b and avb6 integrin act in a common pathway to suppress...

Tumor and Stem Cell Biology

TGF-b and avb6 Integrin Act in a Common Pathway toSuppress Pancreatic Cancer Progression

Aram F. Hezel1,3, Vikram Deshpande1, Stephanie M. Zimmerman1, Gianmarco Contino1, Brinda Alagesan1,Michael R. O'Dell3, Lee B. Rivera4, Jay Harper2, Scott Lonning2, Rolf A. Brekken4, and Nabeel Bardeesy1

AbstractThe TGF-b pathway is under active consideration as a cancer drug target based on its capacity to promote

cancer cell invasion and to create a protumorigenic microenvironment. However, the clinical application of TGF-b inhibitors remains uncertain as genetic studies showa tumor suppressor function of TGF-b in pancreatic cancerand other epithelial malignancies. Here, we used genetically engineered mouse models to investigate thetherapeutic impact of global TGF-b inhibition in pancreatic cancer in relation to tumor stage, genetic profile,and concurrent chemotherapy. We found that avb6 integrin acted as a key upstream activator of TGF-b inevolving pancreatic cancers. In addition, TGF-b or avb6 blockade increased tumor cell proliferation andaccelerated both early and later disease stages. These effects were dependent on the presence of Smad4, acentral mediator of TGF-b signaling. Therefore, our findings indicate that avb6 and TGF-b act in a commontumor suppressor pathway whose pharmacologic inactivation promotes pancreatic cancer progression. CancerRes; 72(18); 4840–5. �2012 AACR.

IntroductionThe transforming growth factor-b (TGF-b) signaling path-

way is an evolutionarily conserved regulator of embryonicpatterning and cell differentiation, and has central roles inwound healing and inflammation (1). The activated TGF-breceptor (TGF-bR1/R2) phosphorylates the Smad2 and Smad3proteins, which modulate transcription in association withSmad4. This pathway has been the subject of intense investi-gation in cancer due to its potential to act in both a pro- andantitumorigenic manner (2). Depending on cross-talk withother pathways, TGF-b can inhibit proliferation and suppresstransformation by modulating expression of cell-cycle regula-tors. Alternatively, TGF-b can promote malignant growththrough multiple mechanisms including enhanced cancercell invasion, survival, matrix remodeling, fibrosis, andimmunosuppression.

As in other epithelial cancers, TGF-b pathway functionin pancreatic ductal adenocarcinoma (PDAC) seems complex.Inactivating mutations in SMAD4 and other pathway compo-

nents are present in approximately 50% of human PDAC andcooperate with activated KrasG12D to promote PDAC in mousemodels (3–6). However, TGF-b ligands are commonly over-expressed in PDAC, and can promote epithelial-to-mesenchy-mal transition (EMT) and invasion in cell lines (7, 8). TGF-b canalso induce angiogenesis, activate tumor-promotingmyofibro-blasts (stellate cells), and attenuate immune surveillance (9,10). In light of these observations, TGF-b inhibitors are underinvestigation as PDAC therapeutics and have shown efficacy inxenograft studies (11, 12).

The multifaceted and cell-type specific effects of TGF-binhibition present problems in fully assessing the clinical utilityof drugs against this pathway. Such effects are likely to be best-understood using native cancer models that appropriatelyrecapitulate tumor–stroma interactions as well as the multi-stage progression that defines human cancers. Here, we inves-tigated the upstream regulation of TGF-b signaling in thepancreas to establish new strategies to target the pathway,and we examined the impact of pharmacologic inactivation ofmultiple TGF-b signaling components using genetically engi-neered mouse (GEM) models of PDAC. These studies, carriedout in the context of sequential tumor stages, different geneticlesions, and combined treatments with cytotoxic chemothera-pies, failed to reveal a therapeutic window. Instead we foundmultiple settings in which disease was exacerbated by TGF-binhibition. This preclinical information does not presentlysupport the utility of broadly targeting this pathway in PDAC.

Materials and MethodsMouse models

All treatment studies were conducted in accordance toUCAR and institutional standards using previously describedmouse strains (5). Littermates were distributed among 1D11

Authors' Affiliations: 1Massachusetts General Hospital, Harvard MedicalSchool, Boston; 2Genzyme Corporation, Framingham, Massachusetts;3James P. Wilmot Cancer Center, University of Rochester School ofMedicine, Rochester, New York; and 4Hamon Center for TherapeuticOncology Research, University of Texas Southwestern Medical Center,Dallas, Texas

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

Corresponding Author: Nabeel Bardeesy, Massachusetts General Hos-pital, Harvard Medical School, 55 Fruit Street, Boston, MA 02114. E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-0634

�2012 American Association for Cancer Research.

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(anti-Tgf-b), 13C4 (IgG isotype control), and 3G9 (anti-avb6)groups (13, 14). Gemcitabine was dosed at 100 mg/kg intra-peritoneally (i.p.) twice weekly. Mice were treated at age 6weeks and euthanized at 12 weeks (PanIN study) or at 9 weeksuntil exhibiting signs of illness (PDAC study). In the PDACcohort, 4 long-lived controls were sacrificed and censored after20 weeks of age when all mice in the experimental cohorts haddied. These animals were free of signs of illness but uponpathologic evaluation were found to have advanced PanIN orearly cancers.

Histologic analysisPanIN/PDAC tumor burden was determined by serial anal-

ysis of more than 3 H&E sections through the longitudinalplain of the pancreas. A gastrointestinal pathologist (V. Desh-pande) determined percentage of pancreas occupied by nor-mal tissue, PanIN, and PDAC, in a blinded fashion.

AntibodiesFor avb6, the mAb 6.2A1 (14) used at 1:100 in human tissue

or the human/mouse chimeric form of 6.2A1 (ch6.2A1) inmouse tissue (15) used at 1:100; for phospho (Ser465/467)-Smad2, catalog number AB3849 (Millipore Corporation); forendothelial cells, the rat endomucin v.7C7 (Santa Cruz) used at1:50; for pericytes, NG2 catalog number AB5320 (Chemicon)used at 1:200; for Ki-67, NCL-Ki67p (Novocastra); for macro-phages, the anti-CD68-M antibody, MCA1957T (Serotech); fortotal T-cells, the anti-CD3 antibody, Catalog number RM-9107-S (Lab vision/Neomarkers); for Foxp3, catalog number14-5773(eBioscience).

Quantification of IHC/IFStaining for CD68, FoxP3 and phospho-Smad2 was quanti-

fied by scanning slides at 20� using the Aperio-XT automatedimaging system. Regions of interest where identifiedwithin thetissues for quantification of DAB positive CD68 and Foxp3stained cells. For phospho-SMAD2 quantification, we used anautomated algorithm to quantify the level of nuclear DABstaining on a scale ranging from 0, þ1, þ2, and þ3. Ki-67stainingwas quantified by pathologic evaluation as the percentof neoplastic cells with positive staining.

Statistical analysisSurvival was determined using the Kaplan–Meier method

and comparisons were determined using the Log-rank test.Animals showing signs of illness and with confirmed cancerswere included as events, whereas animals that died for reasonsother than cancer were censored. Histologic scores for diseaseburden, Ki67, and P-smad2 staining between treatment groupscompared using t-tests. b6 IHC scoring was compared by theMann–Whitney test.

ResultsPDAC evolves from premalignant lesions including acinar-

to-ductal metaplasia (ADM) and pancreatic intraepithelialneoplasia (PanIN; ref. 16). We evaluated the activationstatus of the TGF-b pathway during PDAC progression via

immunohistochemical staining for Serine465/467-phosphory-lated Smad2 (phospho-Smad2) in the Ptf1-Cre;LSL-KrasG12D;p53L/þ (Kras-p53Lox/þ) model. Phospho-Smad2 was elevated inADM and early PanINs compared to normal ductal and acinarcells, and remained at high levels throughout PDAC progres-sion (Fig. 1A, upper panels, yellow arrowheads). Stromal fibro-blasts also showed strong nuclear P-Smad2 staining (Fig. 1A,red arrowheads).

Previous studies have documented increased expression ofTGF-b ligands in PDAC progression (5). Because TGF-b isproduced as a latent complex, additional processes such asproteolytic cleavage or conformational changes are required toactivate signaling. To determine the basis for TGF-b activation,we examined the expression pattern of avb6, a candidateactivator of latent TGF-b that is upregulated in advancedtumors (15, 17). In mice, avb6 was absent in islet and acinarcells and at low to moderate levels in normal ducts, whereasexpression was increased throughout each stage of PDACprogression (Fig. 1A, lower panels). Expression of avb6 wasrestricted to transformed pancreatic ductal epitheliumwith noevidence of staining in the stromal microenvironment. Humanspecimens showed a similar avb6 expression profile, withstaining in low- and high-grade PanIN lesions andmost PDAC,with normal human ducts showing only weak staining(Fig. 1B). The correlation between induction of avb6 expres-sion and phospho-Smad2 in PanIN and PDAC suggests thisintegrin may be important in local activation of TGF-b sig-naling in ductal lesions.

To examine the relationship between avb6 function andTGF-b signaling, we treated Kras-p53Lox/þ mice with an avb6blocking IgG monoclonal antibody (3G9; ref. 14), a pan-TGF-bblocking IgG monoclonal antibody, 1D11 (13), or an isotypecontrol antibody (13C4). Anti-avb6 treatment stronglydecreased phospho-Smad2 expression in PanIN and PDAClesions as well as surrounding stroma (Fig. 1C). Collectively,our data indicate that avb6 is critical for activation of TGF-bsignaling in the neoplastic epithelium.

Targeting TGF-b signaling could limit PDAC growth byblocking TGF-b-mediated protumorigenic effects on themicroenvironment and on the invasiveness of cancer cells.Moreover, avb6 inhibition could serve to inactivate TGF-bsignaling in a restricted manner, limiting the effects of apharmacologic blockade to the diseased pancreas. To testthese possibilities, we used the anti-TGF-b, anti-avb6, andisotype control antibodies in the Kras-p53Lox/þ model. Toevaluate the impact of treatments on progression of preinva-sive lesions antibodies were administered at 5 weeks of age—when the pancreas is largely normal but contains focal earlystage PanINs (schematic in Fig. 2A). Pancreases were evaluatedfor the presence of gross tumors and by correlative histologicand immunohistochemical analysis at 12 weeks. Anti-avb6treated animals had increases in the proportion of the pancreasexhibiting PanIN or PDAC lesions compared with controls(mean ¼ 73% in 3G9 vs. 45% control; P ¼ 0.04; Fig. 2B, upperrow), as well as a higher frequency of invasive PDAC (66%, vs.33% in controls). Comparable increases in neoplasia wereobserved in anti-Tgf-b–treatedmice. Therefore, blockingavb6accelerated the course of PanIN initiation and progression,

TGF-b Inhibition Accelerates Pancreatic Cancer Progression

www.aacrjournals.org Cancer Res; 72(18) September 15, 2012 4841




leading to a larger burden of disease and more advancedtumors.

Acceleration in progression of PanIN lesions among anti-Tgf-b and anti-avb6–treated mice was associated with anincrease in proliferation as reflected by Ki67 staining(Fig. 2B, lower row). Consistent with this we observed that4/5 primary pancreatic ductal cell cultures with activated Krasshowed growth inhibition in responses to TGF-b treatments.We failed to observe significant alterations in stromal compo-nents that can be activated by TGF-b, including the stellatecells (smooth muscle actin), Tregs (FoxP3), macrophages(CD68), the desmoplastic stroma (qRT-PCR analysis for col-lagen-1) and vasculature (endomucin andNG2; SupplementaryFig. S1A–C). Thus, these data indicate that the avb6-TGF-bpathway has a primary role in restraining proliferation andmalignant progression of PanIN epithelial cells.

Because both TGF-b and avb6 signaling have been impli-cated in the induction of EMT and invasive growth of estab-lished cancers, we next sought to test whether the anti-TGF-band anti-avb6 antibodies had a differential impact at later

disease stages. Kras-p53Lox/þ mice were treated beginning at 9to 10 weeks of age, when either high grade PanINs (PanIN-3) orlocally invasive PDAC were present (schema in Fig. 2C, upper).Micewere treated until signs of illness necessitated euthanasia.Notably, overall survival was significantly diminished in theanti-TGF-b and anti-avb6 groups demonstrating a persistentrole for TGF-b in suppressing growth at later stages of disease(Fig. 2C, lower left). Histopathologic analysis revealed treatedtumors to be invasive PDAC showing a range of histologicdifferentiation. Blocking antibodies did not produce significantdifferences in the spectrum of tumor grade and histologicsubtypes (Supplementary Fig. S2A and B).

The PDAC stroma is a potential barrier to effective deliveryof chemotherapeutic agents to tumor cells (18). Based on thepotential function of TGF-b signaling in activating stromalfibroblasts, we tested whether TGF-b blockade influenced theresponse of the Kras-p53Lox/þ model to gemcitabine, a stan-dard chemotherapy. The addition of avb6 or TGF-b blockingantibodies to standard gemcitabine treatment led to a dimin-ished survival as compared with gemcitabine alone (Fig. 2C,

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Figure 1. avb6 activates TGF-b inthe pancreatic epithelium. A, IHCstaining for phospho-Smad2 (toprow,�400) and integrin b6 (bottomrow, �200), during multistageprogression of the Kras-p53Lox/þ

PDAC model. Duct (D), acinar (A),islet (I), metaplastic acini (M), andPanIN (P) cells indicated. P-Smad2þ neoplastic and stromalcells designated by yellow and redarrowheads, respectively. B, IHCfor integrinb6 in normal humanpancreas and in multistage PDACprogression. C, left, phospho-Smad2 IHC in PanIN and PDACfrom Kras-p53Lox/þ mice treatedwith avb6 blocking antibodies orcontrol. Right, phospho-Smad2þnuclei quantified by automatedanalysis.

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Cancer Res; 72(18) September 15, 2012 Cancer Research4842




bottom right). Therefore, avb6 and TGF-b restrain the initi-ation and progression of PDAC, apparently through functionson the neoplastic epithelium; potential positive roles of avb6and TGF-b in stromal regulation may have a less prominentimpact on tumorigenesis.Our work shows that avb6 is a critical component of

the TGF-b-Smad4 tumor suppressor pathway in PDAC. Cor-respondingly, reduced avb6 expression could serve as an

alternative mechanism to SMAD4 mutations as a means toinactivate the TGF-b pathway during PDAC progression. Toexamine this question, we carried out IHC analysis across a setof PDAC specimens derived from Smad4 wild type and Smad4null mouse models using antibodies to avb6. Although all thetumors with Smad4mutations expressed avb6 at higher levelsin invasive tumors compared with PanINs, we found thatapproximately 26% of Smad4 wild-type tumors lost avb6

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Figure 2. TGF-b or avb6 blockade accelerates PDAC initiation and progression in GEMmodels. A and B, evaluation of the impact of anti-avb6 and anti-TGF-bantibodies on early disease in Kras-p53Lox/þ mice. A, schematic indicating the course of PDAC progression in control animals (top) as well as thetreatment interval (red line). Mice were euthanized for analysis at 12 weeks. B, left, representative histologic images to quantify the proportion diseasedpancreas (top) and Ki67 staining to evaluate proliferation of PanIN epithelium (bottom). Right, quantification of% of pancreatic area occupied by PanIN andPDAC lesions; treatment groups have significantly increased disease burden compared with controls (�, P < 0.05). Quantification of Ki67 staining; treatmentgroups have increased epithelial proliferation compared with controls (��, P < 0.005; ��, P < 0.001). C, impact of anti-avb6 and anti-TGF-b antibodieson tumor progression in Kras-p53Lox/þ mice. Schematic, top, treatment was initiated at later stages of disease and continued until clinical signs of illness.Bottom, Kaplan–Meier analysis. Left, survival is shortened in the anti-avb6 (mean 6.6 weeks; P ¼ 0.03) or anti-TGF-b (mean 5.6 weeks; P ¼ 0.007) cohortscompared with isotype control-treated animals (mean 8.9 weeks). Survival of untreated animals is shown for comparison (gray line); �, P < 0.05. Right,anti-TGF-b and anti-avb6 antibody treatments reduce survival of gemcitabine (Gem) -treated mice.






expression (Fig. 3A). Importantly, the absence of avb6 stainingcorrelated with SMAD4 status (P¼ 0.01). The spontaneous lossof expression of avb6 among Smad4 wild-type tumors, butnever in combination with Smad4mutation, supports the viewthat avb6 is a central activator of the TGF-b-SMAD4 tumorsuppressor pathway, and suggests thatmolecular alterations ofboth upstream and downstream components promote PDACtumorigenesis.

To test more directly whether avb6 acts in a common TGF-b/Smad4 tumor suppressor pathway, we assessed the impactof 3G9 on a Kras-driven PDAC model that also has an engi-neered homozygous deletion of Smad4 and therefore hasdefective TGF-b signaling in the pancreatic epithelial cells(5). Treatment was started at the time when focal PDAC ispresent, and maintained until signs of illness required eutha-nasia. In contrast to their effects in the Smad4 wild-type Kras-p53Lox/þ model, avb6 blocking antibodies did not alter thelatency or histopathologic features of Smad4 null tumors (Fig.3B). Therefore, genetic inactivation of TGF-b signaling obviatesthe effect ofavb6 blockade on tumorigenesis, consistent with apredominant action of the blocking antibodies in inhibitingepithelial TGF-b-Smad4 signaling.

DiscussionHere, we show that TGF-b pathway blockade with specific

monoclonal antibodies toavb6 or TGF-b1-3 accelerated PDACprogression in GEM models. This effect was observed whenusing antibodies at early and later disease stages, and as asingle agent or in combination with gemcitabine. Althoughtumorigenesis was not accelerated in a GEM model lackingSmad4, avb6/TGF-b inhibition did not provide any appreci-able benefit in this setting. Although it is possible that avb6/TGF-b pathway inhibition may restrain aspects of malignancysuch as metastatic spread, our data indicate that there may be

risks in broadly targeting this pathway given its primaryfunction as a tumor suppressor.

Recent findings have supported the ability of GEM modelsto recapitulate therapeutic responses seen in patients (19).Our experiments illustrate a number of potential advantagesof GEM models, such as the capacity to evaluate the impactof an intervention at different disease stages includingpreinvasive disease, and in defined tumor genotypes. Where-as preclinical studies in xenografts have supported the use ofTGF-b pathway inhibitors in the treatment of PDAC, ourwork indicates this strategy carries risk in the context ofautochthonous tumors.

It remains possible that there may be contexts in whichinhibition of components of the avb6-TGF-b pathway mayprove beneficial in PDAC treatment. Both avb6 and TGF-bhave been implicated in metastasis, which we were not able toaddress definitively in our studies (1/7 controls and 1/15treated mice exhibited metastasis, which was insufficient toprovide statistical significance). In addition, while direct tar-geting of avb6 or TGF-b may carry risks, it is possible thatsignaling receptors or downstream effectors of the pathwayhave strictly tumor-promoting effects, and thus may be effec-tive targets for pharmacologic blockade. Along these lines arecent study showedTGF-b activates CxC chemokine signalingin a PDAC GEM model and that inhibition delays tumorprogression (20).

Our studies also reveal new insights into the mechanisms ofTGF-b activation in the pancreas and the contributions of thispathway inmultistage PDAC progression. We show that globalinactivation of TGF-b signaling promotes increased prolifer-ation of the PanIN epithelial cells and enhances PDAC initi-ation and progression in a Smad4-dependant manner.Although TGF-b likely has additional functions in regulatingthe PDAC microenvironment, these functions do not seemessential for either the tumor promotion or tumor suppression.

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Figure 3. avb6 functions throughSmad4 tumor suppressor in thepancreas. A, the relationshipbetweenSmad4andb6 expressionwas evaluated in Smad4 wild typeand mutant PDAC models.Percentage of b6-neoplastic cellsin individual tumors (left) andrepresentative IHC images (right)are shown. All Smad4 null tumorsretain b6 expression (left), whereassubsets of Smad4 wild-typetumors either lose (middle) or retain(right) Smad4 expression (�, P <0.05). Insets show highermagnification. B, impact ofanti-avb6 treatment on PDACprogression in theKras-Smad4Lox/Lox; Ink4a/ArfLox/þ

model. Left, schematic showingexperimental design. Right,Kaplan–Meier analysis showingthat avb6 blockade does not affectsurvival (n.s. ¼ not significant).

Hezel et al.

Cancer Res; 72(18) September 15, 2012 Cancer Research4844




Wealso identifyavb6 as a critical upstream regulator of TGF-bsignaling in the ductal epithelium and show that avb6 has apreviously unanticipated function in tumor suppression. avb6blockade attenuated Smad2 activation, produced similar bio-logical effects to TGF-b blockade in ourmousemodels, and didnot affect the progression of Smad4 null tumors. Thus,although avb6 has been shown to promote invasive growthof advanced cancers, our data indicate that the primaryfunction of avb6 in the pancreas is to serve as an upstreamcomponent of the TGF-b tumor suppression program. It isworth noting that PDAC in the Kras-Smad4 model arise fromcystic precursors rather than PanIN (3, 5), which could alsocontribute to the differential response to pathway inhibition inthis setting.In summary, this series of experiments highlight the use of

GEM cancer models to guide in the clinical development ofnovel therapeutics and to elucidate signaling pathway circuitryin vivo.We show thatavb6 andTGF-b act in a commonSmad4-dependent PDAC tumor suppressor pathway. Moreover, weconclude that broad use of TGF-b inhibitors in unselectedpopulations of PDAC patients could have detrimental con-sequences, and in particular, that there is potential for diseaseacceleration in cancers with an intact TGF-b/SMAD4 signalingpathway.

Disclosure of Potential Conflicts of InterestR.A. Brekken received commercial research support from Imclone Systems.

N. Bardeesy received commercial research grant from Genzyme and Stromedix.

Authors' ContributionsConception and design: A.F. Hezel, J. Harper, S. Lonning, N. BardeesyDevelopmentofmethodology:A.F.Hezel, V. Deshpande, J. Harper, N. BardeesyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.F. Hezel, V. Deshpande, S.M. Zimmerman, G.Contino, M.R. O'Dell, L. B. Rivera, N. BardeesyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.F. Hezel, V. Deshpande, S.M. Zimmerman, G.Contino, J. Harper, S. Lonning, N. BardeesyWriting, review, and/or revision of the manuscript: A.F. Hezel, V. Desh-pande, J. Harper, S. Lonning, R.A. Brekken, N. BardeesyAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A.F. Hezel, B. Alagesan, M.R. O'Dell, N.BardeesyStudy supervision: A.F. Hezel, R.A. Brekken, N. Bardeesy

Grant SupportA.F. Hezel is supported by a HHMI Early Career Award and NCI KO8

CA122835-03. N. Bardeesy is supported by grants from NIH (NCI2P01CA117969-06, NCI 1R01 CA133557-01, and P50CA127003), and Lynda Ver-ville Cancer Research Foundation.

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 February 27, 2012; revised June 8, 2012; accepted June 26, 2012;published OnlineFirst July 11, 2012.

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2012;72:4840-4845. Published OnlineFirst July 11, 2012.Cancer Res Aram F. Hezel, Vikram Deshpande, Stephanie M. Zimmerman, et al. Pancreatic Cancer Progression

6 Integrin Act in a Common Pathway to Suppressβvα and βTGF-

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