il22ra1/stat3 signaling promotes stemness and ... · il22ra1/stat3 signaling promotes stemness and...

Tumor Biology and Immunology

IL22RA1/STAT3 Signaling Promotes Stemnessand Tumorigenicity in Pancreatic CancerWeizhi He1, Jinghua Wu1, Juanjuan Shi1, Yan-Miao Huo2,Wentao Dai3,Jing Geng1, Ping Lu1, Min-Wei Yang2, Yuan Fang4,Wei Wang4,Zhi-Gang Zhang5, Aida Habtezion6, Yong-Wei Sun2, and Jing Xue1

Abstract

Chronic inflammation is a feature of pancreatic cancer, butlittle is known about how immune cells or immune cell–relatedsignals affect pancreatic cancer stemness and development. Ourprevious work showed that IL22/IL22RA1 plays a vital role inacute and chronic pancreatitis progression by mediating cross-talk between immune cells and acinar cells or stellate cells,respectively. Here, we find IL22RA1 is highly but heteroge-neously expressed in pancreatic cancer cells, with high expres-sion associated with poor prognosis of patients with pancreaticcancer. The IL22RA1hi population from pancreatic cancer har-

bored higher stemness potential and tumorigenicity. Notably,IL22 promoted pancreatic cancer stemness via IL22RA1/STAT3signaling, establishing the mechanism of regulation of cancerstemness by microenvironmental factors. Moreover, STAT3 wasindispensable for the maintenance of IL22RA1hi cells. Overall,these findings provide a therapeutic strategy for patients withPDAC with high expression of IL22RA1.

Significance: IL22RA1/STAT3 signaling enhances stemnessand tumorigenicity in pancreatic cancer. Cancer Res; 78(12);3293–305. �2018 AACR.

IntroductionPancreatic cancer is a highly aggressive disease with few effec-

tive therapies. Pancreatic ductal adenocarcinoma (PDAC) is themost common form of pancreatic cancer (1–3). Despite recentadvances in surgical and early diagnosis techniques, survival hasnot changedmuch in the past two decades. Pancreatic cancer stemcells (CSC), first identified in 2007, are characterized by theirabilities of self-renewal and differentiation, in vivo tumorigenicity,and the ability to drive metastasis (4, 5). Most importantly, CSCsare believed to contribute to tumor initiation, relapse, and ther-apeutic resistance (6, 7). Therefore, revealing mechanisms regu-

lating CSCs and developing novel CSC-targeted therapies are anurgent need.

IL22, a well-defined ligand for IL22RA1, belongs to theIL10 cytokine family and plays a critical role in host defense,tissue repair, and carcinogenesis in a context-dependent manner(8–10). IL22 signals via the IL22 receptor that consists of IL22RA1and IL10RB (11). IL10RB is expressed ubiquitously in variousorgans (12), whereas IL22RA1 has more restricted expression indifferent tissues and pathologic conditions (13). IL22 andIL22RA1 are expressed on leukocytes and epithelial cells, respec-tively, allowing for cross-talk between the two cell types. Ourprevious studies have elucidated the role of IL22/IL22RA1 sig-naling in acute and chronic pancreatitis (14, 15); in this study, wehave explored the role of this signaling axis in pancreatic cancerdevelopment and progression.

The interactions between tumor cells and immune cells pro-mote tumor development and progression (16–18). It is wellaccepted that both intrinsic and external signals from microen-vironment define the phenotype of CSCs (19–21). Chronicinflammation is a feature of PDAC and is believed to be anindependent risk factor for pancreatic cancer initiation and pro-gression (22, 23). However, how immune cells or immune cell–related signaling modulate CSCs is poorly defined in pancreaticcancer.

Here, we show that high expression of IL22RA1 is associatedwith poor prognosis in pancreatic cancer. PDAC cells with highexpression of IL22RA1 possess stemness characteristics, includingself-renewal, tumorigenicity, and metastasis. Notably, IL22 pro-motes pancreatic cancer stemness via IL22RA1/STAT3 signaling,identifying the mechanism of regulation of cancer stem cells bymicroenvironmental factors. Moreover, STAT3 activation is indis-pensable for the maintenance of IL22RA1hi cells, both in theabsence or presence of IL22, providing a therapeutic target forpatients with PDAC with high expression of IL22RA1.

1State Key Laboratory of Oncogenes and Related Genes, Stem Cell ResearchCenter, Renji Hospital, School of Medicine, Shanghai Jiao Tong University,Shanghai, China. 2Department of Biliary-Pancreatic Surgery, Renji Hospital,School of Medicine, Shanghai Jiao Tong University, Shanghai, China. 3ShanghaiCenter for Bioinformation Technology & Shanghai Engineering Research Centerof Pharmaceutical Translation, Shanghai, China. 4Department of General Sur-gery & Research Institute of Pancreatic Disease, Ruijin Hospital, School ofMedicine, Shanghai Jiao Tong University, Shanghai, China. 5State Key Labora-tory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai JiaoTong University, Shanghai, China. 6Division of Gastroenterology and Hepatol-ogy, Stanford University School of Medicine, Stanford, California.

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

Corresponding Authors: Jing Xue, Renji Hospital, School of Medicine, ShanghaiJiao Tong University, Shanghai 200127, China. Phone: 8621-6838-3922; Fax:8621-6838-3916; E-mail: [email protected]; Yong-Wei Sun, [email protected]; and Aida Habtezion, Department of Medicine, Division of Gastroenterology &Hepatology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford,CA 94305. E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-3131

�2018 American Association for Cancer Research.

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Materials and MethodsPancreatic cell lines and primary tumor cells

Human pancreatic cancer cell lines (BXPC3, SW1990, PANC1,Miapaca-2, AsPC-1, andHPAC) and normal pancreatic ductal celllines (HPDEandhTERT-HPNE)were obtained fromATCCorCellBank of Chinese Academy of Sciences (Shanghai, China). PDACcells were cultured in DMEM or RPMI medium supplemented

with 10% FBS and 1% penicillin–streptomycin in a humidifiedchamber at 37�C with 5% CO2. Pancreatic patient-derived xeno-graft (PDX)-derived cancer cells (PDC), including PDC0034,PDC0049, and PDC0001, were isolated from pancreatic PDXtumors and cultured in complete RPMI medium plus 10 ng/mLEGF and 1% ITS as described previously (24). Primary tumor cellsfrom a patient with PDAC samples were directly isolated by

Figure 1.

IL22RA1 is upregulated in pancreatic cancer, and its level is negatively associated with patient prognosis. A, Immunoblotting analysis of IL22RA1 proteinlevel among human pancreatic cancer cell lines (HPAC, AsPC-1, Miapaca-2, PANC1, SW1990, and BxPC3) and normal pancreatic ductal cells (HPDE andhTERT-HPNE). B, Representative immunofluorescence images of PDAC tissues from three different patients with pancreatic cancer, costained with IL22RA1, CK19,and DAPI (nuclei). Scale bar, 50 mm. C, Flow cytometry analysis for IL22RA1 expression among PDAC cell lines, PDCs, and primary tumor cells (pregatedon liveþEpCAMþ cells). D, The association between IL22RA1 transcript level and patient overall survival. The analyses were conducted in 50 patients withpancreatic cancer (GSE102238; log-rank test, P ¼ 0.031). E, The association between IL22RA1 transcript level and patient overall survival. The analyseswere conducted in 177 patients with pancreatic cancer from TCGA database (log-rank test, P ¼ 0.022).

He et al.

Cancer Res; 78(12) June 15, 2018 Cancer Research3294




collagenase digestion (collagenase IV, 2mg/mL, 30–60minutes).Human pancreatic tissues from patients with PDAC wereobtained from the Renji Hospital (Shanghai, China) with LocalEthics Committee approval and patient consent.

Flow cytometryCells were prepared as a single-cell suspension for FACS

staining. For surface staining, the following antibodies wereused: PE/Cy7-CD24, FITC-CD44, PE-ESA/EpCAM, APC-CD3,

Figure 2.

Pancreatic cancer cells with high IL22RA1 expression harbor stemness properties. A, Morphology of SW1990 cells was grown as monolayer or spheres.Western blot analysis of IL22RA1, NANOG, SOX2, OCT3/4, and b-actin in spheres as compared with adherent cells. B, qPCR analysis of stemness-associatedgenes and IL22RA1 in spheres versus adherent cells among different PDAC cell lines. Data are normalized to GAPDH expression and are presented as foldchange in gene expression relative to adherent cells. Data are mean � SEM from three independent experiments. � , P < 0.05; �� , P < 0.01; �� , P < 0.001;(unpaired two-tailed Student t test). C and D, ALDEFLUOR analyses of IL22RA1hi and IL22RA1� cells from SW1990 and primary PDAC cells. DEAB, a specificinhibitor of ALDH1, was used as a control. ALDH1þ cells were gated as shown. E, IL22RA1hi and IL22RA� cells were sorted with flow cytometer. The relativeexpression of IL22RA1 and stemness-associated genes was determined by qPCR assay. Data are mean � SEM, n¼ 3; � , P < 0.05 (multiple unpaired Student t test).F, IL22RA1hi and IL22RA1� cell population from SW1990 and PDC0034 was sorted for sphere assay. Representative image of sphere and mean number ofsphere are shown. Scale bar, 50 mm. Data, mean � SEM (unpaired two-tailed Student t test). G, Cell viability of IL22RA1hi and IL22RA1� cells was examinedupon titrated dose of gemcitabine treatment for 72 hours. Data represent mean � SD of three replicates from one representative experiment (two-wayANOVA test and Bonferroni post hoc test were performed to compare the doses effects between two groups; � , P < 0.05).

IL22RA1/STAT3 Promotes Stemness in PDAC

www.aacrjournals.org Cancer Res; 78(12) June 15, 2018 3295




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PE/cy7-CD45, and BV421-CCR6 (BioLegend). For IL22RA1staining, cells were stained first with the IL22RA1 antibody(ab5984, Abcam) for 30 minutes at 4�C and second with AF647goat anti-rabbit IgG for 30 minutes at 4�C. For p-STAT3intracellular staining, sorted cells were fixed and permeabilizedwith methanol and stained with FITC-conjugated pSTAT3 anti-body (clone 4/P-STAT3, BD) for 30 minutes at 4�C. ALDHactivity was determined using the ALDEFLUOR Kit and follow-ing the manufacturer's instructions (STEMCELL). For IL22intracellular cytokine staining, immediately after isolation,cells were cultured in RPMI complete medium and stimulatedwith phorbol myristate acetate (50 ng/mL), ionomycin (1 mg/mL), and brefeldin A (10 mg/mL, eBioscience) for 4 hours at37�C. The cells were washed and stained with surface markers(CD45, CD3, and CCR6) and subsequently fixed and permea-bilized using the eBioscience Kit following the manufacturer'sguidelines (14). PE-IL22 (BioLegend) was used for intracellularstaining. Dead cells were excluded from the analysis using aviability stain (Invitrogen). The stained cells were acquired foranalysis or sorting using the LSRFortessa or AriaII (BD Bios-ciences). Flow cytometry data was analyzed with FlowJo soft-ware (Tree Star Inc.).

Sphere formation assayPDAC spheres were generated as described previously (4, 25).

Briefly, cells were suspended in DMEM: F12 medium containing2% B27 (Gibco), 20 ng/mL bFGF (PeproTech), and plated in24-well ultralow attachment plates (Corning). Growth factorswere replenished every 3 days. After 7 days, spheres with adiameter >50 mm were quantified.

Quantitative RT-PCRCells were lysed with TRIzol (Invitrogen) and RNA extracted

following standard protocol. cDNA was synthesized with theHigh-Capacity cDNA Reverse Transcription Kits (Applied Biosys-tems). Specific primers are included in the Supplementary Infor-mation (Supplementary Table S1). Quantitative RT-PCR reactionwas performed using SYBR Green Master Mix (Roche) and theStepOnePlus system (Applied Biosystems). Relative expression oftarget genes was calculated according to the Ct value with nor-malization to GADPH.

ImmunoblottingImmunoblotting was performed using the following anti-

bodies: human STAT3 and phosphorylated STAT3 (Cell SignalingTechnology); OCT3/4, NANOG, SOX2, and IL22RA1 (Abcam);and b-actin (Sigma). Signals were detected by ECL reagents(Thermo Fisher Scientific).

IHC and immunofluorescencePancreas tissues were fixed in 10% buffered formalin or frozen

in Tissue-Tek OCT compound. Fixed tissues were sectioned andused for hematoxylin and eosin (H&E) staining and CK19 IHCstaining (Troma-III, DSHB). Frozen tissueswere also sectioned forimmunofluorescence staining with the following antibodies:rabbit anti-human CK19 (Santa Cruz Biotechnology) andIL22RA1 (Abcam). Cells were mounted with DAPI and ProLongGold Antifade Reagent (Life Technology) before analysis with aconfocal microscope.

Chromatin immunoprecipitationChromatin immunoprecipitation (ChIP) was performed

according to the EZ-ChIP Kit protocol (Millipore). In brief, cross-linking was performed with 1% formaldehyde at room temper-ature for 10minutes; then, glycinewas added to quenchunreactedformaldehyde. Cells were lysed with SDS lysis buffer and soni-cated with the Sonics VibraCell Sonicator for shearing crosslinkedDNA to about 200 to 1,000 bp. Protein andDNA complexes wereprecipitated with specific antibodies against STAT3 and IgG con-trol (Cell Signaling Technology). To reverse DNA–protein cross-links, NaCl was added, and lysates were incubated at 65�C for 4 to5 hours. ChIP-enriched chromatin was used for qPCR with SYBRGreenMasterMix, normalizing to input. Specific primers are listedin the Supplementary Information (Supplementary Table S1).

Lentiviral transductionSeveral lentiviral vectors were used to transduce PDAC cells and

establish stable cell lines.The vectors included pGIPZ lentiviral vector encoding gene-

specific shRNAs for STAT3, IL22RA1, or scrambled shRNA. Thestable cell lines were obtained through GFPþ (coexpressed withthe lentiviral vector) cell sorting or puromycin selection. Knock-down efficiency was assessed by qPCR and immunoblotting.

Cell viability analysisCell proliferation and sensitivity assay to gemcitabine and

STAT3 inhibitors were all performed with CellTiter-Glo 2.0reagent (Promega) according to the manufacturer's instructions.

In vivo tumor formationSerial numbers (103, 104, and 105) of sorted IL22RA1� and

IL22RA1hi SW1990 cells were subcutaneously injected into eachflank of 4-week-old male nude mice (BALB/cA-nu/nu). Tumorincidence was monitored within 8 weeks. KPC1199 mouse pan-creatic cancer cells were sorted into IL22RA1� and IL22RA1hi

subpopulations and injected either orthotopically into the pan-creas tail (104/20 mL) or intraspenically (5 � 104/50 mL) into

Figure 3.IL22RA1hi subpopulation of pancreatic cancer cell possesses higher capability for tumorigenicity and metastasis. A, Tumor-initiation assay was performedand compared between IL22RA1hi and IL22RA� subpopulations with SW1990 cells. Cells from the indicated populations were injected into nude mice after FACSsorting. The number of successful tumor initiations after 8 weeks is shown. Representative images of H&E staining of indicated xenografts are shown.Scale bar, 50 mm. B, Sorted IL22RA1hi and IL22RA� cells from SW1990 were examined by proliferation assay (mean � SD; � , P < 0.05, one-way ANOVA andBonferroni post hoc test). C and D, KPC1199 representative FACS plots of IL22RA1 staining are shown. IL22RA1hi and IL22RA� cells from KPC1199 were sortedand examined for indicated genes expression and proliferation capability. Data, mean � SEM; � , P < 0.05 (unpaired two-tailed Student t test). E, IL22RA1hi

and IL22RA� KPC1199 cells (104 each) were sorted and injected into pancreatic tail of C57BL/6J mice. Representative images and weight of orthotropic tumorsoriginated from indicated group (n ¼ 6 of each group; mean � SEM, unpaired two-tailed Student t test). F, Representative images of H&E, IHC (CK19), andimmunofluorescence (IL22RA1) staining from indicated group are shown. G, IL22RA1hi and IL22RA� cells (5 � 104) from KPC1199 were sorted and intraspleniccells injected into C57BL/6J mice. Representative images of H&E and CK19 staining on the liver of mice injected with indicated cell. Scale bar, 100 mm. Quantificationof CK19þ liver metastatic lesion (per 4� filed) from indicated population. Data are mean � SEM, n ¼ 12 pooled from two independent experiments (unpairedtwo-tailed Student t test).






C57BL/6J mice. For the orthotopic model, tumors were harvested3 to 4weeks after injection. Tumorweightsweremeasured and thetumors processed for H&E, IHC (CK19), and immunofluores-cence (IL22RA1) analysis. For the liver metastasis model, livertissues were harvested 4 to 6 weeks after injection and processedfor H&E and CK19 staining. CK19þ staining was quantified andrepresents the metastatic index.

Statistical analysisPearson correlation together with P value was computed to

assess the degree of association between indicated genes. Survivalcurves were estimated by Kaplan–Meier methods. Two-wayANOVA or one-way ANOVA together with Bonferroni post hoctest was used formultiple groups analysis. Unpaired Student t testwas used to determine statistical significance for the remainingexperiments, and a P value of less than 0.05 was consideredsignificant. Values are expressed as mean � SEM or mean � SD(Prism 6; GraphPad Software). Unless indicated, results are fromat least two or three independent experiments.

ResultsIL22RA1 is upregulated in pancreatic cancer and is negativelyassociated with prognosis of patients with PDAC

In previous studies, we have shown that the IL22/IL22RA1signaling axis plays a critical but distinct role in the pathogenesisof acute and chronic pancreatitis (14, 15). It is widely acceptedthat chronic inflammatory conditions of the pancreas are riskfactors for the development of PDAC (22, 26). To extend ourunderstanding of IL22RA1 in pancreatic disease, here we inves-tigated the function of IL22RA1 in pancreatic cancer. We firstcompared IL22RA1 protein levels between PDAC cell lines andnormal pancreatic ductal cell lines (HPDE and hTERT-HPNE).IL22RA1 was elevated inmost PDAC cells compared with normalductal cells (Fig. 1A). IL22RA1 is mainly expressed by pancreaticacinar cells and stellate cells under homeostatic and inflammatoryconditions, respectively (14, 15). To determine the distribution ofIL22RA1 expression in tumors, human pancreatic cancer tissueswere analyzed by immunofluorescence staining of IL22RA1.IL22RA1was dominantly expressed by PDAC cells, demonstratedby its coexpression with CK19 (Fig. 1B). Moreover, the expressionof IL22RA1 was heterogeneous among the PDAC tumor cells. Toconfirm the intratumoral heterogeneity of IL22RA1, PDAC cellsfrom primary tumors were isolated and analyzed by flow cyto-metry staining for IL22RA1.Consistentwith immunofluorescencestaining, intratumoral heterogeneity of IL22RA1 was shown(about 4%–32% depending on the antibody isotype). In addi-tion, a similar IL22RA1 expression pattern was observed in PDACcell lines and our established pancreatic PDCs (Fig. 1C; Supple-mentary Fig. S1A and S1B; Supplementary Table S2).

Next, we examined the clinical significance of elevated IL22RA1in pancreatic cancer. When patients were divided into "high" and"low/median" IL22RA1 expression based on the median mRNAvalue of IL22RA1, we observed that elevated IL22RA1 expressionwas associated with poor patient survival in the Renji cohort (Fig.1D). This cohort includes 50 patients with pancreatic cancer withclinical andpathologic information. Tumordifferentiation state isalso positively associated with IL22RA1 expression (Supplemen-tary Table S3). Comparable results were observed from TheCancer Genome Atlas (TCGA) pancreatic cancer database, inwhich overall survival was shorter in patients with high IL22RA1

expression (Fig. 1E). Altogether, these data strongly suggest thatincreased tumor IL22RA1 abundance is an important predictor ofpoor survival in pancreatic cancer.

Pancreatic cancer cells with high IL22RA1 expression harborstemness properties

To elucidate the function of IL22RA1 in pancreatic cancerprogression, we first assessed gene coexpression with IL22RA1using the TCGA pancreatic cancer database. We found thatIL22RA1 expressionwas positively correlatedwith developmentaland stemness genes, like PDX1, SOX9, HES1, and CD24 (Supple-mentary Fig. S2A), which indicated that increased IL22RA1expression might be associated with cancer stemness. PancreaticCSCs can be enriched in vitro using the anchorage-independentsphere formation assay (4, 25). Compared with adherent mono-layer cells, IL22RA1 together with multiple core stem cell genes(NANOG, POU5F1, and SOX2) were dramatically elevated insphere cultures (Fig. 2A andB). CD44þCD24þESAþ triple positivecells are characterized as pancreatic CSCs (5); we observed thatIL22RA1 was upregulated in these cells compared with non-CSCs(CD44�CD24�ESA�; Supplementary Fig. S2B). These resultsindicate that IL22RA1 is associatedwith cancer stemness in PDAC.

We determined whether IL22RA1hi cells had cancer stemnessproperties. ALDH1 is a functionalmarker of stemness. IL-22RA1hi

cells possessed higher ALDH activity compared with IL22RA1�

cells in multiple PDAC cell lines and primary tumor cells (Fig. 2Cand D; Supplementary Fig. S3A and S3B). This was comparablewith the analysis of CD44þCD24þESAþ cells as shown in Sup-plementary Fig. S3C. Furthermore, IL22RA1� and IL22RA1hi cellswere sorted from SW1990 and PDC0034 pancreatic cancer celllines and qRT-PCR analysis revealed that the expression of corestemness genes (SOX2, NANOG, POU5F1, STAT3) was upregu-lated in IL22RA1hi cells compared with IL22RA1� cells (Fig. 2E).To support this, the sphere formation assay showed that theclonogenic potential of IL22RA1hi cells was superior to that ofIL22RA1� cells (Fig. 2F). Moreover, IL22RA1hi cells possessedstronger resistance to the chemotherapy drug gemcitabine (Fig.2G; Supplementary Fig. S3D). These results demonstrated thatpancreatic cancer IL22RA1hi cells display stemness properties.

IL22RA1hi subpopulation of pancreatic cancer cells possesseshigher capability for tumorigenicity and metastasis

The in vivo tumorigenicity of IL22RA1hi cells compared withIL22RA1� cells was explored using limiting-dilution tumor assaysin nude mice. IL-22RA1hi cells possessed enhanced tumor-initi-ating capability in vivo (Fig. 3A) despite showing significantlylower proliferative capacity in vitro compared with IL22RA1� cells(Fig. 3B). Xenografts derived from these two groups had noobvious histology differences (Fig. 3A).

IL22, themajor ligand of IL22RA1 is only expressed by immunecells (27). Given the immunodeficiency of nude mice, we furtherexamined the tumorigenicity of IL22RAhi cells using a PDACorthotopic model in immunocompetent C57BL/6J mice. To thisend, IL22RA1hi and IL22RA1� cells sorted from KPC1199, amouse pancreatic cancer cell line generated from KPC mice(C57BL/6J background), were examined by qRT-PCR analysis.Consistent with human PDAC cells, IL22RA1hi cells fromKPC1199 cells expressed higher core stemness genes, includingNanog, Pou5f1, Sox2, and Epcam (Fig. 3C). In line with this, thesecells exhibited lower proliferation in vitro, but enhanced tumor-igenicity in vivo (Fig. 3D and E). Interestingly, the orthotopic

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Figure 4.

IL22/IL22RA1 axis promotes stemness of pancreatic cancer cells. A, SW1990 cells were treated with vehicle (VE) or different IL22RA1 ligands (IL22, IL20, andIL24) for 24 hours before ALDEFLUOR analyses. B, Sphere assay was performed in SW1990 under vehicle or IL22 treatment. Representative images, number, anddiameter of spheres are shown (n � 3; mean � SEM). C, SW1990 and PDC0034 were cultured with vehicle or IL22 for 6 hours. The relative mRNA of indicatedgenes was detected by qPCR assay. Data, mean � SEM (�� , P < 0.01; �� , P < 0.001, compared with vehicle). D, SW1990 cells were treated with vehicle or IL22 forindicated time. The expression of SOX2, NANOG, IL22RA1, and b-actin was examined by immunoblotting. E, Sphere assay was performed with shIL22RA1 or shCON-transfected SW1990 cells in the presence of IL22. Results are shown as sphere images and quantification of sphere number in triplicate (mean � SD). IL22RA1knockdownefficiencywas confirmedwith immunoblotting.F, IL22RA1hi and IL22RA� cells fromSW1990were sorted for sphere assayunder vehicle or IL22 treatment.Representative images and quantification of sphere number are shown (mean � SEM). G, IL22RA1hi and IL22RA� cells from SW1990 were sorted and treatedwith vehicle or IL22. The relative expression of indicated genes was detected by qPCR after 6 hours. Mean � SD; � , P < 0.05; �� , P < 0.01; �� , P < 0.001. H, IL22level in adjacent normal tissue and pancreatic tumor was examined by flow cytometry. Cells were pregated with live/dead and CD45 staining. n ¼ 8–10; dataare mean � SEM; �� , P < 0.01; ns, nonsignificant.






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tumor derived from IL22RA1hi cells possessed similar histologicfeatures with primary pancreatic cancer (Fig. 3F), indicatingIL22RA1hi cells have self-renewal and repopulation abilities inthe pancreatic tumor microenvironment.

Cancer cells with stemness features play a vital role in metas-tasis in a broad spectrum of malignancies (4, 7). We next deter-mined whether PDAC IL22RA1hi cells had higher metastaticcapability compared with IL22RA1� cells. The epithelial–mesen-chymal transition (EMT) is a process by which epithelial cells losetheir cell polarity and cell–cell adhesion to initiate cancer cellmetastasis (25). IL22RA1hi cells showed higher expression ofEMT-related transcript factors than IL22RA1� cells (Supple-mentary Fig. S4). IL22RA1hi and IL22RA1� KPC1199 cells (5� 104) were injected intrasplenically into C57BL/6J mice. After35 days, liver tissues were examined for histology and CK19þ

PDAC lesions. Mice injected with IL22RA1hi cells had more andlarger CK19þ metastatic lesions in the liver when comparedwith mice injected with IL22RA1� cells (Fig. 3G). Taken togeth-er, these results revealed the enhanced malignancy of IL22RA1hi

cells in vivo and prompted us to further characterize theirregulation in vitro.

IL22/IL22RA1 signaling axis promotes stemness of pancreaticcancer cells

IL22RA1 executes its function through activation by variousligands, including IL22, IL20, and IL24. As IL22 has been reportedto promote cancer stemness in colon cancer (28), here wehypothesized that IL22 might enhance stemness in pancreaticcancer. In support of this, IL22 significantly increased ALDHactivity in PDAC cells compared with other ligands (Fig. 4A).Next,we examinedwhether IL22 can enhance sphere formationofPDAC cells. IL22 increased the number and size of spheres for alltested cells (Fig. 4B; Supplementary Fig. S5A and S5B) withoutaltering cell proliferation (Supplementary Fig. S5C). IL22enhanced mRNA (Fig. 4C) and protein (Fig. 4D) expression ofmultiple core stem cell genes, including NONOG, SOX2,and POU5F (OCT3/4), as well as IL22RA1 in SW1990 andPDC0034 cells. Moreover, IL22RA1 knockdown abrogatedIL22-induced sphere formation and core stemcell gene expression(Fig. 4E; Supplementary Fig. S5D). These data showed that theIL22/IL22RA1 signaling axis contributes to stemnessmaintenancein PDAC cells.

We have shown that IL22 promoted pancreatic cancer stemnessvia the IL22/IL22RA1 signaling pathway. We next determinedwhether only IL22RA1hi cells, which have stemness features,could respond to activation by IL22. IL22RA1hi and IL22RA1�

cells fromSW1990 cellswere treatedwith IL22. IL22preferentially

enhanced stemness of cells with high expression of IL22RA1 (Fig.4F and G; Supplementary Fig. S5E). More importantly, theexpression of IL22RA1 could be upregulated by IL22 in IL22RA1hi

cells (Fig. 4G), providing positive feedback for stemness mainte-nance in IL22RA1hi cells.

Having established that IL22 promotes pancreatic cancer stem-ness, we determined whether IL22 was elevated in pancreaticcancer. Elevated IL22 has been reported in primary pancreaticcancer previously (29, 30). Consistentwith these previous studies,we confirmed that IL22þ leukocytes were increased and IL22 wasdominantly expressed by CD4þ T cells in the pancreatic cancermicroenvironment (Fig. 4H; Supplementary Fig. S5F).

IL22 promotes pancreatic cancer stemness and IL22RA1expression via STAT3 activation

Next, we explored the molecular mechanisms by which IL22promotes pancreatic cancer stemness. STAT3 plays a critical rolein bridging the cross-talk between cancer cells and immunecells in the tumor microenvironment (31–34). Moreover,the majority of PDAC show constitutive activation of STAT3(34, 35). In line with previous reports, using immunoblottingand flow cytometry assays, we observed that IL22 activatedSTAT3 in pancreatic cancer cells (Fig. 5A and B). Moreover,STAT3 expression was elevated upon long-term IL22 exposure(Fig. 5C). We sought to examine whether the effect of IL22 inPDAC stemness was STAT3 dependent. To this end, we manip-ulated STAT3 activation (using specific inhibitors) or expres-sion (through shRNA knockdown) in pancreatic cancer cells. Asshown, both inhibition of STAT3 activity and decreased STAT3expression resulted in reduced sphere number upon IL22treatment (Fig. 5D; Supplementary Fig. S6A). Interestingly, IL6activated STAT3 but failed to promote stemness of PDAC cells(Supplementary Fig. S6B–S6D). IL6/STAT3–triggered synthesisof SOCS3 is expected to shut down IL6-mediated (36) but notIL22-mediated STAT3 activation in the long term, which mightresult in the observed differences between IL6 and IL22 (Sup-plementary Fig. S6E). These observations suggest that IL22-mediated STAT3 activation is necessary and relatively specific inpromoting PDAC stemness.

We further investigated the effects of IL22 activation of STAT3on the expression of stemness-associated genes. STAT3 knock-down reduced the expression of core stemness genes in thepresence of IL22 (Fig. 5E). Herein, we speculated that STAT3might directly bind to promoters of stemness-associated genesand subsequently induce their expression. We found severalpredictions for STAT3 binding to SOX2 and NANOG promoterregions (http://www.sabiosciences.com/chipqpcrsearch.php).ChIP

Figure 5.IL22 promotes pancreatic cancer stemness and IL22RA1 expression via STAT3 activation. A, SW1990 cells were treated with IL22 (50 ng/mL) for indicatedtime points. The amount of phosphorylated STAT3, STAT3, and b-actin proteinwas detected by immunoblotting.B, SW1990 cells were treatedwith IL22 (50 ng/mL)for 15 minutes, and phosphorylated STAT3 (Y705) was determined by flow cytometry. Data are representative FACS plot and quantification of p-STAT3 level (mean� SEM). C, SW1990 cells were treated with IL22 (50 ng/mL) for 24 and 48 hours. STAT3 and b-actin protein were detected by immunoblotting.D, Sphere assay wasperformed with shSTAT3 or scrambled shRNA (shCON)-transfected SW1990 cells in the presence of IL22. STAT3 knockdown efficiency was examined byimmunoblotting. Representative sphere images and quantification of sphere number from two independent experiments are shown (mean � SD; �� , P < 0.01).E, SW1990 cells expressing shSTAT3 or shCON were cultured with vehicle (VE) or IL22 for 6 hours. Cells were prefasted before IL22 stimulation. The relative mRNAlevel of NANOG and SOX2 was normalized with vehicle from shCON group. Mean � SD; � , P < 0.05; �� , P < 0.01. F, STAT3-ChIP assay was performed to examineSTAT3 binding at SOX2 and NANOG promoters in SW1990 cells upon vehicle or IL22 treatment. One of two independent experiments is shown. G, IL22RA1 mRNAexpression was detected in SW1990 cells after vehicle and STAT3 inhibitors (stattic or S3I-201) treatment in the presence of IL22. The relative mRNA level ofIL22RA1 was normalized with vehicle from shCON group. Data, mean � SD; � , P < 0.05; �� , P < 0.01. H, STAT3-ChIP assay was performed to examine STAT3binding at IL22RA1 promoters in SW1990 cells upon vehicle or IL22 treatment. One of two independent experiments is shown. Unpaired two-tailed Student t testwas used for all data analyses. ns, nonsignificant.





http://www.sabiosciences.com/chipqpcrsearch.php



He et al.




demonstrated that IL22 increased STAT3 binding to several sitesof the SOX2 and NANOG promoters (Fig. 5F), suggesting thatSTAT3 might directly activate stemness genes. Moreover, IL22failed to induce IL22RA1 expression in the presence of STAT3inhibitors stattic or S3I-201 (Fig. 5G). In addition, severalSTAT3 potential binding sites were predicted on the IL22RA1promoter, and direct binding of STAT3 to the IL22RA1 pro-moter was further verified using ChIP (Fig. 5H). Thus, IL22promotes pancreatic cancer stemness and IL22RA1 expressionvia STAT3 activation.

STAT3 activity is required for IL22RA1hi populationmaintenance

STAT3 is essential for stemness maintenance in pancreaticcancer (37–40). As showed in Fig. 2B, STAT3 mRNA expressionwas elevated in IL22RA1hi populations. Moreover, STAT3 activa-tion was significantly higher in IL22RA1hi PDAC cells (Fig. 6A).Therefore, we next determined whether STAT3 was required forexpression of IL22RA1 in the absence of IL22. The percentage ofIL22RA1þ cellswas significantly reduced by STAT3 inhibitors (Fig.6B). In addition, STAT3 inhibitors downregulated IL22RA1mRNA expression together with core stem cell genes in IL22RA1hi

cells (Fig. 6C). As showed in Fig. 5E, STAT3 inhibitors did notdownregulate basal levels of IL22RA1, which is partially due tolow levels of STAT3 activity caused by serum-free pretreatment.The above results demonstrated that STAT3 activation was indis-pensable for the maintenance of IL22RA1hi cells, both in theabsence or presence of IL22 (Figs. 6B and C and 5G). Thus, STAT3could be a potential target for those cells with high expression ofIL22RA1. We therefore determined whether STAT3 inhibitioncould sensitize IL22RA1hi cells to chemotherapy. The STAT3inhibitor stattic specifically inhibited phosphorylated STAT3(Y705) in a dose-dependent manner (0.1–1 mmol/L; Supplemen-tary Fig. S6F). IL22RA1hi cells were exposed to gemcitabine alone,titrated concentrations of stattic alone, or both. IL22RA1hi PDACcells could be targeted by combined treatment with gemcitabineand stattic in vitro (Fig. 6D). The synergistic effect of gemcitabineand stattic on IL22RA1hi cells was examined in vivo using anorthotopic model. Combined treatment with gemcitabine andstattic significantly reduced IL22RA1hi cell-derived tumor pro-gression in vivo (Fig. 6E) compared with each drug alone. Besidesmaintaining stemness, IL22RA1hi cells would be expected todifferentiate in vivo. Thus, it was not surprising to see the inhib-itory effect of gemcitabine or stattic alone on IL22RA1hi cell-derived tumors. Taken together, these data show the significanceof STAT3 in maintaining the IL22RA1hi population and in medi-ating IL22/IL22RA1–promoted stemness and provides a thera-peutic strategy for eliminating IL22RA1hi PDAC cells as depictedby the schematic model (Fig. 6F).

DiscussionChronic inflammation is a key feature of pancreatic cancer

(22, 23). Many immune cells exist in tumor microenvironment(41), indicating their indispensable role in PDAC initiation andprogression. Elucidation of the cross-talk between immune andPDAC cells, especially pancreatic CSCs, would enable betterunderstanding of pancreatic cancer progression and provide apotential therapeutic strategy for targeting PDAC. In our currentstudy, we aimed to investigate how IL22/IL22RA1 contributed toPDAC development and progression.

IL22 can be produced by Th17 cells, Th22, gd T cells, NKT cells,and innate lymphoid cells (42–45). Knowledge of IL22 biologyhas evolved rapidly since its discovery in 2000 (46), and a role forIL22 has been identified in numerous tissues, including theintestines, lung, liver, kidney, thymus, pancreas, and skin. Theexpression IL22 and its receptor IL22RA1 is limited to hemato-poietic cells and epithelial/stromal cells, respectively (27). TheIL22/IL22RA1 signaling axis enables communication betweenimmune and epithelial/stromal cells. In our previous studies, wefound IL22RA1 is dominantly expressed on pancreatic acinar cellsduring acute pancreatitis, which enabled IL22þ immune cells topromote tissue repair by targeting acinar cells (14). However, wealso found IL22þ immune cells, elevated in patients with chronicpancreatitis with a smoking history, promoted fibrosis via trig-gering pancreatic stellate cell activation (15). In all, we observedthat the function of IL22 under different pathologic conditionscould be different even in the same tissue, which was partlydependent on where IL22RA1 is expressed.

Two recent reports identified IL22 and IL22RA1 were elevatedin pancreatic cancer (29, 30). However, little is known about howthe IL22/IL22RA1 signaling axis is involved in pancreatic cancerdevelopment. Here, we confirmed that IL22RA1 expression waselevated in pancreatic cancer using PDAC cell lines, and elevatedexpression of IL22RA1 was associated with poor survival in bothour Renji and TCGA pancreatic tumor cohorts. To further under-stand how IL22RA1 is involved in PDAC development, we ana-lyzed genes coexpressed with IL22RA1 in the TCGA pancreatictumor database. We observed that multiple stemness-relatedgenes were tightly associated with IL22RA1, which suggested thatIL22RA1 may contribute to cancer stemness. To confirm ourhypothesis, pancreatic CSCs were enriched via sphere formationand CD44þCD24þESAþ cell sorting (5, 25). IL22RA1 togetherwith core stemness genes were upregulated in the enriched CSCs.By sorting and analyzing IL22RA1hi cells, we found IL22RA1hi

PDAC cells harbored stemness features including higher expres-sion of core stemness genes, sphere formation ability, ALDHactivity, and gemcitabine resistance when compared withIL22RA1� cells. In addition, in vivo studies further confirmed thehigher capability for tumor formation and metastasis of

Figure 6.STAT3 activity is required for IL22RA1hi population maintenance. A, Phosphorylated STAT3 (p-STAT3) level was detected in sorted IL22RA1� and IL22RA1hi

cells from SW1990 with intracellular staining. Representative FACS plot is shown. B, Flow cytometry analysis of IL22RA1 expression in SW1990 cells after treatmentof vehicle (VE) and STAT3 inhibitors (stattic 1 mmol/L; S3I-201 50 mmol/L). Representative FACS plots and quantification of IL22RA1þ cells (%) from threeindependent experiments are shown (mean � SEM; � , P < 0.05, unpaired two-tailed Student t test). C, IL22RA1hi cells from SW1990 were sorted for vehicle orstattic treatment (1 mmol/L, 8 hours). The relative expression of indicated genes was detected by qPCR assay. Data, mean � SD (one-way ANOVA test).D, IL22RA1hi cells from SW1990were treatedwith titrated doses of stattic (0–1 mmol/L) together with or without gemcitabine (100 nmol/L) for 72 hours. Percentageof cell viabilitywas determined (mean� SD; � ,P <0.05, one-wayANOVA test). ns, nonsignificant.E, IL22RA1hi sorted fromKPC1199 cells (104 each)were injected intopancreatic tail of C57BL/6J mice. After 8 days, mice were grouped for indicated treatments. Representative images of orthotopic tumors and H&E stainingfrom indicated group are shown. F, The scheme of IL22/IL22RA1/STAT3 signaling on IL22RAhi population maintenance and the targeting strategy for IL22RAhi cellswith CSC-like potentials.






IL22RA1hi PDAC cells compared with IL22RA1� cells. Moreinterestingly, orthotopic tumors from IL22RA1hi cells possessedsimilar histologic features with primary pancreatic cancer, indi-cating IL22RA1hi cells have an ability to repopulate in the pan-creatic tumor microenvironment.

IL22RA1 exerts its function upon directly binding to its ligands(including IL22, IL20, and IL24) and leading to STAT3 signalingactivation (42, 43, 47). Of the ligands tested, IL22 showed thegreatest induction of ALDH activity in PDAC cells, indicating itscritical role in PDAC stemness. Consistent with previous findingsin colon cancer (28), we found IL22 promoted cancer stemness inpancreatic cancer in an IL22RA1-dependent manner. Moreover,IL22's enhancement of stemness was restricted to IL22RA1hi

PDAC cells. Knockdown or inhibition of STAT3 activity blockedincrease of PDAC cell stemness by IL22 treatment. Interestingly,IL22 increased IL22RA1mRNA expression by promoting bindingof STAT3 directly to the IL22RA1 promoter region, therebyenhancing IL22/IL22RA1–STAT3 signaling. STAT3-mediated sig-naling involves multiple cytokines, such as IL6. Unlike IL22, wefound IL6 unable to promote PDAC cell stemness (Supplemen-tary Fig. S6B–S6D), which suggested that other factors mightcoordinate with STAT3 to determine activation of specific genes.STAT3 could regulate gene expression through epigeneticmechanisms (48). Previous studies showed that Dot1L (aH3K79 methyltransferase), together with STAT3, was involvedin IL22-promoted colon cancer stemness (28). In our study, wefound Dot1L-specific inhibitors did not inhibit sphere formationupon IL22 treatment in PDAC cells (Supplementary Fig. S7A–S7C). Identifying potential coordinators, especially epigeneticregulators, is worth investigating in the future.

Establishing the cells that secrete IL22RA1 ligands may providebetter understanding as to how PDAC cells are affected by othercells in the tumor microenvironment. IL22 is dominantlyexpressed by CD4þ T cells under acute and chronic pancreatitis(14, 15). Here, elevated intratumoral IL22 was secreted by pan-creatic leukocytes (CD45þ cells), mainly CD4þ T cells (Supple-mentary Fig. S4). IL22þ CD4þ T cells migrating into colon cancertissue are dependent on the CCR6/CCL20 chemokine axis (28).Consistent with this study, we found the majority ofCD4þCD45ROþ IL22þ cells were CCR6þ (Supplementary Fig.S5F).Whether CCR6/CCL20 is involved in IL22þ T-cell traffickinginto the pancreatic cancer microenvironment needs to be furtherdetermined.

IL22RA1 intratumoral heterogeneity expression may resultfrom various microenvironments. In our study, we found that

STAT3 activation is required for the maintenance of IL22RA1hi

cells.STAT3 inhibition may sensitize IL22RA1hi cells to chemother-

apy.We showed STAT3 inhibition using the inhibitor stattic had asynergistic effect with gemcitabine for treating IL22RA1hi PDACcells both in vitro and in vivo. Furthermore, STAT3 mediatesinflammation-induced tumor development. Multiple pathways,including IL6, G-protein–coupled receptors, Toll-like receptors,andmiRNAs, were identified to regulate STAT3 signaling in cancer(48). In addition to IL22, how other microenvironmental factorsregulate IL22RA1 is worth investigating in future studies, as itmayprovide a therapeutic strategy for patients with PDAC.

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

Authors' ContributionsConception and design: W. He, A. Habtezion, Y.-W. Sun, J. XueDevelopment of methodology: W. He, J. Wu, J. Shi, J. XueAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W. He, J. Wu, J. Geng, P. Lu, M.-W. Yang, W. Wang,Y.-W. SunAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W. He, W. Dai, J. XueWriting, review, and/or revision of the manuscript: W. He, W. Dai, Z.-G.Zhang, A. Habtezion, Y.-W. Sun, J. XueAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Wu, Y.-M. Huo, J. Geng, Y. Fang, Z.-G. Zhang,J. XueStudy supervision: J. Xue

AcknowledgmentsWe thank Prof. Tian from Southern University of Science of Technology

for providing KPC1199 cells. This work was supported by China State KeyLaboratory of Oncogenes and related gene no. 91-15-15 (to J. Xue), Programfor Professor of Special Appointment (Eastern Scholar) at Shanghai Institu-tions of Higher Learning no. TP2015007 (to J. Xue), Shanghai MunicipalEducation Commission-Gaofeng Clinical Medicine Grant Support no.20161312 (to J. Xue), National Natural Science Foundation of China no.81702938, no. 81770628 (to J. Xue), Shanghai Sailing Program no.16YF1408600 (to W.T. Dai), and National Natural Science Foundation ofChina no. 81672736 (to W.T. Dai).

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 October 15, 2017; revised December 13, 2017; accepted March 19,2018; published first March 23, 2018.

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2018;78:3293-3305. Published OnlineFirst March 23, 2018.Cancer Res Weizhi He, Jinghua Wu, Juanjuan Shi, et al. in Pancreatic CancerIL22RA1/STAT3 Signaling Promotes Stemness and Tumorigenicity

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