Interleukin-6 Is Required for Pancreatic Cancer …...Tumor and Stem Cell Biology Interleukin-6 Is Required for Pancreatic Cancer Progression by Promoting MAPK Signaling Activation

Tumor and Stem Cell Biology

Interleukin-6 Is Required for Pancreatic Cancer Progressionby Promoting MAPK Signaling Activation and OxidativeStress Resistance

Yaqing Zhang1, Wei Yan2,5, Meredith A. Collins6, Filip Bednar1, Sabita Rakshit1, Bruce R. Zetter8,Ben Z. Stanger9,10, Ivy Chung11,12, Andrew D. Rhim4, and Marina Pasca di Magliano1,3,7

AbstractPancreatic cancer, one of the deadliest human malignancies, is almost invariably associated with the

presence of an oncogenic form of Kras. Mice expressing oncogenic Kras in the pancreas recapitulate thestepwise progression of the human disease. The inflammatory cytokine interleukin (IL)-6 is often expressedby multiple cell types within the tumor microenvironment. Here, we show that IL-6 is required for themaintenance and progression of pancreatic cancer precursor lesions. In fact, the lack of IL-6 completelyablates cancer progression even in presence of oncogenic Kras. Mechanistically, we show that IL-6synergizes with oncogenic Kras to activate the reactive oxygen species detoxification program downstreamof the mitogen-activated protein kinase/extracellular signal—regulated kinase (MAPK/ERK) signalingcascade. In addition, IL-6 regulates the inflammatory microenvironment of pancreatic cancer throughoutits progression, providing several signals that are essential for carcinogenesis. Thus, IL-6 emerges as a keyplayer at all stages of pancreatic carcinogenesis and a potential therapeutic target. Cancer Res; 73(20); 6359–74.�2013 AACR.

IntroductionPancreatic cancer is one of the deadliest human malig-

nancies, with a 5-year survival rate of less than 6% (1–3).The dismal survival rate has remained essentiallyunchanged over the course of the past 40 years, highlightingthe need for a deeper understanding of the biology of thisdisease, which might lead to new targeting strategies.Recent sequencing studies (4) have confirmed the decadesold observation that KRAS is the most commonly mutatedgene in pancreatic cancer (5, 6). KRASmutations occur early

during disease progression, in pancreatic cancer precursorlesions known as pancreatic intraepithelial neoplasias(PanIN; ref. 7). However, KRAS mutations are found inhealthy pancreata at a much higher rate than the incidenceof pancreatic cancer (8, 9), suggesting that additionalgenetic, epigenetic, or environmental factors are requiredfor tumorigenesis.

Chronic pancreatitis confers a significantly increased life-time risk to develop pancreatic cancer, and is thus one of thehighest known risk factors (10). The relationship betweenacute pancreatitis and carcinogenesis is less well established;however, acute pancreatitis can progress to chronic pancre-atitis in individuals carrying additional risk factors (such assmoking or alcohol abuse; ref. 11). In mice, both chronic andacute pancreatitis synergize with the presence of oncogenicKras to drive formation of PanINs (12, 13). The cytokineinterleukin (IL)-6 is upregulated during pancreatitis in miceand humans (14). IL-6 plays an essential procarcinogenicfunction in colon and liver cancer (15, 16). In contrast, at leastin mice, its role is secondary to the closely related cytokineIL-11 in gastric cancer (17). Thus, the relevance of IL-6 incarcinogenesis is tissue-specific. Previous studies have identi-fied IL-6 and its downstream effector Stat3 as being importantfor pancreatic cancer initiation inmousemodels of this disease(18–20). However, whether IL-6 plays a role in inflammation-driven pancreatic carcinogenesis, as well as its role at laterstages of carcinogenesis, was not known. These questions havetherapeutic relevance, as patients with pancreatitis are a popu-lation where preventive strategies could be successfully usedto avoid progression to cancer. Preventive strategies that

Authors' Affiliations: Departments of 1Surgery, 2Pathology, and 3Cell andDevelopmental Biology, 4Department of Internal Medicine-Gastroenterol-ogy, 5MichiganCenter for Translational Pathology, 6Program inCellular andMolecular Biology, 7Comprehensive Cancer Center, University of Michi-gan, Ann Arbor, Michigan; 8Vascular Biology Program, Department ofSurgery, Karp Family Research Laboratories, Children's Hospital, Boston,Massachusetts; 9Gastroenterology Division, 10Abramson Cancer Center,Perelman School of Medicine, University of Pennsylvania, Philadelphia,Pennsylvania; 11Department of Pharmacology, and 12University of MalayaCancer Research Institute, Faculty ofMedicine, University ofMalaya, KualaLumpur, Malaysia

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

Current address for W. Yan, Department of Pathology, Xijing Hospital,Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.

Corresponding Author: Marina Pasca di Magliano, Department ofSurgery, Cell and Developmental Biology, University of Michigan, 1500EMedical Center Drive, Ann Arbor, MI 48109-5936. Phone: 734-615-7424;Fax: 734-647-9654; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-1558-T

�2013 American Association for Cancer Research.

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block PanIN progression to cancer could conceivably also beuseful in familial pancreatic cancer, as well as to preventrecurrence in patients who have undergone resection of theprimary tumor.

In this study, we set out to determine whether sustainedIL-6 expression was required to initiate pancreatitis-asso-ciated pancreatic cancer. We used a genetically engineeredmouse model of pancreatic cancer, the iKras� mouse, basedon pancreas-specific, inducible, and reversible expressionof oncogenic KrasG12D (Kras�) recently described by ourgroup (21). This model develops pancreatic cancer in a step-wise manner within an intact microenvironment. Our datashow that IL-6 was dispensable for the initiation of pan-creatic cancer precursor lesions in the presence of inflam-mation. However, we uncovered a previously unrecognizedrole for IL-6 in the maintenance of these precursor lesionsand progression to cancer. Thus, our data set the rationalefor exploring IL-6 as a therapeutic target in pancreaticcancer.

Materials and MethodsMouse strains

We generated iKras�;IL-6�/� mice by crossing previouslydescribed triple-transgenic mice iKras� (p48-Cre;R26-rtTa-IRES-EGFP;TetO-KrasG12D; ref. 21) with IL-6–deficient mice(B6;129S6-Il6tm1Kopf; The Jackson Laboratory). Combinations ofsingle- or double-mutant littermates were used as controls.Animals were housed in specific pathogen-free facilities ofthe University of Michigan Comprehensive Cancer Center(Ann Arbor, MI). Studies were carried out in compliancewith University of Michigan University Committee on Use andCare of Animals guidelines. Pdx1-Cre;KrasLSL-G12D/þ;p53fl/þ;Rosa26LSL-YFP/þ (KPCY) mice (22) were housed in a specificpathogen-free facility at the University of Pennsylvania(Philadelphia, PA) and in compliance with Penn InstitutionalAnimal Care and Use Committee guidelines.

Doxycycline treatmentiKras� or iKras�;IL-6�/�mice were treated with doxycycline

to induce KrasG12D expression. Doxycycline was administeredin the low-dose doxycycline chow (50 mg/kg) or drinkingwater, at a concentration of 0.2 g/L in a solution of 5% sucrose,and replaced every 3 to 4 days.

Primary tumor cellsPrimary tumor specimen implantation and preparation of

single-cell suspensions of tumor cells were carried out aspreviously described (23). All samples derived from humansubjects were approved by University of Michigan and theFederal Institutional Review Board. Establishment of primarymouse pancreatic cancer cell line from iKras� p53� mousetumor was previously described (24).

Histopathologic and histologic analysisThe histopathologic analysis was conducted as previously

described (21). The data were expressed as percentage of totalcounted clusters. Error bars represent SE.

Flow cytometrySingle-cell suspensions of fresh spleen and pancreas were

prepared as follows: spleens were crushed and passed througha 40-mm cell strainer, washed once with RPMI/10% fetal calfserum (FCS), and treated with RBC lysis buffer (eBioscience) toeliminate RBC. Pancreata were minced using sterile scalpels,then incubated in 1 mg/mL collagenase (Sigma-Aldrich) inHank's Balanced Salt Solution (HBSS) for 15 minutes at 37�Cbefore passing through a 40-mm cell strainer. Single-cell sus-pensions were stained in HBSS/2% FCS with the followingantibodies: CD3 (17A2), CD4 (RM4-5), CD8a (53-6.7), CD25(PC61), CD11b (M1/70), F4/80 (BM8), CD11c (HL3), Gr-1 (RB6-8C5), Foxp3 (FJK-16s; all from BD Pharmingen), and CD45(MCD4530; Invitrogen). Flow cytometry was conducted using aCyAn ADP Analyzer (Beckman Coulter), and data were ana-lyzed with Summit 4.3 software.

Reactive oxygen species induction and detectionPrimary mouse pancreatic cancer cell line 9805 was treated

with 600 mmol/L H2O2 for 1 hour and the presence of reactiveoxygen species (ROS) was detected using CellROX Greenreagent (C10444; Invitrogen), a fluorogenic probe, accordingto the manufacturer's instructions. Briefly, cells were thenincubated with 10 mmol/L CellROX Green in RPMI with 10%FBS for 30 minutes at 37�C. Cells were then washed in PBS andimaged on a Olympus IX71 inverted microscope. Signal inten-sity was analyzed using Image-Pro Plus software.

In vivo anti-IL-6 treatmentKPCY mice aged 10 weeks were randomized to two treat-

ment arms: anti-IL-6 antibody treatment (25 mg/kg; cloneMP5-20F3; BioXCell) or rat immunoglobulin G1 (IgG1) control(25 mg/kg; BE0088; BioXCell). Mice (3 for each group) receivedtreatments on days 0, 2, 4, and 6 intraperitoneally and werethen sacrificed on day 7 for histologic analysis. PanIN lesions inthree sections per mouse were quantified by grade in a blindedmanner. Data are expressed as number of acinar-ductal meta-plasias (ADM) or PanINs of each grade per medium (10�)powered field. No discernible side effects were noted in eitherof the treatment groups.

Statistical analysisAll data were presented as mean � SEM. Intergroup com-

parisons were conducted using the Student t test. Prism 6 wasused for all statistical analyses, and P < 0.05 was consideredstatistically significant.

Detailed procedures and standard procedures are includedin the Supplementary Methods; detailed antibody informationand primer sequences are listed in Supplementary Tables S1and S2.

ResultsIL-6 is expressed by several cell types within thepancreatic cancer microenvironment

Analysis of human and mouse pancreatic cancer samplesrevealed IL-6 immunostaining in several cell compartments,including epithelial cells, smooth muscle actin (SMA)–positivefibroblasts, and immune cells (Supplementary Fig. S1A and

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S1B). Interestingly, IL-6 was expressed in mouse primarypancreatic fibroblasts only upon incubation with conditionedmedium from pancreatic cancer cells (Supplementary Fig.S1C). Because previous studies had described expressionrestricted to immune cells (18), we sought to confirm theimmunostaining results by quantitative real-time PCR(qRT-PCR), and observed that primary mouse pancreaticfibroblasts in culture expressed Il6 mRNA when exposed toconditioned medium from pancreatic cancer cells (Supple-mentary Fig. S1D). Moreover, one of two primary pancreaticcancer cells and three commercially available pancreatic can-cer cells lines tested expressed IL-6 (Supplementary Fig. S1E).Thus, multiple sources of IL-6 are present within the pancrea-tic cancer microenvironment.

IL-6 is required for PanIN formation in iKras� mice withembryonic Kras activationIn iKras� mice, the expression of oncogenic Kras can be

timed at will by adding or removing doxycycline from theanimal's food or water (21). We previously reported thatactivation of KrasG12D in adult animals leads to PanIN forma-tion with low penetrance and long latency (21). In contrast,embryonic activation of KrasG12D (Supplementary Fig. S1F)resulted in PanIN formation in all the animals by 6weeks of age(Supplementary Fig. S1I). To determine the effect of IL-6inactivation on PanIN formation in iKras� mice, we generatediKras�;IL-6�/� mice (Fig. 1A). iKras�;IL-6�/� and iKras� litter-mates were sacrificed at 6 weeks of age and their pancreatawere harvested (Supplementary Fig. S1F). The expression ofthe KrasG12D transgene was comparable in iKras� and iKras�;IL-6�/� mice, and undetectable in wild-type controls. Il6mRNA was elevated in iKras� pancreata compared with con-trol, and, as expected, undetectable in iKras�;IL-6�/� mice(Supplementary Fig. S1G). Histologic examination of iKras�

pancreata revealed PanIN lesions surrounded by extensivefibro-inflammatory stroma (Supplementary Fig. S1I). Wedetected IL-6 expression, as well as activation of the down-stream effector Stat3, both in the lesions and in the surround-ing stroma. Moreover, we detected elevated expression ofphosphorylated extracellular signal–regulated kinase 1/2(p-ERK1/2), as readout of mitogen-activated protein kinase(MAPK) pathway activity. Similar findings were obtained iniKras�;IL-6þ/� mice. In iKras�;IL-6�/� pancreata, we observeda majority of normal acini with infrequent areas of ADM andrare PanINs (Supplementary Fig. S1I; and histopathologicanalysis in Supplementary Fig. S1H). As expected, IL-6 expres-sion was completely abrogated in these tissues. Interestingly,the residual lesions in iKras� ;IL-6�/� mice had reducedp-ERK1/2 and p-Stat3 compared with lesions in iKras� mice.Thus, in the iKras� model, IL-6 is important for the onset ofPanINs. These findings are consistent with the observationthat IL-6 is important for pancreatic cancer initiation (18).

Pancreatitis-driven PanINs in IL-6–deficient mice havealtered signaling and reduced proliferationIn a next set of experiments, we let the iKras� and iKras;

IL-6�/� mice reach adulthood in absence of doxycycline,thus maintaining Kras� expression off. Doxycycline was then

administered when the animals reached 4 to 6 weeks of age, toinduce Kras� expression, and pancreatitis was induced bytwo series of caerulein injections, for 2 consecutive days,starting 72 hours after doxycycline administration, as previ-ously described (Fig. 1B; refs. 12, 21). One day later, pancreatitiswas evident in control, iKras� and iKras�;IL-6�/� pancreata,with characteristic acinar damage, ADM, edema, and inflam-matory infiltrates, as well as increased expression of p-ERK1/2and p-Stat3 (Fig. 1C). Flow cytometry showed that the numberof infiltrating CD45þ immune cells was similar in all theexperimental groups; although we observed a trend towardreduced infiltration of T cells and a significant increase inmacrophages (but not in the related myeloid-derived suppres-sor cells) in iKras�;IL-6�/�mice (Supplementary Fig. S2A). Theacute response to pancreatitis is accompanied by upregula-tion of the MAPK pathway (25, 26), the PI3K/Akt pathway(27), and by activation of Stat3 (19). In iKras�;IL-6�/�mice, thelevels of p-ERK1/2, p-Stat3 and, to a lesser extent, p-AKTwere reduced (Fig. 1D). Thus, while the absence of IL-6 didnot prevent induction of pancreatitis, it qualitatively changedthe response to the inflammatory stimulus.

To determine whether IL-6 deficiency altered PanIN forma-tion, we dissected tissues 3 weeks post-pancreatitis, when fullrecovery is expected in control animals, and extensive PanINlesions are expected in iKras� mice (21). Upon histopathologicanalysis, both in iKras� and in iKras�;IL-6�/� mice, weobserved pancreas-wide PanIN lesions with accumulation offibro-inflammatory stroma, characteristic periodic acid-Schiff(PAS) staining, and expression of the PanINmarker Claudin 18(Fig. 1E). Quantification of the type and extent of lesionsrevealed no significant changes in iKras�;IL-6�/� comparedwith iKras� mice (Fig. 2E; 3 week time point). Thus, PanINlesions formed even in absence of IL-6, upon induction ofpancreatitis.

Further characterization of the lesions in iKras� andiKras�;IL-6�/� animals revealed striking differences. First,the proliferation index was dramatically reduced in absenceof IL-6, both within the lesions and in the surroundingstroma (Fig. 1E; Ki67 immunostaining). Second, the levelsof p-ERK1/2, p-Akt, and p-Stat3 were reduced in IL-6–deficient pancreata (Fig. 1F).

In mice bearing mutant Kras�, PanIN formation is associ-ated with persistence of inflammatory infiltrates (28). Todetermine whether the absence of IL-6 altered the prevalence,or nature of the inflammatory infiltrates, we conducted flowcytometry for components of both the innate and adaptiveimmune systems in iKras� and iKras� ;IL-6�/� pancreata. Ouranalysis revealed a higher number of inflammatory cells iniKras� pancreata compared with the control 3 weeks post-pancreatitis—consistent with the completed repair of thepancreatic tissue in control mice and with the accumulationof a fibro-inflammatory stroma in iKras� animals. When wecompared iKras� with iKras�;IL-6�/� pancreata, we did notobserve a change in the total immune component, or in thenumber and nature of most infiltrating T-cell subsets (Sup-plementary Fig. S2A). However, we observed a reduction inseveral immune subtypes that have been associated withtumor progression, such as macrophages, myeloid-derived

IL-6 Is Required for Pancreatic Cancer Progression

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Figure 1. IL-6 expression is dispensable for pancreatitis-induced PanIN formation. A, genetic makeup of the iKras�;IL-6�/� mouse model. B, experimentaldesign; n ¼ 4–7. C, hematoxylin and eosin (H&E) and immunohistochemistry staining for p-ERK1/2 and p-Stat3 in control, iKras�, and iKras�;IL-6�/� micepancreata 1 day post-pancreatitis induction. (Continued on the following page.)

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monocyte suppressor cells, and a trend toward a reduction inthe number of other myeloid-derived suppressor cell subsetsand regulatory T cells (Supplementary Fig. S2A). We alsoobserved a dramatic decrease in infiltrating mast cells (Sup-plementary Fig. S2B), a cell type that has been linked topancreatic carcinogenesis (29); however, whether the lack ofmast cells was the cause or effect of the lack of progressionremains to be established.Taken together, our data showed that pancreatitis-driven

PanIN formation was independent of IL-6, possibly mediatedby other cytokines that are released during the induction ofpancreatitis. However, lesions formed in absence of IL-6 werequalitatively distinct. In fact, IL-6 was required for the activa-tion of the MAPK signaling pathway, and, to a lesser extent, forthe activation of Akt and Stat3. Moreover, IL-6–deficientPanINs had a low proliferation index. Thus, while histologicallysimilar, PanINs in iKras and iKras;IL-6�/�mice had significantmolecular differences in the activation of signaling pathwaysunderlying pancreatic cancer progression.

IL-6 is necessary forPanINmaintenance andprogressionWe next compared the kinetics of PanIN progression

between iKras� and iKras�;IL-6�/� mice. We aged cohorts ofeach genotype (n ¼ 4–7/genotype/time point) for 3, 5, 8, or 17weeks following induction of pancreatitis (see scheme in Fig.2A). As previously described, at the 3 weeks time point,histopathologic analysis revealed no significant differencesbetween iKras� and iKras�;IL-6�/� tissues. Over time, however,iKras� mice developed high-grade PanIN lesions with sus-tained MAPK activity (Fig. 2B). We observed PAS-positiveepithelial cells and expansion of a collagen-rich stroma (Fig.2C). In contrast, in iKras�;IL-6�/� tissues, we observed aprogressive decrease in the prevalence of PanINs and stromaand conversely an increase of acinar clusters (Fig. 2B and C).PanIN lesions are characterized by distinct microscopic fea-tures that can be highlighted by transmission electron micro-scopy (TEM): multilobular nuclei, lack of large secretorygranules (which are found in acinar cell), microvilli on theluminal surface, and large, irregular ductal lumen (Fig. 2D);PanINs in iKras� ;IL-6�/� samples had similar features,although the nuclear shape was less irregular. In addition, by5 weeks after the induction of pancreatitis, apoptotic cellswere detected within the ductal structures (Fig. 2D). Withinthe residual lesions, MAPK activation was low, as determinedby p-ERK1/2 immunostaining, and was confined to a smallsubset of cells (Fig. 2B, insets). In addition, we observedreduction in p-Stat3 and p-Akt levels and, as expected, lackof IL-6 expression (Supplementary Fig. S3A–S3C). By 17 weeks,the pancreata of iKras�;IL-6�/� mice were populated by nor-mal acini for more than 80% of the tissue, with rare ADM andsporadic PanIN1A, as confirmed by histopathologic analysis

of de-identified samples (Fig. 2E). Because no lesions wereobserved at this time point, it was not surprising to observelimited p-ERK1/2 and p-Akt staining; occasional p-Stat3 wasobserved in acini and ducts of normal appearance, possiblyindicating a residual inflammatory response in the pancreas(Supplementary Fig. S3C). Contemporary to the changes inthe epithelium, the stroma surrounding the PanIN lesionsalso underwent major remodeling, resulting in very littleresidual fibrosis and conversely in accumulation of adiposetissue (Fig. 2B and C). This process correlated with the upre-gulation of MMP7, MT1-MMP and, to a lesser extent, MMP9(Supplementary Fig. S4A). These proteases might play a rolein the remodeling of the tissue, but future experimental work,beyond the scope of the current study, will be needed toaddress this possibility. Interestingly, the expression of SMA,a fibroblast activation marker, decreased before the remodel-ing of the stroma in iKras�;IL-6�/� mice (SupplementaryFig. S3D). Thus, remodeling of the stroma was preceded bythe return of fibroblasts to a nonactive state, a similar findingto what we previously observed upon inactivation of Kras�

expression in the pancreas (21). We then investigated whetherIL-6 influenced the expression of other inflammatory cyto-kines in tissues harvested 3, 5 to 8, or 17 weeks after pancre-atitis. We did not observe changes in several cytokines andinflammation-related genes such as Il2, Il4, Il10, Il11, Il17, Cox2,Tnfa, and Ifng (Supplementary Fig. S4B). However, in additionto confirming the lack of Il6 expression, we detected lowerlevels of Il1b (a proinflammatory cytokine), Slfn4 a myeloidactivation marker; ref. 30, and GM-CSF (a cytokine that isrequired for pancreatic cancer growth; refs. 31, 32) in iKras�;IL-6�/� mice compared with iKras� (Fig. 3A).

In summary, while pancreatitis-induced lesions in iKras�

mice progressed over time to high-grade PanINs surroundedby abundant stroma, neoplastic lesions in iKras�;IL-6�/� miceregressed, the stroma was remodeled, eventually resulting in anormal pancreatic parenchyma, albeit with some adiposetissue infiltration. The remodeling process in iKras�;IL-6�/�

pancreata resembled the effect of Kras� inactivation in PanIN-bearing iKras� mice (21), indicating that IL-6 is a key player inKras�-driven carcinogenesis.

IL-6 regulates proliferation, survival, anddifferentiationstatus of PanIN cells

Because expression of IL-6 was associated with enhancedproliferation of PanIN lesions (Fig. 3B), we quantified theproliferation index for the different cell populations presentin the tissues, namely ADM/PanIN cells, components of thestroma and acinar cells. In iKras� mice, the PanIN cells had ahigh proliferation index that was maintained over time, andproliferation in the stroma reached a peak at 5 to 8 weeks.Proliferation in both compartments was significantly reduced

(Continued.) Scale bar, 50 mm. D, Western blot analysis for p-ERK1/2, total-ERK1/2, p-Akt, total-Akt, p-Stat3, and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) in untreated, control, iKras�, and iKras�;IL-6�/� mice pancreata 1 day post-pancreatitis induction. E, H&E, PAS, andimmunohistochemistry staining for Claudin 18 and Ki67 in iKras� and iKras�;IL-6�/� mice pancreata 3 weeks post-pancreatitis. Scale bar, 50 mm. F,immunohistochemistry staining and Western blot analysis for p-ERK1/2, p-Stat3, and p-Akt in iKras� and iKras�;IL-6�/� mice pancreata 3 weekspost-pancreatitis. Scale bar, 50 mm. Doxy, doxycycline.






Figure 2. IL-6 is necessary for PanIN progression and maintenance. A, experimental design; n¼ 4–7. B, hematoxylin and eosin (H&E) and p-ERK1/2 staining(insets) of iKras� and iKras�;IL-6�/� mice pancreata 3, 5 to 8, and 17 weeks following pancreatitis. Scale bar, 50 mm. C, Gomori Trichrome staining and PASstaining (insets) of iKras� and iKras�;IL-6�/� mice pancreata. Scale bar, 50 mm. D, TEM image of iKras� and iKras�;IL-6�/� mice pancreata 5 weeks followingpancreatitis. Scale bar, 10 mm. E, pathologic analysis. Data representmean�SEM; n¼ 3–5. The statistical difference between iKras� and iKras�;IL-6�/�miceat the same timepoint per lesion typewasdeterminedby two-sidedStudent t test. �,P<0.05; ��,P<0.01; ��,P<0.0001; #, not significant.Doxy, doxycycline.

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Figure 3. IL-6 regulates proliferation and survival of PanIN cells. A, qRT-PCR for Il6, Il1b, GM-CSF, and Slfn4 expression inWT, iKras�, and iKras�;IL-6�/�micepancreata. B,Ki67 immunohistochemistry stainingof iKras� and iKras�;IL-6�/�micepancreata 3, 5 to 8, and17weeks followingpancreatitis. Scale bar, 25mm.C, TUNEL (red) and coimmunofluorescence staining for CK19 (green), SMA (gray), and 40,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 25 mm.D and E,Ki67 proliferation index (D) in ADM/PanIN, stroma, and acinar cells and quantification (E) of apoptotic cells in iKras� and iKras�;IL-6�/� mice pancreata.Data representmean�SEM; n¼ 3. The statistical differencewas determined by two-sidedStudent t test, comparing the different genotypemice at each timepoint for each category considered (acini, stroma, and ADM/PanIN). �, P < 0.05; ��, P < 0.01; ��, P < 0.001; ��, P < 0.0001. Doxy, doxycycline.






in iKras�;IL-6�/� lesions. Interestingly, when we compared theproliferation of the acinar compartment between the twogenotypes, we observed a significant increase (P < 0.05) iniKras� ;IL-6�/� compared with iKras� at the 5 to 8 weeks and atthe 17 weeks time points (Fig. 3B and D). Taken together, ourdata indicate that IL-6 promotes proliferation of the meta-plastic/dysplastic cells, and possibly prevents acinar prolifer-ation, a normal aspect of the tissue recovery followingpancreatitis.

In iKras�;IL-6�/�mice, this decrease in proliferation poten-tially explains the lack of progression in the lesions, but notthe progressive elimination of the lesions. To investigate thisaspect, we investigated two possible mechanisms of regres-sion: apoptosis of PanIN cells, as well as redifferentiation ofPanIN cells into normal acini. First, we determined thatPanIN cells have severely compromised survival in absenceof IL-6. Apoptotic cells [as determined by terminal deoxy-nucleotidyl transferase–mediated dUTP nick end labeling(TUNEL) or cleaved caspase-3 staining] were occasionallypresent in iKras� tissues; the overall level of apoptosis was low(less than 1% of the epithelial cells), and it decreased overtime (Fig. 3C and E and Supplementary Figs. S4C and S5A). Incontrast, iKras� ;IL-6�/� PanINs had elevated levels of apo-ptosis both within the epithelium and in the surroundingstroma (Fig. 3C and E and Supplementary Figs. S4C and S5A).In addition to the immunostaining analysis, we conductedqRT-PCR to determine the relative ratio between the med-iators of apoptosis Bak and Bax and the antiapoptotic genesBcl-2 and Bcl-x. At the 5- to 8-week time point, when mostof the changes within the tissue were taking place, iKras� ;IL-6�/� tissues, compared with iKras�, had a higher proapop-totic ratio, for Bak/Bcl-x and for Bax/Bcl-x (SupplementaryFig. S5B). In a second set of experiments, we conducted co-immunofluorescent staining with CK19, a ductal and PanINmarker, and amylase, an acinar marker. In iKras� tissues, asexpected, we observed almost exclusive expression of CK19 inepithelial cells (Fig. 4A and B). In iKras�;IL-6�/� tissues, CK19was prevalent at 3 weeks post-pancreatitis, but by 5 to 8weeks post-pancreatitis up to one third of the epithelial cellscoexpressed CK19 and amylase, and a significant amylase-positive population was observed (Fig. 4A and B). By 17 weeks,CK19-only cells were rare, whereas amylase-positive cellsrepresented the majority of the epithelial population, andsome cells coexpressing CK19 and amylase were still present(Fig. 4A and B). The transcription factor Mist1 is a keyregulator of acinar cell differentiation (33), and its expressionis lost during PanIN formation. Importantly, inactivation ofMist1 in the context of oncogenic Kras accelerates pancreaticcarcinogenesis (34), whereas conversely forced expression ofMist1 prevents Kras-driven carcinogenesis (35). As expected,we did not observe any Mist1 expression in the lesions ofiKras� mice (Fig. 4C). Similarly, in iKras�;IL-6�/� lesions at3 weeks post-pancreatitis, Mist1 expression was absent. How-ever, by 5 weeks post-pancreatitis, Mist1 was expressed inthe residual ductal/PanIN structures, thus confirming theirmixed differentiation status (Fig. 4C). At later time points,Mist1 expression was confined to acini and excluded fromthe ducts, as in the normal pancreas.

Taken together, our data indicate that PanIN elimination isunderscored by a combination of apoptotic cell death, redif-ferentiation of cells toward the acinar lineage, and increasedproliferation of acinar cells, mechanisms of pancreas repairthat we have previously observed upon inactivation of onco-genic Kras in PanIN-bearing mice (21).

IL-6 is required for Nrf2 upregulation and oxidativestress resistance

The onset of PanIN formation is accompanied by the acti-vation of a ROS detoxification program (36). PanINs thatform in mice deficient for Nrf2, a key factor in the ROSdetoxification pathway, are nonproliferative (36). Expressionof Nrf2 has been shown to be activated downstream of onco-genic Kras (37) via the MAPK/ERK signaling pathway (36, 38).Given that iKras�;IL-6�/� mice had a defect in MAPK/ERKactivation, we decided to investigate whether Nrf2 expressionwas defective in these animals. Indeed, we found robustexpression of Nrf2 in PanINs of iKras� mice at all the timepoints studied, but drastically reduced Nrf2 expression iniKras� ;IL-6�/� lesions at those time points when lesions couldstill be detected (Fig. 5A). Thus, the presence of oncogenicKras� was not sufficient to activate the ROS detoxificationprogram, and additional inflammatory signals were required.

To investigate in more detail the effect of IL-6 depletion onthe ability of tumor cells to cope with oxidative damage, weused the primary mouse pancreatic cancer cell line, 9805,derived from iKras�;p53� mice (24). The in vitro approachallowed us to investigate the effect of IL-6 on the tumor cells,independently from possible indirect effects mediated bycomponents of the stroma. Of note, 9805 cells express the Il6receptor (Il6r) in a Kras�-dependent manner, and are thus ableto respond to IL-6 signaling (Fig. 5C). The cancer cells wereseeded to chamber slides at 50% to 60% confluency andcultured in complete medium containing doxycycline at 1mg/mL overnight. Then, the cells were subdivided into fourgroups: control, treatedwithH2O2 alone to induce intracellularROS accumulation, treated with H2O2 in the presence ofrecombinant IL-6, or IL-6 preincubated with an IL-6–blockingantibody—as these cells express low levels of IL-6 in a Kras-dependent manner (Fig. 5C). The experiment was carried outin presence of doxycycline (thus, with expression of onco-genic Kras�) or without doxycycline (thus, with no oncogenicKras). First, we confirmed the notion that oncogenic Kras�

protects the cells fromROSaccumulation, as bothROS stainingand the number of dead cells were higher in absence ofdoxycycline. Second, we determined that treatment with IL-6had a protective effect both on ROS accumulation and on celldeath; this effect was observed independently from Kras�

expression, and was abrogated by the anti-IL-6 antibody(Fig. 5B; quantification in 5D; Supplementary Fig. S5C). Todetermine whether the MAPK pathway was affected by induc-tion of oxidative stress, we conducted Western blot analysisfor p-ERK1/2 in our samples. Of note, even though 9805 cellsexpressed some IL-6, exogenous IL-6 was effective in increas-ing the activation of downstream pathways such as MAPK,phosphoinositide 3-kinase (PI3K), and p-Stat3. We observedthat treatment with H2O2 caused a modest increase in p-

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ERK1/2, even in the absence of exogenous IL-6. However, therewas a strong increase of p-ERK1/2 in cells treated with H2O2

in the presence of IL-6, which was reversed by the anti-IL-6antibody. A similar effect was observed with p-Akt and Nrf2,whereas p-Stat3 did not seem to be affected by the induction

of oxidative stress (Fig. 5E). Thus, our data indicate that IL-6provides protection from oxidative stress.

To determine whether the failure to activate the ROSdetoxification program in iKras�;IL-6�/� mice could explainthe lack of PanIN progression, we used a ROS scavenger, N-

Figure 4. IL-6 inhibition results in ductal-to-acinar redifferentiation of PanIN cells. A, coimmunofluorescence staining for CK19 (green), amylase (red),SMA (magenta), and 40,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 25 mm.B, quantification of CK19 and amylase single- or double-positive epithelialcells in iKras� and iKras�;IL-6�/�micepancreata. Data representmean�SEM;n¼3. The statistical differencewasdeterminedby two-sidedStudent t test. Foreach time point, the number of single- or double-positive cells was compared between genotypes. �, P < 0.05; ��, P < 0.01; ��, P < 0.0001. C, Mist1immunohistochemistry staining. Scale bar, 25 mm. Doxy, doxycycline.






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acetyl-L-cysteine (NAC). In brief, iKras�;IL-6�/� mice weretreated with NAC starting 2 weeks after induction of pancre-atitis, and then for 3 weeks (Supplementary Fig. S5D). Themicewere harvested at 5 weeks post-pancreatitis. Compared withuntreated iKras�;IL-6�/� mice (Fig. 2B), NAC-treated iKras�;IL-6�/� mice revealed partial rescue of carcinogenesis. Theirpancreata had extensive PanIN lesions with elevated p-ERK1/2and p-Stat3 levels, and rescue of proliferation both withinthe epithelial and the stroma compartments (SupplementaryFig. S5E). In conclusion, the requirement for IL-6 duringpancreatic carcinogenesis might be explained, at least in part,by preventing ROS accumulation.

IL-6 prevents tissue repair following Kras inactivationThe iKras� mouse allows us to turn off Kras� expression at

will, thus it is a suitable model to study pancreatic repairfollowing inactivation of the Kras oncogene. To study the roleof IL-6 during pancreatic repair, we carried out the followingexperiment: adult mice were kept on doxycycline for 3 weeksfollowing pancreatitis induction, then some of the animalswere harvested, and the others were placed on doxycycline-free chow and water. Of note, 4 to 5 mice per genotype werethen harvested 1 day, 3 days, and 2 weeks later (Fig. 6A), basedon our previous characterization of the dynamics of pancreasrepair in this model (21). In iKras� mice, IL-6 was expressedthrough most of the repair process, though with decreasingexpression levels over time (Supplementary Fig. S6B and S6D).Whole tissue analysis by Western blot indicated that inacti-vation of oncogenic Kras� led to rapid downregulation ofp-ERK1/2 and p-Akt levels within 1 day and to a transientupregulation of cleaved caspase-3 levels, indicating cell death.However, p-Stat3 remained high initially, possibly as a result ofits expression in the inflammatory cells that infiltrate the tissueduring the repair process (Supplementary Fig. S6C). In fact,immunohistochemistry revealed abundant p-Stat3–positivecells within the stroma compartments (Supplementary Fig.S6E). iKras�;IL-6�/� mice had lower levels of p-ERK1/2, p-Akt,and p-Stat3 than iKras� mice; these pathways were furtherdownregulated upon Kras� inactivation. In contrast, thesemice had a higher basal level of cleaved caspase-3, and itfurther increased in a transient manner following Kras� inac-tivation. However, cleaved caspase-3 became undetectable asearly as 3 days following Kras� inactivation (SupplementaryFig. S6C).In iKras� mice, the repair process was complete by 2 weeks

(Fig. 6B and quantification in 6C). Consistently, PAS positivitywas still abundant 1 day after Kras� inactivation, whereas by3 days, the dysplastic ducts were mostly lined by PAS-negativecells (Supplementary Fig. S7A). Coimmunostaining for CK19,amylase, and SMA highlighted some limited expression of

amylase already 1 day after Kras� inactivation, and clustersof CK19/amylase–positive cells with ductal morphology 3days after Kras� inactivation (Fig. 6D and quantification in6E). The stroma was still SMA-positive 3 days after Kras�

inactivation (Fig. 6D, top, magenta staining; SupplementaryFig. S7B), and trichrome staining for collagen deposition wasstill abundant (Supplementary Fig. S7C). iKras�;IL-6�/� micehad an accelerated repair process, with several acinar clustersalready evident 1 day after Kras� inactivation, and almostcomplete recovery as early as 3 days after doxycycline removal(Fig. 6B and C). Consistently, mixed differentiation acinar-ductal cells were common 1 day after Kras� inactivation, and amajority of normal acini, amylase-positive, and CK19-negativepopulated the tissue by 3 days after Kras� inactivation (Fig. 6Dand E). The appearance of mixed acinar-ductal differentiationcells is preceded by expression of the transcription factorMist1. Interestingly, we observed earlier reexpression of Mist1in iKras�;IL-6�/� pancreata (1 day after inactivation of Kras�

compared with 3 days after inactivation in iKras� mice),possibly explaining the accelerated recovery of the pancreasparenchyma (Supplementary Fig. S8A). Remodeling of thestroma also occurred in an accelerated manner: both SMAand trichrome staining were clearly downregulated already 1day after Kras inactivation, and then abrogated in large areasof the pancreas by 3 days (Fig. 6D, bottom, magenta staining;Supplementary Fig. S7B and S7C).

In pancreata that have been exposed to Kras� activity for alimited period of time, Kras inactivation results in rediffer-entiation of PanINs to acinar cells and proliferation of thenewly formed acini (21). Upon Kras� inactivation, there is adecrease in proliferation both in the CK19(þ) epithelial cellcompartment and in the stroma; in contrast, CK19(�) epithe-lial cells, corresponding to the newly formed acinar cells, enterthe cell cycle, as part of the repair process. In iKras�;IL-6�/�

pancreata, acinar cells were formed and entered the cell cyclesooner upon Kras inactivation, possibly contributing to thefaster recovery process (Supplementary Fig. S8B and S8C;quantification in Supplementary Fig. S8D). Thus, while IL-6was required for proliferation of PanIN cells and stroma, it wasdispensable for—and possibly inhibited—proliferation of nor-mal acinar cells. Our data indicate that IL-6 has a negativeeffect on pancreatic tissue repair, possibly inhibiting pancre-atic acinar differentiation and acinar cell proliferation.

Therapeutic inhibition of IL-6 causes PanIN regressionIn a final set of experiments, we investigated the effect of

blocking IL-6 signaling in KPCY (22, 39) mice using an anti-IL-6–blocking antibody, a strategy that has therapeutic rele-vance. The KPCY model was chosen because of its provenrelevance to clinical response in patients (40), and for its

Figure 5. IL-6 inhibits ROS induction in pancreatic cancer cells. A, Nrf2 immunohistochemistry staining of iKras� and iKras�;IL-6�/� mice pancreata 3, 5 to8, and17weeks followingpancreatitis. Scale bar, 25mm.B,ROSstaining andbright field imagesof primarymousepancreatic cancer cell line 9805 treatedwith600 mmol/L H2O2 alone, or in the presence of IL-6 (50 ng/mL), or IL-6 preincubated with anti-IL-6 (8 mg/mL) for 1 hour. Scale bar, 50 mm. C, qRT-PCR of Kras�,Il6ra, and Il6 expression in 9805 cells with or without doxycycline (Doxy) treatment. Data represent mean � SEM; n ¼ 3. ��, P < 0.01; ��, P < 0.001.D, quantification of ROS signal intensity. Data represent mean � SEM; n ¼ 3. �, P < 0.05. Each category was compared between the two genotypes foreach time point. E,Western blot analysis for p-ERK1/2, total-ERK1/2, p-Akt, total-Akt, p-Stat3, total Stat3, Nrf2, and b-actin in 9805 cells 30minutes followingROS induction.






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predictable disease progression. Our goal was to treat KPCYmice after PanIN lesions had developed, to determine whetherIL-6 was required for PanINmaintenance. Ten-week-old KPCYmice were randomized to two treatment arms, and adminis-tered respectively anti-IL-6 antibody or isotype control. Theanimals were sacrificed 7 days after the beginning of the

treatment (see scheme in Fig. 7A). The tissue was then pre-pared for histopathologic examination. In the control group(n ¼ 3), mice presented with PanINs interspersed withinnormal exocrine tissue and areas of ADM, as expected on thebasis of the disease progression in this model (Fig. 7C).Although the majority of PanINs were of grade 1A-2, PanIN3

Figure 7. In vivo anti-IL-6 treatment reverses PanIN lesions. A, experimental design; n ¼ 3. B, pathologic analysis. Data represent mean � SEM; n ¼ 3.C and D, hematoxylin and eosin (H&E) staining of KPCY mice pancreata treated with isotype control (C) or anti-IL-6 antibody (D) 1 week followingthe beginning of treatment. Scale bar, 100 mm. E, working model indicating the requirement of IL-6 during the maintenance and progression ofpancreatic cancer in mice.

Figure 6. IL-6 prevents tissue repair following Kras� inactivation. A, experimental design; n¼ 4–5. B, hematoxylin and eosin (H&E) staining of iKras� and iKras�;IL-6�/�mice pancreata 3 weeks post-pancreatitis and 1 day, 3 days, and 2 weeks following Kras� inactivation. Scale bar, 50 mm. C, pathologic analysis. Datarepresent mean � SEM; n ¼ 3–4. The statistical difference was determined by two-sided Student t test. �, P < 0.05; ��, P < 0.01; #, not significant. D,coimmunofluorescence staining for CK19 (green), amylase (red), SMA (magenta), and 40,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 25 mm. E,quantification of CK19 and amylase single- or double-positive epithelial cells in iKras� and iKras�;IL-6�/�mice pancreata. Data representmean�SEM; n¼ 3.The statistical difference was determined by two-sided Student t test. �, P < 0.05; ��, P < 0.01; ��, P < 0.001. Doxy, doxycycline.






lesions were also observed (Fig. 7B). In contrast, in the anti-IL-6–treated group, the tissue was largely populated by normalacini, with occasional areas of ADM and virtually no PanINs(Fig. 7D and quantification in Fig. 7B). Thus, these resultsindicate that IL-6 signaling is required for PanIN maintenanceonce initiated, mimicking the findings obtained in iKras� miceusing genetic inactivation of IL-6. Notably, the requirementfor IL-6 was maintained even in presence of a loss-of functionallele of the tumor suppressor p53.

DiscussionThe formation of PanINs, the most common precursor

lesions of pancreatic cancer, is accompanied by the accu-mulation of a desmoplastic stroma with abundant immuneinfiltrates (28) and by expression of several inflammatorycytokines, including IL-6 (18). It has been suggested thatinflammatory environment is an important component ofpancreatic cancer (41); however, our mechanistic under-standing of the effect inflammation during cancer progres-sion is incomplete. Recent evidence indicates that, at least inmice, the most common cell of origin for pancreatic canceris an acinar cell that has dedifferentiated to a duct-like cell,often in response to local tissue injury (42, 43). The process ofADM occurs in the pancreas in response to damage such asthe induction of acute or chronic pancreatitis. However, thewild-type pancreas is able to undergo tissue repair rapidlyand effectively within a short time from the cessation of theinjury stimulus. In contrast, pancreata bearing an oncogenicform of Kras are unable to undergo tissue repair, and get"locked" in the ADM status, and progress to PanIN lesions(12). We have previously shown that inactivation of onco-genic Kras at early stages of carcinogenesis allows tissuerepair, by allowing redifferentiation of duct-like cells intoacinar cells (21). We have also shown that IL-6 is expressed ina Kras-dependent manner in the iKras� pancreas. Ras-depen-dent expression of IL-6 was previously described in multiplecell types (44). Progression toward cancer is accompaniedby high levels of IL-6, whereas the repair process correlateswith repression of IL-6 expression (21).

Given the important role that IL-6 plays during the ini-tiation of pancreatic cancer (18), we decided to investi-gate whether it constitutes a key player in modulating thebalance between tissue repair and carcinogenesis. Our find-ings showed that IL-6 was dispensable for pancreatitis-drivenPanIN formation, but necessary for the maintenance andprogression of the lesions. In fact, in absence of IL-6, thelesions were progressively cleared from the tissue, by acombination of cell-intrinsic mechanisms within the epi-thelial cells, and extrinsic mechanisms involving epithelial-mesenchymal interactions (see scheme in Fig. 7E).

Within the epithelial cells, we observed extensive apoptoticcell death in absence of IL-6, consistent with previous reportsassociating IL-6 with cell survival (15), as well as redifferentia-tion of PanIN cells into normal acini. Moreover, PanIN lesionsthat formed in the absence of IL-6 failed to activate theKras effector pathways MAPK and PI3K/Akt and had reducedlevels of p-Stat3. The failure to activate the MAPK and PI3K

pathways was somewhat surprising, as it could be expectedthat the presence of constitutively active, oncogenic Kras�

would bypass any upstream signal. However, our finding isconsistent with several recent reports indicating the need fora positive feedback loop to ensure full activation of oncogenicKras� (45–47). Importantly, activation of the MAPK pathway isboth sufficient and necessary for pancreatic carcinogenesisin mice (45, 48). An important target of the MAPK signalingpathway in pancreatic cancer isNrf2 (36), a transcription factorthat drives the ROSdetoxification program (49). ROShave beenassociated with carcinogenesis both as a tumor promoter,given the connection between ROS and genome instability(50), and as a barrier to carcinogenesis as ROS accumulationis toxic for the cells (51). Nrf2 deletion in mouse models ofpancreatic cancer prevents tumor progression, indicating thatthe ROS detoxification is an essential step during tumorigen-esis (36). Here, we show that, even in the presence of mutantKras, IL-6 signaling was essential to upregulate Nrf2 expressionin PanIN lesions. We then showed, using a primary mousepancreatic cancer cell line, that failure to upregulate Nrf2caused oxidative damage and led to tumor cell death. In fact,the failure of iKras�;IL-6�/� lesions to progress could berescued, at least partially, by treating the mice with the ROSscavenger NAC. Thus, our results indicate that IL-6 signalingis essential to activate the ROS detoxification program inpancreatic cancer.

In addition to the epithelial changes, important differencesalso occur within the stroma. Lack of IL-6 affected both thefibroblasts within the stroma, which became unable to maintaintheir activation status, and the inflammatory microenviron-ment. In particular, the cytokines IL-1b and granulocyte mac-rophage colony-stimulating factor (GM-CSF) were expressed atlower levels, and fewer myeloid-derived suppressor cells accu-mulated within IL-6–deficient tissues. IL-1b can directly driveoncogenesis and suppress cancer immunosurveillance mechan-isms via mobilizing myeloid-derived suppressor cells (52).GM-CSF is overexpressed by pancreatic cancer cells and pro-motes tumor growth by creating a permissive immune environ-ment (31, 32). Thus IL-6 emerges as a key regulator of multipleimmune signals that are important for tumor growth.

Our working model is that IL-6 promotes proliferation andsurvival of neoplastic cells, whereas inhibiting proliferation ofacinar cells, as evidenced by our tissue repair experiments.The iKras� mouse model has the unique feature of allowinginactivation of oncogenic Kras� at will (21). Mice lacking IL-6expression had accelerated recovery of acinar cells and increas-ed acinar cell proliferation. Thus, IL-6 switches the balancebetween tissue-repair and carcinogenesis in the pancreas.

Our results have potential therapeutic implications by pro-viding a rationale for the use of IL-6 inhibitors in pancreaticcancer, at a time when several inhibitors are reaching theclinic (for review see ref. 53). In fact, our proof-of-principle testin KPCY mice showed that IL-6 blocked PanIN progressionin this model. Expression patterns of IL-6 in human tissuesand blood should be explored in a large panel of humancancers, as the current published findings relied on a relativelysmall sample set (14), and compared with the conclusionsmade in mouse models. Further work will also be needed to

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determine the best potential use of IL-6 inhibitors in thisdisease, either for treatment or to prevent progression oflatent lesions in high-risk individuals or to prevent relapse inpatients following resection, and possibly as an adjuvant com-bined with standard-of-care chemotherapy.

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

DisclaimerThe funding sources of this study had no role in study design, data collection

and analysis, decision to publish, or preparation of the article.

Authors' ContributionsConceptionanddesign:Y. Zhang, B.R. Zetter, B.Z. Stanger, I. Chung, A.D. Rhim,M.P. di MaglianoDevelopment of methodology: Y. Zhang, M.P. di MaglianoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Zhang, M.A. Collins, F. Bednar, S. Rakshit, B.R.Zetter, A.D. Rhim, M.P. di MaglianoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Zhang, W. Yan, F. Bednar, B.R. Zetter, A.D. Rhim,M.P. di MaglianoWriting, review, and/or revision of the manuscript: Y. Zhang, M.A. Collins,F. Bednar, B.R. Zetter, B.Z. Stanger, I. Chung, A.D. Rhim, M.P. di MaglianoStudy supervision: B.R. Zetter, B.Z. Stanger, M.P. di Magliano

AcknowledgmentsThe authors thank Esha Mathew, Arthur L. Brannon III, and Dr. J€org

Zeller for scientific discussion and critical reading of the article, and MarshaM. Thomas for laboratory support. The p48-Cre mouse was a generous giftfrom Dr. Chris Wright (Vanderbilt University, Nashville, TN). The authorsalso thank Dr. Stephen Konieczny (Purdue University, West Lafayette, IN)for providing the Mist1 antibody and Dr. Diane M. Simeone for providinghuman pancreatic cancer samples and primary cell lines. The CK19 anti-body (Troma III) was obtained from the Iowa Developmental HybridomaBank.

Grant SupportThis project was supported by the University of Michigan Biological Scholar

Program, the University of Michigan Cancer Center and NCI-1R01CA151588-01.A.D. Rhim was supported by National Institutes of Diabetes, Digestive andKidney Diseases (NIDDK; K08DK088945) and used the Morphology and Molec-ular Biology Cores of the PennCenter forMolecular Studies inDigestive andLiverDiseases (P30-DK050306). M.A. Collins was supported by a University of Michi-gan Program in Cellular and Molecular Biology training grant (NIH T32GM07315) and by a University of Michigan Center for Organogenesis TrainingGrant (5-T32-HD007515).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received June 7, 2013; revised July 18, 2013; accepted July 27, 2013;published OnlineFirst October 4, 2013.

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2013;73:6359-6374. Published OnlineFirst October 4, 2013.Cancer Res Yaqing Zhang, Wei Yan, Meredith A. Collins, et al. ResistancePromoting MAPK Signaling Activation and Oxidative Stress Interleukin-6 Is Required for Pancreatic Cancer Progression by

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Interleukin-6 Is Required for Pancreatic Cancer …...Tumor and Stem Cell Biology Interleukin-6 Is Required for Pancreatic Cancer Progression by Promoting MAPK Signaling Activation

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