Genetic and Pharmacological Inactivation of the Purinergic ... · Thierry Juhel1,2,4, Xavier Hebuterne1,2,5, Eric Gilson1,2,4,6, Annie Schmid-Alliana2,7, Olivier Boyer8,9, Sahil Adriouch8,9,

Microenvironment and Immunology

Genetic and Pharmacological Inactivation of thePurinergic P2RX7 Receptor DampensInflammation but Increases Tumor Incidence in aMouse Model of Colitis-Associated CancerPaul Hofman1,2,3,4, Julien Cherfils-Vicini1,2,4, Marie Bazin1,2,4, Marius Ilie1,2,3,4,Thierry Juhel1,2,4, Xavier H�ebuterne1,2,5, Eric Gilson1,2,4,6, Annie Schmid-Alliana2,7,Olivier Boyer8,9, Sahil Adriouch8,9, and Val�erie Vouret-Craviari1,2,4

Abstract

Colitis-associated cancer (CAC) is a complication of inflamma-tory bowel disease (IBD). Binding of extracellular ATP to thepurinergic receptor P2RX7 has emerged as a critical event incontrolling intestinal inflammation, acting to limit elevation ofproinflammatorymast cells and cytokines andpromote survival ofregulatory T cells (Treg) and enteric neurons. In this study, weinvestigated the effect of P2RX7 blockade in an established mousemodel of CAC. Using genetic and pharmacologic tools, we foundunexpectedly thatwhile P2RX7mediated inflammatory responses,it also acted at an early time to suppress CACdevelopment. P2RX7

blockade enhanced proliferation of intestinal epithelial cells andprotected them from apoptosis. The proliferative effects of P2RX7blockade were associated with an increased production of TGFb1that was sufficient to stimulate the proliferation of intestinalepithelial cells. Finally, P2RX7 blockade also altered immune cellinfiltration and promoted Treg accumulation within lesions of thedigestive system. Taken together, ourfindings reveal anunexpectedrole for P2RX7 inpreventingCAC, suggesting cautions in the use ofP2RX7 inhibitors to treat IBD given the possibility of increasingrisks CAC as a result. Cancer Res; 75(5); 835–45. �2015 AACR.

IntroductionPurinergic receptors form a family of adenosine (P1) and ATP

(P2) receptors involved in a complex signaling network thataffects various cellular functions such as cell proliferation, celldifferentiation and cell death, but also exocrine and endocrinesecretion, immune responses, and inflammation (1). They arewidely expressed throughout the digestive tract, where they areinvolved in the regulation of intestinal motility. Moreover, it wasshown that this family of receptors regulated several neuronal andnon-neuronal gut functions, visceral sensation, and the immunecell activity (2). Therefore, it is now widely accepted that pur-

inergic receptors actively cooperate in the maintenance of guthomeostasis.

P2X receptors (P2RX) display a conserved topology with twotransmembrane spanning regions, a large extracellular regionsensitive to the ligand and intracellular amino- and carboxyl-termini that contain signaling binding motifs. Upon gating byeATP, P2RX7, also referred to as P2X7R, forms a poorly selectivechannel leading to membrane depolarization, potassiumefflux, and calcium and sodium influx (3). P2RX7 triggeringalso induces the formation of larger membrane pores that allowthe passage of molecules reaching up to 900 Da. This particularfeature has been linked to the presence of a long cytoplasmicC-terminal tail and to the possible activation of a pore-formingmolecular partner suggested to be pannexin-1 (4, 5). Impor-tantly, both P2RX7 activation and pannexin-1 mediate NLRP3inflammasome assembly, caspase-1 (casp-1) activation, andmaturation of the proinflammatory cytokines IL1B (IL1b) andIL18 (6).

At the level of the gastrointestinal tract, P2RX7 is expressedon immune and nonimmune cells (7, 8). Its low level ofsensitivity to eATP limits its activation within inflammatorysites and in the tumor microenvironment where eATP concen-trations are in the 100 mm range (9). We previously reportedthat P2RX7 is differentially expressed in the mucosa of patientswith active and quiescent inflammatory bowel disease (IBD)and that its activation could be triggered in response to neu-trophil transepithelial migration (8). These results, togetherwith published data based on the use of P2RX7 antagonists orP2rx7�/� mice, support the notion that P2RX7 participates inthe initiation as well as in the regulation of the inflammatory

1Institute for Research on Cancer and Aging, Nice, France; IRCANU1081 UMR CNRS 7284, Nice Cedex, France. 2University of Nice-SophiaAntipolis, Nice, France. 3Laboratoryof Clinical andExperimen-tal Pathology and Biobank, Pasteur Hospital, Nice, France. 4CentreAntoine Lacassagne, Nice, France. 5Laboratory of Gastroenterology,Archet II Hospital, Nice, France. 6Department of Medical Genetics,Archet 2 Hospital, CHU of Nice, Nice, France. 7iBV, UMR7277 CNRS-UMR1091 INSERM, Nice, France. 8INSERMU905, Rouen, France. 9Insti-tute for Research and Innovation in Biomedicine (IRIB), NormandyUniversity, Rouen, France.

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

Corresponding Author: Val�erie Vouret-Craviari, IRCAN, Inserm U1081, UMR7284 CNRS/Universit�e Nice-Sophia Antipolis, 28 Avenue de Valombrose, Nice06108, France. Phone: 33-492-031-223; Fax: 33-492-031-241; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-14-1778

�2015 American Association for Cancer Research.

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response notably via the processing and release of IL1B(10–12).

It is widely accepted that chronic inflammation can promotetumor development (13). In this scenario, the proinflammatoryrole of P2RX7 in immuneand epithelial cellswouldbe expected tofavor carcinogenesis. However, P2RX7 also regulates cell prolif-eration and/or cell death (14–17). Moreover, adding to thecomplex interplay between inflammation and carcinogenesis, astudy has revealed the role of the proinflammatory P2RX7/NLRP3/casp-1 cascade in the priming of dendritic cells and inthe immunogenicity of dying tumor cells following chemother-apy, which favors the emergence of an endogenous antitumorimmune response (18). Finally, P2RX7 appears to be overex-pressed in a variety of neoplastic disorders and tumors (19–21)and P2RX7 expression increases the tumorigenic properties ofhuman embryonic kidney cells (16). Hence, based on the appar-ently contradictory evidence, the precise role of P2RX7 in vivo inthe context of inflammation-associated carcinogenesis needs tobe carefully addressed.

In this study, we investigated the role of P2RX7 in CAC andwe showed, using P2rx7�/� mice as well as the P2RX7-specificpharmacologic antagonists, that the P2RX7 activity suppressedtumor development. While deciphering the cellular and molec-ular events leading to this suppressive function, we demon-strated that P2RX7 inactivation leads to: (i) overproduction ofTGFB1, (ii) recruitment of Tregs within inflammatory lesions,and (iii) TGFB1-mediated proliferation of colonic epithelialcells.

Materials and MethodsAnimal strains

This study was approved by the Institutional Care and UseCommittee of the University of Nice-Sophia Antipolis (Nice,France). Animal protocols were approved by the committee forResearch and Ethics of the PACA region (CIEPAL azur, #PEA 12-125) and followed the European directive 2010/63/UE. Thegeneration of C57BL/6 mice harboring targeted disruption of theP2rx7 gene has been described previously (10). Control C57BL/6Jmice were supplied by Harlan Laboratories.

Induction of colitis and inflammation-driven tumor formationAcute inflammation was induced as followed: intraperitoneal

injection of azoxymethane at day 0 before 5% dextran sulfate(DSS) treatment fromday 1 to day 5. CACwas induced by a singleintraperitoneal injection of azoxymethane followed by 3 cycles of3% DSS in the drinking water. Each cycle lasted 5 days and wasseparated by 2 weeks, as illustrated in Fig. 4A. We used thefollowing semiquantitative clinical score, which was adaptedfrom ref. 22. Body weight loss was scored as follows: 0, no loss;1, loss < 5%; 2, loss < 10%; 3, loss < 20%; and 4, loss > 20%. Thescores for stool consistency were measured as 0, normal; 1, loosestools; 2, watery diarrhea; or 3, severe watery diarrhea. Rectalbleeding was scored as 0, no blood; 1, presence of petechia; 2,stools with a trace of blood; or 3, bleeding. Inflammation scorewas performed by trained pathologists. Briefly, the severity ofinflammation (none, mild, moderate, severe), extent of inflam-mation (none, mucosa, mucosa, and submucosa, transmural),crypt damage (none, basal to 1/3, basal to 2/3, crypt loss, crypts,and epithelium loss), and percentage of tissue affected by inflam-mation (0, 25, 50, 75, and 100%) were scored.

Macroscopic polyp analysis and histopathologyColons (cecum to rectum) were removed from animals and

processed for histopathology as described in the SupplementaryMaterials and Methods.

Serum, colon organ cultures, and ELISABlood was collected with a heparin-treated needle from the tail

vein and serum was obtained by centrifugation (15 minutes,3,000 rpm at 4�C). After resection, colon tissues were culturedovernight inDMEM, supernatants were collected, and productionof IL1B, TGFB1, CXCL1, and CXCL2 assayed by an ELISA asdescribed by the manufacturers (see Supplementary Materialsand Methods).

Quantitative real-time PCR and protein gel blottingTotal RNA and protein were isolated from colonic tissues using

TRI Reagent following manufacturer's instructions and processedas described by manufacturers (see Supplementary Materials andMethods). We used the following antibodies: phospho-STAT3(#9131), STAT3 (#9139), and BCL2 (Bcl2, #50E3) antibodieswere from Cell Signaling Technology, anti-BCL2L1 (Bcl-XS/L, sc-634), and anti-BAX (Bax, sc-526) were from Santa Cruz Biotech-nology and the anti-ACTB (Actin, clone AC40) antibodywas fromSigma Aldrich.

Immunohistochemical analyses of mouse colon tissuesResected mouse colon tissues were fixed in 10% formalin,

paraffin-embedded, and processed for immunohistochemicalanalyses, as described previously (8) and SupplementaryMaterialsand Methods. We used the following antibodies: anti-PCNA (Epi-tomics), anti-cleaved-caspase-3 (Imgenex), anti-CD3 (Abcam),anti-Foxp3-biotin (eBioscience), anti-F4/80 (Abcam), and anti-Ly6G (Abcam).

Treatment with the P2RX7 antagonists and depletingantibodies

Competitive P2RX7 antagonists A438079 and A740003 anddepleting antibodies were given intraperitoneally during the DSStreatment as indicated in the Supplementary Materials andMethods.

Statistical analysisAll data are represented asmean values and error bars represent

SEM. The unpaired Mann–Whitney t test and ANOVA were usedto evaluate the statistical significance between groups.

ResultsGenetic and pharmacologic inactivation of P2RX7 dampenDSS-induced colonic inflammation

The inflammatory environment generated by DSS is associatedwith the release of eATP. We reasoned that P2RX7 inactivationshould impact the overall inflammatory response andwe used theacute DSS colitis model to test this hypothesis. After AOMinjection, WT and P2rx7�/� mice were fed a 5% DSS solutionduring 5 days and then clean drinking water for 4 or 9 days. Asexpected, when considering that P2RX7 triggered inflammasomeassembly and proinflammatory cytokine release, P2rx7�/� micewere less susceptible to this treatment (Fig. 1A). P2rx7�/� micesurvived better and lost less weight. Furthermore, they showedmilder signs of colitis with less diarrhea and less rectal bleeding.

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We also observed a less dramatic shortening of the colon length atnecropsy. Such protection required themutation of the two allelessince heterozygous P2rx7þ/�mice showed the same phenotype asWTmice (Supplementary Fig. S1). Interestingly, these results werereproduced on WT mice treated with competitive P2RX7 antago-nists (Fig. 1B and Supplementary Fig. S2). Systemic blockade ofP2RX7 with A438079 or A740003 improved the survival of themice and reduced all the hallmarks of colitis. Indeed, 9 days afterDSS treatment, the loss of body weight and the disease activityscore decreased by around 25%, whereas the colon lengthincreased, as observed with P2rx7�/� mice.

Histologic analyses of colon tissues were performed to evaluatethe degree of inflammation. In the nontreated condition, bothWTand P2rx7�/� mice showed a normal colonic mucosa, with thecrypts being straight, well defined, and sitting on the muscularismucosa (Fig. 2A). At the end of the DSS regimen (day 5),histologic colonic transversal tissue sections of both WT andP2rx7�/� mice showed diffuse hemorrhagic walls with multipleulcerations, mucosal edema, transmural neutrophil infiltration,and the presence of large areas of erosion. Scoring for inflamma-tion, which demonstrated no difference between WT and KOmice, nearly reached the maximum (which was scored as 14). Incontrast, 4 days after the end of the DSS challenge (day 9),histologic mid colon tissue sections of WT mice still showed ahigh level of inflammation (12 � 0.7), whereas that of P2rx7�/�

mice showed signs of re-epithelialization, as indicated by better

delineated crypts and milder inflammation of the mucosa(inflammatory index of 5.1 � 1.5). Finally, most of the colonicmucosa of P2rx7�/� mice presented a normal aspect 9 days afterDSS treatment (day 14). In contrast, recovery of WT mice waspartial with a mean inflammatory index of 9 � 0.9 as comparedwith 2.2� 0.6 for KOmice. These differences were reproduced inWT mice treated with the P2RX7 antagonists (Fig. 2B and Sup-plementary Fig. S2). In particular, mice that received the compet-itive antagonist showed weak inflammatory infiltrates with nomucosal erosion and no epithelial cell defects within the crypts.Furthermore, histologic scoring of mid colon tissue sections ofantagonist-treated mice demonstrated a very low inflammatoryindex, which was statistically significant as compared with thecontrol group.

Taken together, these results demonstrate that both genetic andpharmacologic inactivation of P2RX7 dampen DSS-inducedcolonic inflammation. Notably, a time course analysis demon-strated that mice deficient in P2RX7 activity recovered morerapidly from inflammatory lesions than WT mice.

P2rx7�/� mice exhibit increased colonic epithelial cellproliferation and decreased apoptosis after AOM/DSStreatment

The prompt recovery of P2rx7�/� mice to acute AOM/DSSchallenge led us to evaluate whether P2RX7 regulated colonicmucosal epithelial cell proliferation. For that, colon tissue

Figure 1.Genetic and pharmacologic inactivation of P2RX7 decreases susceptibility to develop colitis. A, survival [P � 0.01, log-rank (Mantel–Cox) test], body weightloss, stool consistency, intestinal bleeding, and colon length from cohorts of 8–10 WT and P2rx7�/� mice subjected to colitis. Data are presented as means� SEM; � , P � 0.05; �� , P � 0.01. B, effect of selective P2RX7 antagonist A438079 on survival [P � 0.01, log-rank (Mantel–Cox) test], body weight loss, stoolconsistency, intestinal bleeding, and colon length. Data are presented as means � SEM; � , P � 0.05.

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sections were stained for proliferating cell nuclear antigen(PCNA). In nontreated WT and P2rx7�/� animals, quantificationof PCNA-positive cells per 30 to50well-formed crypts showed5%to 10% PCNA-positive cells (Supplementary Fig. S3A). Five daysafter acute AOM/DSS treatment, no well-defined crypts werevisible (Fig. 2B). Therefore, we evaluated cell proliferation at days9 and 14. The proliferation indices of AOM/DSS-treated animalswere significantly higher in P2rx7�/� as compared with WT mice(Fig. 3A). Such an effect could directly depend on P2RX7expressed by colonic epithelial cells (8) and (Supplementary Fig.S3B), or alternatively, depends on infiltrating immune cells. Toaddress this point, we evaluated whether this effect could bereproduced in vitroon cultivated colonic epithelial cells expressingP2RX7. Indeed, blockade of P2RX7 enhanced the in vitro prolif-eration of the T84 colonic epithelial cell line (Supplementary Fig.S3C). The effect of P2RX7 blockade on epithelial cell proliferationwas further confirmed in vivo as A438079 administration toAOM/DSS-treated mice increased colonic epithelial cell proliferation(Supplementary Fig. S3D).Wenext evaluatedwhether P2RX7alsoregulates apoptosis of intestinal epithelial cells. For this, inflam-matory tissue lesions were stained with cleaved caspase-3 anti-body. We observed significantly less apoptotic cells in P2rx7�/�

mice at day 9 and their complete absence at day 14 after AOM/DSS

treatment (Fig. 3B). We then examined the expression of anti-apoptotic genes. No changes were observed in the expression ofBAX. In contrast, a significant increase in the expression of thegenes encoding BCL2 and BCL2L1 (Bcl-xL) was observed in thewhole colon of P2rx7�/� mice at day 14 after AOM/DSS admin-istration (Fig. 3C). Finally, the increase in epithelial cell prolifer-ation correlated with dysplasia in the colonic mucosa of P2rx7�/�

mice. We consistently found abnormal crypts characterized bymisalignment of the epithelial nuclei and a partial or total loss ofmucosal secretion (Fig. 3D). P2rx7�/� mice were particularlyprone to develop dysplasia, as they were also present in micethat received a short and low dose ofDSS (Supplementary Fig. S4)that induced no signs of colitis and only a very low inflammatoryindex. Taken together, these results demonstrate that the func-tional activity of the P2RX7 receptor is required to control the levelof intestinal epithelial cell proliferation and apoptosis in responseto an inflammatory insult and suggest that alteration in P2RX7functionality may favor possibly tumor progression.

P2RX7 mediates gastrointestinal tumorigenesis in the colitis-associated cancer model

To investigate the effect of P2RX7 on inflammation-inducedcolorectal cancer, azoxymethane-challenged mice received 3

Figure 2.Mice deficient in the P2RX7 activity recovered more rapidly from inflammatory lesions than WT mice. A, WT and P2rx7�/� were submitted to colitis. Colonswere removed either at the end of the DSS treatment (day 5), 4 days after (day 9), or 9 days after (day 14) treatment. Tissue sections were stained with hematoxylinand eosin and analyzed microscopically (scale bar, 100 mm). Histologic scoring of mid colon tissue sections was calculated as indicated in the SupplementaryMaterials and Methods. Data are presented as means � SEM; � , P � 0.05. B, nontreated and A438079-treated WT mice were submitted to colitis. Colontissue sections were removed and stained with hematoxylin and eosin either at the end of the DSS treatment or 4 days after (day 9) treatment. Histologic scoring ofmid colon tissue sections is shown. Data are presented as means � SEM; �� , P � 0.01.

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cycles of DSS to mimic chronic colitis (Fig. 4A). Both WT andP2rx7�/� mice survived azoxymethane-DSS treatment andshowed comparable disease activity indices during the first 10weeks of treatment. However, P2rx7�/� mice displayed a higherdisease activity scores at the end of the protocol (SupplementaryFig. S5). Unexpectedly, despite their lower susceptibility to acuteDSS-induced inflammation, P2rx7�/� mice developed signifi-cantly higher numbers of macroscopic polyps (mean of 9 polypsper mouse) as compared with WT mice (mean of 2 polyps permouse). As expected for the CAC model, tumors were located atthe distal end of the colon inWTmice, whereas they invaded up tothe medial part of the colon in P2rx7�/� mice (Fig. 4A). Inaddition, the 4-fold increase in tumor burden in P2rx7�/� micewas accompanied by a 2-fold increase in polyp size (Fig. 4B).Histologic examination of colons from WT and P2RX7-deficientmice showed that 80% of the KOmice developed colonic lesionsranging fromhyperplasia and dysplasia to adenocarcinoma (Sup-plementary Fig. S6A). Adenocarcinoma was observed in morethan 60% of KO mice, whereas only 25% of the WT cohortdisplayed a carcinoma.

Furthermore, we investigated the pharmacologic effect ofP2RX7 blockade in the CAC model using the A438079-compet-itive antagonist. Changes in bodyweightwere followedduring theentire protocol and the colonic tumor burden was monitored 10weeks after AOM injection. As observed for WT and P2rx7�/�

mice, antagonist-treated and nontreated mice displayed compa-rable body weight variations during the course of the treatment(Supplementary Fig. S5, bottom). However, A438079-treatedmice harbored higher numbers of macroscopic polyps andtumors were significantly larger in size than in control mice (Fig.4C). Collectively, these results demonstrate that alteration of theP2RX7 functionality enhanced tumor promotion and/or progres-sion. Interestingly, tumor progression was also found to becontrolled by P2RX7 in an unrelated tumor mouse model basedon subcutaneous injection of Lewis Lung Carcinoma (LLC) cells.As shown in Supplementary Fig. S6B, mice injected with LLC cellsdeveloped tumors within 2 weeks. Importantly, excised tumorsfrom P2rx7�/� mice were bigger than tumors from WT miceconfirming that P2RX7 behaved as a tumor suppressor in thistumorigenic mouse model.

Figure 3.P2RX7-dependent signaling prevented early excessive colonic epithelial cell proliferation following AOM/DSS treatment. A, cohorts of 8–10 WT and P2rx7�/� micewere treated for colitis and epithelial cell proliferation was evaluated by PCNA staining performed on colon tissue 4 and 9 days after DSS treatment (day 9and day 14, respectively). Representative results are shown on the panels on the left and the number of proliferating cells (PCNA positive, arrows) per cryptwas determined at day 9 and 14 (right). Arrows, crypts containing epithelial cells with PCNA nuclear staining. A higher magnification is shown in the inset. B,detection of the cleaved form of caspase-3 (arrows). Representative pictures are shown on the left. Quantification of the number of apoptotic cells at day 9 and 14 isshown on the right. A higher magnification is shown in the insets. C, immunoblot analysis of BCL2, BCL2L1 (Bcl-XL), and BAX expression in the colon tissues ofWT andP2rx7�/� mice, treated for colitis, were performed at day 14. The total actin (ACTB) level was monitored as a control for equal protein loading. D, quantificationof the number of low dysplasia in animals. Data, shown as arbitrary unit, are representative of two independent experiments (mean� SEM). � , P� 0.05; �� , P� 0.01.






P2RX7 shapes the inflammatory microenvironmentP2RX7 stimulation is known to trigger inflammasome assem-

bly and casp1 activation and to favor the release of the processedmature form of IL1B, thereby initiating the inflammatoryresponse (4, 10). As the early level of inflammation can influencethe quality and the quantity of recruited immune cells, we nextcharacterized the inflammatory microenvironment before tumoronset.We first checked the capacity of colonic explants from acuteDSS-treatedmice to secrete IL1B. After in vivoDSS treatment, IL1Bwas readily produced ex vivo in colon tissue cultures derived fromWT mice (Fig. 5A). In contrast, and not surprisingly, very littleIL1B was produced by colonic explants from DSS-treatedP2rx7�/� mice. In agreement with their lowered inflammatoryresponse, we also observed significantly lower activation of STAT3in P2RX7-deficient mice at day 14 after DSS treatment as com-pared with WT control mice (Fig. 5B).

We next evaluated how the lower P2RX7-dependent IL1Bproduction influenced the composition of the inflammatoryinfiltrate in the colon of DSS-treated mice. For that, colon tissuesections from acute DSS-treated mice were stained for markersspecific for T lymphocytes (CD3), Treg lymphocytes [FOXP3(Foxp3)], macrophages [EMR1 (F4/80)], and neutrophils(LY6G). Colonic-lamina propria tissue sections from P2rx7�/�

mice displayed statistically more CD3þ (9% vs. 3%), FOXP3þ

(15% vs. 3%), and EMR1þ (F4/80) (16% vs. 5%) cells as com-pared with WT animals (Fig. 5C). In contrast, no differencebetween the two groups of mice was observed in regard to

LY6G-positive cells. However, we noticed that colon tissue sec-tions from both groups harbored a significantly high number ofneutrophils (reaching 8% to 10% of total immune cells). Thisobservation is particularly relevant to the field of IBD pathogen-esis. Indeed, acute inflammatory lesions of patients with IBD arecharacterized by a large influx of neutrophils within the intestinalmucosa (23). Keeping in mind the recent concept of the role ofneutrophils in tumor biology (24), we wondered whether theP2RX7 activity participated in the polarization of tumor-associ-ated neutrophils (TAN). To assess this question, we analyzed theexpression of genes encoding chemokines that have been used todistinguish TAN from other tumor-associated myeloid cells (25).As illustrated in Fig. 5D, the P2RX7 deficiency resulted inincreased expression of the Ccl17 transcript and decreased expres-sion of Cxcl10, a molecular profile that has been associated withgenuine TAN (24, 25).

Enhanced production of TGFB1 in the colonic mucosa ofAOM/DSS-treated P2rx7�/� mice

TGFB1 is frequently associated with poor prognosis in patientswith cancer (26). This immunoregulatory cytokine can be pro-duced by immune as well as tumor cells and has been implicatedin the polarization of various immune cell subsets but also in theestablishment of a suppressive microenvironment that favorstumor growth (27). The accumulation of immunosuppressivecells within the inflammatory lesions of mutant mice could

Figure 4.The P2RX7 activity impeded tumorigenesis in the colitis-associated cancer model. A, schematic representation of CAC. B, CAC-treated WT and P2rx7�/� micewere euthanized and colons were resected and opened longitudinally to visualize angiogenic (arrows) and nonangiogenic polyps (star). Macroscopic polypswere then counted, sized, and colon tissue sections were fixed in formalin and stained with hematoxylin/eosin for histologic examination. C, the CAC protocol wasapplied to cohorts of ten nontreated and A438079-treated mice. Colons were resected and macroscopic polyps were counted and sized. Data are presentedas means � SEM; � , P � 0.05; �� , P � 0.01.

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possibly be associated with the local production of TGFB1.Indeed, a 3-fold induction of TGFB1 production was observedin acute inflammatory of colon explant culture from P2rx7�/�

versus WT mice (Fig. 6A). These results were confirmed at latertime points, as P2rx7�/� mice subjected to the chronic AOM/DSStreatment displayed an overexpression of Tgfb1 and Tgfb2 tran-scripts, and their cultured explanted colonic tissue showedincreased secretion of the TGFB1 cytokine (Fig. 6B).

As TGFB1 is known to promote the suppressive activity ofFOXP3þ Tregs, we further characterized the composition of thecellular infiltrate in colon tissue biopsies from chronic AOM/DSS-treated mice. As described before for the inflammatory colonicmucosa, a significant increase in the content of FOXP3þ cells wasobserved within tumors derived from P2rx7�/� mice (Fig. 6C),whereas there was no difference in the numbers of macrophages(EMR1, F4/80þ cells) nor total T lymphocytes (CD3þ cells). AsTGFB1 has been proposed to control differentiation of TAN inmice (24), we next investigated the presence of neutrophil infil-tration in their colonic mucosa. We found an enrichment in theproportion of LY6Gþ cells in P2rx7�/� mice (Fig. 6C), in agree-ment with the higher concentration of CXCL1 and CXCL2, twochemokines involved in neutrophil recruitment, in the serum ofthese animals (Supplementary Fig. S7A). Interestingly, neutrophilenrichment appeared to be restricted to colonic tumor tissues asanalysis of bone marrow and spleen cells did not show anydifference in the composition of immune cells (SupplementaryFig. S7B). Moreover, the cytokine profile observed in tumor

lesions of P2rx7�/�mice is compatible with the molecular profileassociated with genuine TAN (Supplementary Fig. S7C; ref. 25).

In addition to its immunosuppressive function, TGFB1 hasbeen reported to increase proliferation of colonic epithelial cells(28). Therefore, overproduction of TGFB1 in inflammatory andtumor lesions of P2rx7�/� mice may account for the increasedproliferation of epithelial cells observed in this strain (Fig. 3). Totest this hypothesis, neutralizing anti-TGFB antibody was injectedin AOM/DSS-treated P2rx7�/� mice. As shown in Fig. 7A, bodyweight and colon length were not significantly different in micetreated with isotype control or with the anti-TGFB antibodies. Incontrast, the number of colonic epithelial cells positive for PCNAstaining was reduced by 2-fold in mice that received the neutral-izing anti-TGFB antibody (Fig. 7B). Indeed, while 35% � 4% ofintestinal epithelial cells were PCNAþ in mice treated with theisotype control antibody, only 15%� 2%of cells were detected tobe positive in mice treated with the neutralizing antibody, a levelcomparable to what was observed in WT mice (see Fig. 3).

Taken together, these results suggest that enhanced secretion ofTGFB1 in P2RX7�/� favors the establishment of an immunosup-pressive microenvironment and stimulates epithelial cell prolif-eration, thereby enhancing the development of colon cancer.

DiscussionP2RX7 is an ATP gated ion channel that can induce multiple

cellular responses. In the early 1990s, studies demonstrated cell-permeabilizing and cytolytic effects of P2RX7 in response to high

Figure 5.P2RX7 shapes the inflammatorymicroenvironment. A, cohorts of 8–10WT and P2rx7�/� mice were treatedfor colitis. Expression levels of Il1bmRNA and of the correspondingsecreted protein (IL1B) weredetermined by RT-qPCR and ELISA. B,proteins were extracted from the midto distal colon tissue sections of DSS-treated WT and P2rx7�/� mice. Theactivation of STAT3 was analyzed byimmunoblotting using an anti-phospho-STAT3 antibody. The totalactin (ACTB) level was monitored as acontrol for equal protein loading. Dataare presented as means � SEM;� , P � 0.05. C, immunohistochemistrywas performed on day 9 after DSStreatment. The quality of infiltratingcells was evaluated using antibodiesdirected against characteristic cellmarkers: anti-CD3 (T cells), anti-FOXP3 (Foxp3, Tregs), EMR1 (F4/80,macrophages), and anti-LY6G(Ly6G, neutrophils). Results arepresented as means � SEM;� , P � 0.05; �� , P � 0.01. D, theexpression of Ccl17 and Cxcl10(IP-10) was examined by RT-qPCR.Data are presented as means� SEM; � , P � 0.05.






doses of eATP (29, 30). A few years later, it appeared thatstimulation of P2RX7 by low doses of eATP enhanced prolifer-ation of human primary cells (31). This observation was furtherconfirmed in vitro using heterologous expression systems (32) aswell as in vivo using xenograft tumor models (16). P2RX7appeared to be overexpressed in a variety of malignant tumors(reviewed in ref. 33).However, P2RX7-dependent signaling couldalso favor an antitumor effect in vivo. Indeed, P2RX7 participatesin inflammasome assembly and may thereby alert the immunesystem through processing and liberation of the proinflammatoryIL1B cytokine (6). It has been demonstrated that P2RX7 plays acrucial role in the immunogenicity of dying tumor cells inresponse to chemotherapy (a process named immunogenic celldeath) through the activation of dendritic cells and the conse-quent promotion of an adaptive antitumor immune responsethat prevents tumor relapse (18). Of particular interest, in spo-radic breast cancer treated with anthracyclines, patients bearingloss-of-function alleles of P2RX7 (rs3751143, NM_002562.4:

c.1487A>C) are more prone to develop metastases than patientswithout mutation. Such results fuel the hypothesis that P2RX7plays an important role in the antitumor response.

In this study, we conducted experiments in the context of acuteand chronic colitis with the aim of examining the global in vivocontribution of P2XR7 signaling to inflammation and cancerprogression. Using a mouse model of acute AOM/DSS-inducedcolonic inflammation, we found that P2rx7�/� mice displayedreduced signs of inflammation. This finding is in agreement withrecent studies demonstrating that prophylactic systemic blockadeof the P2RX7 activity prevented TNBS-induced colitis in rats (12)and that overexpression of P2RX7 in the intestinal mucosa wasassociated with the pathogenesis of CD (34). In the current study,we found that P2RX7 inactivation using selective antagonists aswell as genetic deletion of the P2rx7 gene attenuated the weightloss and the overall disease activity score (Fig. 1). However,whereas the histologic inflammatory score ofDSS-treated animalswas similar in bothWT and P2rx7�/�mice at day 5 (i.e., at the end

Figure 6.Increased expression of TGFB1 in colontumor biopsies from P2rx7�/�mice. A,cohorts of 8–10WT and P2rx7�/�micewere treated for colitis. Secretion ofTGFB1 was assayed by ELISA onsupernatants from organocultures.Data are expressed as mean� SEM; � , P� 0.05. B, Tgfb1 and Tgfb2transcripts were analyzed by RT-qPCRand the levels of secreted TGFB1 byELISA from CAC-treated cohorts of8–10 WT and P2rx7�/� mice. Data areexpressed asmean� SEM; � , P� 0.05.B, the number of CD3-, FOXP3(Foxp3)-, EMR1 (F4/80)-, and LY6G(Ly6G)-positive cells was determined.Arrows, positive cells. Data arepresented as means � SEM;� , P � 0.05 (scale bar, 100 mm).

Hofman et al.





of DSS treatment), we paradoxically observed a faster re-epithe-lialization of the digestive mucosa of P2rx7�/� mice during therecovery phase (Fig. 2).

Considering that the faster recovery of P2rx7�/�mice correlatedwith enhanced epithelial cell proliferation, reduced apoptosis,and higher expression of BCL2L1 (Bcl-xL) and BCL2 survivalfactors (Fig. 3), our results support the notion that P2RX7 playsa role in the control of epithelial cell proliferation and/or apo-ptosis in response to injury. We indeed confirmed in vivo inP2rx7�/� mice as well as in WT mice treated with P2RX7 phar-macologic inhibitors, that P2RX7 inactivation increases epithelialcells proliferative index (Fig. 3 and Supplementary Fig. S3D).Concordantly, we confirmed in vitro using the human T84 epi-thelial cell line derived from a colon cancer that pharmacologicinhibition of P2RX7 significantly stimulates cell proliferation(Supplementary Fig. S3C). This results support the notion thatP2RX7 activity directly inhibit epithelial cell proliferation.Wheth-er immune cells, that we found to be recruited at higher level inP2RX7-inactivated mice (i.e, FOXP3þ Tregs and LY6Gþ neutro-

phils) also contribute to the control of epithelial cell proliferationneed to be further address in future studies.

Our finding, which demonstrated that a lack of P2RX7 is linkedto increased epithelial cell proliferation, may explain, at leastpartly, the increased tumor burden observed in chronic AOM/DSS–treated P2rx7�/� mice (Fig. 4). Another nonexclusive mech-anistic explanation may reside downstream of IL1B signaling. Asexpected, little if any IL1B was detected from the colonic mucosaof P2rx7�/� mice at early time points following AOM/DSS treat-ment (Fig. 5). This was associated with reduced STAT3 phosphor-ylation, whichmay indicate lower activation of immune cells andmay favor the establishment of an immunosuppressive microen-vironment prone to tumor development. However, downregula-tion of IL1B and STAT3 phosphorylation could not represent thesolemechanismas it has been reported that genetic inactivation ofIL1BR or of STAT3 in the intestinal epithelial cell compartment(DIECStat3 mice) decreased tumor development (35, 36).

In addition,we found that FOXP3þ Tregs, a cell population thathas been associated with colon cancer progression (37), was

Figure 7.Treatment with anti-TGFB antibodyinhibited colon epithelial cellproliferation in AOM/DSS–treatedP2rx7�/� mice. Cohorts of 6 P2rx7�/�

mice were injected with depletingantibodies and treated for colitis. A,body weight loss and colon lengthwere analyzed as described in thelegend of Fig. 1. Data are presented asmeans � SEM. Results are notstatistically different. B, epithelial cellproliferation was evaluated by PCNA-staining performed on colon tissuesections 9 days after DSS treatment.Representative results are shown onthe panels on the left. The number ofproliferating cells is shownon the rightpanel. A highermagnification is shownon the insets. Data are presented asmeans � SEM. �� , P � 0.01.






increased in the colonic mucosa of acute AOM/DSS-treatedP2rx7�/� mice (Fig. 5C) in association with lower STAT3 phos-phorylation (Fig. 5B) and higher TGFB1 production (Fig. 6).Interestingly, we previously demonstrated that P2RX7 activationat the surface of Tregs inhibited their suppressive function andinduced cell death (38, 39). Therefore, inactivation of P2RX7should protect Tregs from the deleterious effect of endogenousP2RX7 ligands (i.e., eATP and NAD) and should promote theirearly accumulation in the damaged colonic mucosa, as observedin the current study (Fig. 5C). In addition, local production ofTGFBmay further enhance their suppressive activity andmay evenparticipate in the conversion of conventional CD4þ T cells toinduced Tregs (iTregs). Of note, several reports have linked TGFB1to negative regulation of tumor-specific cytotoxic T lymphocyteresponses (40–44). Therefore, upregulation of TGFB productionin the colonic mucosa of AOM/DSS-treated P2rx7�/� mice mayalso inhibit tumor-specific cytotoxic T cells activity and therebyfurther participates in tumor promotion. In addition to its immu-nosuppressive role, TGFB has been reported to enhance prolifer-ation of epithelial cells (28). Concordantly, we show here thattreatment of DSS-treated P2rx7�/� mice with an anti-TGFB anti-body significantly reduced the proliferation of colonic epithelialcells (Fig. 7B).

During the course of this study, we also observed the recruit-ment of LY6Gþ neutrophils within tumor lesions of P2rx7�/�

mice. These cells shared the characteristics of genuine TAN (Sup-plementary Fig. S7), a population of cell that has been recentlyassociated to tumor progression (24). Therefore, we cannotexclude that TAN also participate in the reinforcement of theimmunosuppressive environment in the colonic mucosa ofAOM/DSS-treated P2rx7�/�mice and in the stimulation of tumorgrowth.

We demonstrate in our study that P2RX7 inactivation dampensinflammation but paradoxically enhances tumor susceptibility.Interestingly, we reproduced this finding in an independenttumor mouse model consisting of subcutaneous injection of LLCcells into C57BL/6 mice (Supplementary Fig. S6B). Several poly-morphisms have been described for the P2RX7 gene and theirimpact on the P2RX7 function, the mechanisms and relationshipwith diseases were characterized for a number of mutants (45–47). Surprisingly, none of these variants were found in a meta-analysis of IBD (CD and UC) that included a Genome-WideAssociation study (48). However, this analysis highlighted theimportance of TGFB signaling in IBD and reinforced the link withcolorectal cancer. Furthermore, P2RX7 was reported to be overexpressed in chronic B lymphocytic leukemia (19), prostate cancer

and adjacent tissue (21), papillary thyroid carcinoma (49) andgastric, colon, kidney, and ovarian cancers (16). However, despitethe large number of P2RX7 mutants and of P2RX7 variants, noattempts were made to characterize their functionality in tumorlesions, highlighting the need for additional studies to provide abetter understanding of the role of P2RX7 in cancer onset.

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

Authors' ContributionsConception and design: P. Hofman, X. H�ebuterne, S. Adriouch, V. Vouret-CraviariDevelopment of methodology: P. Hofman, T. Juhel, E. Gilson, V. Vouret-CraviariAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Cherfils-Vicini, M. Bazin, O. BoyerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Hofman, J. Cherfils-Vicini, M. Bazin, M. Ilie,V. Vouret-CraviariAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Bazin, E. GilsonWriting, review, and/or revision of the manuscript: P. Hofman, J. Cherfils-Vicini, M. Ilie, X. H�ebuterne, O. Boyer, S. Adriouch, V. Vouret-CraviariAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Schmid-Allilana, V. Vouret-CraviariStudy supervision: P. Hofman, V. Vouret-Craviari

AcknowledgmentsThe authors thank Salom�e Lalvee and Oliver Bordone (LPCE, Nice, France)

for their expert technical assistance and Ludovic Cervera (Cytomed platform,funded by the CG06, INSERM and FEDER) for his expertise in flow cytometry.The authors thank Sandrine Marchetti for helpful discussion. The authorsacknowledge the IRCAN's PICMI, CYTOMED, and Animal core facilities.

Grant SupportThis workwas supported by grants from "InstitutNational de la Sant�e et de la

RechercheM�edicale," the "Associationpour la Recherche contre leCancer" (ARCGrants SL220110603478), the Canc�eropole PACA, the "Centre National de laRecherche Scientifique," the "Ligue Nationale contre le Cancer (E. Gibson:Equipe lab�elis�ee), and the FrenchGovernment (National ResearchAgency, ANRthrough the "Investments for the Future" LABEX SIGNALIFE: program reference#ANR-11-LABX-0028-01).

The costs of publication of this articlewere defrayed inpart 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 13, 2014; revised November 5, 2014; accepted November 26,2014; published OnlineFirst January 6, 2015.

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2015;75:835-845. Published OnlineFirst January 6, 2015.Cancer Res Paul Hofman, Julien Cherfils-Vicini, Marie Bazin, et al. a Mouse Model of Colitis-Associated CancerReceptor Dampens Inflammation but Increases Tumor Incidence in Genetic and Pharmacological Inactivation of the Purinergic P2RX7

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