integrin high cell population functions as an initiator in tumorigenesis and relapse...

Therapeutic Discovery

Integrin a6high Cell Population Functions as an Initiator inTumorigenesis and Relapse of Human Liposarcoma

Lu Wang1, Lingxian Wang1, Yanhong Gu1,2, Yongqian Shu2, Yan Shen1, and Qiang Xu1

AbstractThe relapse and resistance to chemo- and radiotherapy are main problems in the treatment of human

liposarcoma. It is important to find a functional marker existing in the liposarcoma cells for targeting. In this

article, we established a new sub-cell line SW872-S cells with high tumorigenicity from human liposarcoma

SW872 cells by repeated inoculation approach. The characteristic of the sub-cell line is linked to the high levels

of integrin a6 on the surface. The integrin a6high cells show much higher tumor initiation and self-renewal

potential in vivo than integrin a6low cells do. Targeting integrin a6 with its specific short interfering RNA and

antibody significantly inhibits the cell adhesion to laminin and the tumor growth in vitro and in vivo,

respectively. Interestingly, integrin a6 marks almost all of the surgical biopsy specimens of patients with

liposarcoma relapse. Moreover, integrin a6 is found to coexpress with CD13, which might contribute to the

antiapoptosis ability of integrin a6high cells. Consistently, integrin a6high cells are more sensitive to the CD13

inhibitor bestatin, and 61% of 23 other human tumor cell lines also contain integrin a6high CD13high subgroup.

These results provide evidence, for the first time, to our knowledge, that integrin a6 and CD13 can serve as

functionalmarkers of the tumor-initiation subcell population in human liposarcomaaswell as other cancers for

therapeutic targeting. Mol Cancer Ther; 10(12); 2276–86. �2011 AACR.

Introduction

Liposarcoma is the most common soft tissue sarcomaand accounts for at least 20% of all sarcomas in adults (1).The 2 major locations of liposarcoma are the extremitiesand the retroperitoneum (2). Surgery almost serves as thesole therapy for localized liposarcoma, and there are feweffective treatment options (3). For high-grade liposar-coma such as lipoblastic sarcoma, 100%of local recurrencewill be present within 5 years and half of them had adistant relapse. Moreover, liposarcoma is highly resistantto chemotherapy and radiotherapy. All these findingssuggest the existence of a subgroup of cells with highertumorigenicity in human liposarcoma.

Extracellular matrix (ECM) proteins are key structur-al components of the perivascular niche. The proteinsare known to play a role in the proliferation and migra-tion of normal stem cells and tumor cells (4) and in

liposarcoma progression (5). One of the functions ofECM in regulating cell behavior is through their recep-tors on the cell surface called heterodimer integrins,which consist of a and b subunits (6). Integrin a6, thereceptor for the ECM protein laminin, forms heterodi-mers with integrin b1 or b4. Integrin a6 is highlyexpressed in gastrointestinal stromal tumors (7), embry-onic, hematopoeitic, and neural stem cells (8). Recentstudies suggest that integrin a6 is important for thetumorigenicity of stem cell–like subpopulation withinbreast cancer cells (9). It is also known that integrin a6–positive cells are enriched in adult prostatic progenitor/stem cells (10). Justin D. Lathia and colleagues showedthat integrin a6 may serve as a functional marker ofhuman glioblastoma stem cells (11).

In this study, for the first time, we established a sub-cellline (SW872-S) derived from human liposarcoma cellsSW872 by repeated inoculation approach in nude mice,which showed high tumor initiation ability in vivo. TheSW872-S cells revealed higher adhesion ability to lamininbut not to fibronectin than SW872 cells did. This resultwasthen linked to the upregulated expression of integrina6 inSW872-S cells, which is rarely expressed in normal adi-pose-derived cells (12). Furthermore, integrin a6high cellsshowedmuch higher tumor formation inmice than integ-rin a6low cells did. Consistently, integrin a6–positive cellswere confirmed in tumor tissues of patients who sufferedfrom liposarcoma relapse within 2 years after surgery.Moreover, the cohigh expression of integrin a6 withaminopeptidase N (CD13) was observed and the func-tions were examined. The findings obtained here provide

Authors' Affiliations: 1State Key Laboratory of Pharmaceutical Biotech-nology, School of Life Sciences, Nanjing University; and 2Department ofClinical Oncology, The First Affiliated Hospital of Nanjing Medical Univer-sity, Nanjing, China

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Qiang Xu, State Key Laboratory of Pharmaceu-tical Biotechnology, School of Life Sciences, Nanjing University, 22 HanKou Road, Nanjing 210093, China. Phone: 86-25-8359-7620; Fax: 86-25-8359-7620; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0487

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 10(12) December 20112276

on June 19, 2018. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst October 6, 2011; DOI: 10.1158/1535-7163.MCT-11-0487

http://mct.aacrjournals.org/

a novel characteristic of the new sub-cell line of humanliposarcoma.

Materials and Methods

AnimalsSix-week-old NCR-nu/nu (nude) female mice were

purchased from the Shanghai Laboratory Animal Center.They weremaintained in pathogen-free condition at 22�C� 2�C and kept on a 12-hour light–dark cycle. Animalwelfare and experimental procedures were carried outstrictly in accordance with the Guide for the Care andUseof Laboratory Animals (The Ministry of Science andTechnology of China, 2006) and the related ethical regula-tions of our university. All efforts were made to minimizethe animals’ suffering and to reduce the number of ani-mals used.

Isolation of high tumorigenicity subcell populationby repeating inoculation approachSW872 cells at the logarithmic growth phase were

suspended in ice-cold PBS to a final density of 107/mLand injected into the right flank of nudemice with 100 mL.Six months later, neoplasm formed in 2 of 30 mice. Themice with tumor were then sacrificed, and the isolatedcells from the tumor tissue were cultured as methodmentioned above. These cellswere reinoculated into nudemice after in vitro culture for 5 generations.After 3 times ofthe repeated inoculation approach, the sub-cell line withhigh tumorigenicity in vivowasdesignedas SW872-S cells.

Cell cultureHuman liposarcoma cell line SW872 cells were pur-

chased from the American Type Culture Collection(ATCC). Cells were maintained in L-15 Medium supple-mented with 10% FBS (Life Technologies), 100 U/mLpenicillin, and 100 mg/mL streptomycin and incubatedat 37�C.Humanmelanoma cell lines SKMEL-1, A875, andA375; human liver cancer cell lines HepG2 and BEL-7402;humanbreast cancer cell linesMCF-7 andMDA-MB-435S;human ovarian cancer cell lines CAOV3 and SKOV3;human renal cell carcinoma cell line 786-O; human coloncancer cell lines Colo 205, HT29, SW620, and HUT-116;human fibrosarcoma cell line HT1080; human pancreaticcancer cell line PANC-1; human lung cancer cell lineA549; human cervical cancer cell line Hela; and humanprostate cell line PC3 were obtained from ATCC. Humanmelanoma cell line M14, human liver cancer cell lineSMMC7221, human renal cell carcinoma cell line OS-RC2, and human gallbladder carcinoma cell line GBC-SDwerepurchased from theCell InstituteCountryCell Bank,Institute of Biochemistry and Cell Biology, ChineseAcademy of Sciences, Shanghai, China. These cells weremaintained with Dulbecco’s Modified Eagle’s Medium(DMEM) containing 10% FBS, at 37�C with 5% CO2. Noindependent authentication of these cells was doneby the authors. Liposarcoma tissues from surgery (sup-plied by/obtained from The First Affiliated Hospital of

Nanjing Medical University) were washed extensivelywith ice-cold PBS to remove the debris and blood cells.Then, the tissues were treated with 0.15% collagenase(type I) containing 0.1% bovine serum albumin in PBSfor 30 minutes at 37�C with gentle agitation. The cellularpellet digested from tissues was then resuspended in lowglucose DMEM/10% FBS. Human sample investigationswere preceded by local institutional review boardapproval.

Cell adhesion assayThe cell adhesion assaywas conducted as before (13). In

brief, 96-well, flat-bottom culture plates were coated with50 mL of fibronectin (10 mg/mL), laminin (10 mg/mL;Calbiochem), or collagen type I (50 mg/mL; Sigma Chem-ical Co.) in PBS overnight at 4�C, respectively. Plates werethen blockedwith 0.2% bovine serum albumin for 2 hoursat room temperature followed by washing 3 times withDMEM. The cells were harvested with trypsin/EDTA,washed with ice-cold PBS twice, and resuspended inDMEM medium. Cells (2 � 104 per well) were added toeach well in triplicate and incubated for 30 minutes at37�C. Plates were then washed 3 times with DMEMmedium to remove unbounded cells. Cells remainingattached to the plates were quantified with MTT assay.

Real-time PCRQuantitative PCR was carried out with the ABI Prism

7000 sequence detection system (Applied Biosystems)with SYBR Green I dye (Biotium, Inc.), and thresholdcycle numbers were obtained by ABI PRISM 7000 SDSsoftware (version 1.0). Conditions for amplification were1 cycle of 94�C for 5 minutes followed by 35 cycles of94�C for 30 seconds, 59�C for 35 second, and 72�C for45 seconds. The primer sequences used in this studywereas follows: integrin a1: forward 50-GGTTCCTACTTTGG-CAG-30, reverse 50-AACCTTGTCTGATTGAGAG-30; inte-grin a2: forward 50-GGAACGGGACTTTCGCAT-30,reverse 50-GGTACTTCGGCTTTCT-30; integrin a3: for-ward 50-AAGGGACCTTCAGGTGCA-30, reverse 50-TG-TAGCCGGTGATTTACCAT-30; integrin a4: forward 50-GCTTCTCAGATCTGCTCGTG-30, reverse 50-GTCACT-TCCAACGAGGTT-30; integrin a5: forward 50-TGCAG-TGTGAGGCTGTGTACA-30, reverse 50-GTGGCCACC-TGACGCTCT-30; integrin a6: forward 50-TTGAATA-TACTGCTAACC-30, reverse 50-TCGAAACTGAACTC-TTGAGGA-30; integrin a7: forward 50-CTGTTTC-AGCTACATTGCA-30, reverse 50-GCCTGGTGCTTG-GGTT-30; integrin av: forward 50-AATCTTCCAATT-GAGGATA-30, reverse 50-AAAACAGCCAGTAGCAA-30; integrin a11: forward, 50-GGAGGAAGACTTGCG-30,reverse 50-CACAGGTTCCCCAGTA-30; CD9: forward50-ACTGTTCTTCGGCTT-30, reverse 50-AAAATCCCA-AAAATCT-30; CD10: forward 50-TGTGGCCAGATT-GATT-30, reverse, 50-TTGTAGGTTCGGCTGA-30; CD13:forward 50-CCCAAGATGTCCACGTA-30, reverse 50-GGTGCTGATGGCATTAACCT-30; CD44: forward 50-CAACTCCATCTGTGCAGCA-30, reverse 50-GTAACC-

Integrin a6 Marks Stem Cell–like Cells in Human Liposarcoma

www.aacrjournals.org Mol Cancer Ther; 10(12) December 2011 2277




TCCTGAAGTGCT-30; CD55: forward 50-AAGTAATC-TTTGGCTGTAAGG-30, reverse 50-TTCACCAGCATGT-TTTACCTTT-30; CD59: forward 50-CGTAGTCCGCTT-TCTCTTG-30, reverse 50-CTGCCAGAAGTCCTCTA-30;CD90: forward 50-CTAGTGGACCAGAGC-30, reverse50-GCCCTCACACTTGACCAG-30; CD166: forward 50-CCAAGAAGGAGGAGGAATA-30, reverse 50-CCAT-TCCTGTACCAT-30; b-actin: forward 50-GCTGTGCT-ACGTCGC-30, reverse 50-GGAGGAGCTGGAAGC-30.

RNA interferenceChemically synthesized 21-nucleotide sense and anti-

sense RNA oligonucleotides were obtained from Invitro-gen. Cells were plated on 6-well plates at 3� 105 cells perwell and transfected with 100 pmol of short interferingRNA (siRNA) duplex per well with Oligofectamine (Invi-trogen). CD13 siRNA sequences were as follows: 50-AAC-GAUCUCUUCAGCACAUCA-30. Luciferase siRNA wasused asdescribedbefore (14). Integrina6 specific SMART-pool siRNA reagent (Dharmacon) was used to knock-down endogenous integrin a6 in cells.

Immunohistochemistry and hematoxylin and eosinstaining assay

Immunostaining of integrin a6 was done with RealEnvision Detection Kit (Gene Tech Company) accordingto the manufacturer’s instructions. Hematoxylin andeosin (H&E) staining in tumor tissues was carried outfollowing manufacturer’s protocol.

Western blotWestern blot assay was conducted as described (14).

Antibodies to Src, phosphorylated Src(Tyr416), Akt, phos-phorylated Akt (Ser473), phosphorylated ERK, Bcl-2,cleaved caspase 3, cleaved PARP (Cell Signaling Technol-ogy); antibody to integrin a6, glyceraldehyde-3-phos-phate dehydrogenase, CD13 (Santa Cruz); antibody toFAK, phosphorylated FAK (Tyr397; BD Biosciences),respectively, were used for blotting, and detection wascarried out by enhanced chemiluminescence (AmershamPharmacia Biotech).

Colony formation in soft agarTwo hundred SW872 or SW872-S cells were embedded

in 0.3% Noble agar in DMEM containing 10% FBS. Thenthe clotted agarwas plated on a 0.6%agar base in the samemedium. Fourteen days later, the plates were photo-graphed using aNikon invertedmicroscope, and colonies(>50 cells) were counted.

Flow cytometry and cell sortingLaminin-specific integrins (integrin a1, a2, a3, a6, b1,

and b4) were measured with the Laminin-Specific Integ-rins Investigator Kit (ECM420; Millipore), isotype-matched mouse immunoglobulins served as control. Forflowcytometry, sampleswere analyzedbyaFACSCaliburflow cytometer and CellQuest software (BD Biosciences).For cell sorting by flow cytometry, sampleswere analyzed

and sorted on aBDFACSVantage SE (BDBiosciences). Forthe integrina6 high and lowpopulations, the stained cellswith the upper 30% or lower 30%of fluorescence intensitywere selected, respectively.

Statistical analysisData are expressed asmean� SD. The Student t testwas

used to evaluate thedifference between 2groups.Kaplan–Meier method was used to evaluate the survival test.P < 0.05 was considered to be significant.

Results

A new sub-cell line derived from human liposarcomacells SW872 is established and characterized withmuch stronger tumor-initiating potential in mice

SW872 cells (3� 105)were injected into the right flank ofnudemice (n¼ 30). Sixmonths later, themicewith tumors(2 in 30)were sacrificed, and the tumor cells collectedwerereinoculated into the naive nude mice. After 3 rounds ofselection as mentioned in Materials and Methods, weobtained a sub-cell line of SW872 cells (SW872-S) withhigh aggressive tumor formation potential in nude mice,whereas SW872 cells only showed a slight tumor growth(Fig. 1A). H&E staining revealed that the tumor tissuesformed by both cell lines were composed by lipoblastomacells (Fig. 1B), indicating that both cell lines belong to thesame classification. In soft agar, SW872-S cells formedbigger and more clones and possessed 5-fold colonyformation numbers (>50 cells) than SW872 cells (Fig.1C). As compared with no visible tumor in SW872 cell–inoculated nudemice,we observed tumor formation in allof the mice (n ¼ 8) injected with the same amount ofSW872-S cells (1� 105). The smaller amount of 1� 104 andeven as few as 1� 103 SW872-S cells formed tumor in 2 of8, and 1 of 8 nude mice, respectively (Fig. 1D).

SW872-S cells showaspecifically increased adhesionto laminin with an elevated expression of integrin a6

In order to detect the underlying mechanism of tumor-initiating potential between the 2 cell lines, we comparedthe adhesion ability of SW872 and SW872-S cells. SW872-Scells showed higher adherent ability to laminin but not tofibronectin or collagen (Fig. 2A), suggesting the involve-ment of laminin-specific integrins in the sub-line cells.Fluorescence-activated cell sorting (FACS) analyzingrevealed that among all the binding integrins to laminin,integrin a6 was the only high expressing one on cellsurface of SW872-S cells, whereas there was a decreaseof integrin a1 (Fig. 2B). Figure 2C shows that amongintegrin a family, integrin a6, the only higher expressionone in SW872-S cells, indicating integrin a6 may contrib-ute largely to the high adhesion ability on laminin ofSW872-S cells. Consistent with the result, blocking withintegrin a6–specific antibody (Clone: CoH3) significantlyinhibited the adhesion of SW872-S cells to laminin in adose-dependent manner (Fig. 2D).

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Integrin a6 contributes to the increased liposarcomagrowth in vitro and in vivoAs shown in Fig. 3A, SW872-S cells grew faster than

SW872 cells in vitro. To examine the relation of suchenhanced cell growth to the increased expression of integ-rin a6, we divided SW872-S cells into 2 compartmentsaccording to integrin a6 levels (low and high) by FACSsorting and used them for comparison. After 72-hourculture, the a6high cells proliferated 2 times as a6low cells(Fig. 3B) and knocking down of integrin a6 with itsspecific smartpool siRNA significantly decreased thegrowth of SW872-S cells (Fig. 3C). We then inoculatedthe right flankof nudemicewith 1� 106 SW872-S cells thatwere preincubated with control antibody or integrin a6monoclonal antibody for 30 minutes, respectively. Asshown in Fig. 3D, treating with integrin a6 antibodysignificantly inhibited the growth of SW872-S cells inmice. In this case, the tumor formation rate of SW872-Scellswas also reduced to 60%, as comparedwith the rate of100% of control.

Integrin a6 expression associates with humanliposarcoma relapsePatients with high-grade liposarcoma always suffered

fromhigh relapse frequencywithin 5 years. This fact leadsus to presume that there exists a subgroup of cells with

high tumorigenicity in human liposarcoma. To test thisassumption, we collected liposarcoma specimens from 30patients who suffered from recurrence within 2 years, 6samples of well-differentiated liposarcoma with goodprognosis, and 3 samples of human normal fat tissues.Weexamined the involvement of integrin a6 in human lipo-sarcoma recurrence by means of immunohistochemistry.As the result, 90% (27 in 30) of recurrent samples containedintegrin a6–positive cells and the representative picturesfrom dedifferentiated, pleomorphic, and round cell lipo-sarcoma, respectively, although we could not detectremarkable integrin a6–positive staining cells in well-dif-ferentiated liposarcoma samples as well as the normal fattissuesamples.Accordingly, FACSanalysis revealedabout6.9% of integrin a6–positive cells in human primary lipo-sarcoma whereas human normal adipose-derived cells(ADC) contained no integrin a6–positive cells (Fig. 4B).

Integrin a6high liposarcoma cells show highself-renewal and tumorigenicity with activatedFAK/Src/ERK signaling

When cultured in serum-free medium, SW872-S cellswith integrin a6 high expression still proliferate well andthe cell numberswere increased by 6.8 and 15.1 times after24 and 48 hours, respectively (Fig. 5A). The SW872-S cellsshowed an asymmetrical distribution of the integrin a6

Figure 1. Establishment andcharacterization of a new sub-cellline derived from humanliposarcoma cells SW872 withmuch stronger tumor-initiatingpotential in vivo. A, a total of 3� 106

of SW872 and SW872-S cells wereinjected into the right flank of nudemice.Sevenweeks later, thegrowthof tumor formed in mice. Data aremean � SD of 5 nude mice in eachgroup. B, tumor sample sectionsfrom each group were stained withH&E. C, 200 SW872 and SW872-Scells were seeded in soft agar asdescribed in Materials andMethods. Two weeks later, theclones formed were counted(bottom), and the representativepicture of clones are shown(top). Data are mean � SD of3 independent experiments.�, P < 0.05; ��, P < 0.01 versus thecolony number of SW872 cells.D, SW872 and SW872-S cellswere diluted from 106 to 103 andinjected into the right flank of nudemice and allowed to grow for2 months; the tumor formation rateis presented.






expression in a cell as well as in divided daughter cellsduring cell division (Fig. 5B, left). Further analysis wasconducted to test the self-renewal ability of integrin a6cells. Thea6high anda6low cells at 106were inoculated intothe right flank of nude mice and allowed to grow for 4weeks. Interestingly, a totally reduced integrin a6 expres-sion was observed in the tumor tissues from integrina6high cells inoculated mice by real-time PCR (Fig. 5B,middle) or FACS (right), and in this case, there was agreatly enhanced tumor growth in the mice. In soft agar,integrin a6high cells have stronger colony formation than

integrin a6low cells (Fig. 5C). When injected into nudemice, 5,000 a6high cells initiated tumors in 6 of 8 animals,while the same amount ofa6low cells only initiated in 2 of 8mice. Similarly, 500 a6high cells and a6low cells formedtumors in 2 of 4 and 0 mice, respectively (Fig. 5D). InSW872-S cells,we found increasedphosphorylation levelsof FAK (tyr925), Src (Tyr416), ERK, and Akt (ser473) aswell as upregulated expression of Bcl-2. Consistently, thesubpopulationwith integrina6high isolated fromSW872-Scells also showed stronger activation of FAK/Src/ERKpathway and expression of Bcl-2 (Fig. 5E).

Figure 2. Increased adhesion to laminin and elevated expression of integrin a6 in SW872-S cells. A, the ability of adhesion of SW872 and SW872-S cells tofibronectin, laminin, and collagen type I, respectively, was measured. B, the surface expressions of laminin-specific integrins of SW872 and SW872-S cellswere measured by FACS more than 3 times and representative result is presented. C, the mRNA levels of the integrin a family members betweenSW872 and SW872-S cells were qualified by real-time PCR. b-Actin was used as invariant control. D, SW872-S cells were incubated with various amounts ofintegrin a6 monoclonal antibody for 30 minutes, and the adhesion ability to laminin, fibronectin, and collagen was measured. Data are mean � SD of3 independent experiments. �, P < 0.05; ��, P < 0.01 versus control.

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Integrin a6high liposarcoma cells express high levelsof aminopeptidase N (CD13)For liposarcoma is a malignant tumor that arises in fat

cells, we measured several markers that have beenreported to express on the surface of human ADCs(12) by real-time PCR. The results revealed that therewere no significant changes in the expressions of CD44,CD55, and CD59 between SW872 and SW872-S cells.However, the expressions of CD9, CD10, and CD13were significantly elevated in SW872-S cells whereasthose of CD90 and CD166 were decreased in the cells(Fig. 6A). We further identified that in SW872-S cells, thea6high group displayed 3 times CD13 expression asa6low group, whereas there were no significant or justslender changes in the other markers (Fig. 6B). The

cohigh expression of integrin a6 and CD13 was foundin SW872-S cells about 12 times than in SW872 cells (Fig.6C, top). The high expression of CD13 could also beobserved in integrin a6high cells (Fig. 6C, bottom). Inaddition, we found that in 23 other human cancer celllines, 61% (Supplementary Fig. S1) of which containedintegrin a6 and CD13 double–positive subgroups (Sup-plementary Fig. S2). The integrin a6high CD13high cellsshowed the highest level of phosphorylated Src aswell as the expression of Bcl-2 as compared witha6howCD13low, a6highCD13low, and a6lowCD13high cells(Fig. 6D). Knocking down of CD13 expression inducedapoptosis in SW872-S cells (Fig. 6E, top) as well as insome other malignant cancer cells (Supplementary Fig.S3). CD13-specific inhibitor bestatin also induced

Figure 3. Identification of integrina6high cells in liposarcoma cells'growth in vitro and in vivo. A, theproliferation of SW872 and SW872-S cells was determined by MTTassay. B, the proliferation of integrina6high and integrin a6low cells ofSW872-S was determined by MTTassay. C, SW872-S cells weretransfectedwith various amounts ofintegrin a6 smartpool siRNA orluciferase siRNA for 72 hours,respectively. The surfaceexpression of integrin a6 wasmeasured by FACS (left). Theproliferation rate of cellstransfected by integrin a6 siRNA orluciferase siRNA was determinedby MTT assay (right). D, a total of106 SW872-S cells preincubatedwith control antibodyor integrina6–specific blocking antibody (clone:CoH3)were inoculated into the rightflank of nude mice, respectively.Time course of tumor growth (left),representative pictures (right) werepresented. Data are mean � SDof 3 independent experiments.�, P < 0.05; ��, P < 0.01 versuscontrol.






apoptosis in SW872-S cells in dose-dependent manner(Fig. 6E, bottom). The integrin a6high cells were moresensitive to the bestatin treatment than integrin a6low

cells, in which the proliferation of both cells were dosedependently inhibited (Fig. 6F). Consistent with the invitro experiment, 20 mg/kg/d treatment of bestatincould also inhibit the SW872-S cells growth in nudemice (Fig. 6G).

Discussion

Patients with liposarcoma always suffered from highrisk of relapse, and 100% of local recurrence will bepresent within 5 years such as lipoblastic liposarcoma

despite of thorough surgery treatment (15). This fact letsus to think if there is a subgroup of cells with hightumorigenicity in human liposarcoma tissues.

In this study, we tried to generate the possibly existingsubpopulation by an in vivo repeated inoculationapproach with a small amount of cells as mentioned inMaterials and Methods. After 9-month selection, a sub-cell line (SW872-S)wasobtained fromhuman liposarcomaSW872 cells,which have beenpreviously shownunable ordifficult to initiate tumors in nudemice (16). The SW872-Scells obtained revealed extremely high tumorigenicity inthe nude mice. The elevated tumorigenicity of SW872-Scells was also exhibited in the assays of colony formation(Fig. 1C), and specific cell adhesion to laminin but not to

Figure 4. Integrina6–positive cells inhuman tumor tissues withliposarcoma relapse.Immunohistochemical stainingassays were conducted for integrina6 with human liposarcomaspecimens. Paraffin section ofhuman liposarcoma tissues werecollected from 30 patients who hadundergone liposarcoma relapsewithin 2 years; 6 samples frompatients underwent well-differentiated liposarcoma withgood prognosis; 3 samples ofnormal fat tissue. A, therepresentative pictures fromdedifferentiated, pleomorphic,round cell, well-differentiatedliposarcoma, and normal fat tissue,respectively, were shown. B, FACSanalysis between human normalADC cells and human primaryliposarcoma cells isolated fromtumor tissues with relapse.

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fibronectin or collagen type I (Fig. 2A). This result suggestsan involvement of laminin-specific integrins in the func-tions of the sub-line cells. In order to prove this assump-tion, we compared the expressions of various integrinsassociatedwith lamininadhesionon the surfaceof SW872-S cells with SW872 cells and found that integrina6 but notall the other integrins was elevated. The decrease ofintegrin a1 in SW872-S cells seemed to be in less relation-ship with cell adhesion ability on laminin, at least in oursystem, for integrin a1 antibody could not inhibit neitherSW872 cells nor SW872-S cells adhesion on laminin (data

not shown). Furthermore, blocking with integrin a6–spe-cific antibody (Clone CoH3) significantly inhibited theadhesion of SW872-S cells to laminin in vitro (Fig. 2D) andthe tumor growth in vivo (40.77% of inhibition rate; Fig.3D). These findings strongly suggest that the high tumor-igenicity of the subcell population could be linked tointegrin a6 at least as one of the important factors.

In previous studies, integrin a6 has been reported assubunit form heterodimers with either b1 or b4 integrins,which both function as receptors for the laminin family ofECM. Activated a6b1 or a6b4 components then bring

Figure 5. Multiple tumor formationpotential in integrin a6high cells withactivated FAK/Src/ERK signaling.A, SW872-S cells were cultured inserum-free medium for 24 and 48hours. Integrin a6 level wasmeasured by FACS, and the foldinduction of integrin a6high

subgroup is shown. B, single-cellsuspension of SW872-S cells wasseeded at the concentration of1,000/mL, the surface expressionof integrin a6 was analyzed byimmunofluorescence (left). Integrina6high and integrin a6low cells(106 cells per mouse) were s.c.inoculated into the flank of nudemice, respectively. Four weekslater, the tumor xenografts werecollected and integrin a6expression was detected by real-time PCR (middle) and FACS (right),respectively, for comparison withthe tumor size. C, the colonyformation ability of integrin a6high

and integrina6low cells divided fromSW872-S was measured. D,integrin a6high and integrin a6low

cells divided from SW872-S cellswere diluted from 50,000 to 500,respectively, and injected into theright flank of nudemice and allowedto grow for 2 months. The tumorformation rate (top) andrepresentative pictures of tumorformed (bottom) are presented. E,the protein levels of total integrina6, FAK, Src, ERK, Akt, Bcl-2 aswell as phosphorylated FAK, Src,ERK, Akt of SW872, SW872-S,integrin a6high, and integrin a6low ofSW872-S cells were determined byWestern blot. FITC, fluoresceinisothiocyanate.






extracellular signal to active PI3K/Akt pathway or hyp-oxia-inducible factor-dependent pathway, respectively(17). High expression of the a6 subunit in women withbreast cancerhas been shown tobe correlated significantlywith reduced survival times (9). Recently, integrin a6 hasbeen suggested as a potential marker of cancer stem cells(CSC) in humanglioblastoma (11). In addition, integrina6is critical to the self-renewal and tumor formation of theglioblastoma stem cells, and knocking down or blockingwith a6-specific antibody could significantly inhibit glio-blastoma growth in vitro and in vivo (11). This report is inconsistence with our data shown in Fig. 2, suggesting that

integrin a6 may be crucial for initiating tumor growth insome kinds of cancers.

As reported by D-S. Kim, and colleagues, rapid adher-ing cells of human epidermal keratinocytes with highlevels of integrin a6 show a smaller size, which have highability of colony formation and skin equivalents recon-struction (18). In our study, we noticed that in SW872-Scells, integrin a6high subgroup could grow in serum-freemedium at least more than 72 hours (Fig. 5A), and the sizeof proliferated integrin a6high cells were also smaller thanthe others according to FACS analysis (Fig. 5A). Theunderlying mechanism, however, remains unknown.

Figure 6. Coexpression ofaminopeptidase N (CD13) inintegrin a6high cells and increasedsensitivity to CD13 inhibitorbestatin. A, the mRNA levels ofCD9, CD10, CD13, CD44, CD55,CD59, CD90, and CD166 betweenSW872 and SW872-S cells. B, themRNA levels of the above markersbetween SW872-S-integrin a6high

and SW872-S-integrin a6low cells.C, flow cytometric analysis forexpressions of CD13 and integrina6 in SW872 and SW872-S cells(top). The protein level of CD13 wasalso determined in a6low and a6high

cells (bottom). D, SW872-S cellswere sorted into 4 groups: a6low

CD13low, a6low CD13high, a6high

CD13high, and a6high CD13low; theprotein levels of phosphorylatedSrc, Src, and Bcl-2 weredetermined by Western blot. E,SW872-S cells were transfectedwith control siRNA and CD13-specific siRNA for 72 hours. Theprotein levels of CD13, cleavedPARP, and cleaved caspase 3 weredetermined by Western blot (top).SW872-S cells were incubated withvarious amount of bestatin for24 hours, the protein level of integrina6, cleaved PARP, caspase 9,cleavedcaspase3weredeterminedby Western blot (bottom). F, the 72-hour inhibition rates of bestatin onSW872-S-integrin a6high andSW872-S-integrin a6low cells weredetermined by MTT assay. G, themice with tumor formed of SW872-S cells were treated with PBS or 20mg/kgbestatin daily. The tumor sizewasmeasured everyweek. Data aremean � SEM of 5 mice in eachgroup. �, P < 0.05; ��, P < 0.01versus control. FITC, fluoresceinisothiocyanate.

Wang et al.





Nevertheless, we have evidenced that integrin a6 wasdistributed asymmetrically on the surface of a cell and theexpression level was quite different in divided daughtercells during cell division. This finding suggests that integ-rina6high cells could differentiate into both integrin a6high

and a6low cells. In fact, the integrin a6high cells showedmuch higher proliferation in mice than a6low cells. Per-haps due to the dilution of increased a6low cells after 4-week growth inmice, the total content of integrina6 in thetumor tissues was reduced (Fig. 5B). Although cancerstem cells are believed to have stem cell characteristics(19), such as tumor initiation, proliferation (20), and self-renewal (21), which may lead to metastasis and relapseafter therapy (22), it still lacks the gold standard definitionof cancer stem cells in all types of cancers. Our resultshighlight the involvement of integrina6 in the self-renew-al capacity, colony formation ability, and tumor initiationin vivo of liposarcoma cells. All these results revealed thetumor initiation function of integrina6high subgroup cells,at least in human liposarcoma.It should be noted that in some experiments mentioned

above,wedidnot observe the complete blockade of tumorproliferation or growth by silencing integrin a6 or usingits specific monoclonal antibody. This could be partiallyexplained by incomplete silence due to technique restric-tion. We can also assume that it might be because ofanother factor involved in integrin a6-mediated tumorinitiation upon these data. As the evidence for this, Fig. 6Band C showed that integrin a6 cohighly expressed withCD13, which shows a variety of functions of one memberof this family, aminopeptidase N (23).CD13 has been proved to be a transcript target of Ras

signaling pathways (24), playing an important role intumor progression in ovarian carcinoma cells (25). It wasalso reported that CD13 could block the apoptosis induc-tion by tumor necrosis factor-alpha (26) and enhancesensitivity of tumor cells to cisplatin (27). CD13 was alsoproved to be involved in cell adhesion (28) as well as inendothelial invasion (29). Recent study made by Haragu-chi and colleagues proved that CD13may be a therapeutictarget in human liver cancer stem cells and inhibition of

CD13bymonoclonal antibody could induce cell apoptosis(30). In this study, knocking down of CD13 with itsspecific siRNA induced apoptosis in SW872-S cells (Fig.6E), and small-molecule inhibitor ofCD13 such as bestatinshowed higher inhibition rate on SW872-S-integrin a6high

cells (Fig. 6F). All these results suggest that CD13 may bean essential cooperator in integrin a6high subgroup ofcells, which defends cells from apoptosis induction. Inorder to confirm the implication of these 2 proteins simul-taneously appearing in the cells, we measured the coex-pression of both proteins in 23 other human cancer celllines. As the result, we found that 61% (14 in 23) of themcontained CD13 and integrin a6 double-positive sub-group cells (Supplementary Fig. S1). Knocking down ofCD13 inducedapoptosis inA375,A549, andBel-7,402 cellswith CD13 and integrin a6 cohigh expression (Supple-mentary Fig. S3). These results indicate a possibility thatboth integrin a6 and CD13 may serve as functional mar-kers of not only liposarcoma but also various humancancers and sarcomas, which may be new therapeutictargets for the clinical cancer treatment.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors sincerely consecrate this article to Prof. Ting Chenwho hadbeen our colleague and had struggled with retroperitoneum liposarcomafor 15 years, and who is also the donor of some of the samples used in thisstudy.

Grant Support

This study was supported by National Natural Science Foundation ofChina (Nos. 30730107 and 90913023), Science Fund for Creative ResearchGroups (No. 30821006), and Natural Science Foundation of Jiangsu Prov-ince (BK2008022).

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 July 7, 2011; revised September 7, 2011; accepted September 20,2011; published OnlineFirst October 6, 2011.

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Wang et al.





2011;10:2276-2286. Published OnlineFirst October 6, 2011.Mol Cancer Ther Lu Wang, Lingxian Wang, Yanhong Gu, et al. Tumorigenesis and Relapse of Human Liposarcoma

Cell Population Functions as an Initiator inhigh6αIntegrin

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