cytokine induction of tumor necrosis factor receptor 2...

Signaling and Regulation

Cytokine Induction of Tumor Necrosis Factor Receptor 2 IsMediated by STAT3 in Colon Cancer Cells

Kathryn E. Hamilton, James G. Simmons, Shengli Ding, Laurianne Van Landeghem, and P. Kay Lund

AbstractThe IL-6/STAT3 and TNFa/NFkB pathways are emerging as critical mediators of inflammation-associated

colon cancer. TNF receptor (TNFR) 2 expression is increased in inflammatory bowel diseases, the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colitis-associated cancer, and by combined interleukin (IL) 6 andTNFa. Themolecular mechanisms that regulate TNFR2 remain undefined. This study used colon cancer cell linesto test the hypothesis that IL-6 and TNFa induce TNFR2 via STAT3 and/or NFkB. Basal and IL-6 þ TNFa–induced TNFR2 were decreased by pharmacologic STAT3 inhibition. NFkB inhibition had little effect on IL-6þTNFa–induced TNFR2, but did inhibit induction of endogenous IL-6 and TNFR2 in cells treated with TNFaalone. Chromatin immunoprecipitation (ChIP) revealed cooperative effects of IL-6 þ TNFa to induce STAT3binding to a�1,578 STAT response element in theTNFR2 promoter but no effect onNFkB binding to consensussites. Constitutively active STAT3 was sufficient to induce TNFR2 expression. Overexpression of SOCS3, acytokine-inducible STAT3 inhibitor, which reduces tumorigenesis in preclinical models of colitis-associatedcancer, decreased cytokine-induced TNFR2 expression and STAT3 binding to the �1,578 STAT responseelement. SOCS3 overexpression also decreased proliferation of colon cancer cells and dramatically decreasedanchorage-independent growth of colon cancer cells, even cells overexpressing TNFR2. Collectively, these studiesshow that IL-6- and TNFa-induced TNFR2 expression in colon cancer cells is mediated primarily by STAT3and provide evidence that TNFR2may contribute to the tumor-promoting roles of STAT3.Mol Cancer Res; 9(12);1718–31. �2011 AACR.

Introduction

Patients with inflammatory bowel diseases (IBD), such asCrohn's disease and ulcerative colitis, have an increasedlifetime risk of developing inflammation-associated colorec-tal cancer (CRC; refs. 1–3). Chronic increases in prolifer-ation of intestinal epithelial cells (IEC) driven by proin-flammatory factors have been shown to promote tumori-genesis. The IL-6/STAT3 (4–9) and TNFa/NFkB (10–12)pathways are both major mediators of inflammation-asso-ciated CRC, and recent studies show that TNFa blockadedecreases intestinal tumor formation in mice (13, 14).TNFa signals through 2 receptors, TNFR1 and TNFR2.

TNFR1 exerts proapoptotic functions due to its intracellulardeath domain (15). TNFR2, which lacks a death domain,has been linked to increased proliferation of IEC in animalmodels of colitis and colon cancer cells (16, 17). Thissupports a concept that TNFR2 may mediate protumori-

genic effects of TNFa. The role of TNFR2 in inflammation-associated cancer is a topic of increasing interest, as recentstudies indicate that TNFR2 is upregulated in IBD and inthe azoxymethane/dextran sodium sulfate (AOM/DSS)model of inflammation-associated cancer (14, 16, 18). Invitro studies have shown that TNFR2 is induced in coloncancer cells by TNFa and IL-6 combined but neithercytokine alone (16). Other studies have shown TNFR2induction by IFNg (19). These findings suggest that theSTAT pathways activated by IL-6 or IFNg and/or NFkBpathways typically activated by TNFa may interact toinduce TNFR2 expression. In support of this possibility,the human TNFR2 promoter contains 2 consensus STAT-binding sites as well as 2 consensusNFkB-binding sites (20).The present study tested the hypothesis that IL-6 andTNFainteract to induce TNFR2 expression by activation ofSTAT3, NFkB, or both STAT3 and NFkB.Suppressors of cytokine signaling (SOCS) proteins are

negative feedback inhibitors of the JAK/STAT pathway(21). IEC-specific SOCS3 gene deletion increased tumorload in the AOM/DSSmodel of colitis-associatedCRC (22).This effect was associated with enhanced activation of bothSTAT3 and NFkB (22). In vitro, SOCS3 overexpressionreduced proliferation of colon cancer cell lines and inhibitedboth IL-6–induced STAT3 activation and TNFa-inducedNFkB activation (22). Furthermore, SOCS3 genes aresilenced by promoter hypermethylation in various human

Authors' Affiliation: Department of Cell and Molecular Physiology, Uni-versity of North Carolina School of Medicine, Chapel Hill, North Carolina

Corresponding Author: P. Kay Lund, University of North Carolina Schoolof Medicine, 111 Mason Farm Road CB#7545, Chapel Hill, NC 27599.Phone: 919-966-1490; Fax: 919-966-6927; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-10-0210

�2011 American Association for Cancer Research.

MolecularCancer

Research

Mol Cancer Res; 9(12) December 20111718

on June 19, 2018. © 2011 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst October 12, 2011; DOI: 10.1158/1541-7786.MCR-10-0210

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cancers, including CRC (23–26). Together these data pro-vide strong evidence that SOCS3 normally acts as a sup-pressor of inflammation-associated CRC. The current studytested the hypothesis that SOCS3 overexpression limits thecytokine induction of TNFR2 and/or growth-promotingeffects of TNFR2 in colon cancer cells.Our studies reveal a novel pathway where IL-6 and TNFa

cooperatively induce TNFR2 in colon cancer cells by inter-actions at multiple levels. TNFa, acting through NFkB,induces endogenous IL-6, but combined effects of IL-6 andTNFa to induceTNFR2 gene expression depend on STAT3activation. We also provide direct evidence that TNFR2promotes growth of colon cancer cells. Overexpression of theknown STAT3 inhibitor SOCS3 decreases TNFR2 expres-sion, decreases STAT3binding to theTNFR2 promoter, andpotently inhibits proliferation and anchorage-independentgrowth of colon cancer cells.

Materials and Methods

Cell culture and cytokine treatmentsThe 2 colon cancer cell lines primarily used in this study

were SW480 and COLO205 cells, both of which expresslow levels of endogenous SOCS3. SW480 cells were used forthe majority of studies because our prior studies showed thatthese cells are responsive to both IL-6 and TNFa, whichrobustly activate STAT3 and NFkB, respectively (22).COLO205 cells were used as an independent cell line toconfirm cytokine induction of TNFR2. Because COLO205cells grow well in soft agar, they were also used to addresseffects of TNFR2 and SOCS3 on anchorage-independentgrowth. SW480, COLO205, and Caco2 cells were obtainedfrom the American Type Culture Collection. The humanintestinal epithelial cell line (HIEC)was generously providedby Dr. Jean-Francois Beaulieu (University of Sherbrooke,Sherbrooke, Quebec, Canada). Cells were grown in RPMI-1640 media (Gibco) supplemented with 10% heat-inacti-vated FBS, 50 U/mL penicillin, and 50 mg/mL streptomy-cin. Given prior findings that both IL-6 and TNFa wererequired to induce TNFR2 (16), cells were treated withrecombinant human IL-6 and/or TNFa (Peprotech) at50 ng/mL in serum-free medium. Cells were harvested atvarious times after cytokine treatment for evaluation ofTNFR2 mRNA and protein, or STAT3 and NFkB bindingto consensus regions in the TNFR2 promoter.

Semiquantitative real-time PCR analysesTotal RNA was extracted from cell lines using the

RNeasy Mini Kit (Qiagen) according to manufacturer'sinstructions. Reverse-transcription was carried out usingAMV-RT (Promega). PCR and analyses were completedon the Rotorgene 2000 (Qiagen) using Invitrogen Plat-inum qPCR Supermix-UDG and the following TaqManprimer–probe sets (Applied Biosystems): human TNFR2Hs00961755_m1, human TNFR1 Hs00533568_g1,human IL-6 (Hs00985639_m1), human ICAM-1 (pos-itive control as NFkB-induced gene). Hydroxymethylbi-lane synthase (human HMBS) Hs00609297_m1 was used

as an invariant control. Non–reverse-transcribed sampleswere used as negative controls. Gene expression wascalculated using the R ¼ 2(�DDCt) method, where changesin Ct values for the genes of interest were normalized toHMBS. In all cases, gene expression for particular treat-ment groups was expressed as fold change versus meanvalues for no treatment control. Real-time PCR reactionswere carried out in triplicate and replicated in at least 3independent experiments.

ELISA for TNFR2Soluble TNFR2 levels in cell supernatants were measured

using Quantikine ELISA system (R&D System) accordingto manufacturer's instructions. Samples were normalized tototal protein as measured by BCA protein assay (Pierce).

Flow cytometric analysis of surface TNFR2 expressionCell surface expression of TNFR2 was assessed using flow

cytometry as previous described (16). SW480 cells weretrypsinized, washed with serum-free RPMI-1640 media(Gibco), and incubated in medium alone or medium plusIL-6 and TNFa (50 ng/mL) for 10 hours at 37�C withrotation in 15mL conical tubes. Cells were then resuspendedin wash buffer [PBS supplemented with 1% bovine serumalbumin and 1mg/mLDNase (Roche)], and Fc-blockedwith1 mg human IgG (R&D Systems) for 15 minutes. Cells wereincubated with fluorescein-conjugated anti-TNFR2 (R&DSystems) or isotype control (BD Pharmingen) for 45 minutesat 4�C. Following antibody incubation, cells were washed andresuspended in 2% paraformaldehyde. Flow cytometric anal-ysis of surface TNFR2 expression was then conducted using aCyAn flow cytometer (Beckman-Coulter-Dako). Effect ofcytokine treatment on TNFR2 surface expression was mea-sured on the basis of fluorescein intensity.

STAT3 and NFkB inhibitionTo test whether cytokine induction of TNFR2 mRNA

requires STAT3 and/or NFkB activation, SW480 cells weretreated with the STAT3 inhibitor Cucurbitacin I (Tocris)at 20 mmol/L or IkB kinase inhibitor Bay 11-7082 at5 mmol/L. Bay 11-7082 was kindly provided by Dr. AlbertBaldwin (University of North Carolina, Chapel Hill, NC).Inhibitor doses were based onmaximum effective doses usedin pilot and published studies (27–29). SW480 cells wereseeded in serum-free media, grown for 24 hours, and treatedwith inhibitors and/or IL-6, TNFa, or both cytokines at 50ng/mL for 10 hours prior to mRNA extraction. No treat-ment controls were treated with vehicle [dimethyl sulfoxide(DMSO)]. Intercellular adhesion molecule (ICAM) 1,which is known to be upregulated by cytokine inductionof NFkB (30), was measured by TaqMan quantitativereverse transcription PCR (qRT-PCR) to confirm effective-ness of Bay 11-7082.

Chromatin immunoprecipitation to assess STAT3 andNFkB binding to the TNFR2 promoterFor chromatin immunoprecipitation (ChIP), SW480

cells were serum deprived overnight followed by treatment

STAT3-Dependent TNFR2 Induction in Colon Cancer Cell Lines

www.aacrjournals.org Mol Cancer Res; 9(12) December 2011 1719




with IL-6, TNFa, or both cytokines (50 ng/mL) for 5 to 60minutes. After treatment, cells were cross-linked with 1%formaldehyde for 10 minutes. Subsequent steps were con-ducted as specified in the ChIP-IT Express (Active Motif)user manual. Briefly, cross-linked cells were lysed andsonicated, followed by overnight immunoprecipitation withanti-STAT3 (SC-483x; Santa Cruz Biotechnology) andanti-p65 NFkB (SC-372) or negative control IgG antibody(Active Motif). Eluted, reverse cross-linked protein:DNAcomplexes were digested with proteinase K for 1 hour.Digested samples were then purified using QIAquick col-umns according to manufacturer's instructions (QIAquickPCR Purification Kit, Qiagen). PCR amplification wassubsequently carried out using primers specific to STAT3and NFkB-binding elements within the TNFR2 promoter.Oligomers used to amplify these binding sites were pur-chased from Sigma, and sequences are shown in Table 1.Densitometry was conducted to quantify the PCR-amplifiedtranscription factor–binding sites.

Western blot for activated STAT3 in nuclear extractsWestern blot analyses were conducted on nuclear extracts

fromSW480 cells to determinewhether IL-6 combinedwithTNFa enhanced levels of activated or total nuclear STAT3.Nuclear extracts were prepared as previously described (31).Briefly, SW480 cells were grown to confluence, serum-deprived overnight, and treated with IL-6 and TNFa(50 ng/mL) for 30 minutes. Cells were then pelleted inlysis buffer containing 10 mmol/L HEPES, pH 7.9, 10mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA,1 mmol/L dithiothreitol (DTT), 2 mg/mL aprotinin, and 1mmol/L phenylmethylsulfonylfluoride (PMSF). Nucleiwere obtained by adding 10% NP40 and centrifuging for5 minutes at 15,000 � g. Pellets were then resuspended inbuffer containing 20mmol/LHEPES, pH 7.9, 400mmol/LNaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/LDTT, and 1 mmol/L PMSF to release nuclear proteins. Thenuclear suspension was centrifuged at 15,000 � g for 5minutes, and supernatants containing nuclear extracts werethen subjected to immunoprecipitation and immunoblotwith the following antibodies: anti-phospho-tyrosineSTAT3: rabbit polyclonal pTyr705 (#9131; Cell Signaling);anti-STAT3 (total): rabbit polyclonal SC-7179 (Santa Cruz

Biotechnologies). Coomassie-stained protein gels verifiedequivalent amounts of nuclear protein in samples used forimmunoprecipitation.

Constitutive activation of STAT3Constitutively active STAT3 (CA-STAT3) adenovirus

was kindly provided by Dr. Christian Jobin (University ofNorth Carolina, Chapel Hill, NC). This vector is constitu-tively activated due to C661A and C663N mutations andhas been functionally characterized in prior studies (32, 33).SW480 cells were treated with CA-STAT3 at a multiplicityof infection (MOI) of 50 or with IL-6 and TNFa for 10hours prior to mRNA extraction for evaluation of TNFR2expression.

TNFR2 and SOCS3 expression constructsCells treated with TNFR2 or SOCS3 expression con-

structs were used to directly evaluate the proliferative andgrowth-promoting effects of TNFR2 overexpression and testwhether SOCS3 could inhibit TNFR2 expression or itsgrowth-promoting effects. Retroviral expression vectorpQCXIP was obtained from BD Biosciences Clontech.pQCXIP containing c-myc–tagged human TNFR2 waskindly provided by Dr. Daniella M€annel, University ofRegensburg, Germany. HEK293 cells were cotransfectedwith retroviral vectors and packaging vector as previouslydescribed (34) using jetPei (Polyplus Transfection) accord-ing to manufacturer's instructions. Media containingTNFR2 retrovirus was collected from transfected HEK293cells and used to treat SW480 orCOLO205 cells for 24 to 48hours. TNFR2 overexpression was confirmed by Westernimmunoblot (data not shown). Plasmid pBIG2i expressinghuman SOCS3 and empty vector were kindly provided byDrs. Richard Furlanetto and Peter Nissley. These plasmidswere used to generate adenovirus expressing human SOCS3or empty vector control as previously described (22). Ade-noviruses were used at an MOI of 100 unless otherwisenoted. Cells were treated with adenovirus for 24 to 48 hoursin complete media and switched to serum-free media over-night prior to cytokine stimulation. Adenovirus-mediatedoverexpression of SOCS3 was confirmed by Northern blotanalysis and qRT-PCR (data not shown).

Table 1. Oligomers used in ChIP assays

Site within TNFR2 promoter Oligomers Product size, bp

STAT (�1,578) F-50-CTGCAGTGAGCTATGGGTGA-30 223R-50-GAGGGTGTGGCTGGTATGAC-30

STAT (�364) F-50-CTGCAGTGAGCTATGGGTGA-30 172R-50-GGGTGAGGCACTAATTTGGA-30

NFkB (�1,890) F-50-TTGAATTCGTTCCCAGGATG-30 171R-50-CTAGTTGTCCCCCACACACC-30

NFkB (�1,517) F-50-AAGGCTCTGTGGGTCATACCAG-30 228R-50-GGCTGCCTGAAGAGGTACAG-30

Hamilton et al.

Mol Cancer Res; 9(12) December 2011 Molecular Cancer Research1720




TNFR2 or SOCS3 knockdownSW480 cells were grown to approximately 50% to 70%

confluence in RPMI-1640 medium (Gibco, with 10% FBS,plus antibiotics). Cells were trypsinized and counted. A totalof 5 � 104 cells per condition were transfected usingNucleofector Technology according to manufacturer'sinstructions (Kit V, Lonza) or by using Lipofectamine2000 (Invitrogen) in Opti-MEM (Gibco) with scramblecontrol, TNFR1, or TNFR2 siRNAs (Applied Biosystems)according to manufacturer's instructions. AdditionalTNFR2 knockdown experiments were carried out usingthe Santa Cruz plasmid sc-36689-SH. Knockdown wasconfirmed using qRT-PCR. Caco2 cells, which are knownto express SOCS3 and show cytokine-induced SOCS3expression (30), were used to test the effects of siRNA-mediated SOCS3 knockdown using siRNAs (sense 50-CCAAGAACCUGCGCAUCCAdTdT-30, antisense, 50-UGGAUGCGCAGGUUCUUGGdTdT-30; Dharmacon)and methods previously described for prostate cancer cells(35).

Analysis of cell proliferation and anchorage-independentgrowthAssays of [3H]-thymidine incorporation into DNA were

used to assess S-phase of the cell cycle as one measure orproliferation and were conducted as previously described(36). Briefly, SW480 cells were plated in 24-well plates at adensity of 1 � 104 cells per well and treated with TNFR2and/or SOCS3 expression constructs for 24 hours. Mediumwas then supplemented with 2 mCi/mL [3H]-thymidineovernight, and thymidine incorporation into DNA wasmeasured using scintillation counting. Values are expressedas fold change compared with empty vector control. As anindependent measure of effects of TNFR2 knockdown oncell growth,WST-1 assays (Roche), whichmeasure numbersof viable cells, were conducted according to manufacturer'sinstructions. Briefly, cells were transfected with control orTNFR2 siRNAs in 96-well plates, then switched to mediacontaining IL-6 and TNFa (50 ng/mL) for 48 hoursfollowed by WST-1 assay.COLO205 cells, which show robust colony formation in

soft agar, were used to test the effects of overexpression ofTNFR2 and/or SOCS3 on anchorage-independent growth.Cells were treated with empty vector, TNFR2, SOCS3, orbothTNFR2 and SOCS3 expression constructs. COLO205cells were trypsinized and suspended in complete culturemedia supplemented with 0.3% agar followed by plating in

6-well culture dishes coated with 5% agar. Cells were treatedwith expression constructs at days 1, 7, and 14. At day 21,viable cells were stained with MTT for 4 hours and colonieswere quantified using NIH ImageJ software (37).

Evaluation of TNFR2 in colon of AOM/DSS-treatedmice with IEC-specific disruption of SOCS3 genesC57BL6 mice homozygous for pLox-modified SOCS3

alleles were crossed with mice hemizygous for the villin-Cretransgene (Jackson Laboratories) as previously described(22). Study mice are denoted as VC-SOCS3D/D and WT-SOCS3fl/fl controls with pLox-modified, but intact SOCS3alleles. Genotyping primers are listed in Table 2. All pro-cedures were conducted with Institutional Animal Care andUse Committee approval and in accordance with the NIHguidelines for use of live animals.VC-SOCS3D/D and WT-SOCS3f l/f l were treated with

AOM/DSS as previously described (22). Briefly, 8- to 10-week-old mice were given an intraperitoneal injection of 10mg/kg AOM. After 7 days, mice were given 3 cycles of 2.5%DSS in the drinking water for 5 days, allowing 14 days ofrecovery between each DSS treatment. Animals were eutha-nized 80 days after initial AOM injection. Colon sampleswere collected and fixed in 10% zinc-buffered formalin forimmunohistochemical staining.Immunostaining for TNFR2 was conducted on paraffin-

embedded sections of distal colon tumor and nontumortissues. Only VC-SOCS3D/D had histologically detectabletumors in these experiments. After deparaffinizing andhydrating, sections were pretreated with 0.05 mol/L TrisTriton epitope retrieval solution at room temperature for 30minutes. Endogenous peroxidase activity was blocked with3% H2O2 for 10 minutes, followed by blocking in 5%normal goat serum (NGS)/TT buffer for 1 hour. Thesections were incubated with TNFR2 antibody (NBP1-03130, Novus Biologicals) at a dilution of 1:250 in 5%NGS/TT buffer overnight at 4�C in a humidity chamber.After washing 3 times in 0.05 mol/L Tris buffer, the sectionswere incubated with biotinylated goat anti-rabbit IgG (11-065-114, Jackson ImmunoResearch) for 1 hour at roomtemperature. ABC (Vector Labs) was applied for 1 hour,followed by adding 3,30—diaminobenzidine (DAB;Zymed)for 10 to 14 minutes and the reaction was stopped in dH2O.Counterstaining was conducted with hematoxylin for 25seconds and coverslips were mounted inDPXmount (VWRInternational Ltd.). Images were taken by Imager A2 micro-scope (Zeiss).

Table 2. Genotyping oligomers for VC-SOCS3D/D mice

Target Oligomers Product size, bp

VC transgene F-50-CTGCCACGACCAAGTGACAGC-30 324R-50-CTTCTGTAGACCTGCGGTGCT-30

Wild-type or pLox-modified SOCS3 alleles F-50-GAGTTTTCTCTGGGCGTCCTCCTA-30 402 or 534R-50-TGGTACTCGCTTTTGGAGCTGAA-3






StatisticsValues are expressed asmean� SE. Comparisons between

cell treatments were analyzed using Student t test forcomparisons between 2 treatments or one-way ANOVAfor comparisons of multiple treatments followed by post hoc,pairwise comparisons using the Fisher protected least sig-nificant difference (PLSD) test. A value of P < 0.05 wasconsidered statistically significant for all experiments.

Results

IL-6 andTNFa induceTNFR2 inCOLO205 and SW480cellsPrior studies in colon cancer cells indicate that TNFR2

expression is increased by combined IL-6 or TNFatreatment, with only modest effects due to either cytokinealone (16). Figure 1A and C confirm elevation of TNFR2mRNA levels by combined IL-6 and TNFa in SW480and COLO205 cells. TNFR1 mRNA levels were notsignificantly altered with cytokine treatment. Evaluation

of TNFR2 protein by ELISA and flow cytometry (Fig.1C–E) verified these data. In SW480 cells, treatment withTNFa alone induced TNFR2 to levels similar to thoseobserved with combined IL-6 and TNFa. Because TNFahas been reported to induce IL-6 in cancer cell lines fromother organs (38–42), we tested whether TNFa upregu-lated endogenous IL-6 mRNA levels. As shown in Fig. 1F,TNFa treatment and TNFa combined with IL-6 signif-icantly increased endogenous IL-6 mRNA. This resultsuggests that autocrine or paracrine actions of TNFa-induced IL-6 may contribute to the ability of TNFa toinduce TNFR2.

A predominant role of STAT3 in mediating basal andcytokine-induced TNFR2 expressionTo assess the functional role of STAT3 or NFkB in

regulating TNFR2 expression, we examined the effects ofa STAT3 (cucurbitacin) or NFkB (Bay 11-7082) inhibitoron basal and IL-6 þ TNFa–induced TNFR2 mRNA (Fig.2A). On the basis of published and pilot studies, we used

A COLO205

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Figure 1. Increased TNFR2 mRNAand protein levels in colon cancercell lines treated with IL-6 andTNFa. Histograms (A–D) showlevels of TNFR2 mRNA or solubleTNFR2 in SW480 and COLO205cells treated with 50 ng/mL IL-6plus 50 ng/mL TNFa for 10 hours.TNFR2 mRNA was normalized toHMBS. sTNFR2 was measured byELISA on cell supernatants andvalues were normalized to totalprotein. All values are expressed asfold change (mean � SE) versusmean levels in untreated controls.(�, P � 0.05 compared with notreatment controls). TNFR2 mRNAwas significantly increased by IL-6and TNFa treatment in both celllines (A and C). Consistent withfindings for TNFR2 mRNA, proteinlevels of soluble TNFR2 weresignificantly increased with IL-6and TNFa treatment (B and D). E,representative histogram ofsurface TNFR2 expression asmeasured by flow cytometry. Cellstreated with IL-6 and TNFa exhibitan increase in surface TNFR2expression. Negative controlsinclude isotype and no antibody(Ab) controls for each condition. F,histograms show IL-6mRNA levelsin SW480 cells. Note that TNFaalone or TNFa þ IL-6 elicit similar,significant increases as IL-6 mRNA(�, P � 0.05 compared with notreatment). For all experiments, n�3 independent experiments werecarried out in duplicate. FITC,fluorescein isothiocyanate.

Hamilton et al.





maximally effective doses of 20 mmol/L cucurbitacin or 5mmol/L Bay 11-7082. Cucurbitacin significantly inhibitedbasal TNFR2 mRNA and TNFR2 mRNA induction bycombined IL-6 and TNFa. In contrast, Bay 11-7082 had nosignificant effect on basal or IL-6þTNFa–induced TNFR2mRNA. Furthermore, effects of combined inhibition ofSTAT3 and NFkB on basal or IL-6 þ TNFa–inducedTNFR2 were not significantly different than the inhibitoryeffect of cucurbitacin alone. These data suggest a predom-inant role of STAT3 compared with NFkB pathways ininduction of TNFR2 by combined IL-6 and TNFa. Con-firmatory studies were conducted in cells treated with IL-6 orTNFa alone plus STAT or NFkB inhibitors. IL-6 alone didnot induce TNFR2 mRNA above basal levels, but cucurbi-tacin, and not Bay 11-7082, reduced TNFR2 mRNA inIL-6–treated cells.

Analyses of the effects of cucurbitacin andBay 11-7082 onTNFR2 mRNA in cells treated with TNFa alone revealedthat either the NFkB or the STAT3 inhibitors significantlyattenuated TNFa induction of TNFR2, but combinedinhibitors did not have a more dramatic effect than eitherinhibitor alone. These results are consistent with our find-ings that TNFa induces IL-6 and suggest that NFkB likelymediates TNFa induction of IL-6 in SW480 cells. Todirectly test this, we measured IL-6 mRNA levels in cellstreated with TNFa or TNFaþ IL-6 in the presence of Bay11-7082 or DMSO control. Treatment with Bay 11-7082dramatically decreased IL-6 mRNA in TNFa- or TNFa þIL-6–treated cells, confirming that TNFa induction of IL-6in SW480 cells is mediated through NFkB (Fig. 2B).Because it has been established that cytokines induceICAM-1 through NFkB-dependent mechanisms, we also

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Figure2. Effects ofSTAT3orNFkB inhibitors andconstitutively activatedSTAT3onTNFR2, andeffectsofNFkB inhibitor on IL-6 and ICAM.A, histograms showlevels of TNFR2mRNA inSW480cells treatedwith vehicle, IL-6þTNFa, IL-6 alone, or TNFa alone, for 10hours in the absence (�) or presence (þ) of theSTAT3inhibitor cucurbitacin (20 mmol/L) or the NFkB inhibitor Bay 11-7082 (5 mmol/L). Values are expressed asmean fold change versus basal values in the absenceof inhibitor; �, P � 0.05 compared with basal; ��, P �0.05 for effect of STAT or NFkB inhibitor; NS, not significant. Note the dramatic inhibitory effect ofcucurbitacin on both basal and IL-6 þ TNFa–induced TNFR2 compared with Bay 11-7082. Note that IL-6 alone did not induce TNFR2 but cucurbitacinreduced TNFR2 mRNA in IL-6–treated cells. TNFa alone induced TNFR2, and this was inhibited by Bay 11-7082 or cucurbitacin, with no greater inhibitionwhen both STAT3 and NFkB inhibitors were combined. B, IL-6 mRNA levels were measured in SW480 cells treated with TNFa or IL-6þ TNFa in the absence(�) or presence (þ) of Bay 11-7082 (5 mmol/L). Note that Bay 11-7082 completely abolished TNFa or IL-6 þ TNFa–mediated increases in IL-6 mRNA levels.�, P � 0.05 compared with no treatment; ��, P � 0.05 for effect of NFkB inhibitor. For all experiments, n � 3 independent experiments were carried out induplicate. C, ICAM mRNA induction in SW480 cells treated with IL-6 þ TNFa in the absence (�) or presence (þ) of Bay 11-7082 (5 mmol/L). Bay 11-7082completely abolished IL-6þ TNFa-mediated increases in ICAMmRNA levels. �,P� 0.05 comparedwith no treatment; ��,P� 0.05 for effect of NFkB inhibitor.D, SW480 cells were treatedwith adenovirus to overexpress constitutively active STAT3 (CA-STAT3) for 10 hours followed bymRNA collection. Expression ofCA-STAT3 significantly increased TNFR2mRNA levels to the same level as that foundwith IL-6 and TNFa treatment. �,P� 0.05 comparedwith empty vector.






verified the efficacy of Bay 11-7082 by showing that IL-6and TNFa induced a 4.4 � 0.6-fold increase in ICAM-1mRNA, and treatment with Bay 11-7082 potently inhibitedthis effect (Fig. 2C). Thus, the modest effects of NFkBversus STAT3 inhibitor on TNFR2 expression in cellstreated with IL-6 and TNFa were not due to a lack ofeffective Bay 11-7082 dosing. Together these findingssuggest a predominant role of STAT3 in mediating cyto-kine-induced TNFR2 expression.To confirm a role for STAT3 in TNFR2 expression, we

treated SW480 cells with a constitutively active STAT3(CA-STAT3) adenovirus. Expression of CA-STAT3 in theabsence of cytokine treatment induced TNFR2 mRNAlevels to the same degree as observed with combined IL-6and TNFa treatment (Fig. 2D). Together, the data withSTAT3 inhibitor and constitutively activated STAT3 sug-gest that STAT3 is necessary and sufficient for basal and IL-6/TNFa-induced TNFR2 expression.

IL-6 and TNFa induce STAT3, but not NFkB binding,to the TNFR2 promoterWeverified a predominant role of STAT3 in the control of

TNFR2 expression byChIP assays that allowed us to directlyassess the effect of cytokines on STAT3 and NFkB bindingto putative binding elements in the TNFR2 promoter(Fig. 3A). Combined IL-6 and TNFa treatment inducedbinding of STAT3 to both of the putative STAT-bindingsites, with maximal binding at 30 and 60 minutes for�364and �1,578 sites, respectively (Fig. 3B). Densitometryrevealed that IL-6 and TNFa induced 2.0 � 0.4-fold and3.8� 1.6-fold and increases in STAT3 binding to the�364and �1,578 sites, respectively. There was a small degree ofbasal NFkB binding, but this was not enhanced by IL-6 andTNFa treatment.To delineate the individual contribution of IL-6 and

TNFa on STAT3 binding to the TNFR2 promoter, wetreated cells with either cytokine alone or in combination

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Figure 3. IL-6 and TNFa induce STAT3 binding to the TNFR2 promoter. A, schematic shows the locations of 2 putative STAT-binding elements and 2 putativeNFkB-binding elements in the human TNFR2 promoter. B, PCR products from ChIP assays of STAT3 or NFkB binding to consensus STAT3 or NFkB sites inuntreated cells or cells treated with IL-6 and TNFa for the indicated times. IL-6 and TNFa treatment leads to a time-dependent increase in STAT3 binding atboth consensus sites. Cytokine treatment has no effect on NFkB binding. Densitometric analysis revealed a 3.8 � 1.6-fold and 2.0 � 0.4-fold increase inoverall STAT3 binding at the �1,578 and �364 site, respectively. �, P � 0.05 compared with no treatment; n ¼ 3. C, PCR products from ChIP assaysin SW480 cells treated with IL-6 or TNFa alone, or in combination (30 minutes). Note the dramatically enhanced effect of combined IL-6 and TNFa on STAT3binding to the �1,578 site, but not for the �364 site. n ¼ 3. D, Western immunoblots on nuclear extracts from SW480 cells treated with IL-6, TNFa,or both cytokines for 30minutes. Top, immunoblots for tyrosine-phosphorylated (pY) and total STAT3. IL-6 treatment induced pY-STAT3 and increased totalnuclear STAT3, whereas TNFa alone had no detectable effect, and IL-6 and TNFa combined gave a similar effect as IL-6 alone. Bottomconfirms equal proteinloading by Coomassie stain of proteins in nuclear extracts. Blot is representative of total 3 experiments.

Hamilton et al.





and conducted ChIP for STAT3. Treatment with IL-6 andTNFa alone for 30 minutes modestly induced STAT3binding to the �1,578 STAT–binding site, but both cyto-kines combined induced dramatic increases in STAT3binding to the �1,578 binding site (Fig. 3C). PutativeSTAT3-binding site �364 differed in that IL-6 alone, butnot TNFa, induced STAT3 binding and combined cyto-kines had similar effects as IL-6 alone. Thus, the cooperativeeffects of IL-6 and TNFa to activate STAT3 appear selectivefor the �1,578 STAT3–binding site.Because combined IL-6 and TNFa led to a dramatic

increase in STAT3 binding to the�1,578 STAT3–bindingsite in the TNFR2 promoter, we assessed whether the 2cytokines in combination enhanced tyrosine phosphoryla-tion of STAT3 relative to IL-6 or TNFa alone.Western blotanalysis on nuclear extracts revealed that IL-6 increasedphosphorylated and total STAT3 in the nucleus whereasTNFa alone had no effect (Fig. 3D). Both cytokinestogether did not dramatically augment tyrosine phosphor-ylation of STAT3 or total nuclear STAT3 as compared withIL-6 alone. Thus, enhanced tyrosine phosphorylation ofSTAT3 does not appear to account for the combinatorialeffects of IL-6 and TNFa on STAT3 binding to the�1,578STAT3–binding site. It is important to note that thetreatment time points used to examine effects of TNFaalone on STAT3 binding by ChIP or STAT3 tyrosinephosphorylation are much shorter than the time points(10 hours) needed for TNFa to induce IL-6. Thus, theWestern immunoblot and ChIP data together indicate that

IL-6 alone enhances STAT3 activation and nuclear locali-zation, but interaction with TNFa is required to promoteIL-6–induced binding of STAT3 to the TNFR2 promoter.

Effects of TNFR2 overexpression or knockdown onproliferation and cell numberPrior in vivo studies suggest that TNFR2-null mice show

reduced crypt proliferation during intestinal inflammation(16). To directly test the effects of TNFR2 on colon cancercell proliferation, we overexpressed TNFR2 in SW480 andCOLO205 cells and measured [3H]-thymidine incorpo-ration into DNA. TNFR2 overexpression significantlyenhanced [3H]-thymidine incorporation in both cell lines(Fig. 4A). In complementary experiments, we knockeddown TNFR1 or TNFR2 using siRNA (Fig. 4B) andmeasured [3H]-thymidine incorporation over 24 hours. Themaximal knockdown achieved after testing multipleTNFR2-targeted siRNAs was a 60% reduction in TNFR2mRNA levels (Fig. 4B). However, this was a specific effectbecause expression of TNFR1mRNA was unaffected by theTNFR2 siRNA. Similarly, TNFR1 siRNA had no effecton TNFR2 mRNA but inhibited TNFR1 expression by80%. TNFR2 knockdown resulted in a modest but signif-icant decrease (14% � 2.5%) in [3H]-thymidine incorpo-ration in SW480 cells (Fig. 4C). WST-1 assays, whichmeasure number of viable cells rather than just S-phase,revealed that knockdown of TNFR1 significantly increasedcell number, whereas knockdown of TNFR2 reduced cellnumber up to 40% compared with control siRNA and up to

Figure 4. Effects of TNFR2 overexpression orsiRNA-mediated knockdown on colon cancercell proliferation. A, histogram of [3H]-thymidineincorporation into DNA as a measure ofCOLO205 or SW480 cell proliferation after 24-hour overexpression of TNFR2. TNFR2overexpression significantly increased cellproliferation. �, P � 0.05 compared with emptyvector. B, SW480 cells were treated with control,TNFR1, or TNFR2 siRNA for 24 hours. Histogramshows fold change in expression of TNFR1 orTNFR2 mRNA. TNFR2 siRNA led to a 60%decrease in TNFR2 mRNA, with no effect onTNFR1 mRNA. �, P � 0.05 compared with othertreatments. C, untreated SW480 cellstransfected with TNFR2 siRNA exhibit a modest,but significant (�, P � 0.05) decrease in [3H]-thymidine incorporation into DNA. D, cytokine-treated SW480 cells with TNFR2 knockdownexhibited a significant decreases cell numbermeasured by WST-1 assay, whereas TNFR1knockdown significantly increased cell number.�, P � 0.05 compared with control siRNA;��, P � 0.05 compared with cells treated withTNFR1 siRNA. n ¼ 3.

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70% compared with cells with knockdown of TNFR1 (inwhich TNFR2 is the predominant TNFR; Fig. 4D). Wehave observed similar effects of TNFR2 knockdown inCOLO205, Caco2, and HIECs (Supplementary TablesS1–S3). The more dramatic effect of TNFR2 knockdownon cell number than [3H]-thymidine incorporation suggeststhat TNFR2 knockdown likely impacts cell survival and/orother phases of cell cycle than S-phase.

SOCS3 inhibits cytokine-induced TNFR2 expression,STAT3 binding to the �1578 consensus site, andproliferation and anchorage-independent growth ofcolon cancer cellsNegative regulation of STAT3 by SOCS3 is well estab-

lished (43–45). To test whether SOCS3 inhibits TNFR2expression, we treated COLO205 and SW480 cells withSOCS3 adenovirus or empty vector control and examinedTNFR2 mRNA. As anticipated, SOCS3 overexpressionsignificantly inhibited cytokine-induced TNFR2 in bothcell lines (Fig. 5A). ChIP assay also revealed that SOCS3overexpression dramatically inhibited cytokine-inducedSTAT3 binding to the �1,578 site but had variable andnonsignificant effects on STAT3 binding to the �364 site(Fig. 5B).We have previously reported that SOCS3 overexpression

reduces proliferation of SW480 cells (22). We used thesecells to test whether SOCS3 could prevent the increase inproliferation resulting fromTNFR2 overexpression. SOCS3overexpression reduced [3H]-thymidine incorporation inSW480 cells transfected with empty vector or TNFR2expression construct (Fig. 5C). However, the magnitude ofthe increase in [3H]-thymidine incorporation resulting fromTNFR2 overexpression was similar in SOCS3 overexpres-sing and control cells suggesting that SOCS3 overexpressioncannot reverse TNFR2-associated increases in DNAsynthesis.SOCS3 overexpression had more potent effects on

anchorage-independent growth of COLO205 cells.COLO205 cells overexpressing TNFR2 showed a small butsignificant increase in colony formation when comparedwith empty vector controls. SOCS3 overexpression dramat-ically decreased (>70%) colony formation in both emptyvector and TNFR2 overexpressing cells (Fig. 5D and E).

Increased TNFR2 immunostaining in colon tumors ofmice with IEC-specific deletion of SOCS3Previously published work showed a dramatic increase in

AOM/DSS- induced tumor number and size in mice withvillin-cre (VC)-mediated SOCS3 gene disruption in IEC(VC-SOCS3D/D) compared with controls with floxed butintact SOCS3 genes (WT-SOCS3fl/fl).We therefore assessedTNFR2 expression in colon of these AOM/DSS-treatedmice. In normal colon tissue, TNFR2 immunostaining wasweak in bothWT-SOCS3fl/fl and VC-SOCS3D/Dmice (Fig.6A–F). Only VC-SOCS3D/D mice showed histologicallydetectable tumors, and these tumors showed dramaticincreases in TNFR2 immunostaining (Fig. 6G–I). Thus,the increase in tumor load in VC-SOCS3D/D mice is asso-

ciated with increased expression of TNFR2 within thetumors.

Short-term SOCS3 knockdown is not sufficient toupregulate TNFR2We used Caco2 colon cancer cells, which are known to

have higher basal SOCS3 expression than SW480 andCOLO205 cells and show cytokine mediated upregulationof SOCS3 (ref. 30 and unpublished data), to test whetherSOCS3 knockdown was sufficient to increase basal orcytokine-stimulated TNFR2 expression. Caco2 cells weretransfected with control or SOCS3 siRNA followed bycytokine treatment for 24 hours. As shown in Supplemen-tary Fig. S1A, SOCS3 siRNA treatment reduced the basaland cytokine-induced levels of SOCS3 mRNA, but we wereunable to completely abolish the increase in SOCS3 mRNAresulting from cytokine treatment. SOCS3 siRNA did notenhance but rather reduced TNFR2 expression in these cells(Supplementary Fig. S1B). Thus, while SOCS3 overexpres-sion is sufficient to inhibit basal and TNFR2 overexpression,short-term knockdown of SOCS3 expression was itself notsufficient to increase basal or cytokine-stimulated TNFR2expression.

Discussion

The etiology of inflammation-associated CRC is basedstrongly on themodel that chronically upregulated cytokinesdrive excessive proliferation of IECs, tumor initiation, andprogression. TNFR2 has recently emerged as a proprolifera-tive factor that is upregulated in IBD and in the AOM/DSSmodel of IBD-associated cancer (14, 16). Mechanismsregulating TNFR2 expression in IBD or CRC are not fullydefined, although prior studies suggest that combined effectsof IL-6 and TNFa promote TNFR2 expression (16). Thecurrent study provides novel evidence that IL-6 and TNFaact predominantly through STAT3 and a consensusSTAT3-binding site in the TNFR2 promoter to induceTNFR2 in CRC. We also show that SOCS3 overexpressioninhibits cytokine induction of TNFR2 and STAT3 bindingto this STAT3 consensus site and can dramatically decreaseanchorage-independent growth of colon cancer cells, eventhose overexpressing TNFR2.Mizoguchi and colleagues provided the first evidence that

TNFR2 was upregulated during acute DSS-colitis and thatthis was preceded by IL-6/STAT3 activation. They alsoshowed that mice with global TNFR2 gene disruptionexhibited decreased IEC proliferation in the T-cell receptora (TCRa)-null model of colitis (16). Furthermore, TCRamice with disruption of both IL-6 alleles showed reducedcolitis severity and decreased TNFR2 expression comparedwith TCRamice with intact IL-6 (16). While these studiessuggested an association between IL-6/STAT3 and TNFR2,the ability of STAT3 to directly regulate TNFR2 expressionhas not been previously reported. Prior in vitro studiesindicated that both IL-6 and TNFawere required to induceTNFR2 in CRC cells, suggesting that TNFR2 inductionrequires a specific microenvironment of multiple cytokines,

Hamilton et al.





as found in IBD or IBD-associated CRC. The current studyconfirmed induction of TNFR2 mRNA and protein bycombined IL-6 and TNFa in 2 different colon cancer celllines. Importantly, we provide novel evidence for a model of

IL-6 and TNFa interaction in regulating TNFR2 in coloncancer cells (Fig. 7). Our studies show a predominant role ofSTAT3 in TNFR2 induction by IL-6 and TNFa, whichinvolves cooperative effects of IL-6 and TNFa to induce

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Figure 5. SOCS3 overexpression decreases TNFR2 mRNA, STAT3 binding to the �1,578 binding site on the TNFR2 promoter, cell proliferation, andanchorage-independent growth. A, histogram shows levels of TNFR2mRNA in SW480 or COLO205 cells in the absence (�) or presence (þ) of IL-6 and TNFaand/or SOCS3 adenovirus. Cytokine treatment significantly increased TNFR2 mRNA and SOCS3 overexpression attenuated this effect. �, P � 0.05 versusvehicle empty vector; ��, P � 0.05 versus empty vector plus cytokine treatment. n � 3 independent experiments were carried out in duplicate. B, top, PCRproducts from ChIP assays of SW480 cells stimulated with IL-6 and TNFa for 30 to 60 minutes. Histograms indicate fold change in STAT3 binding withSOCS3 overexpression compared with empty vector. SOCS3 decreased STAT3 binding to the TNFR2 promoter at the �1,578 site and had variable,nonsignificant effects on binding at the�364 site. �, P� 0.05 compared with empty vector. n¼ 3. C, histogram of [3H]-thymidine incorporation into DNA as ameasure of SW480 cell proliferation after 24-hour overexpression of TNFR2, SOCS3, or both. TNFR2 overexpression significantly increased cell proliferation,and SOCS3 overexpression limited this effect. �, P � 0.05 compared with empty vector; ��, P � 0.05 compared with TNFR2 overexpressing cells. D,representative photographs of individual wells of COLO205 cells grown in 0.3% soft agar and overexpressing TNFR2 and/or SOCS3. Images arerepresentative of at least 3 independent experiments. E, colonies were stained withMTT and quantified using NIH ImageJ software. Cells treated with TNFR2retrovirus exhibited a small but significant increase in colony number (�, P <0.05 vs. empty vector), and treatment with SOCS3, or SOCS3 combined withTNFR2, caused a dramatic (>70%) decrease in colony number. (��, P � 0.05 compared with empty vector or cells overexpressing TNFR2 alone).






STAT3 binding to a consensus element within the TNFR2promoter. Our studies also reveal that TNFa acts throughNFkB to induce endogenous IL-6 and promote TNFR2induction via autocrine effects of IL-6 (Fig. 7).The TNFR2 promoter contains 2 putative STAT-bind-

ing sequences and NFkB-binding sequences (20), butfunctional effects of these binding elements have not beenreported. Because TNFa typically activates NFkB and IL-6 typically activates STAT3, we hypothesized that IL-6and TNFa induction of TNFR2 would be mediated byactivation of both of these transcription factors. Weprovide several independent pieces of evidence to indicatethat STAT3, rather than NFkB, is the predominantmediator of TNFR2 induction by IL-6 and TNFa.Specifically, STAT3 inhibition reduced basal TNFR2expression and completely reversed the induction ofTNFR2 by IL-6 and TNFa. In contrast, NFkB inhibitionhad no effect on basal TNFR2 expression and onlynonsignificantly reduced induction of TNFR2 by com-bined IL-6 and TNFa. This was despite data verifyingthat the NFkB inhibitor potently and completely reversedcytokine induction of ICAM-1 mRNA, whose expressionis known to be dependent on NFkB. Importantly, com-bined STAT3 and NFkB inhibitors did not further reducebasal or cytokine-induced TNFR2 expression comparedwith STAT3 inhibitor alone. Constitutively activated

STAT3 was able to induce TNFR2 to a similar extentas IL-6 and TNFa. Together these findings indicate apredominant role of STAT3 in mediating TNFR2 induc-tion and show that STAT3 activation alone is sufficient tomimic cytokine effects on TNFR2 expression.ChIP assays also confirmed that IL-6 and TNFa induced

STAT3 binding to two putative STAT3-binding sites onTNFR2 promoter but had no effect on NFkB binding. Thiswas despite the fact that TNFa is known to induce phos-phorylation of NFkB in this same cell system (22). It is alsonotable that combined IL-6 and TNFa more potentlyinduced STAT3 binding to the �1,578 binding site in theTNFR2 promoter than the �364 site. Interestingly, the�1,578 STAT3-binding site also showed dramatic cooper-ative effects of IL-6 and TNFa to induce STAT3 binding,whereas either cytokine alone only modestly induced STAT3binding to this site. These combinatorial effects of IL-6 andTNFa to dramatically enhance STAT3 binding to the�1,578 site were not associated with effects of combinedIL-6 and TNFa to increase tyrosine phosphorylation ornuclear levels of STAT3. Collectively, these observationsprovide compelling evidence that IL-6 and TNFa interactto promote maximal STAT3 binding to the TNFR2 pro-moter and TNFR2 induction, and that this cooperative effectappears to occur primarily at the�1,578 STAT-binding site.The specific mechanisms by which TNFa promotes STAT3

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Figure 6. Increased TNFR2immunostaining in colon tumorsfrom mice with VC-mediatedSOCS3 gene deletion in IEC cells.Immunohistochemical analysis ofTNFR2 in nontumor tissue fromWT-SOCS3fl/fl (B and E) and VC-SOCS3D/D (C andF)micecomparedwith negative control, whichcorresponds to no primaryantibody control (A and D). A–C,10� magnification; D–F, 40�magnification. Note that TNFR2staining is weak in nontumor tissuefrom colon of both VC-SOCS3D/D

and WT-SOCS3fl/fl mice. TNFR2immunostaining is enhanced intumor tissue from VC-SOCS3D/D

mice. G, 4�; H, 10�; I, 40�magnification.

Hamilton et al.





binding to the TNFR2 promoter remain undefined and willrequire further study. We cannot rule out the possibility thatNFkB binds to other regions in the TNFR2 gene than theconsensus sites tested, but observations that IL-6 and TNFadid not induceNFkB binding to these NFkB consensus sitesand theminimal effects ofNFkB inhibitor on basal or IL-6þTNFa–induced TNFR2 expression support a novel mech-anism of TNFa and IL-6 interaction to induce TNFR2 bypredominant effects on STAT3.Recent and increasing evidence implicates TNFR2 as a

mediator of colitis-associated cancer. TNFR2has been shownto increase migration of colon cancer cell lines and is upre-gulated in mouse models and patients with IBD (16, 17). Inaddition, disruption of TNFR2 genes was associated withdecreased proliferation of crypt epithelial cells in colitismodels (16, 17). Recent studies in the AOM/DSS modelof inflammation-associated CRC revealed that TNFR2 ispreferentially upregulated over TNFR1 and that treatmentwith the anti-TNFa monoclonal antibody MP6-XT22reduced the number and size of tumors, although colitisseverity was unchanged (14). In a separate study, anti-TNFaantibodies given at late stages of the AOM/DSS modelreduced tumor load (9). To our knowledge, a direct effectof TNFR2 on CRC proliferation or transformed phenotypehas not been previously shown. Our current study shows in 2independent colon cancer cell lines that TNFR2 overexpres-sion directly enhances proliferation. In COLO205 cells,TNFR2 overexpression increased anchorage-independentgrowth. Although effects of TNFR2 overexpression on pro-liferation and anchorage-independent growth might be con-sidered relativelymodest, it is important to emphasize that thecolon cancer cell lines used exhibit high rates of basalproliferation and are generally refractory to increases in

growth in response to exogenous stimuli. Indeed, we haveexamined effects of serum deprivation and serum supple-mentation on these cells and serum, typically a potent growthinducer, does not significantly increase proliferation (unpub-lished observations). Thus, even the small increases in pro-liferation and anchorage-independent growth support a directeffect of TNFR2 to promote colon cancer cell proliferationand the concept that TNFR2 is a protumorigenic factor. Thisis further supported by data showing that siRNA-mediatedTNFR2 knockdown modestly decreased proliferation ofmultiple colon cancer cell lines. A limitation of our studyis that the impact of TNFR2 knockdown on cancer cellproliferationwasmodest.Thismay reflect the fact that despitetesting of multiple siRNAs, the maximum knockdownachieved was 60%, which may reflect the existence ofmultiple TNFR2 splice variants (34). Future experimentswill be required to definitively establish the role of normallevels of endogenously expressed TNFR2 in IEC or coloncancer cell proliferation.SOCS3 is induced by cytokines, is a known endogenous

negative feedback inhibitor of STAT3, and is epigeneticallysilenced in lung, liver, and squamous cell cancers or cancercell lines (25, 46–49). Our previous study showed that IEC-specific deletion of SOCS3 led to an increase in tumor loadin the AOM/DSS model, supporting the hypothesis thatSOCS3 may act as a suppressor of colitis-associated cancer(22). This same study revealed that loss of IEC-SOCS3resulted in enhanced activation of both STAT3 and NFkB.We report here that SOCS3 overexpression limits TNFR2expression in colon cancer cell lines and limits STAT3binding to �1,578 consensus element in the TNFR2 pro-moter. A predominant effect of SOCS3 on the �1,578 siteversus�364 STAT3–binding site further supports a criticalrole of this region of the TNFR2 promoter in regulation ofTNFR2 expression levels. SOCS3 overexpression reducedproliferation of colon cancer cells and dramatically decreasedanchorage-independent growth. Also, increased TNFR2wasobserved in tumors that develop in AOM/DSS-treated micewith specific SOCS3 gene disruption in IECs. Together,these findings support a role of SOCS3 as a potent inhibitorof colon cancer cell growth and suggest that these effectsmay, at least in part, involve the ability of SOCS3 to limitcytokine-induced TNFR2 expression. The findings alsoindicate that loss of SOCS3 in IECs may promote colontumors by facilitating increases in cytokine-induced TNFR2expression in a setting of chronic intestinal inflammation.However, short-term siRNA-mediated reductions in basal orcytokine-induced SOCS3 mRNA did not enhance TNFR2expression, indicating that SOCS3 knockdown alone or inthe short term is itself not sufficient to cause increasedTNFR2 expression.Anti-TNFa therapies are widely used in the treatment of

human IBD (50–54). The effect of anti-TNFa therapy onrisk of colitis-associated cancer or other cancers is not welldefined and is a topic of intensive investigation (55, 56). Ourfindings that TNFa cooperates with IL-6 to induceTNFR2,an inducer of colon cancer cell growth, suggests that TNFR2may prove useful as a biomarker of the potential effects of

NF B IL-6

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Figure 7. Model of interactionsbetween IL-6 and TNFa in regulating TNFR2expression in colon cancer cells. IL-6 and TNFa cooperatively induceSTAT3 binding to the�1,578 site of the TNFR2 promoter to induce TNFR2expression. TNFa also induces endogenous IL-6 expression throughNFkB. SOCS3 overexpression limits STAT3 binding to the TNFR2promoter, TNFR2 expression, and proliferation of colon cancer cells.






anti-TNFa on colitis-associated cancer risk or could repre-sent a specific target to decrease colon cancer risk in IBD.Indeed, increased circulating TNFR2 was recently reportedto be associated with enhanced CRC risk in humans, andthose with higher plasma TNFR2 exhibit reduced CRC riskwith use of anti-inflammatory drugs (57). Furthermore, thefinding that STAT3 is a mediator of TNFR2 induction bycombined TNFa and IL-6 adds to the growing evidence forSTAT3 as a key mediator of colitis-associated cancer andsupports further investigation of STAT3 inhibitors as poten-tial cancer therapies.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Drs. Richard Furlanetto and Peter Nissley for providing thehuman SOCS3 plasmid; Daniela M€annel for the human TNFR2-expressing

retroviral construct; Christian Jobin for CA-STAT3 adenovirus; and AlbertBaldwin for NFkB inhibitors. They also thank Kirk McNaughton and KimberlyEtzel for help with TNFR2 staining. They also thank members of KEH doctoraldissertation committee Drs. James Anderson, Susan Henning, John Rawls, andRobert Sandler for useful input; Agostina Santoro, Amanda Mah, Eric Blue,Lauren Parker, Drs. Rachael Rigby, Marcus Muehlbauer, and Paul Campbell fortechnical assistance; and Dr. Jean-Francois Beaulieu (University of Sherbrooke) forthe HIEC line. Finally, they also thank Dr. Anil Rustgi for helpful suggestions.Further assistance was obtained from the UNC CGIBD ImmunotechnologiesCore DK-24987 and UNC Vector Core.

Grant Support

The National Institute of Diabetes and Digestive and Kidney DiseaseGrant DK-47769 (P.K. Lund), a grant from the UNC University CancerResearch Fund (P.K. Lund), and a Howard Hughes Medical Institute–spon-sored Med-Into-Grad predoctoral training grant (K.E. Hamilton) supportedthis work.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received May 12, 2010; revised October 3, 2011; accepted October 7, 2011;published OnlineFirst October 12, 2011.

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2011;9:1718-1731. Published OnlineFirst October 12, 2011.Mol Cancer Res Kathryn E. Hamilton, James G. Simmons, Shengli Ding, et al. Mediated by STAT3 in Colon Cancer CellsCytokine Induction of Tumor Necrosis Factor Receptor 2 Is

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