Smad7 Expression in T cells Prevents Colitis-Associated CancerMicroenvironment and Immunology Smad7...

Microenvironment and Immunology

Smad7 Expression in T cells Prevents Colitis-AssociatedCancer

Angelamaria Rizzo1, Maximilian J. Waldner2, Carmine Stolfi1, Massimiliano Sarra1, Daniele Fina1,Christoph Becker2, Markus F. Neurath2, Thomas T. Macdonald3, Francesco Pallone1, Giovanni Monteleone1,and Massimo C. Fantini1

AbstractPatients with inflammatory bowel disease (IBD) have an increased risk of developing colorectal cancer due to

chronic inflammation. In IBD, chronic inflammation relies upon a TGFb signaling blockade, but its precisemechanistic relationship to colitis-associated colorectal cancer (CAC) remains unclear. In this study, weinvestigated the role of the TGFb signaling inhibitor Smad7 in CAC pathogenesis. In human colonic specimens,Smad7 was downregulated in CD4þ T cells located in the lamina propria of patients with complicated IBDcompared with uncomplicated IBD. Therefore, we assessed CAC susceptibility in a transgenic mouse modelwhere Smad7 was overexpressed specifically in T cells. In this model, Smad7 overexpression increased colitisseverity, but the mice nevertheless developed fewer tumors than nontransgenic mice. Protection was associatedwith increased expression of IFNg and increased accumulation of cytotoxic CD8þ and natural killer T cells in thetumors and peritumoral areas. Moreover, genetic deficiency in IFNg abolished the Smad7-dependent protectionagainst CAC. Taken together, our findings defined a novel and unexpected role for Smad7 in promoting aheightened inflammatory response that protects against CAC. Cancer Res; 71(24); 7423–32. �2011 AACR.

Introduction

Chronic inflammation is believed to be a leading causes ofcancer in various organs (1). Indeed, chronic release of oxygenand nitrogen reactive species induces genomic damage thuscontributing to tumor initiation and promotion, whereasproinflammatory cytokines stimulate dysplastic cell prolifer-ation and neoangiogenesis thereby supporting tumorprogression.TGFb1, hereafter indicated as TGFb, is a crucial negative

regulator of inflammation and the TGFb knockout mice devel-op a fatal multiorgan inflammatory disease (2). The anti-inflammatory properties of TGFb rely, at least in part, on itsability to suppress T-lymphocyte activities. TGFb prevents theactivation and differentiation of naive T cells in proinflamma-tory T-helper (Th) 1 and Th2 cells while inducing them to

acquire a suppressive phenotype [i.e., regulatory T cells(Treg); 3, 4]. Moreover, TGFb suppresses proliferation andeffector/memory T-helper cell activity (5).

TGFb signaling is tightly regulated in T cells. The binding ofTGFb with its specific receptor complex (i.e., TGFb receptortype I and type II) causes the phosphorylation of the cyto-plasmic molecules Smad2 and Smad3 which in turn migrateinto the nucleus where they regulate the expression of severalproinflammatory genes. TGFb signal transduction is negative-ly regulated by Smad7 that prevents Smad2/3 phosphorylationby recruiting on the intracellular domain of the TGFb receptortype I, the GADD34 complex, and the protein phosphatase 1 todephosphorylate it (6). Moreover, Smad7 has been shown topromote the ubiquitination and proteasomal degradation ofthe TGFb receptor complex (7–10).

Smad7-mediated suppression of the TGFb signaling hasbeen well documented in the inflamed gut of patients withCrohn disease and ulcerative colitis, the two major forms ofinflammatory bowel diseases (IBD), which are associated withan increased risk to develop colitis-associated colorectal can-cer (CAC). Downregulation of Smad7 with a specific antisenseoligonucleotide restores TGFb signaling thus suppressingproinflammatory cytokine synthesis in vitro (11). Moreover,Smad7 overexpression makes T cells resistant to the suppres-sive activity of Tregs, and Smad7 downregulation restoresTreg-mediated suppression (12). In vivo, the downregulationof Smad7 attenuates IBD-like experimental colitis in mice (13).

Although the above findings suggest that high Smad7 canfacilitate colorectal cancer progression by sustaining chronicinflammation, studies in mice with a nonphysiologic block ofTGFb activity in T cells operated by the dominant-negative

Authors' Affiliations: 1Department of Internal Medicine, University ofRome Tor Vergata, Rome, Italy; 2Medical Department 1, Friedrich-Alexan-der University Erlangen-Nuremberg, Erlangen, Germany; and 3Bart's andthe London School of Medicine and Dentistry, London, United Kingdom

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

G. Monteleone and M.C. Fantini share the senior authorship of themanuscript.

Corresponding Author: Massimo C. Fantini, Department of InternalMedicine, University of Rome Tor Vergata, Via Montpellier 1, Rome 00133,Italy. Phone: 390672596150; Fax: 390672596391; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-11-1895

�2011 American Association for Cancer Research.

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TGFb receptor have provided divergent results on the role ofTGFb in cancer cell growth and metastasis (14, 15). Therefore,this study investigated the role of Smad7-induced block ofTGFb signaling on the initiation and progression of CAC. Here,we show that the development of CAC in patients with IBD isassociated with a significant downregulation of Smad7 inCD4þ T cells. Moreover, we have identified a novel andunexpected protective role of Smad7 against CACdevelopmentin a transgenic mouse model in which Smad7 is selectivelyoverexpressed in T cells.

Materials and Methods

Cell linesYAC-1 lymphoma cell cells were obtained from the Amer-

ican Type Culture Collection (ATCC-TIB-160). The abovemen-tioned cell lines were procured more than 6 months ago andhave not been tested recently for authentication in ourlaboratory.

MiceC57BL/6 CD2-Smad7Tg mice were generated as previously

published (12), C57BL/6 IFNg�/� mice were purchased fromJackson Laboratories, and IFNg�/þ Smad7Tg were hosted inthe SPF Animal Facility at the University of Rome TorVergata (Rome, Italy). See also Supplementary Materials andMethods.

Azoxymethane/dextran sodium sulphate protocolAll mice were male and 6 to 8 weeks old and experimental

protocols were conducted according with the local institu-tional guidelines. To induce CAC, mice were injected intra-peritoneally with a single dose (10 mg/kg) of the alkylatingagent azoxymethane (AOM; Sigma-Aldrich) followed by 3cycles of 2.5% dextran sodium sulphate (DSS; MW, 36,000–50,000 Da; MP Biomedicals) given in the drinking water for 3days. Mice were kept on water for 2 weeks after each DSS cycle.In some experiments, mice were treated with DSS only toinduce chronic colitis but not CAC.

Endoscopic proceduresTumor development and colitis were screened by a minia-

turized endoscope (Coloview System). Tumors were countedand scored as previously described (16). Colitis was gradedaccording to the mouse endoscopic index of colitis severity(MEICS).

Histologic analysis and immunohistochemistryColonic sections from mice were obtained for hematoxylin

and eosin (H&E) staining and analyzed by light microscope(Olympus). Colitis was scored as previously published (17). Insome cases, colonic sections were used for indirect immuno-fluorescence using rat anti-mouse CD8 or CD4 (BD Pharmin-gen) or CD68 (Acris) or Smad7 (Santa Cruz Biotechnology) orFOXP3 (eBioscience). Antigen detection was obtained withTyramide [Cy3 and fluorescein isothiocyanate (FITC)] accord-ing to the manufacturer's guidelines (PerkinElmer). Paraffin-embedded colonic sections from patients with active IBD, IBD-

related CAC, and controls were obtained from the Institute ofPathology (Bayreuth, Germany) as previously reported (18).Patients' characteristics are reported in SupplementaryTable S1. Sections were used for H&E staining or stained withanti-human CD4 (BD Pharmingen) and anti-human Smad7–specific antibodies (R&D System) and analyzed by confocalmicroscope as previously published (18). The number of pos-itive cells was quantified by ImageJ software (NIH, Bethesda,MD).

Isolation of lamina propria mononuclear cells andtumor-infiltrating lymphocytes

Lamina propria mononuclear cells (LPMC) and tumor-infil-trating lymphocytes (TIL) were isolated accordingly to stan-dard protocols. See Supplementary Material and Methods.

Flow cytometryThe following anti-mouse conjugated antibodies were used:

CD4-FITC, CD8-phycoerythrin (PE), CD49b (DX5)-PE, CD3-FITC, IL-17A-PE, IFNg-APC (BD Pharmingen), and Annexin V-PE (BD Pharmingen). Interleukin (IL)-17A- and IFNg-expres-sing intracellular staining on LPMCs and TILs were analyzedafter 5-hour stimulation with phorbol-12-myristate-13-acetate(PMA; 40 ng/mL) and ionomycin (1 mg/mL; Sigma-Aldrich), inthe presence ofmonensin (2mmol/L; eBioscience) according tostandard protocols. Stained cells were analyzed by flow cyto-metry (FACScalibur; BD).

Cytotoxicity assayCytotoxic activity was measured by flow cytometry–based

assay as previously described (19). See SupplementaryMaterialand Methods for detailed description.

RNA extraction, cDNA preparation, and real-time PCRTotal RNA was isolated using TRIzol reagent (Invitrogen)

and retrotranscribed with the M-MLV reverse transcriptase(Invitrogen). Real-time PCR was carried out using a SYBRGreen–based PCR using iQ SYBR mix (Bio-Rad). b-Actin wasused to normalize gene expression results. Specific primerswere designed on the cDNA sequence provided by ENSEMBLdatabase (Supplementary Table S2).

Statistical analysisThe Student t test and the nonparametric Mann–Whitney

test were used to calculate statistical significance betweengroups. Spearman rank correlation test was used to calculatecorrelation between inflammation grade and tumor score.

Results

Smad7 is downregulated in inflamed colon of patientswith IBD-related CAC

To assess Smad7 expression in patients with CAC, sectionsfrom patients with IBD with or without CAC and controls(Fig. 1A) were stained for Smad7. A marked accumulation ofSmad7-expressing cells was seen in the lamina propria ofpatients with Crohn disease and ulcerative colitis withoutCAC in comparison to controls. In contrast, the number of

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Smad7-positive cells did not differ between patients with IBDwith CAC and controls (Fig. 1B and C). As shown in Fig. 1D, themajority of the Smad7-expressing cells coexpressed the CD4.These data indicate that IBD not complicated by CAC ischaracterized by accumulation of Smad7þCD4þ T cells in thelamina propria and that the number of these cells is signifi-cantly reduced in patients with IBD with CAC.

Smad7Tg mice develop severe DSS-mediated colitisTo investigate the functional role of Smad7 in CAC devel-

opment, we used a T-cell–specific Smad7 transgenic(Smad7Tg) mouse as previously described (12). We initiallyassessed whether, analogously to human IBD, high expression

of Smad7 in T cells was associated with enhanced mucosalinflammation. To this end, 6- to 8-week-old wild-type andSmad7Tg mice received 3 cycles of 2.5% DSS to mimic humanchronic-relapsing colitis (Fig. 2A). Endoscopy conducted at theend of the experimental protocol (day 75) showed colitis inboth wild-type and Smad7Tg mice (Fig. 2B). However,Smad7Tg mice had a more severe colitis as shown by theendoscopic score of colitis (Fig. 2C). Histologic analysis ofcolonic specimens obtained at the end of the experimentconfirmed the presence of inflammation in both wild-typeand Smad7Tgmice (Fig. 2D), even though Smad7Tgmice had asignificant higher histologic score (Fig. 2E). Both DSS-treatedwild-type and Smad7Tg mice were characterized by highexpression of the proinflammatory cytokines IFNg , TNFa,

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Figure 1. Smad7 is downregulated in colonic samples of patients with IBDcomplicated by CAC. A, H&E staining of colonic samples from patientswith IBD [both ulcerative colitis (UC) and Crohn disease (CD)] with orwithout CAC and control (Ctrl) patients. Scale bars, 100 mm (top) and 20mm (bottom). B–D, immunofluorescent staining of the previouslydescribed colonic samples with anti-Smad7 and CD4-specific antibodysingularly or in combination. Scale bars in B and D represent 100 mmand7.5 mm in the left and right, respectively. C, bars represent mean � SEMlamina propria Smad7þ cells (n ¼ 5 per group, Crohn disease andulcerative colitis have beenpooled,n¼10). Positive cellswere counted inat least 5 high-power fields (HPF) per section. �, significant differencesbetween the groups (P < 0.05).

Figure 2. Smad7Tg mice develop more severe colitis than wild-type micein the chronic DSS model of colitis. A, experimental protocol of chronicDSS colitis. B, representative endoscopic images collected at day 75. C,mouse endoscopic index of colitis severity (MEICS) applied to wild-typeand Smad7Tg mice at day 75. Data were obtained from 2 independentexperiments.D,H&Estainingof representativecolonic sectionscollectedfrom wild-type and Smad7Tg mice at the end of the experiment. Scalebars, 100 mm. E, histologic score of colonic sections obtained from thedifferent groups. Horizontal bars indicate median value. �, significantdifferences between the groups (P < 0.05). F, mRNA expression (left) andfold change relative to the wild-type (right) of cytokines in Smad7TgLPMCs. Bars, mean � SD. Wt, wild-type.

Smad7 Overexpression Prevents Colitis-Associated Cancer

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IL-6, and IL-17A, although these cytokines are more expressedin the Smad7Tg mice (Fig. 2F). Moreover, Smad7Tg mice werecharacterized by a higher number of CD4þ and CD8þ T cellsaccumulating in the lamina propria as compared with wild-typemice (Supplementary Fig. S1), with no differences in termsof CD68-expressing macrophages. Finally, as observed inhuman IBD, the stronger inflammation observed in theSmad7Tg was associated with the accumulation of a highernumber of FOXP3þ regulatory T cells (20). These data indicatethat, similar to humans, high Smad7 expression in T cellsmakes mice more susceptible to colitis.

Smad7Tg mice are largely protected against CACTo investigate whether the more severe inflammation

observed in Smad7Tg mice was associated with an increasedsusceptibility to CAC, mice underwent the AOM/DSS model ofCAC (Fig. 3A) which is characterized by the induction ofadenomas with histologic signs of high-grade dysplasia by 9to 10 weeks (16, 21).

As expected, at the end of the experiment, wild-type micedeveloped multiple colonic tumors (Fig. 3B). As observed inhumans, Smad7was highly expressed by LPMCs of peritumoral

areas. In contrast, Smad7 was mostly expressed by dysplasticepithelial cells, with significantly fewer positive cells infiltrat-ing the tumor stroma than peritumoral LPMCs (Supplemen-tary Fig. S2A). Surprisingly, Smad7Tg mice developed fewertumors than wild-type (Fig. 3C). The tumor score, which takesinto account not only the number of tumors but also their size,was significantly higher in wild-type than Smad7Tg mice (Fig.3D). Histologic analysis of peritumoral areas showed higherinflammatory cell infiltrate in the mucosa and submucosaassociated with modest mucosal hyperplasia, conserved cryptarchitecture, and minimal goblet cell depletion in wild-typemice [Fig. 3E (i)–(iii)]. In contrast, Smad7Tg mice were char-acterized by dense inflammatory cell infiltrate both in themucosa and submucosa, mucosal hypertrophy, crypts distor-tion and rarefaction, and strong depletion of goblet cells [Fig.3E (iv)–(vi)]. These differences were confirmed by the histo-logic score of sections of wild-type and Smad7Tgmice (Fig. 3F)and this inversely correlated with the tumor score of bothgroups of mice (Fig. 3G). Accordingly, CD4þ and CD8þ T cellsseen in both tumor and peritumoral areas were more in theSmad7Tg than in the wild-type, whereas no differences wereobserved in CD68þ macrophages (Supplementary Fig. S2B).

Figure 3. Smad7Tg mice areprotected from tumor developmentin the AOM/DSS model of CAC. A,experimental protocol of AOM/DSS model of CAC. B,representative endoscopic imagescollected at day 75. C, number oftumors and (D) tumor score of wild-type and Smad7Tgmice at the endof the experiment. Pooled resultsfrom 3 independent experimentsare shown.Horizontal bars indicatethe median value. �, significantdifferences between the groups(P < 0.01). E, H&E staining ofrepresentative colonic sections ofwild-type (a) and Smad7Tg (d),magnification of wild-type (b) andSmad7Tg (e) tumors, wild-type (c)and Smad7Tg (f) peritumoral areas.Outlining indicates dysplasticareas. Scale bars, 100mm. F,histologic score of colitis wasobtained from each animal of the 2groups. Horizontal bars indicatemedian value. �, significantdifference between the groups(P < 0.05). G, correlation betweenhistologic score and tumor score inthe wild-type and Smad7Tg micepooled together, rs ¼ �0.72;P ¼ 0.003. i.p., intraperitoneal; Wt,wild-type.

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To assess whether the higher inflammation and lower tumorincidence observed in the Smad7Tg mice were related to adefect in Treg accumulation, the expression of the Treg-specific transcription factor FOXP3 and TGFb was evaluatedin peritumoral LPMCs and TILs. Although TGFb expressionwas higher in TILs than in LPMCs isolated from both thegroups, there was no significant difference between wild-typeand Smad7Tg mice (Supplementary Fig. S3A, left). FOXP3 wasmore expressed in the LPMCs of highly inflamed Smad7Tgmice than those of the wild-type, whereas no significantdifference was observed among TILs of the two groups asshown by real-time PCR and immunofluorescent staining(Supplementary Fig. S3A–S3C). Altogether these results indi-cate that the higher inflammation seen in the Smad7Tg micemight interfere with CAC, despite the accumulation of FOXP3-expressing cells in the peritumoral areas.

Smad7Tg mice exhibit a marked intratumoral Th1-mediated immune responseCytokine expression analysis in the LPMCs and TILs of wild-

type and Smad7Tg mice showed IFNg , TNFa, IL-6, and IL-17Ato be the most expressed cytokines in both wild-type andSmad7Tg mice (Fig. 4A and B, left). In keeping with the moresevere inflammation observed in the Smad7Tg, IFNg , IL-6, andIL-17A mRNA levels were nearly 8-fold higher in the Smad7TgLPMCs than in wild-type mice (Fig. 4A, right). In contrast, inthe tumors, only IFNg wasmore than 4-fold higher in Smad7Tgmice while all the other cytokines were downregulated ascompared with the wild-type (Fig. 4B, right). These datasuggest that the peritumoral areas of both wild-type andSmad7Tg mice are characterized by a similar cytokine profile,whereas in the tumors of Smad7Tg, there is a prevalent IFNg-associated immune response. Consistently, both Tbet andRORgt, the Th1- and Th17-specific transcription factors, wereoverexpressed in Smad7Tg LPMCs (Fig. 4C), whereas in theSmad7Tg TILs, there was a higher Tbet and lower RORgtexpression than wild-type (Fig. 4D). Consistent with themRNAdata, the number of IFNg-expressing CD4þ T cells resulted inabout 10-fold higher expression in the Smad7Tg than in thewild-type at both LPMC and TIL level (Fig. 4E). In contrast, thenumber of IL-17A–expressing cells in the Smad7Tg was higherin the LPMC but lower in the TILs as compared with wild-type.These data indicate that in contrast to the Wt, a prevalent Th1immune response develops in the tumor microenvironment ofSmad7Tg mice.

A critical role of IFNg in mediating protection ofSmad7Tg against CACBecause IFNg is upregulated in Smad7Tg mice, we next

assessed the functional role of this cytokine in tumor protec-tion in these mice. To this end, IFNg-deficient Smad7Tg(IFNg�/�Smad7Tg) mice were used in the AOM/DSS model.In the IFNg�/�Smad7Tgmice, tumor incidencewas restored tolevels observed in the wild-type (Fig. 5A and B). However, thetumor score was still significantly lower than wild-type (Fig.5C) thus suggesting that factors other than IFNg contribute tosuppress tumor growth in Smad7Tg mice. Histologic analysisof colonic sections from the 3 groups of mice showed severe

inflammation in both Smad7Tg and IFNg�/�Smad7Tg mice,although in the latter, many dysplastic areas were detected(Fig. 5D, outlined). TNFa, IL-6, and IL-17A were the mostexpressed cytokines in both LPMCs and TILs isolated fromIFNg�/�Smad7Tg mice (Fig. 5E and F, left). However, levels ofTNFa, IL-6, and IL-17A expressed by both LPMCs and TILswere reduced in the IFNg�/�Smad7Tg as compared with wild-type mice (Fig. 5E and F, right). These data indicate that colitisdevelops in Smad7Tg mice independently of IFNg but its

Figure 4. Smad7Tg mice are characterized by Th1 immune response inthe tumor microenvironment. Relative mRNA expression of cytokines inthe peritumoral LPMCs (A, left) and TILs (B, left) isolated from wild-typeand Smad7Tg mice. A and B, right, the fold change of cytokine mRNAexpression in Smad7Tg LPMCs and TILs relative to thewild-type. C, Tbetand RORgt mRNA relative expression (left) in LPMCs from wild-type andSmad7Tgmice and their fold change in the Smad7Tg relative to the wild-type (right). D, Tbet and RORgt mRNA relative expression (left) in TILsfromwild-type and Smad7Tgmice and their fold change in the Smad7Tgrelative to the wild-type (right). Bars indicate mean value � SD whereindicated. E, flow cytometric analysis of LPMCs and TILs from wild-typeand Smad7Tgmice at the end of the experiment. Frequency of IFNg- andIL-17A–expressing cells was obtained gating on CD4þ cells. Numbers inthe quadrants indicate the percentage of positive cells. Representativedot plots of 3 independent experiments showing similar results areshown. Wt, wild-type.






expression is required for protection against CAC in thesemice.

Smad7Tg mice are characterized by IFNg-dependentaccumulation of CD8þ and natural killer T cells into thetumors

To investigate how IFNg regulates tumor growth inSmad7Tg mice, we evaluated the percentages of CD4þ,CD8þ, CD3-DX5þ natural killer (NK), and CD3þDX5þ NKT

cells in LPMCs and TILs isolated from wild-type, Smad7Tg,and IFNg�/�Smad7Tg at the end of the AOM/DSS protocolby flow cytometry. Although not statistically significant, weobserved an IFNg-dependent accumulation of CD8þ andNKT cells in the LPMCs of Smad7Tg (Fig. 6A). However,the number of NKT and CD8þ cells in the TILs of Smad7Tgmice was significantly higher than in wild-type (Fig. 6B). Incontrast, the percentage of both intratumoral CD8þ andNKT cells in IFNg�/�Smad7Tg mice was similar to wild-type.

Figure 5. IFNg is required to protectSmad7Tg mice from tumors. A,representative endoscopicimages of wild-type, Smad7Tg,IFNg�/�Smad7Tg mice at the endof AOM/DSS protocol. Number oftumors (B) and tumor score (C)obtained in the different groups atthe end of the experiment. Barsindicate the median value.�, significant differences betweenthe groups as indicated (P < 0.01).Data were obtained pooling 4independent experiments. D, H&Estaining of representative colonicsections collected from thedifferent groups. Outliningindicates dysplastic areas. Scalebars, 100 mm. mRNA expressionand fold change relative to thewild-type of cytokines in Smad7Tgand IFNg�/�Smad7Tg LPMCs (E)and TILs (F). Bars indicate meanvalue � SD where indicated. ND,not determined; ns, not significant;Wt, wild-type.

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No significant differences were observed in CD4þ and NKcell accumulation in both LPMCs and TILs of the 3 groups ofmice. Similar results on CD4þ and CD8þ T-cell accumulationin the tumor and peritumoral areas of wild-type, Smad7Tg,and IFNg�/�Smad7Tg mice were obtained by immunofluo-rescent staining of colonic sections (Supplementary Fig. S4).

IFNg increases CD8þ and NK/NKT cell–mediatedcytotoxicity in the tumoral and peritumoral areas ofSmad7Tg miceTh1 cells are required to activate cytotoxic lymphocytes

and to generate antitumor immune response in differentexperimental systems. Accordingly with the dominant Th1-mediated immune response observed in the Smad7Tg mice,the cytotoxicity-related markers Perforin1, Granzyme B, andFasL mRNA were upregulated in the TILs and LPMCs ofthese mice in an IFNg-dependent manner (Fig. 7A). To assesswhether CD8þ and NK/NKT cells isolated from Smad7Tgmice were more cytotoxic, we cocultured CD8þ and DX5þ

cells sorted from mesenteric lymph nodes of wild-type,

Smad7Tg, and IFNg�/�Smad7Tg mice with YAC-1 lympho-ma cell line. CD8þ and DX5þ cells isolated from Smad7Tgmice induced more YAC-1 cell death (Fig. 7B). The highercytotoxic activity of Smad7Tg effector cells was completelyreverted by the loss of IFNg . Consistently, tumors developedin the Smad7Tg mice exhibited more terminal deoxynucleo-tidyl transferase–mediated dUTP nick end labeling(TUNEL)-positive cells than neoplastic lesions developed inwild-type and IFNg�/�Smad7Tg mice (Fig. 7C).

Discussion

Patients with IBD have an increased risk to develop CAC,which is related to the duration and extent of inflammation.Although circumstantial evidence suggests that severity ofcolitis could also affect the development of CAC, the roleof inflammation in the initiation, promotion, and progressionof CAC remains poorly characterized (22–24).

The Smad7-dependent block of the TGFb signaling isthought to sustain chronic inflammation in IBD (11). Data of

Figure 6. IFNg expressioninfluences the accumulation ofimmune cells into colonic mucosa.Fluorescence-activated cell-sorting(FACS) analysis of CD4þ, CD8þ,CD3þDX5þ (NKT), and CD3-DX5þ

(NK) cells accumulated in peritumoralLPMCs (A) and TILs (B) of wild-type,Smad7Tg, and IFNg�/�Smad7Tgmice. Representative dot plots from4 independent experiments areshown. Percentages of the differentcell populations are reported in thequadrants (left). Pooled results from4independent experiments arereported for each cell populationanalyzed (right). Bars indicateaverage � SEM obtained from 4independent experiments.�, significant differences between thegroups (P < 0.01). Wt, wild-type.






the present study confirm and expand our knowledge on thisby showing that Smad7-expressing CD4þ T cells that highlyinfiltrate the intestinal lamina propria of patients with IBDare markedly reduced in CAC, thus suggesting that low Smad7in T cells might contribute to CAC development in thesepatients.

In our mouse model, and similarly to human IBD, Smad7overexpression in T cells caused more severe colitis as com-pared with wild-type. However, when we used Smad7Tg micein the well-established AOM/DSS model of CAC, surprisingly,we observed a significant and highly reproducible reduction oftumor incidence even in the presence of more severe colitis.Analysis of the cytokines and transcription factors showedhigher expression of the Th1-related markers IFNg and Tbet inthe peritumoral areas and evenmore in the tumors of Smad7Tgmice than in the wild-type. In contrast, the Th17-relatedmarkers IL-17, IL-6, and RORgt were relatively downregulatedin the tumors of Smad7Tg mice thus indicating that a Th1-mediated immune response prevails in the transgenics.

Both Th1 and Th17 cells contribute to intestinal inflamma-tion in different models of colitis and their differentiation isinfluenced by TGFb (25–27). TGFb prevents the differentiationof Th1 cells by directly inhibiting the expression of Tbet andIFNg (3). In contrast, in the presence of IL-6, TGFb promotesTh17 cell differentiation (28, 29). Moreover, recent studiesindicate that the TGFb-mediated suppression of Th1 cells andIFNg expression promotes the generation of Th17 (30). Thesedata fit well with our results showing that Smad7-mediatedblock of the TGFb promotes the accumulation in the gut of Th1cells and increases IFNg expressionwhile reducing the numberof Th17 cells especially in the tumor microenvironment.

TGFb has been also involved in the differentiation of Tregs, asubset of cells implicated in the suppression of the immuneresponse against cancer. As observed in human IBD, the moresevere inflammation observed in the Smad7Tg was associatedwith accumulation of Tregs in both the tumor and peritumoralareas thus ruling out the possibility that less FOXP3þ cells inthe Smad7Tgmice may enhance the immune response againstcancer (20).

Loss of IFNg restored susceptibility to CAC in Smad7Tg.IFNg has been shown to play a pivotal role in the immuneresponse against cancer. In vivo, IFNg expression protects fromtumor in different tumormodels (31–33). In amurinemodel ofCAC, Osawa and colleagues showed that IFNg-deficient miceare more susceptible to develop CAC (34). IFNg expression isalso essential for CD8-mediated antitumor activity. This effectcan be partially explained by the capacity of IFNg to induce Fason dysplastic target cells surface thus inducing cell death byFas ligand–Fas interaction (35, 36). Moreover, IFNg increasesthe expression of MHC class I expression on target cells thuspromoting recognition of tumor-associated antigens (37). IFNgmight also contribute to NK andNKT cell activation and killingactivity. However, it is worth noting that the tumor score in theIFNg�/�Smad7Tg was still lower than in the wild-type thusindicating that even in the absence of IFNg , Smad7 over-expression in T cells can restrain tumor growth.

In the attempt of identifying the mechanism by which IFNgdetermines protection against tumors in the Smad7Tg, weobserved several effects linked to high expression of thiscytokine. Indeed, the higher accumulation and cytotoxic activ-ity of CD8þ and NKT cells observed in the Smad7Tg mice wasreverted in the IFNg�/�Smad7Tg mice. The high IFNg-depen-dent activation and cytotoxic activity of CD8þ and DX5þ NK/NKT cells in the Smad7Tg might be sustained by the prevalent

Figure 7. Smad7Tg mice show higher IFNg-dependent cytotoxicity. A,relative expression of Perforin1, Granzyme B, and FasL mRNA inperitumoral LPMCs and TILs isolated from wild-type, Smad7Tg, andIFNg�/�Smad7Tg mice. Bars indicate mean value � SD. Representativesimilar results from 1 of 4 experiments carried out are shown. B, in vitroflow cytometry–based cytotoxicity assay: target (T) cells, CD8þ or DX5þ

effector (E) cells. Killing activity is expressed as percentage of AnnexinV–positive target cells in the different groups. Points indicate average �SEM of Annexin V–positive target cells. Results from 3 independentexperiments are shown. �, significant differences between the groups(P < 0.05). C, representative TUNEL staining of colonic sections obtainedfrom different groups of mice at the end of the experiment. Arrowheadsindicate TUNEL-positive cells. Scale bars, 100 mm. TUNEL, terminaldeoxynucleotidyl transferase–mediated dUTP nick end labeling; Wt,wild-type.

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Th1 immune environment operating in these mice. Alterna-tively, it might be secondary to the Smad7-mediated block ofthe TGFb signaling in these subsets of cells as previouslyreported (38, 39).Althoughwewere unable to provide direct demonstration of

a stronger cytotoxic activity toward dysplastic epithelial cellsin vivo, due to the difficulties encountered in culturing thesecells ex vivo, these data strongly sustain the central role of IFNgin the protection from colonic tumor development in ourmodel.We have previously shown that the block of the TGFb

signaling in mice overexpressing the dominant-negative formof the TGFb receptor II (dnTGFbRII) in T cells develop moreinflammation and more CAC (14). The opposite outcomesobtained in the dnTGFbRII and Smad7 transgenic mice mightbe explained considering the level at which the block of theTGFb signaling occurs. Indeed, while the expression ofdnTGFbRII blunts all the downstream TGFb-dependent intra-cellular signals, the result of the Smad7-mediated block atintracellular level is less clear. In agreement with publisheddata, we have previously shown that Smad7 prevents theactivation of Smad2/3 (12). However, TGFb is known toactivate SMAD-independent pathways mediated by mito-gen-activated protein kinases (i.e., p38, Akt, extracellular sig-nal-regulated kinase), and the activation of these pathways isnot affected by Smad7 expression (40). Accordingly, whilednTGFbRII transgenic mice develop a rapidly fatal multiorganautoimmune disease, Smad7Tg mice did not (41). Therefore, apossibility is that in primary tumors, Smad7 might modulate,rather than fully inhibit, the TGFb signaling in T cells and thatthe net result of Smad7 overexpression is an antitumorimmune response instead of the generation of a tumor-pro-moting environment as in the case of dnTGFbRII transgenicmice. However, it is worth noting that in a different system, theblock of TGFb signaling obtained with the overexpression ofthe dnTGFbRII in CD4þ T cells protected mice from livertumor metastasis (15), thus suggesting that the TGFb intra-

cellular signals required in T cells to favor tumor progressionmight be tumor stage dependent.

Another possibility to explain the different results obtainedwith the dnTGFbRII and the Smad7Tg mice relies on the factthat Smad7 overexpression could inhibit other members of theTGFb superfamily [e.g., activin and bone morphogenetic pro-tein (BMP)] whose role in the tumor immunity is currentlypoorly understood.

In our system, a persisting Th1-mediated immune responsecharacterized by high expression of IFNg was protectivetoward the development of CAC. This observation is in agree-ment with the emerging concept that an indolent but chronicinflammatory process sustains cancer promotion and progres-sion rather than a highly detrimental acute inflammation.Translating this model into humans, one could envision thatwhile acute flares of colitis are required to generate dysplasticcells, a phase of persisting subacute inflammation, allowed bylow Smad7 expression, might be more effective at sustainingCAC growth. It is tempting to speculate that the Smad7 over-expression in T cells might block the evolution of the inflam-matory process thus maintaining a condition resembling anacute flare in which emerging dysplastic cells are destroyed bythe immune system rather than supported in their expansion.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This work was supported by the "Fondazione Umberto di Mario," Rome, the"Associazione Italiana per la Ricerca sul Cancro" AIRC, MFAG-9353, and GiulianiSpA, Milan, Italy.

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 3, 2011; revised September 24, 2011; accepted October 13, 2011;published OnlineFirst October 25, 2011.

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2011;71:7423-7432. Published OnlineFirst October 25, 2011.Cancer Res Angelamaria Rizzo, Maximilian J. Waldner, Carmine Stolfi, et al. Smad7 Expression in T cells Prevents Colitis-Associated Cancer

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