060, P.R. China, Department of Biochemistry, The Hong Kong

University of Science and Technology, Hong Kong, P.R. China, and Research Center of Basic Medical Science,Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, P.R. China

Received May 24, 2009

Multidrug resistance (MDR) is the major cause of failure in cancer chemotherapy. Recent reports evensuggest that MDR is associated with elevated invasion and metastasis of tumor cells. In the currentstudy, we used a proteomic approach to identify genes that play an important role in MDR inducedcell migration. 2D-PAGE and MALDI-TOF/MS-based proteomics approach were used to separate andidentify differentially expressed proteins between MCF-7 and MCF-7/ADR, a p-glycoprotein-overex-pressing adriamycin-resistance breast cancer cell line. Annexin a2 (Anxa2) was identified as highlyexpressed in MCF-7/ADR cells, but not in MCF-7 cells. Small interference RNA-mediated genesuppression demonstrated that Anxa2 was required for enhanced cell proliferation and invasion of theMCF-7/ADR cells. Down-regulation of Anxa2 alone was not sufficient to revert the cell sensitivity toadriamycin, suggesting that Anxa2 was not required for MDR phenotype. Taken together, our resultsshowed that expression of Anxa2 is enhanced when cancer cells, MCF-7, acquired drug resistance andit plays an essential role in MDR-induced tumor invasion.

Keywords: breast cancer Anxa2 metastasis MDR 2-dimensional gel electrophoresis massspectrometry small interference RNA

1. Introduction

Breast cancer is one of the leading causes of death amongwomen. Chemotherapy plays an important role in the treat-ment of breast cancer at various stages. However, long-termtreatment often resulted in chemoresistance and even multi-drug resistance (MDR), a phenomenon in which cross resis-tance of tumor cells to several structurally unrelated chemo-therapeutic agents was developed after exposure to a singlecytotoxic drug.1,2 MDR is a major cause of treatment failureand mortality for most cancer patients. Previous studies haverevealed several mechanisms contributing to drug resistance,but it still remains as a formidable obstacle to reverse drugresistance for effective treatment of human cancer.3-6

Recent studies showed that acquisition of MDR phenotypeis often associated with an elevated invasion/metastasis.7-10

Yang et al. observed increased motility, invasion, and metastasisof certain P-glycoprotein-overexpressing MDR cancer cells

treated with P-gp transportable drugs;11 Li et al. reported thatup-regulation of CD147 (also knows as extracellular matrixmetalloproteinase inducer, EMMPRIN) and matrix metallopro-teinase-2, -9 were induced by P-glycoprotein substrates inmultidrug resistant breast cancer cells, and expression of CD147and MMPs correlated with the invasiveness of the tumorcells.12,13 Stephen Hiscox showed that, following acquisitionof tamoxifen resistance, breast cancer cells displayed an alteredgrowth rate associated with increased aggressive behavior invitro,14-16 and elevated invasion and metastasis properties ofdrug resistant cancer cell lines have also been demonstratedin animal models.17-19 Taken together, development of MDRnot only prohibits effective chemotherapy, but also exacerbatesthe metastatic symptom of cancer patients.

Invasion/metastasis is a multistep process, including detach-ment of tumor cells from the primary sites, intravasation intocirculation, spread of tumor cells through circulation, extrava-sation to the secondary sites, and growth into new tumors.20,21

Spectra of genes are up-regulated to orchestrate the invasionand metastasis of tumor cells to the secondary sites. Amongthem, several reports have revealed that the cytosolic level ofAnnexin A2 (Anxa2), a calcium dependent phospholipid bind-ing protein, is enhanced in metastatic cancers.22-26 Overex-pression of Anxa2 has been proposed to be a potential markerfor cancer diagnosis and prognosis.27-32 However, the role ofAnxa2 in cancer metastasis remains unclear.

* To whom correspondence should be addressed. Ning Zhang, TianjinMedical University, Research Center of Basic Medical Sciences & CancerInstitute and Hospital, Tianjin, 300060 China; e-mail, nzhangchina@yahoo.com.Ruifang Niu, Tianjin Medical University Cancer Institute and Hospital,Tianjin. 300060, China; e-mail, Niurf1982@yahoo.com.cn.

Key Laboratory of Breast Cancer Prevention and Treatment, TianjinMedical University Cancer Institute and Hospital.

The Hong Kong University of Science and Technology. Research Center of Basic Medical Science, Tianjin Medical University

Cancer Institute and Hospital.

10.1021/pr900461c CCC: $40.75 2009 American Chemical Society Journal of Proteome Research 2009, 8, 50415047 5041Published on Web 09/18/2009

In this study, we investigate the molecular mechanism ofenhanced invasion/metastasis in MDR cancer cells. It has beendocumented that MCF-7/ADR, a p-glycoprotein overexpressingadriamycin-resistant breast cancer cell, shows elevated inva-sion/metastasis in comparison with its parental MCF-7 cell.33-36

A 2D-PAGE based proteomic approach was deployed to screenfor the proteins with altered level of expression upon cellularacquisition of MDR, and small interference RNA technique wasused to further verify the functional relevance. Our studyidentified Anxa2 as an essential factor which was responsiblefor the enhanced invasiveness of the MCF-7/ADR cells.

2. Materials and Methods

2.1. Cell Culture. MCF-7 human breast cancer cells (adria-mycin-sensitive) and their MCF-7/ADR multidrug-resistantvariant (adriamycin-resistance) were obtained from Dr. Zi-Zheng Hou of the Detroit Hospital, Detroit, MI. The cells werecultured in RPMI 1640 supplemented with 10% (v/v) fetalbovine serum (Hyclone, Logan, UT), 100 U/mL penicillin and100 U/mL streptomycin, maintained in a humidified atmo-sphere at 37 C under 5% CO2, and were passaged every 2-3days when digestion was made by a mixture of 0.025% trypsinand 0.01% EDTA (Gibco BRL, Rockville, MD). The MCF-7/ADRcells were continuously exposed to 0.5 M adriamycin for themaintenance of the MDR phenotype, but cultured in drug-freemedium for at least 1 month before use in any experiment.

2.2. Two-Dimensional Gel Electrophoresis and ImageAnalysis. For sample preparation, cells were harvested, washedthree times with ice-cold PBS without Ca2+ or Mg2+ to removeserum proteins and stored as cell pellets in aliquots of 1 107cells/tube at -80 C until use. The cell pellets were resuspendedin lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS,0.5% IPG buffer (pH 3-10 NL), 20 mM tris, 40 mM DTT, 1 mMPMSF and a mixture of protease inhibitors at room temperaturefor 1 h. The lysates were centrifuged at 20 000g for 1 h at 4 C.Protein concentrations were determined using the modifiedBio-Rad protein assay kit. Fifty micrograms or 500 g of proteinwas loaded onto IPG strips for analytical or preparative gels,respectively. The strips were rehydrated at 20 V for 14 h at 20C and isoelectric focusing was performed sequentially at 500,1000, 3000, and 5000 V, for 1 h each plus 8000 V for 5 h. Thestrips were then equilibrated for 15 min in buffer I (50 mMTris-HCl, pH 8.8, 6 M urea, 30% glycerol, 100 mM DTT and 2%SDS) and 15 min in buffer II (buffer I with 250 mg of

iodoacetamide instead of DTT). The second dimension wasperformed on homogeneous 12% SDS-PAGE gels, and theseparated proteins were detected by silver staining for analyticalgels and coomassie blue staining for preparative gels. Allsamples were pools of three independent cell preparations andanalyzed for at least three times. Image acquisition wasperformed by UMAX image scanner at 300 dpi and the imageswere analyzed using the ImageMaster 2D Elite software. Onlyprotein spots which consistently showed at least 3-fold differ-ence were considered to be differentially expressed.

2.3. Trypsin Digestion, MALDI-TOF-MS and ProteinIdentification. Coomassie blue-stained protein spots wereexcised using a clean ophthalmic scalpel and transferred to an

Figure 1. 2-D protein profiles from human breast cancer cell lineMCF-7 and its multidrug resistant subline MCF-7/ADR. Proteinswere run on IPG DryStrips (pH 3-11 NL) and further separatedusing SDS-PAGE (12%) gels and visualized by silver staining. (A)Representative images of 2-D gel for human breast cancer cellline MCF-7 and MCF-7/ADR. (B) Representative images of en-larged view of the spots differentially expressed between the twocell lines. (C) Anxa2 was overexpressed in MCF-7/ADR cellscompared with MCF-7 cells.

Table 1. Proteins with Altered Expression in MCF-7 and MCF-7/ADR Cells

no.accessionnumber protein name MW (Da) pI

Mascotscorea

peptidesmatch/total

sequencecoverage (%)

Down-Regulated Proteins in MCF-7/ADR Cells1 gi|1384068 NADPH-flavin reductase 22105.4262 7.13 110 11/46 552 gi|182311 fructose-1,6-bisphosphatase 36804.8007 6.54 125 14/55 44

Up-Regulated Proteins in MCF-7/ADR Cells3 gi|8923900 cytidine 5-monophosphate

N-acetylneuraminicacid synthetase

48348.6472 8.02 68 7/25 24

4 gi|45786109 Anxa2 38579.8085 7.57 216 17/26 535 gi|13623417 Ubiquitin carboxyl-terminal esterase L1 24808.4609 5.53 100 8/31 376 gi|386854 type II keratin subunit protein 52752.5195 5.31 83 9/36 217 gi|35440 unnamed protein product 23497.8203 5.43 73 6/19 298 gi|265222 beta 2-microglobulin 11431.0336 5.86 79 8/28 289 gi|4504893 kininogen 1 47883.2079 6.62 68 6/25 22

10 gi|1335012 beta-gonadotropin 15487.9648 8.43 74 7/21 26

a Protein scores greater than 66 are significant (p < 0.05).

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5042 Journal of Proteome Research Vol. 8, No. 11, 2009

Eppendorf tube; stained gel slabs were cut into small piecesand destained using washing buffer (25 mM ammoniumbicarbonate, 50% acetonitrile) for 15 min. Washing step wasrepeated for at least three times until no color was visible. Thegel pieces were dehydrated with 100% acetonitrile and driedin a Speedvac centrifuge. Digestion was performed withsequencing grade trypsin. After tryptic digestion, the peptidefragments were extracted three times with 20 L of 5% TFA in50% acetonitrile, dried, and redissolved in 10 L of 0.5% TFA.The samples were desalted with ZipTipC18 according to themanufactures instructions and eluted with 2.5 L of 50%acetonitrile containing 0.5% TFA and 3 mg/mL R-cyano-4-

hydroxycinnamic acid (CHCA). Samples were spotted ontostainless steel MALDI target plates and then analyzed using the4700 Proteomics Discovery System MALDI-TOF mass spec-trometer (Applied Biosystems, Framingham, MA) in the positiveion reflector mode. Peptide masses were obtained for the range700-4000 Da and the acquired peptide mass fingerprints wereused to search through the NCBI nonredundant (NCBI nr)Protein Data Base with the Mascot software (www.matrix-science.com). After removal of known contamination peakssuch as keratin and autoproteolysis peaks, the following searchparameters were used in all mascot searches: human species,monoisotopic peptide masses, tolerance of one missed cleav-age, and a maximum error tolerance of 100 ppm, carbami-domethylation and oxidation of methionine as fixed andvariable modification. Protein scores greater than 66 wereconsidered as significant (p < 0.05).

2.4. Western Blotting and Immunodetection of Anxa2.Cellular proteins were extracted with RIPA cell lysis buffer and20 g of lysates was separated by 10% SDS-PAGE and trans-ferred onto a PVDF membrane. The membrane was incubatedin a blocking solution consisting of 5% milk in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% Tween 20 at roomtemperature for 1 h. The mouse monoclonal antibody againstAnxa2 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) wasthen added followed by incubation overnight at 4 C andwashing with TBST three times for 10 min. Detection was madeusing horseradish peroxidase (HRP)-linked goat anti-mouse IgGsecondary antibody (1:5000, Santa Cruz Biotechnology, SantaCruz, CA) and ECL kit (Pierce Biotechnology, Rockford, IL)according to the manufacturers protocol. Mouse monoclonalantibody against -actin (1:2000, Santa Cruz Biotechnology,Santa Cruz, CA) was used as a loading control.

2.5. SiRNA Design, Construction and Transfection. TheSiRNA expression vector pGCsilencer H1/hygro was obtainedfrom Genechem company (Genechem, Shanghai, China). Thetargeting sequence 5-GGTCTGAATTCAAGAGAAA-3 againstAnxa2 was designed and verified to be specific by Blast searchagainst the human genome, and a sequence of similar GCcontent which does not match any known human codingsequence was used for negative control. Transfection wasmediated with Lipofectamine 2000 (Invitrogen, Carlsbad, CA)according to the manufacturers instruction, and three separateMCF-7/ADR stable transfectants were generated after 14 daysselection by 300 g/mL Hygromycin for both Anxa2 siRNA(pGCsi-Anxa2) and the control (pGCsi-control). The transfec-tants were routinely maintained under selection but withdrawnof the drug before performing drug-sensitivity assays.

2.6. RT-PCR Analysis of the Stable Transfectants. For RT-PCR, total RNA was Trizol-extracted, reverse-transcribed usingthe Reverse Transcription System (Invitrogen, Carlsbad, CA),and PCR amplified using a Taq DNA polymerase kit (TakaraBio., Dalian, China). The PCR primers 5-CTCCCGCAGT-GAAGTGGA CAT-3 (forward) and 5-TTG AAA GCA GGG CCACAA AGT-3 (reverse) were synthesized by Augct BiotechnologyCo. (Augct Biotechnology Co., Beijing, China), which generateda fragment of 466 bp in length. -Actin was amplified with theprimers 5-GGC CGG GAC CTG ACT GAC TAC-3 (forward) and5- GCC GCC AGA CAG CAC TGT GTT-3 (reverse) with productfragment of 363 bp. The PCR reaction conditions were asfollows: initial at 94 C for 5 min, followed by 35 cycles of 94C (1 min), 60 C (30 s) and 72 C (30 s), and a final extensionat 72 C for 7 min. The amplified fragments were separated by1% (w/v) agarose gel electrophoresis, stained by 0.3 mg/mL

Figure 2. Down-regulation of Anxa2 decreased cell proliferation.(A) RT-PCR and Western blotting analysis of Anxa2 in MCF-7/ADR cells, control cells and three stable clones of si-Anxa2/MCF-7ADR cells. The Control cells were transfected with a siRNAplasmid containing a scrambled sequence in MCF-7/ADR cells.-Actin was used as an internal control. Anxa2 expression wassignificantly decreased both at mRNA and protein levels in threestable clones. (B) Cell proliferation activity was decreased afterknockdown of Anxa2 in MCF-7/ADR cells; each point and barshows the mean ( SD for triplicates. Statistical analysis wascarried out with one-way ANOVA, *P < 0.05 vs control cells. (C)Down-regulation of Anxa2 induced a decrease in the proportionof G2/M + S phase cells compared to the control. All theexperiments were repeated at least three times.

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ethidium bromide and analyzed using a Kodak 2D image-analysis software.

2.7. Cell Proliferation Assay. Both flow cytometer and MTTmethods were used to determine the cell proliferation. For flowcytometer analysis, cells were harvested, washed three timeswith ice-cold PBS and fixed in ice-cold 70% ethanol overnight.The cells were then washed with PBS and incubated withpropidium iodide (50 g/mL) and RNase (1 g/mL) at 37 Cfor 30 min. Flow cytometric analysis was performed on aBeckman Coulter EPICS analyzer. The cell cycle phase distribu-tion was calculated from the resultant DNA histogram usingMulticycle AV software. The Proliferation Index (PI) wascalculated using the following equation: PI ) (S + G2/M)/[(G0/G1) + S + (G2/M)] 100%. The results were the averages of 3experiments. For MTT, 2 103 cells were plated in 96-wellmicrotiter plates per well. Upon analysis, 20 L of 5 mg/mLMTT in PBS was added to each well and the cells wereincubated for another 4 h at 37 C. The supernatant was thenremoved, and 200 L of DMSO was added to each well. Theabsorbency of each sample was measured with a micro ELISAreader using test and reference wavelength of 570 and 630 nm.The surviving cells were measured every day for 5 consecutivedays.

2.8. Adriamycin Sensitivity Assay. MTT-based cytotoxicityassay was used to determine the cell sensitivity to adriamycin.Five 103 cells were seeded in 96-well plates and different

concentrations of adriamycin were added accordingly 24 hlater. Three replicates were prepared for each treatment andthree blank wells containing only media without drug were alsoincluded as control in all experiments. After 72-h incubation,cell viability was examined by the ability of viable cells toreduce MTT dye to formazan. The relative drug resistance wasdetermined by comparing the IC50 values of experiment andcontrol groups. The IC50 was defined as the concentration ofthe drug causing 50% inhibition of cell growth, as comparedto the untreated control and the IC50 values were calculatedby linear regression of percent survival versus drug concentration.

2.9. Cell Invasion Assay. Invasion assays of MCF-7/ADR ortransfectants were performed in 24-well Boyden chamber plateswith polycarbonate membrane filter inserts with 8 m pores(Corning Costar Corporation, Cambridge, MA). The abovesurface of the porous membrane of transwell inserts werecoated with matrigel (BD Biosciences, San Jose, CA). Cellsuspension (1 104 to 1 105) in 200 L of 0.1% bovine serumalbumin (BSA)-DMEM was added into each of the upperchamber, and the lower chamber was filled with 500 L of 10%FBS-DMEM. After incubation for 6-36 h, noninvaded cells inthe inserts were removed with cotton swabs. Invaded cells onthe bottom side of the membrane were fixed and stained withthe 3 Step Stain Set kit (Richard-Allen Scientific, Kalamazoo,MI) according to the manufacturers instructions. The stainedmembranes were cut and placed on a glass slide, and the

Figure 3. Down-regulation of Anxa2 decreased MCF-7/ADR cell invasion. (A) Same amounts of cell suspension with serum-free mediawere loaded into the upper, matrigel coated surface of the transwell inserts, and FBS-containing media were loaded in the lower wellof the assay chamber. Then, the number of cells invaded and attached to the bottom of the membrane was counted. (B) The numberof cells invaded was evaluated in three fields for each experimental group; each column and bar shows the mean ( SD for triplicates.The experiments were repeated at least three times, and statistical analysis was carried out with one-way ANOVA, *P < 0.05 vs controlcells.

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number of invaded cells on the bottom surface of the mem-brane was counted using a bright field light microscope. Allassays were performed in triplicates. Statistical analysis wasdone using one-way ANOVA, and P-value was calculated basedon two-tailed test.

2.10. Statistics. Statistics were conducted using SPSS soft-ware. All experiments were carried out at least 3 times, theresults are presented as mean ( standard errors (SEM), andone-way ANOVA was used for data analysis. A value of P < 0.05was considered to be statistically significant.

3. Results

3.1. MCF-7/ADR Cells Exhibits a Different 2D-PAGEPattern from MCF-7 Cells. Reproducible protein expressionpatterns were obtained with a majority of proteins visiblebetween pH 5-8 in our triplicate analysis. As shown in Figure1A, a panel of representative gel images selected from threereplicates, more than 900 protein spots are visible in each gel.

Most of the spots correlated well between MCF-7 and MCF-7/ADR cells, showing equivalent intensity between the two setsof gels. Quantitative analysis by ImageMaster 2D Platinumsoftware revealed that 67 spots showed differentiated intensity,with p-values less than 0.05. Among them, 10 spots wereselected, cut out, and subjected to in-gel digestion. Eight spotsshowed increased intensity and two with decreased intensityin MCF-7/ADR cells. MALDI-TOF MS analysis followed by adatabase search revealed the identity of these proteins as listedin Table 1. Several proteins have been previously reported tobe differentially expressed in drug resistance cell lines, indicat-ing that our method is suitable for this study.37-39 Within these10 proteins, Anxa2 was significantly up-regulated in MCF-7/ADR cells (Figure 1B). Western blotting analysis further con-firmed that Anxa2 level was significantly up-regulated in MCF-7/ADRcells(Figure1C).Anxa2,acalcium-dependentphospholipidbinding protein, has been implicated in a number of biologicalresponses, such as cell proliferation, angiogenesis, ion channelactivation, and cell-cell communication.40,41 We speculate thatAnxa2 may also play a role in an elevated invasion of MDRcancer cells.

3.2. Down-Regulation of Anxa2 Decreased CellProliferation. A small RNA interference technique was usedto down-regulate the expression of Anxa2 in MCF-7/ADR cells(Figure 2A). Semiquantitative PCR results showed that siRNA,not control vector, significantly reduced the mRNA levels ofAnxa2 in MCF-7/ADR cell. Western blotting analysis of Anxa2showed a more than 10-fold decrease at protein level, furtherconfirming the efficacy of siRNA techniques (Figure 2A).Suppression of Anxa2 expression exhibited a marked inhibitionin cell proliferation (Figure 2B). The cell growth curves indi-cated that the three Anax2 suppression clones grew significantlyslower than MCF-7/ADR and the control siRNA cells (p < 0.05).Cell cycle analysis showed that down-regulation of Anxa2induced a decrease in the proportion of G2/M + S phase cellscompared to the control (Figure 2C).

3.3. Down-Regulation of Anxa2 Decreased MCF-7/ADRCell Invasion. A previous study demonstrated that Anxa2 wasoverexpressed in invasive breast cancer and contributed totumor invasion and progression;25 we investigated the role ofAnxa2 in MDR induced invasiveness by using a BoydenChamber based assay. As shown in Figure 3A,B, MCF-7/ADRshowed a more than 4-fold increase in invasiveness in com-parison with its parental MCF-7 cells, consistent with previousreport.33,34,36 In three Anxa2 knockdown clones, the invasionproperties were significantly blocked up to 70%, suggesting thatAnxa2 played an essential role in invasion of MCF-7/ADR cells(p < 0.05).

3.4. Down-Regulation of Anxa2 Did Not Reverse theDrug Resistance Phenotype of MCF-7/ADR Cells. To examinewhether Anxa2 is required to convey drug resistance phenotypeof MCF-7/ADR cells, MTT assay was used to check theadriamycin cytotoxic effect on MCF-7/ADR cells depleted ofAnxa2. As shown in Figure 4A and Table 2, MCF-7/ADR is 10-fold less sensitive to the treatment with adrianmycin, consistentwith previous report.12 All three Anxa2 knockdown clones, aswell as vector control transfected cells, did not show detectabledifference at IC50 of adriamycin in comparison with MCF-7/ADR cells (p > 0.05) (Figure 4B, Table 2). Taken together, ourresults suggest that elevated expression of Anxa2 is associatedwith acquisition of multidrug resistance phenotype, but notrequired for multidrug resistance.

Figure 4. Down-regulation of Anxa2 did not reverse the drugresistance phenotype of MCF-7/ADR cells. (A) MCF-7 cells weresignificantly more sensitive to the treatment compared with MCF-7/ADR cells. (B) All of the three Anxa2 knockdown clones retainedadriamycin-resistant property compared with control and MCF-7/ADR cells; each point and bar shows the mean ( SD fortriplicates. All the experiments were repeated at least three times.

Table 2. IC50 of Adriamycin in MCF-7, MCF-7/ADR, Anxa2Knockdown Clones and Their Negative Controla

cell type IC50 (M)

MCF-7 3.72 ( 0.38*MCF-7/ADR 39.11 ( 4.19Control 35.12 ( 6.49Control 1 38.90 ( 8.83Control 2 36.14 ( 5.14Control 3 41.48 ( 3.97

a IC50 values are presented as mean ( SD from at least threeexperiments performed in triplicate, *P < 0.05 vs. control cells.

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4. Discussion

Recent studies demonstrated that there might be a functionallink between MDR and tumor invasion/metastasis. One rep-resentative example was the fact that enhanced properties ofinvasion and metastasis were found in a drug resistant strainof MCF-7 cells, suggesting that drug resistance and invasion/metastasis might be inseparable cellular events during theprogression of malignant tumors.10,12-16,34 We speculate thatacquisition of drug resistance and metastasis is a complexprogress, involving changes of multiple cellular components.Thus, in order to possess a relatively full spectrum of moleculesaffected upon the acquisition of MDR, we employed a powerful2D-PAGE coupled with MALDI-TOF/MS technique to study thedifferential protein expression patterns between the MCF-7 andMCF-7/ADR cells. Our results showed that Anxa2 was up-regulated in the drug resistant MCF-7/ADR cells, in comparisonto MCF-7 cells. When the expression of Anxa2 in MCF-7/ADRwas reduced to a low level, the drug resistant MCF-7/ADR cellslost their invasiveness, indicating that Anxa2 played a criticalrole in the invasion/metastasis of MCF-7/ADR cells.

Among the identified proteins, there were metabolic proteins(fructose-1, 6-bisphosphatase and NADPH-flavin reductase),structure proteins (type II keratin subunit protein), and signal-ing intermediates (Anxa2 and -2-microglobulin). These pro-teins may play an important role in acquisition of drugresistance or in the new more malignant phenotype of the drugresistant strains. Interestingly, we also identified ubiquitincarboxy-terminal hydrolase l (UCHL1), whose overexpressionwas closely correlated with advanced progression and invasionof various cancers including breast cancer,42-45 as expressedat a much higher level in the MCF-7/ADR cells. Its role ininvasion/metastasis is still under investigation.

Down-regulation of Anxa2 by siRNA did not reverse adria-mycin resistance in MCF-7/ADR cells. One plausible explana-tion is that Anxa2, up-regulated along with the acquisition ofMDR phenotype, was not required for drug resistance. Theother possibility is that a number of proteins played redundantroles in MDR; therefore, knockdown of Anxa2 alone wasinsufficient to reverse the drug resistance. Taken together, ourresults suggest that Anxa2 is not required and is not sufficientin the acquisition of MDR phenotype.

In conclusion, our results confirm the fact that MDR isclosely associated with enhanced invasion/metastasis of tumorcells. Furthermore, our study revealed Anxa2 as a criticalcomponent in MCF-7/ADR cells that mediated the invasion/metastasis. We speculate that Anxa2 may be used as a biom-arker for MDR and may be developed into an antimetastasisdrug target.

Acknowledgment. This research was supported bygrants from NFSC (30772529), Tianjin Commission ofScience and Technology (06TXTJJC14502) and 973 Project(2010CB933900).

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Pivotal Role of Anxa2 in MDR Breast Cancer Cells research articles

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