Integrin avb6 Promotes an Osteolytic Program in Cancer ... · Cell adhesion and migration assays...

Tumor and Stem Cell Biology

Integrin avb6 Promotes an Osteolytic Program in CancerCells by Upregulating MMP2

AninditaDutta1,2, JingLi1,4,HuiminLu1,2, JacquelineAkech1,4, JiteshPratap1,4, TaoWang5,BradJ. Zerlanko1,2,Thomas J. FitzGerald5, Zhong Jiang6, Ruth Birbe1,3, John Wixted1,7, Shelia M. Violette8, Janet L. Stein1,9,Gary S. Stein1,9, Jane B. Lian1,9, and Lucia R. Languino1,2

AbstractThe molecular circuitries controlling osseous prostate metastasis are known to depend on the activity of

multiple pathways, including integrin signaling. Here, we demonstrate that the avb6 integrin is upregulated inhuman prostate cancer bonemetastasis. In prostate cancer cells, this integrin is a functionally active receptor forfibronectin and latency-associated peptide-TGF-b1; it mediates attachment and migration upon ligand bindingand is localized in focal contacts. Given the propensity of prostate cancer cells to formbonemetastatic lesions, weinvestigated whether the avb6 integrin promotes this type of metastasis. We show for the first time that avb6selectively induces matrix metalloproteinase 2 (MMP2) in vitro in multiple prostate cancer cells and promotesosteolysis in vivo in an immunodeficientmousemodel of bonemetastasis through upregulation ofMMP2, but notMMP9. The effect of avb6 onMMP2 expression and activity is independent of androgen receptor in the analyzedprostate cancer cells. Increased levels of parathyroid hormone–related protein (PTHrP), known to induceosteoclastogenesis, were also observed in avb6-expressing cells. However, by using MMP2 short hairpin RNA, wedemonstrate that theavb6 effect on bone loss is due to upregulation of solubleMMP2 by the cancer cells, not dueto changes in tumor growth rate. Another related av-containing integrin, avb5, fails to show similar responses,underscoring the significance of avb6 activity. Overall, these mechanistic studies establish that expression of asingle integrin, avb6, contributes to the cancer cell—mediated program of osteolysis by inducing matrixdegradation through MMP2. Our results open new prospects for molecular therapy for metastatic bone disease.Cancer Res; 74(5); 1598–608. �2014 AACR.

IntroductionMore than 80% of patients with prostate cancer, at autop-

sy, have metastatic foci in the bone that constitute animportant negative prognostic factor for end-stage malig-nancy (1). When present in the bone, metastatic prostatecancer cells produce osteolytic (2, 3), in addition to the well-characterized osteoblastic, lesions (4). Both types of histo-pathology often occur in the same bone area, but the

molecular underpinnings of such mixed lesion formationand the effector molecules participating in this dynamicprocess are still largely elusive. Prostate cancer osteolyticmetastases cause rapid disease progression as rapid degra-dation of bone by osteoclasts provides space for the tumorcells to grow (5). In contrast, the osteoblastic nature of bonemetastases contributes to a slower progress of the disease ascompared with osteolytic metastases, as the initial increasein bone volume could limit the space available to cancercells, and therefore help to confine tumor growth.

Likely, mediators/activators of this osteolytic pathwayinclude members of the integrin family of cell surfacereceptors and their extracellular matrix (ECM) ligandstogether with the backdrop of a complex bone microenvi-ronment affected by a plethora of regulatory cytokines(3, 6, 7). Integrins are transmembrane receptors that com-prise an a- and b-subunit and are known to be deregulatedas prostate cancer progresses to advanced stages (8). In thiscontext, there is now compelling evidence that signalsoriginating from integrin ligand binding orchestrate keymechanisms of tumor progression, including cell survival,adhesion, proliferation, gene expression, and modulation ofmigratory/invasive phenotypes (8). These properties areexploited in prostate cancer especially as it progresses toan advanced disease status (8). Although expression ofintegrins in human prostate cancer bone metastasis has

Authors' Affiliations: 1Prostate Cancer Discovery and DevelopmentProgram; Departments of 2Cancer Biology and 3Pathology, Thomas Jef-ferson University, Philadelphia, Pennsylvania; Department of 4Cell Biology,5Radiation Oncology, 6Pathology, and 7Orthopedics, University of Mas-sachusetts Medical School, Worcester; 8Biogen Idec, Inc., Cambridge,Massachusetts; and 9Department of Biochemistry, The University of Ver-mont, Burlington, Vermont

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

A. Dutta and J. Li share first co-authorship of this article.

Current address for J. Pratap: Department of Anatomy and Cell Biology,Rush University Medical Center, Chicago, Illinois.

Corresponding Author: Lucia R. Languino, Thomas Jefferson University,233 S. 10th Street, BLSB 506, Philadelphia, PA 19107. Phone: 215-503-3442; Fax: 215-503-1307; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-1796

�2014 American Association for Cancer Research.

CancerResearch

Cancer Res; 74(5) March 1, 20141598

on February 6, 2021. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst January 2, 2014; DOI: 10.1158/0008-5472.CAN-13-1796

http://cancerres.aacrjournals.org/

never been shown, a causal role for integrins in this type oflesion has been reported: avb1, avb3, avb5, or a2b1 havebeen shown to promote tumor growth in bone (9–11), anda6b1 (12) as well as avb3 (11) have been demonstrated tocontribute respectively to osteolytic and osteoblastic lesions.Overexpression of avb6 integrin has been reported to pro-

mote the metastatic potential of HT29 colon cancer cells (13).avb6, an epithelial-specific integrin, is an ideal target, yet to bevalidated, for therapeutic intervention in metastatic disease(14), as this molecule is largely undetectable in most normaltissues but abundantly expressed in primarymalignancies (15–18). Furthermore, this integrin has been shown to bind latency-associated peptide (LAP)-TGF-b1 and in terms of signalingresponses, facilitates the release of active TGF-b1, which is aprometastatic cytokine (17).Our data show that the avb6 integrin promotes matrix

metalloproteinase (MMP) 2 and parathyroid hormone–relatedprotein (PTHrP) upregulation and demonstrate the interplaybetween integrin expression and bone remodeling mechan-isms in intraosseous metastatic prostate cancer. We haveestablished that expression of a single integrin, avb6, is suf-ficient to execute a cancer cell–mediated program of osteolysiscentered on upregulation of MMP2, as well as of PTHrP, andconsequent increased MMP2 catalytic activity that completesthe final matrix degradation stage of osteolytic bone disease.

Materials and MethodsReagents and antibodiesBovine serum albumin (BSA) was from Sigma-Aldrich, type I

collagen and 40,6-diamidino-2-phenylindole (DAPI) were fromInvitrogen; LAP-TGF-b1 was from R&D Systems.We used the following rabbit antibodies against: extracel-

lular signal—regulated kinase 1/2 (ERK1/2) and AKT fromSanta Cruz Biotechnology; avb6 (a gift of Dr. V. Quaranta,Vanderbilt University, Nashville, TN) for immunohistochem-istry; b3 and b5 cytoplasmic domains from Millipore forimmunoblotting; MMP2 from Millipore for immunoblottingusing tumor lysates or from Cell Signaling Technology forimmunoblotting using cell lysates and immunohistochemistry.We used the followingmousemonoclonal antibodies (mAb):

2A1 to b6 for immunoblotting (16, 19) and immunofluores-cence; LFMb-14 to osteopontin (OPN) from Santa Cruz Bio-technology; clones AE1 and AE3 to cytokeratin fromMillipore.IC10, irrelevant mAb and rabbit immunoglobulin G (IgG; fromSigma) were used as negative controls.Rabbit anti-mouse Alexa Fluor 488 secondary antibody for

immunofluorescence was from Invitrogen.

Human tissue specimensFourteen human bone biopsies and one additional speci-

men, dissected from a bone metastasis, of patients withosteolytic prostate cancer were obtained from the Departmentof Pathology, University of Massachusetts Medical School(Worcester, MA) or Cooperative Human Tissue Network(CHTN, other investigators may have received specimens fromthese same tissues) and were processed according to Institu-tion-approved protocols. Thirteen human bone biopsies andthree specimens, dissected from prostate cancer bone metas-

tases (all were osteolytic), were obtained from the Departmentof Pathology, Thomas Jefferson University (Philadelphia, PA).Two human osteolytic prostate cancer bone biopsies wereobtained from CHTN. The specimens were formalin-fixed andparaffin-embedded.

ImmunohistochemistryImmunohistochemical staining was performed as described

previously (20–22).

Cells and culture conditionsAuthentication of the cell lines was provided with their

purchase fromUroCore Inc. (C4-2B) or American Type CultureCollection (PC3 and RWPE); the cells were used in our labo-ratory for less than 6 months. Two PC3 sublines, PC3-1 andPC3-2, previously designated PC3-H and PC3-L, respectively(23), were used; PC3-1 (PC3-H) are positive for b6 expressionand PC3-2 (PC3-L) are negative for b6 expression (23). BPH1cells were provided by Dr. Simon W. Hayward (VanderbiltUniversity) and authenticated as previously described (24). C4-2B and BPH1 cells were maintained in RPMI supplementedwith 5% FBS, 2 mmol/L glutamine, 100 mg/mL streptomycin,and 100 U/mL penicillin.

Viral constructs and cell transfectionConstructs and transfection of PC3-1, PC3-2, and C4-2B cells

were performed as detailed in the Supplementary Methods.

Immunofluorescence and confocal microscopyImmunofluorescence analysis on PC3-1 cells was performed

as described previously (25). To perform immunofluorescenceon tissue sections, antigen retrieval was performed on rehy-drated formalin-fixed paraffin-embedded sections fromhuman prostate cancer osteolytic samples by incubation inpepsin for 20 minutes at 37�C. The sections were blocked withPBS/5%BSA.avb6 Stainingwas performed incubating sampleswith 2A1 antibody for 1 hour, followed by incubation withAlexa Fluor 488-rabbit anti-mouse antibody for 30 minutes.Slides were analyzed on an inverted confocal microscope(LSM510; Carl Zeiss). DAPI was used for nuclear staining.

Cell adhesion and migration assaysCell adhesion and migration Assays were performed as

described previously (25). Chi-square test was used for statis-tical analysis.

Flow cytometryFluorescence-activated cell sorting (FACS) analysis was

performed to determine integrin expression by using mAbs:L230 to av, PIH5 to a2, GoH3 to a6, TS2/16 to b1, AP3 to b3,P1F6 to b5, or 10D5 to b6. IC10 and nonimmune mouse IgGwere used as negative controls.

ImmunoblottingPC3-1, PC3-2, and C4-2B cell lysates were prepared, sepa-

rated by SDS-PAGE gel, and analyzed by immunoblotting asdescribed before (26). Frozen tumor tissues collected frombone injection sites were homogenized and analyzed by immu-noblotting, as described previously (20).

avb6 Integrin–Mediated Osteolysis

www.aacrjournals.org Cancer Res; 74(5) March 1, 2014 1599




Gelatin zymographySerum-free culture media collected from Parental, Mock,

avb6- or avb3-expressing C4-2B cells, and from avb6-PC3-2cells stably transfected with shMMP2 or shTROP2 were ana-lyzed by gelatin zymography as described before. Serum-freeconditionedmedium fromBPH1was used as control forMMP2and MMP9 (27).

Intratibial injectionAnimal studies were conducted in accordance with

approved Institutional Animal Care and Use Committee pro-tocols and the NIH Guide for the Care and Use of LaboratoryAnimals. Tibia intramedullary injections of Mock-PC3-2 cellsor avb5- or avb6-PC3-2 stable transfectants were carried outon isofluorane-anesthetized 4- to 6-week-old male severecombined immunodeficient (SCID) mice (The Jackson Labo-ratory) by using described techniques (28). Briefly, mice wereanesthetized with 0.15 mg ketamine/0.015 mg xylazine intra-peritoneally (i.p.) per gram body weight. A medial parapatellarincision was created and a needle was placed in the intrame-dullary canal of the tibia, by aid of fluoroscopy (XiScan 1000-1;XiTec). Tumor cells (1 � 105 in 100 mL of PBS) were slowlyinjected into the tibia and the incision was closed with 5-0chromic suture (Ethicon Inc.). Mice were given 0.1 mg/kgbuprenorphine subcutaneously postoperatively. The forma-tion of osteolytic lesions in bones was assessed by radiographyusing a Faxitron MX-20 (Faxitron X-ray). Bone radiographswere collected on X-Omat TL film (Kodak) using an exposureof 25 kV for 60 seconds. Lytic areas from epiphysis tometaphysis were quantitated by using ImageJ software, a

public domain Java image processing program inspired byNIH Image. Triplicate values were measured and theaverages � SD are shown.

Micro-computed tomography and bone histologyA detailed analysis of micro-computed tomography (mCT) is

described in the Supplementary Methods.Bones were dissected for fixation in 4% paraformaldehyde

for 24 hours and either demineralized in 18% EDTA for paraffinembedding or embedded in methyl methacrylate for exami-nation of sections of mineralized tissues. Bone sections werestained with hematoxylin and eosin (H&E). The size of avb6-and avb5-PC3-2 bone tumors, isolated 8 weeks after injection,was evaluated visually by Drs. Languino, Lian, and Li using aZeiss Axioskop 40 and AxioCam HRC camera. The softwareused was AxioVision Rel. 4.7.

Quantitative real-time PCR analysisRNA isolated from tumors using TRizol reagent was per-

formed as previously described (29). The sequences for theprimers used for human mRNA transcripts for PCR amplifi-cation are detailed in the Supplementary Methods.

Statistical analysisStatistical significance between datasets was calculated

using t test and all graphs were generated using MicrosoftExcel. The error bars were calculated and represented in termsof mean � SD. A two-sided P value of less than 0.05 wasconsidered statistically significant.

Figure 1. avb6 is expressed in human prostate cancer bone metastases. A, prostate cancer osteolytic metastases were stained for avb6 byimmunofluorescence. Thepanels show two representative imagesofavb6 immunostaining in prostate cancer bone (B)metastases.DAPIwasused for nuclearstaining.Magnification,�40. B, expression ofavb6 in prostate bone (B)metastaseswas analyzed by immunohistochemistry using an antibody specific for b6(left panel). Serial sections were stained using antibodies to IgG (middle) or cytokeratins (CK; right). Two representative examples are shown.

Dutta et al.

Cancer Res; 74(5) March 1, 2014 Cancer Research1600




Resultsavb6 integrin is expressed in human prostate cancerbone metastases and is functionally active in humanprostate cancer cellsA detailed analysis of avb6 integrin expression in human

prostate cancer osteolytic metastases shows that this receptoris detected in 22 of 23 specimens. Representative images ofavb6 integrin staining performed by immunofluorescence orby immunohistochemistry are shown in Fig. 1A and B, respec-tively, using an antibody specific for avb6. In contrast, acontrol nonbinding IgG is unreactive (Fig. 1). Consistent withthe epithelial origin of metastatic tumor cells, all specimens inthis series immunostain positive for cytokeratin expression(Fig. 1B).

Given the above observation, we investigated the functionalstatus of theavb6 integrin in humanprostate cancer cells uponligand binding. Our immunofluorescence data show thatintegrin avb6 localizes to focal contacts when PC3-1 cellswere allowed to attach to fibronectin (Fig. 2A); the a partnerof the b6 integrin subunit, av, was used as a marker forfocal contacts. Furthermore, we show that avb6 promotesligand-dependent prostate cancer cell adhesion and migrationof PC3-1 cells and that cells transfected with b6-shRNA hadreduced activities, whereas b5-shRNA transfectants were notaffected (Fig. 2B). The cells were seeded on BSA-, type Icollagen-, or LAP-TGF-b1–coated Transwell plates. Parentalor shb5 PC3-1 cells attach and consequently migrate on LAP-TGF-b1 to a significantly higher extent than shb6 PC3-1 cells.

Figure 2. avb6 expressionpromotes adhesion andconsequently migration. A,expression of b6 and av wasobserved at the focal contacts inPC3-1humanprostate cancer cells(magnification,�63 ). IgGwasusedas a negative control(magnification, �40). B, adhesionand migration assays wereperformed using Parental, shb5, orshb6 PC3-1 cells seeded on BSA,type I collagen, or LAP-TGF-b1–coated Transwell chambers,respectively. The experiment wasrepeated at least three times, andsimilar results were observed. Theresults from a representativeexperiment are shown in the bargraph. Error bars, SEM. Thedifferences between adhesion andmigration, of shb5 and shb6 cellson LAP-TGF-b1 are statisticallysignificant. �, P ¼ 0.004 and��, P ¼ 0.002. C, FACS analysis ofdifferent av-associated b-subunits(b1, b3, b5, and b6) in PC3-1 cells isshown. IgGwas used as a negativecontrol antibody (Ab). D, Parental,shb5, or shb6 PC3-1 cells wereserum starved for 24 hours and celllysates were analyzed by SDS-PAGE and probedwith an antibodyto b5 or 2A1 antibody to b6. ERKwas used as a loading control.






On the other hand, Parental, shb6 PC3-1, and shb5 PC3-1 cellsmigrated equally well on type I collagen (Fig. 2B). Character-ization of the cells used in these experiments was performed byusing FACS and immunoblotting analysis. Parental cellsexpress high levels of b6 and b1, moderate levels of b5, anda negligible amount of b3 (Fig. 2C). Immunoblotting analysisconfirms successful downregulation of the b6 and b5 subunitsin PC3-1 transfectants (Fig. 2D). Overall, these results indicatethatavb6 is functionally active in human prostate cancer cells.

avb6 integrin promotes early onset of osteolytic lesionsTo elucidate a potential causal role for avb6 in promoting

metastatic bone disease, we used PC3-2 cells stably transfectedwith b6 or control vector (Mock). To prove the specificity of theeffect of b6, we selected clones ofavb6 stably transfected PC3-2cells, which did not show changes in expression of otherintegrins, such as av, a2, a6, b1, b3, or b5 molecules, asevaluated by FACS analysis (Supplementary Fig. S1). Injectionof PC3-2 transfectants expressing avb6 into the tibial medullarcavity of immunocompromised mice gives rise to extensive

osteolysis, with complete loss of trabecular bone at the site ofinoculation followed by erosion of the cortical bone, resulting intumor invasion into the surrounding muscle (SupplementaryFig. S2A). In contrast, intratibial injection of control transfec-tants (Mock) is associated with only mild osteolysis and char-acterized by irregular patterns of woven bone tissue, suggestingthat bothmixed osteolytic and osteoblastic lesions occur underthese conditions. The extent of osteolytic lesions was quantifiedfrom radiograph images of darkened areas of individual bonelesions, representing the absence of mineralized bone (Supple-mentary Fig. S2B). This analysis confirmed that avb6-expres-sing cells significantly increase and sustain the osteolytic dis-ease compared to Mock cells. To further validate that b6 isdriving the osteolytic disease, lysates were prepared from thetumors excised from the limbs from each group (b6 and mock);the results show that osteolysis correlates with high b6 proteinlevels (Supplementary Fig. S2C), whereas there is no differencein the expression of a2, a6, or b3 integrin subunits (data notshown). In these experiments, tumor cell expression of avb6seems to be associated with increased osteolysis and decreased

Figure 3. Specific effect of avb6on osteolytic lesions. A,representative mCT images of bonelesions caused by avb6-PC3-2(top) and avb5-PC3-2 cells(bottom) at 2-, 4-, and 8-week timepoints after intratibial injection.Two representative bones fromn ¼ 8 are shown. B, quantificationof mCT images for net bone loss inthe avb6-PC3-2 and avb5-PC3-2bone tumors at 2, 4, and 8 weeks isshown. Quantitation of bonevolume from three-dimensionalimages at a threshold range 220 to1,000 was performed. �, P < 0.004.

Dutta et al.





formation ofwovenbone, as comparedwithMock transfectants(Supplementary Fig. S2D). Histologic analysis of limb lesionsshows extensive loss of trabecular and cortical bone in tumorspecimens generated by avb6 integrin or Mock transfectants(Supplementary Fig. S2D). However, Mock transfectants exhibitmore new woven bone formation. These osteoblastic lesionsinitiate within the tumor mass at the margins of tumor (T) andcortical bone (CB; NB; Supplementary Fig. S2D). Because somewoven bone is observed in the avb6 group, this suggestscompetency for osteoblastic lesions; however, avb6 may beinducing osteolyticmechanisms that readily degrade thewovenbone. Overall, these data show that extensive loss of trabecularand cortical bone in tumor specimens is generated by avb6integrin-expressing cells.

The effect of avb6 integrin on osteolysis is specificTo determine whether the integrin-mediated causal role in

osteolysis is specific for avb6, we injected PC3-2 cells stablytransfected with a related integrin avb5 (30) in mouse tibiae,and characterized the associated tumor lesions by quantitativemCT (Fig. 3A).Tumors generated in the avb6 group promote extensive

osteolytic lesions from 2 to 8 weeks (Fig. 3), consistent with thedata presented above. Conversely, bone lesions generated by

avb5-expressing transfectants are predominantly osteoblasticlike the PC3-2-Mock transfectants. In the avb6 group, boneerosion starts at 2 weeks and continues with minimal replace-ment by woven bone until 8 weeks with extensive loss of bonevolume at comparable time points (Fig. 3B). In contrast, limbsinjected with avb5-expressing cells exhibit an initial, minorosteolytic response at 2 weeks, as observed with the avb6group albeit with no statistical difference in bone volumebetween the two groups (Fig. 3B). By 4 weeks, these lesionsprogress toward an osteoblastic phenotype, with woven boneoccupying the tumor inoculated region of the tibia and sig-nificant increase in tibial bone volume compared with theavb6 group. The temporal appearance of the osteoblasticlesions, evident by 4 weeks after inoculation of avb5-expres-sing cells, exhibits disorganized woven bone within the med-ullary cavity as well as within the tumor growing on theperiosteal side of the eroded cortex (Fig. 3A).

We also examined PC3-2 cells stably transfected with avb6or avb5 in mouse tibiae by radiography (Supplementary Fig.S3A). The rate of tumor growth and erosion through thecortical bone is indistinguishable between avb6 or avb5transfectants 4 weeks after injection (Supplementary Fig.S3B, low magnification image). These results indicate that theb subunit that associates with av determines the specific

Figure 4. MMP2 is inducedbyavb6.A, b6, MMP2, and OPN proteinlevels (left) andMMP2 activity wereanalyzed by immunoblotting (IB) orgelatin zymography (Zg; right) inavb6- and avb5-PC3-2 bonetumors isolated 8 weeks afterinjection. For MMP2immunoblotting, intervening laneshave been spliced out. As apositive control for active MMPs,conditioned medium of BPH1 cellswas used. B, MMP2 expression(left) and activity (right) in Parental,avb5-PC3-2, and two clonesofavb6-PC3-2 cells were analyzedby immunoblotting (12.5% SDS-PAGE) or Zg, respectively. C,MMP2 expression (left) and activity(right) in Parental, shb5-, and shb6-PC3-1 were analyzed byimmunoblotting (10% SDS-PAGE)or Zg, respectively. AKT (A) andERK (A–C) were used as loadingcontrols.






osteolytic effect observed in response to avb6 expression.Analogous to normal bone remodeling (31), prostate tumorcells secrete factors that facilitate the coupling between oste-oclast resorptive activity and bone formation due to osteo-blast-like activity (6, 7). Therefore, we examined whetherosteoclast activity is induced by avb6. Immunohistochemicaldetection of active osteoclasts by TRAP staining identifiesrobust bone resorption at 2 weeks in the avb6 group, com-pared with mice injected with avb5 transfectants; quantifica-tion of TRAP staining indicates a significant increase inosteoclast number in the avb6 compared with avb5 tumors(Supplementary Fig. S3C).

MMP2 and PTHrP are upregulated upon avb6 integrinexpression

When analyzed in tumor lysates, MMP2 is induced andfound catalytically active in extracts prepared from avb6- butnot avb5-expressing tumors (Fig. 4A). In contrast, the levels ofOPN, a molecule described to mediate prostate cancer celladhesion and migration in bone (32), remain constant in bothgroups (Fig. 4A). Consistent with this in vivo observation, avb6expression in PC3-2 cells increases MMP2 at the protein andactivity levels compared with avb5-expressing PC3-2 cells invitro (Fig. 4B). Also, we used PC3-1 cells because they expresshigh endogenous levels of avb6. In PC3-1 cells, MMP2 expres-

sion as well as its activity is reduced significantly upon shorthairpin RNA (shRNA)–mediated downregulation of b6 com-pared with downregulation of b5 (Fig. 4C). Similar results wereobtained in another prostate cancer cell line, RWPE,which alsoexpresses high levels of avb6 (Supplementary Fig. S4).

To identify avb6 targets related to the tumor phenotype inbone, we screened a panel of markers in PC3-2 cells expressingb6 for potential expression of genes associated with osteolyticor osteoblastic lesions (Fig. 5; refs. 23, 33–35). mRNA levels ofthe following factors were not changed: MMP9, interleukin-8(IL-8), osteocalcin, dickkopf WNT signaling pathway inhibitor1 (DKK1), receptor activator of NF-kB ligand (RANKL), runt-related transcription factor 2 (Runx2), VEGF, secreted frizzled-related protein 1 (SFRP1), lymphoid enhancer-binding factor 1(LEF1), and transcription factor 4 (TCF4). Conversely, mRNAlevels of MMP2 and PTHrP were consistently upregulated inavb6-PC3-2 tumors (Fig. 5A) and cells (Fig. 5B).

MMP2 mediates osteolysis caused by avb6 integrinexpression

We investigated whether MMP2 activity induced by avb6-expressing tumors significantly contributed to the osteolyticlesions, as the causal role of PTHrP in mediating the viciouscycle of osteolytic disease and tumor growth in bone (36) iswell established. We generated stable PC3-2 transfectants

Figure 5. avb6 expressionselectively upregulates MMP2and PTHrP. A, mRNA levels ofosteolytic (DKK1, IL-8, MMP2,MMP9, osteocalcin, PTHrP,RANKL, Runx2, SFRP1, and VEGF)and osteoblastic factors (LEF1,Runx2, SFRP1, and TCF4) inavb6- and avb5-PC3-2 bonetumors were analyzed 8 weeksafter injection by qRT-PCR. B,MMP2, PTHrP, MMP9, DKK1,RANKL, and IL-8 mRNA levelswere analyzed in avb6- and avb5-PC3-2 cells by qRT-PCR. mRNAexpression levels were normalizedto GAPDH. �, statisticallysignificant differences in mRNAexpression levels between the twogroups.

Dutta et al.





expressingMMP2-shRNA or a negative control shRNAdirectedagainst TROP2. In these experiments, shRNA-mediated down-regulation of MMP2 causes dramatic suppression of prostatecancer osteolytic lesions in the intratibial model of metastaticdisease (Fig. 6A). Zymographic analysis shows successfulreduction of MMP2 activity upon shRNA-mediated downre-gulation (Fig. 6B). Consistent with these findings, MMP2silencing also results in a significant reduction of bone loss,compared with control lesions (Fig. 6C). This phenotype isquantitatively associated with a significant preservation oftotal bone, and mature bone in MMP2-silenced lesions, ascompared with tumors expressing TROP2-shRNA (Fig. 6D).Because previous studies showed that metastatic prostate

cancer contains high levels of androgen receptor (AR; ref. 37),we also evaluated our proposed avb6-MMP2 pathway in AR-positive cells in our proposed avb6–MMP2 pathway. To per-form this study in cells expressing AR, the avb6-negative

prostate cancer cells, C4-2B, were stably transfected with b6cDNA. Another av-associated integrin avb3 was used as acontrol. FACS analysis shows successful transfection of b6 orb3 (Supplementary Fig. S5). MMP2 expression and activity areconsistently found to be induced upon avb6, but not avb3,expression in C4-2B cells (Fig. 7A and B). As analyzed byquantitative real-time PCR (qRT-PCR), MMP2 and PTHrPmRNA levels are also increased in avb6-C4-2B transfectantscompared with Parental cells, Mock, or avb3-C4-2B transfec-tants (Fig. 7C). Reproducible results were obtained using oneclone and one population for each transfectant. DKK1, RANKL,and IL-8 levels were undetectable in these cells (data notshown).

Given the above observations in vivo and in vitro, weanalyzed 11 humanprostate cancer–mediated osteolytic speci-mens to study the expression of avb6 and MMP2. Our resultsshow that avb6 positively correlates (r ¼ 0.6787; P ¼ 0.0048)

Figure 6. MMP2 mediates avb6-induced osteolysis in vivo. A,comparison of mCT images fromavb6-PC3-2-shMMP2 (top) and-shTROP2 (bottom) groups oftibiae 4 weeks after injection (n¼ 5mice/group). A representativeimage of each group showing frontand back side of the tibia, scannedat 10 mm resolution, revealsosteolysis in the shTROP2 group,although osteolysis was notdetectable in the shMMP2 group.B, MMP2 activity was analyzed inthe culture supernatant of avb6-PC3-2-shMMP2 and avb6-PC3-2-shTROP2 cells by gelatinzymography (Zg). Serum-freeculture medium from BPH1 cellswas used as a control for activeMMPs.C, bone loss in tumor tibiae.As a reference point forquantitating the inhibited osteolyticdisease by shMMP2, thecontralateral tibia of each mousewas imaged and bone volumequantitated at the 275 to 1,000threshold based on mature high-density bone (HD). Bone loss wascalculated by subtracting the bonevolume of the tumor bearing limbfrom thebone volumeof the normaltibia and the difference ispresented as a percentage loss ofthe contralateral limb. n ¼ 3, avb6-PC3-2 control group; n ¼ 5,shMMP2 group; and n ¼ 5,shTROP2 group. D, calculations ofbone volume include 300 10-mmslices from the beginning growthplate performed at two thresholds175 to1,000 to includewovenboneand 275 to 1,000 to encompass themature bone. A group of mice(n¼ 3) injected with the avb6-PC3-2 cell line (Control) was included forcomparison to evaluate the effectof the shTROP2 or shMMP2.






with MMP2 expression in human osteolytic disease (Supple-mentary Fig. S6). Overall, our data establish that MMP2,through its matrix-degrading activity, promotes osteolysis inavb6-expressing prostate tumors.

DiscussionIn this study, we describe a cancer cell–mediated pathway

that promotes osteolysis and is mediated by the avb6 integrinand its downstream effector MMP2.

The present study shows a unique effect of theavb6 integrinon osteolysis; as avb3 promotes osteoblastic lesions (11), wepropose that the formation of tumor-derived bone lesionsmight be controlled by the relative expression of avb6 andavb3. On the other hand, a differentav integrin,avb5, does notincrease bone lysis. Our results also confirm that the observedeffects are due to b6 expression as other integrin subunits, a2anda6, known to promote bone lesions, are not affected in our

bone model. The results also indicate that a2b1 and a6b1 areinactive or poorly active in these cells as minimal bone loss isobserved in the Mock groups. Finally, our data indicate thatavb6 has a dominant negative effect onavb3 as this integrin isexpressed in PC3-2 cells and promotes the osteoblastic phe-notype of the Mock groups.

Our results show that avb6 predominantly promotes osteo-lytic lesion formation without affecting tumor growth in ourintratibial model. Finally, while PC3-1 cells (avb6þ) have beenreported to cause aggressive osteolytic lesions upon intraboneinjection (36), DU145 and MDA-PCa cells, which lack avb6expression (data not shown), fail either to cause bone lesions orcause osteoblastic lesions (38, 39). These results indicate thatavb6 expression correlates with the osteolytic phenotype ofthe cell type analyzed.

Our results highlight a new specific function of integrins inupregulating MMP2, which consequently causes osteolysis. Asin our report, a previous study has shown that expression ofavb6 in cancer tissues results in enhanced levels of pro-MMPs, specifically MMP2 (40). In another cell type, Morganand colleagues reported that the b6 subunit promotesavb6-mediated invasion in aMMP9-dependent fashion in vitro(41). In our study, the results seem to be independent of the celltype used and of the expression of AR. It remains to beinvestigated whether MMP2 enzymatic activity is maintainedby the balance between MMP2 and its natural inhibitor, tissueinhibitor of metalloproteinase 2 (TIMP2). Reduced levels ofTIMP2 expression, which result in activation of pro-MMP2(42), in conjunction with the observed increase in MMP2protein levels, may conceivably further shift the MMP2/TIMP2ratio toward increased MMP2 activity.

A study by Corey and colleagues shows that administrationof zoledronic acid, under prevention or treatment regimens,reduces MMP2 and 9 expression which correlates with sup-pression of osteolysis caused by PC3 cells in SCID mice (43).The mechanistic requirements of this pathway have not beencompletely delineated, but a role for MMPs, including MMP2and MMP9 in increasing the number of osteoclasts withconcomitant bone resorption, has been shown (44). Inhibitionof MMP activity has been shown to prevent mineralized bonebreakdown induced by the addition of PC3 prostate cancercells to an in vitro coculture system with bone organs (44) andto prevent osteoclast recruitment within bone metastases. Inour study, mRNA levels of an osteolytic factor PTHrP, whichinduces osteoclastogenesis when released during the boneremodeling process, are also found to be increased, whereasthe levels of other osteolytic factors such as DKK1, IL-8,osteocalcin and MMP9 remain unaltered. Therefore, in ourmodel, avb6-mediated osteolysis could result from a cumu-lative effect of increased MMP2 enzymatic activity, which bydegrading ECM facilitates osteoclast-activated bone resorp-tion, and PTHrP secretion (33).

This model is in general agreement with clinical data,implicating MMP2 and 9 as independent predictors ofprostate cancer metastasis and MMP2 association withreduced disease-free survival (45). In addition, although astudy by Thiolloy and colleagues shows that MMP2 releasedfrom the host osteoblasts promotes production of mature

Figure 7. MMP2 is induced upon avb6 expression in ARþ prostate cancercells in vitro. A and B, MMP2 protein levels (A) and activity (B) wereanalyzed by immunoblotting (A) and zymography (Zg, B), respectively, inParental, vector transfected (#A, Mock), avb6- or avb3-transfectedC4-2Bcells.Oneclone andonepopulation (pop.) for eachavb6 (#1, #9) oravb3 (#88, #89) transfectedC4-2Bcellswere used (B). ERKwasusedasaloading control (A). C, MMP2, PTHrP, and MMP9 mRNA levels inParental,Mock,avb6, andavb3-C4-2Bcellswere analyzedbyqRT-PCR.mRNA expression levels were normalized to GAPDH. �, statisticallysignificant difference in mRNA expression levels between the groups.

Dutta et al.





osteoclasts (46), our system implicates a mechanism inwhich MMP2 is released from cancer cells rather than hostcells. In addition, it is conceivable that as a lack of MMP2leads to a reduced number of osteoclasts as shown inMMP2�/� mice (47), the cancer cell–mediated signalingsupplements the need of the local environment to indirectlyactivate osteolytic pathways (47). A potential role of integ-rins in the osteolytic pathway had not been previouslyinvestigated. Our study provides new insights into thefunctions of tumor cells and integrins in the process thatoccurs in metastatic osteolysis (1, 3).The role of AR is important in bonemetastasis given a recent

report thatMDV3100, anARantagonist, stabilizes bone disease(48) and most metastatic androgen-independent prostatecancers express high levels of AR gene transcripts (37). How-ever, in our model, the presence of AR neither alters nor isrequired for avb6-mediated induction of MMP2.Designing new therapeutic approaches for prostate cancer

based on inhibiting integrin function or integrin downstreamsignaling offers novel strategies to cure this cancer. In ourmodel, we propose that increased expression of avb6 causesupregulation of MMP2 and consequently promotes osteolysis.Thus, by inhibiting avb6-MMP2 signaling pathways, we arelikely to prevent bone metastasis associated with advancedprostate cancer. Conjugation of a drug or toxin to an antibodyor to a peptide that selectively bindsav integrins has been usedto enhance the antitumor effect of the drug (49) and a similarapproach may prevent osteolysis associated with metastaticprostate cancer.

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

DisclaimerThe Pennsylvania Department of Health (H.R.) specifically disclaims respon-

sibility for any analyses, interpretations, or conclusions.

Authors' ContributionsConception and design: A. Dutta, J. Li, T.J. FitzGerald, G.S. Stein, J.B. Lian, L.R.LanguinoDevelopment of methodology: A. Dutta, J. Li, H. Lu, J. Akech, J. Pratap, B.J.Zerlanko, T.J. FitzGerald, J. Wixted, S.M. Violette, J.B. LianAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Dutta, J. Li, H. Lu, J. Pratap, T. Wang, B.J. Zerlanko,T.J. FitzGerald, R. Birbe, J.B. LianAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):A. Dutta, J. Li, T.Wang, Z. Jiang, J.L. Stein, G.S. Stein, J.B. Lian, L.R. LanguinoWriting, review, and/or revision of themanuscript: A. Dutta, J. Li, T. Wang,T.J. FitzGerald, Z. Jiang, R. Birbe, S.M. Violette, J.L. Stein, G.S. Stein, J.B. Lian, L.R.LanguinoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J. Li, T. Wang, T.J. FitzGerald, R. BirbeStudy supervision: L.R. Languino

AcknowledgmentsThe authors thank Drs. C. Fedele, A. Sayeed, andM. Trerotola for constructive

discussion. The authors also thankDrs. L.W. Chung for providing PC3-1 cell lines;A. Cress for antibody to a6; I. Hart for pBabe-b6 integrin construct; S. Haywardfor BPH1 cells; C. Hsieh and Qin Liu for statistical analysis; V. Quaranta forantibody to b6. The authors appreciate Tiziana DeAngelis for immunofluores-cence analysis; Cheryl Morris assistance for tissue procurement; Jennifer Colbyfor intratibia injection procedures; Stacy Russell and Sadiq Hussain for imagingand bone histologic analyses in the University of Massachusetts mCT Core.

Grant SupportThis work was supported by the following grants: NIH R01 CA89720 (L.R.

Languino); PO1 CA140043 (L.R. Languino, G.S. Stein, and J.B. Lian); Our DannyCancer Research Foundation P00010003300000 (T. Wang) and P60010008300000(J. Li); NIH S10 RR023540 (J.B. Lian) and R37DE012528 ARRA Merit Award NCIPO1CA82834 (G.S. Stein and J.B. Lian). This project is also funded, in part, under aCommonwealth University Research Enhancement Program grant with thePennsylvania Department of Health (H.R.). Histology core resources weresupported by National Institutes of Diabetes, Digestive and Kidney Diseases(NIDDK) grant DK32520 and the Kimmel Cancer Center Cancer HistologyResources supported in part by National Cancer Institute (NCI) grantP30CA56036, which also supports the Kimmel Cancer Center Cancer GenomicsResource used in this study.

The costs of publication of this article were defrayed in part 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.

Received June 25, 2013; revised November 21, 2013; accepted December 11,2013; published OnlineFirst January 2, 2014.

References1. Mundy GR. Metastasis to bone: causes, consequences and thera-

peutic opportunities. Nat Rev Cancer 2002;2:584–93.2. Morrissey C, Lai JS, Brown LG, Wang YC, Roudier MP, Coleman IM,

et al. The expression of osteoclastogenesis-associated factors andosteoblast response to osteolytic prostate cancer cells. Prostate2010;70:412–24.

3. Keller ET, Brown J. Prostate cancer bone metastases promote bothosteolytic and osteoblastic activity. J Cell Biochem 2004;91:718–29.

4. Roudier MP, Morrissey C, True LD, Higano CS, Vessella RL, Ott SM.Histopathological assessment of prostate cancer bone osteoblasticmetastases. J Urol 2008;180:1154–60.

5. ChoueiriMB, TuSM, Yu-Lee LY, Lin SH. The central role of osteoblastsin the metastasis of prostate cancer. Cancer Metastasis Rev2006;25:601–9.

6. CicekM,OurslerMJ. Breast cancer bonemetastasis and current smalltherapeutics. Cancer Metastasis Rev 2006;25:635–44.

7. Weilbaecher KN, Guise TA, McCauley LK. Cancer to bone: a fatalattraction. Nat Rev Cancer 2011;11:411–25.

8. Goel HL, Li J, Kogan S, Languino LR. Integrins in prostate cancerprogression. Endocr Relat Cancer 2008;15:657–64.

9. Bisanz K, Yu J, Edlund M, Spohn B, Hung MC, Chung LW, et al.Targeting ECM-integrin interaction with liposome-encapsulated small

interfering RNAs inhibits the growth of human prostate cancer in abone xenograft imaging model. Mol Ther 2005;12:634–43.

10. Hall CL, Dai J, van Golen KL, Keller ET, Long MW. Type I collagenreceptor (a2b1) signaling promotes the growth of human prostatecancer cells within the bone. Cancer Res 2006;66:8648–54.

11. McCabe NP, De S, Vasanji A, Brainard J, Byzova TV. Prostate cancerspecific integrin avb3 modulates bone metastatic growth and tissueremodeling. Oncogene 2007;26:6238–43.

12. Ports MO, Nagle RB, PondGD, Cress AE. Extracellular engagement ofa6 integrin inhibited urokinase-type plasminogen activator-mediatedcleavage and delayed human prostate bone metastasis. Cancer Res2009;69:5007–14.

13. Yang GY, Xu KS, Pan ZQ, Zhang ZY, Mi YT, Wang JS, et al. Integrinavb6 mediates the potential for colon cancer cells to colonize in andmetastasize to the liver. Cancer Sci 2008;99:879–87.

14. Breuss JM, Gallo J, DeLisser HM, Klimanskaya IV, Folkesson HG,Pittet JF, et al. Expression of the b6 integrin subunit in development,neoplasia and tissue repair suggests a role in epithelial remodeling.J Cell Sci 1995;108(Pt 6):2241–51.

15. Azare J, Leslie K, Al-Ahmadie H, Gerald W, Weinreb PH, Violette SM,et al. Constitutively activated Stat3 induces tumorigenesis andenhances cell motility of prostate epithelial cells through integrin b6.Mol Cell Biol 2007;27:4444–53.






16. VanAarsen LA, LeoneDR,HoS,Dolinski BM,McCoonPE, LePageDJ,et al. Antibody-mediated blockade of integrin avb6 inhibits tumorprogression in vivobya transforming growth factor-b–regulatedmech-anism. Cancer Res 2008;68:561–70.

17. Sheppard D. In vivo functions of integrins. Matrix Biology 2000;19:203–9.

18. Garlick DS, Li J, Sansoucy B, Wang T, Griffith L, FitzGerald TJ, et al.avb6 Integrin expression is induced in the POET and PTENpc�/�

mouse models of prostatic inflammation and prostatic adenocarcino-ma. Am J Transl Res 2012;4:165–74.

19. Weinreb PH, Simon KJ, Rayhorn P, YangWJ, Leone DR, Dolinski BM,et al. Function-blocking integrin avb6 monoclonal antibodies: distinctligand-mimetic and nonligand-mimetic classes. J Biol Chem 2004;279:17875–87.

20. Fornaro M, Tallini G, Zheng DQ, Flanagan WM, Manzotti M, LanguinoLR.p27(kip1) acts asadownstreameffector of and is coexpressedwiththe b1C integrin in prostatic adenocarcinoma. J Clin Invest 1999;103:321–9.

21. Farabaugh SM, Micalizzi DS, Jedlicka P, Zhao R, Ford HL. Eya2 isrequired to mediate the pro-metastatic functions of Six1 via theinduction of TGF-b signaling, epithelial–mesenchymal transition, andcancer stem cell properties. Oncogene 2012;31:552–62.

22. Vidal M, Salavaggione L, Ylagan L, Wilkins M, Watson M,WeilbaecherK, et al. A role for the epithelial microenvironment at tumor boundaries:evidence from Drosophila and human squamous cell carcinomas. AmJ Pathol 2010;176:3007–14.

23. Akech J, Wixted JJ, Bedard K, van der Deen M, Hussain S, Guise TA,et al. Runx2 association with progression of prostate cancer inpatients: mechanisms mediating bone osteolysis and osteoblasticmetastatic lesions. Oncogene 2010;29:811–21.

24. Franco OE, Jiang M, Strand DW, Peacock J, Fernandez S, JacksonRSII, et al. Altered TGF-beta signaling in a subpopulation of humanstromal cells promotes prostatic carcinogenesis. Cancer Res 2011;71:1272–81.

25. Trerotola M, Jernigan DL, Liu Q, Siddiqui J, Fatatis A, Languino LR.Trop-2 promotes cancer metastasis by modulating b1 integrin func-tions. Cancer Res 2013;73:3155–67.

26. Goel HL, FornaroM,Moro L, Teider N, Rhim JS, KingM, et al. Selectivemodulation of type 1 insulin-like growth factor receptor signaling andfunctions by b1 integrins. J Cell Biol 2004;166:407–18.

27. Chantrain CF, Shimada H, Jodele S, Groshen S, Ye W, Shalinsky DR,et al. Stromal matrix metalloproteinase-9 regulates the vascular archi-tecture in neuroblastoma by promoting pericyte recruitment. CancerRes 2004;64:1675–86.

28. Pratap J, Wixted JJ, Gaur T, Zaidi SK, Dobson J, Veeraraj KD, et al.Runx2 transcriptional activation of indianhedgehogandadownstreambone metastatic pathway in breast cancer cells. Cancer Res 2008;68:7795–802.

29. Pratap J, Javed A, Languino LR, van Wijnen AJ, Stein JL, Stein GS,et al. The Runx2 osteogenic transcription factor regulates matrixmetalloproteinase 9 in bone metastatic cancer cells and controls cellinvasion. Mol Cell Biol 2005;25:8581–91.

30. Sangaletti S, Di Carlo E, Gariboldi S, Miotti S, Cappetti B, ParenzaM, et al. Macrophage-derived SPARC bridges tumor cell–extracel-lular matrix interactions toward metastasis. Cancer Res 2008;68:9050–9.

31. Khosla S, Westendorf JJ, Oursler MJ. Building bone to reverse oste-oporosis and repair fractures. J Clin Invest 2008;118:421–8.

32. Cooper CR, Chay CH, Pienta KJ. The role of avb3 in prostate cancerprogression. Neoplasia 2002;4:191–4.

33. Akhtari M,Mansuri J, NewmanKA,Guise TM, Seth P. Biology of breastcancer bone metastasis. Cancer Biol Ther 2008;7:3–9.

34. Sottnik JL, Hall CL, Zhang J, Keller ET. Wnt andWnt inhibitors in bonemetastasis. Bonekey Rep 2012;1:1–7.

35. Monroe DG, McGee-Lawrence ME, Oursler MJ, Westendorf JJ.Update on Wnt signaling in bone cell biology and bone disease. Gene2012;492:1–18.

36. Pratap J, Akech J, Wixted JJ, Szabo G, Hussain S, McGee-LawrenceME, et al. The histone deacetylase inhibitor, vorinostat, reduces tumorgrowth at the metastatic bone site and associated osteolysis, butpromotes normal bone loss. Mol Cancer Ther 2010;9:3210–20.

37. Zhang X, Morrissey C, Sun S, Ketchandji M, Nelson PS, True LD, et al.Androgen receptor variants occur frequently in castration resistantprostate cancer metastases. PLoS ONE 2011;6:e27970.

38. Yin JJ,MohammadKS, Kakonen SM, Harris S,Wu-Wong JR,WessaleJL, et al. A causal role for endothelin-1 in the pathogenesis of oste-oblastic bone metastases. Proc Natl Acad Sci U S A 2003;100:10954–9.

39. Li ZG, Mathew P, Yang J, Starbuck MW, Zurita AJ, et al. Androgenreceptor-negative human prostate cancer cells induce osteogenesisin mice through FGF9-mediated mechanisms. J Clin Invest 2008;118:2697–710.

40. Ahmed N, Pansino F, Baker M, Rice G, Quinn M. Association betweenavb6 integrin expression, elevated p42/44 kDa MAPK, and plasmin-ogen-dependent matrix degradation in ovarian cancer. J Cell Biochem2002;84:675–86.

41. Morgan MR, Thomas GJ, Russell A, Hart IR, Marshall JF. The integrincytoplasmic-tail motif EKQKVDLSTDC is sufficient to promote tumorcell invasion mediated by matrix metalloproteinase (MMP)-2 or MMP-9. J Biol Chem 2004;279:26533–9.

42. Kawamata H, Nakashiro K, Uchida D, Harada K, Yoshida H, Sato M.Possible contribution of active MMP2 to lymph-node metastasis andsecreted cathepsin L to bone invasion of newly established humanoral-squamous-cancer cell lines. Int J Cancer 1997;70:120–7.

43. Corey E, Brown LG, Quinn JE, Poot M, Roudier MP, Higano CS,et al. Zoledronic acid exhibits inhibitory effects on osteoblastic andosteolytic metastases of prostate cancer. Clin Cancer Res 2003;9:295–306.

44. Nemeth JA, Yousif R, HerzogM,CheM,Upadhyay J, Shekarriz B, et al.Matrix metalloproteinase activity, bone matrix turnover, and tumor cellproliferation in prostate cancer bone metastasis. J Natl Cancer Inst2002;94:17–25.

45. Trudel D, Fradet Y, Meyer F, Harel F, Tetu B. Significance of MMP-2expression in prostate cancer: an immunohistochemical study.CancerRes 2003;63:8511–5.

46. Thiolloy S, Edwards JR, Fingleton B, Rifkin DB, Matrisian LM, LynchCC. An osteoblast-derived proteinase controls tumor cell survival viaTGF-b activation in the bone microenvironment. PLoS ONE 2012;7:e29862.

47. Mosig RA, Dowling O, DiFeo A, Ramirez MC, Parker IC, Abe E, et al.Loss of MMP-2 disrupts skeletal and craniofacial development andresults in decreased bone mineralization, joint erosion and defectsin osteoblast and osteoclast growth. Hum Mol Genet 2007;16:1113–23.

48. Scher HI, Beer TM, Higano CS, Anand A, Taplin ME, Efstathiou E, et al.Antitumour activity of MDV3100 in castration-resistant prostate can-cer: a phase 1-2 study. Lancet 2010;375:1437–46.

49. Sugahara KN, Teesalu T, Karmali PP, Kotamraju VR, Agemy L, GirardOM, et al. Tissue-penetrating delivery of compounds and nanoparti-cles into tumors. Cancer Cell 2009;16:510–20.

Dutta et al.





2014;74:1598-1608. Published OnlineFirst January 2, 2014.Cancer Res Anindita Dutta, Jing Li, Huimin Lu, et al. Upregulating MMP2

6 Promotes an Osteolytic Program in Cancer Cells byβvαIntegrin

Updated version

10.1158/0008-5472.CAN-13-1796doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2014/01/06/0008-5472.CAN-13-1796.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/74/5/1598.full#ref-list-1

This article cites 49 articles, 18 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/74/5/1598.full#related-urls

This article has been cited by 9 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/74/5/1598To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-13-1796

http://cancerres.aacrjournals.org/content/suppl/2014/01/06/0008-5472.CAN-13-1796.DC1

http://cancerres.aacrjournals.org/content/74/5/1598.full#ref-list-1

http://cancerres.aacrjournals.org/content/74/5/1598.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/74/5/1598


Integrin avb6 Promotes an Osteolytic Program in Cancer ... · Cell adhesion and migration assays...

Documents

Transcript of Integrin avb6 Promotes an Osteolytic Program in Cancer ... · Cell adhesion and migration assays...