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Angiogenesis, Metastasis, and the Cellular Microenvironment

Anti–IL-20 Monoclonal Antibody Alleviates Inflammationin Oral Cancer and Suppresses Tumor Growth

Yu-Hsiang Hsu1, Chi-Chen Wei3, Dar-Bin Shieh2, Chien-Hui Chan1, and Ming-Shi Chang1

AbstractInterleukin-20 (IL-20) is a proinflammatory cytokine involved in rheumatoid arthritis, atherosclerosis, and

osteoporosis. However, little is known about the role of IL-20 in oral cancer. We explored the function of IL-20 inthe tumor progression of oral cancer. IL-20 expression levels in tumorous and nontumorous oral tissue specimensfrom 40 patients with four different stages oral cancer were analyzed with immunohistochemistry (IHC) stainingand quantitative real-time PCR (qRT-PCR). Expression of IL-20 and its receptor subunits was higher in clinical oraltumor tissue than in nontumorous oral tissue. The role of IL-20 was examined in two oral cancer cell lines (OC-3andOEC-M1). In vitro, IL-20 promotedTNF-a, IL-1b,MCP-1, CCR4, andCXCR4 and increased proliferation,migration, reactive oxygen species (ROS) production, and colony formation of oral cancer cells via activated STAT3and AKT/JNK/ERK signals. To evaluate the therapeutic potential of anti–IL-20 monoclonal antibody 7E fortreating oral cancer, an ex vivo tumor growth model was used. In vivo, 7E reduced tumor growth and inflammationin oral cancer cells. In conclusion, IL-20 promoted oral tumor growth, migration, and tumor-associatedinflammation. Therefore, IL-20 may be a novel target for treating oral cancer, and anti–IL-20 monoclonalantibody 7E may be a feasible therapeutic. Mol Cancer Res; 10(11); 1430–9. �2012 AACR.

IntroductionWith approximately 500,000 new cases annually, squa-

mous cell carcinomas of the head and neck (HNSCC), areone of the 6 most common cancers in the world (1). Oralcancer comprises nearly 30% of all HNSCC. Of these,approximately 90% of cases are squamous cell carcinomas(2). Oral squamous cell carcinoma (OSCC) is a major healthproblem worldwide, and patients have a particularly poor 5-year survival rate (3). The staging of oral cancer is animportant step in defining the natural history and prognosisof the disease and affects treatment decisions. On the basis ofthe tumor–node–metastasis (TNM) classification with var-ious modifications, stages are determined according to theAmerican Joint Committee on Cancer (AJCC) and theUnion for International Cancer Control (UICC) classifica-tion system for oral cancer (3). Each stage is a progression

through a primary tumor and progressively nodal, adjacent,and distant metastasis.Several mechanisms involved in oral cancer have been

extensively studied; however, the poor prognosis may reflectan incomplete knowledge of the mechanisms underlying themalignant progression of this cancer type. Oral cancer startswith uncontrolled cell proliferation, and over time becomesable to multiply without limit (immortalization; ref. 4).Next, oral cancer cells acquire the ability to migrate fromtheir original site, invade nearby tissue, and metastasize todistant sites. The accumulation of changes in certain mole-cules results in these progressive changes from slightlyderegulated proliferation to full malignancy.Prolonged alcohol and tobacco use, betel nut chewing,

and infection with the human papillomavirus (HPV) areknown risk factors and have suggested the involvement ofchronic inflammation and oxidative stress in the develop-ment of oral cancer (5, 6). Reactive oxygen species (ROS) areformed in the human oral cavity after they have been exposedto the above stimulants. ROS induce DNA damage andactivate intracellular signals and transcription factors (7).These alterations in turn affect the production and functionof certain molecules for regulating the cell cycle, growth,differentiation, migration, invasion, and inflammation (8).Inflammation in the tumor microenvironment has manytumor-promoting effects (9). It aids in the proliferation andsurvival of malignant cells, promotes angiogenesis andmetastasis, disrupts adaptive immune responses, and altersresponses to hormones and chemotherapeutic agents (10–12). Among these agents, proinflammatory cytokines, suchas interleukin-1b (IL-1b), IL-6, andTNF-a, are essential for

Authors' Affiliations: 1Department of Biochemistry and Molecular Biol-ogy, 2Institute of OralMedicine andDepartment of Stomatology, College ofMedicine, National Cheng Kung University, Tainan; and 3Department ofMedical Research and Development, Show Chwan Health Care System,Changhua, Taiwan

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Ming-Shi Chang, Department of Biochemistryand Molecular Biology, College of Medicine, National Cheng Kung Uni-versity, Tainan 70428, Taiwan. Phone: 886-6-235-3535, ext. 5677; Fax:886-6-274-1694; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-12-0276

�2012 American Association for Cancer Research.

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regulating the immune response and may have prognosticsignificance in cancer. The serum levels of IL-1b, IL-6, andTNF-a are higher in patients with oral cancer than inhealthy persons, and increased serum levels seem to becorrelated with the clinical stage of the disease (13). Leu-kocyte trafficking, which is critically regulated by chemo-kines and their receptors, shares many of the characteristicsof tumor cell infiltration and metastasis. Increasing evidencesuggests a pivotal role for CXCL12/CXCR4 in the estab-lishment of lymph node and distant metastasis in OSCC(14, 15) by inducing epithelial–mesenchymal transition(16).A number of downstream signaling events, such as the

Ras/Raf/MAPK, the STAT3, and the PI3K/AKT/mTORpathways, contribute to the malignant growth and meta-static potential of OSCC (17–19). They are often persis-tently active in oral cancer cells.IL-20 is a member of the IL-10 family, which includes

IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26 (20), whichshare 18% to 25% amino acid identity with IL-10. IL-20is expressed in monocytes, epithelial cells, and endothelialcells. It acts on multiple cell types by activating a hetero-dimer receptor complex of either IL-20R1/IL-20R2 or IL-22R1/IL-20R2 (21). It is also involved in various inflam-matory diseases, such as psoriasis (22, 23), rheumatoidarthritis (24, 25), atherosclerosis (26), and renal failure(27).We previously showed (26, 28) that IL-20 not only

promoted angiogenesis but also increased tumor vascu-larization. Moreover, IL-20 is regulated by hypoxia andcontributes to brain injury in an ischemic stroke model,which indicates that IL-20 is a promoting factor in theinflammation-associated disease (29). Most solid tumorcells grow under hypoxic conditions, which may induceIL-20 overexpression. In addition, hypoxia-inducible fac-tor-1a (HIF-1a) is an independent prognostic marker inOSCC and is important in the pathogenesis of oral cancer(30). Little is known about the involvement of IL-20 inthe pathogenesis of oral cancer. Therefore, we studied IL-20 expression and its biologic function in oral cancer cells.We also evaluated the therapeutic effects of anti–IL-20monoclonal antibody (mAb) 7E to alleviate or abrogatekey malignant phenotypic characteristics of oral cancer invitro and in vivo.

Materials and MethodsParticipantsForty patients with oral cancer (stage I, n¼ 10; stage II, n

¼ 10; stage III, n¼ 10; and stage IV, n¼ 10) were enrolledin this study.Oral tumor and nontumorous tissue from thesepatients was analyzed. Nontumorous oral tissue was takenfrom nonpathologic areas distant from tumors in surgicalspecimens, and histologic examinations confirmed that theywere nontumorous. Biopsies were taken after obtaininginformed consents for the study from all participants. Thestudy was approved by the Ethics Committee of NationalCheng Kung University Hospital (Tainan, Taiwan).

Cell linesOC-3 cells (31), from a cell line isolated from a Taiwanese

patient with OSCC, were maintained in Dulbecco's mod-ified Eagle's medium (DMEM) with 10% FBS (Life Tech-nologies), 2 mmol/L of L-glutamine (Life Technologies),100mg/mL of streptomycin, and 100U/mL of penicillin in ahumidified 5% CO2 atmosphere at 37�C. OEC-M1 cells(32), from a cell line derived from the gingival epidermalcarcinoma of a Taiwanese patient, were maintained inRPMI-1640 medium.

Immunohistochemistry and immunocytochemistryParaffin-embedded tissue samples were used for immu-

nohistochemistry (IHC) staining with anti–IL-20 (7E),anti–IL-20R1, anti–IL-20R2, or anti–IL-22R1 mAb (R&DSystems) at 4�C overnight as previously described (33).Incubating paraffin tissue sections with mouse IgG1 isotype(clone 11711; R&D Systems) instead of primary antibodywas the negative control. We used 3 mg/mL as the workingconcentration for each primary antibody and for the controlmouse IgG1. At least 5 sections from each patient's specimenwere analyzed and examined by 2 investigators trained in oralpathology and blinded to patient details. Immunocytochem-ical staining of IL-20 and its receptors in OC-3 and OEC-M1 cells was done using the same protocol as describedearlier.

Quantitative real-time PCRTo examine the oral cancer cell lines and clinical speci-

mens, OC-3 cells, OEC-M1 cells, oral tumor cells, andnontumorous oral tissue specimens from 40 patients wereanalyzed. Total RNA was isolated (Invitrogen). Reversetranscription was done with reverse transcriptase (Clon-tech). Human IL-20, IL-20R1, IL-20R2, and IL-22R1expression was then amplified on a thermocycler (LC 480;Roche Diagnostics), with SYBR Green (Roche Diagnos-tics) as the interaction agent. Quantification analysis ofmRNA was normalized with human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) used as the house-keeping gene. Relative multiples of change in mRNAexpression was determined by calculating 2�DDCt. Toexamine the expression of TNF-a, IL-1b, and MCP-1,OC-3 and OEC-M1 cells were incubated with IL-20 (200ng/mL) for the indicated times, and then total mRNA wasisolated and analyzed. The expression levels of these geneswere analyzed using quantitative real-time PCR (qRT-PCR) with specific primers following the same protocol asdescribed earlier.

Cell proliferation assayOC-3 cells (3� 104) and OEC-M1 cells (4� 104) were

cultured overnight and then exposed to IL-20 (50–200ng/mL) for 72 hours in medium containing 1% FBS. Toshow the specific activity of IL-20, 7E (2 mg/mL) wasadded to the culture system, either alone or together withIL-20 at a 10:1 concentration ratio (7E:IL-20). OC-3 andOEC-M1 cells responses to IL-20 stimulation wereassessed using 3H-thymidine incorporation assays. Briefly,

IL-20 and Oral Cancer

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after 54 hours of incubation, 1 mCi of 3H-thymidine(NEN Life Sciences) was added, and cells were pulse-labeled for 18 hours. The degree of proliferation wasdetermined as counts per minutes, detected using ascintillation counter (Beckman Coulter).

Cell migration assayOC-3 and OEC-M1 cells were assayed using a 48-well

chamber (modified Boyden chamber) housing a polycar-bonate filter with 8-mmpores (Nucleopore). The upper wellswere loaded with OC-3 (2 � 104) or OEC-M1 (3 � 104)cells. The lower chambers were filled with IL-20 (100 and200 ng/mL), 7E (2 mg/mL), or IL-20 plus 7E in mediumcontaining 0.1%FBS.Mediumwith 0.1%FBSwas used as anegative control. 7E against IL-20 was used to specificallyneutralize the activity of IL-20. The chambers were incu-bated for 6 hours at 37�C. Cells adhering to the lower side ofthe filter were fixed with methanol and stained in a Giemsasolution (Diff-Quick; Baxter Healthcare) for counting. Thenumber of OC-3 and OEC-M1 cells on the lower surface ofthe filter was determined by counting 12 randomly selectedfields under a microscope at �100 magnification. Theexperiment was done 3 times using quadruplicate wells, andmigration was determined as the mean of the total cellscounted per field.

Flow-cytometric analysisWe used fluorochrome, 5,6-carboxy-2,7-dichlorofluores-

cein diacetate (DCFH-DA) to measure the intracellulargeneration of ROS. OC-3 cells (1 � 106) and OEC-M1(1.5 � 106) cells were stimulated by the indicated concen-trations of IL-20 in serum-free medium for 30 minutes. TheOC-3 andOEC-M1 cells were harvested after treatment andstained for 30 minutes at 37�C with 10 mmol/L of DCFH-DA (Molecular Probes). After they had been washed, thecells were kept in the dark and immediately analyzed in acytometer (FACSCalibur; BD Biosciences). For each anal-ysis, 10,000 events were recorded.The expression ofCCR4 andCXCR4on the surface of IL-

20–treatedOC-3 andOEC-M1cells was determined using aflow-cytometric assay. OC-3 (1 � 106) and OEC-M1 (1.5� 106) cells were treated with IL-20 (100 and 200 ng/mL)for 24 hours. To show the specific activity of IL-20, 7E (2mg/mL) was added to the culture system, either alone or togetherwith IL-20. After they had been treated, the OC-3 andOEC-M1 cells were dissociated into cell suspension using anenzyme (TrypLE; Invitrogen) and harvested for stainingwith anti–CCR4 and -CXCR4 monoclonal antibodies (BDBiosciences) at 4�C for 30 minutes. Some OC-3 and OEC-M1 cells were also left unstained and labeled with nonspe-cific isotype antibodies as controls. Subsequently, the stainedcells were washed once with PBS containing 1% FBS, andthen subjected to a standard flow-cytometric analysis in thecytometer.

Western blottingOC-3 cells (5 � 105) were stimulated with IL-20 (200

ng/mL; R&D Systems) for the indicated time periods.

Western blotting used antibodies specific for phosphory-lated AKT, c-jun-NH2-kinase (JNK), extracellular signal–regulated kinase (ERK), and STAT3 (Cell SignalingTechnology). a-Tubulin was detected using a specificantibody (Cell Signaling Technology) as an internalcontrol.

Colony formationOC-3 (2,500/well) and OEC-M1 (3,500/well) cells were

suspended in 0.5%Bacto Agar and plated onto a layer of 1%Bacto Agar in medium containing 5% FBS in 6-well tissue-culture plates (Corning). The agar-containing cells wereallowed to solidify overnight at 37�C in a 5% CO2-humid-ified atmosphere. Additional mediumwith 5%FBSwith IL-20 (200 ng/mL) was overlaid on the agar, and the cells wereallowed to grow undisturbed. The culture medium waschanged every 2 days. To show the specific activity of IL-20, 7E (2 mg/mL) was added to the culture system, eitheralone or together with IL-20. The cells were cultured for 10days, fixed in methanol, and stained with Giemsa. Visiblecolonies (>50 cells) were counted with the aid of a dissectingmicroscope.

Oral cancer-bearing tumor modelAll animal experiments were carried out according to the

protocols based on the Taiwan National Institutes of Health(Taipei, Taiwan) standards and guidelines for the care anduse of experimental animals. The research procedures wereapproved by the Animal Ethics Committee of NationalCheng Kung University. Eight-week-old female severe com-bined immunodeficient (SCID) mice were used in allexperiments. The left mammary fat pad of each mouse wassubcutaneously injected with OC-3 cells (2 � 106 cells) orOEC-M1 cells (4� 106 cells). Themice were then randomlyassigned to 3 groups (n ¼ 6 per group), and treated withPBS, 7E (5 mg/kg; s.c.), or mouse immunoglobulin G (IgG;5 mg/kg; s.c.) every 3 days for the duration of the treatmentregimen. The antibody was injected (s.c.) into the peripheryof the growing tumor in tumor-bearing SCIDmice. Healthycontrols were not injected with tumor cells. Fifty days afterthe tumor cells had been injected, the mice were killedand their tumor tissue was harvested and weighed. Thetumor tissues isolated from the 6 mice in each group werefixed in 3.7% formaldehyde for hematoxylin and eosin(H&E) staining. To analyze the expression levels ofIL-20, TNF-a, IL-1b, and MCP-1, the tumor tissue wasplaced in PBS solution and homogenized, and then RNAwas extracted. Levels of cytokines were detected usingqRT-PCR with specific primers as described earlier.

Statistical analysisPrism 5.0 (GraphPad Software) was used for the statistical

analysis. A one-way ANOVA nonparametric test (Kruskal–Wallis test) was used to compare the data between groups.Post hoc comparisons were done using Dunn multiplecomparison test. Results are expressed as mean � SD.Significance was set at P < 0.05.

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ResultsHigher expression of IL-20 and its receptors in oralcancerTo examine whether IL-20 is involved in the pathogenesis

of oral cancer, IHC staining was used to analyze the expres-sion levels of IL-20 in tumorous and nontumorous oral tissuespecimens from 40 patients with oral cancer. IL-20 and itsreceptors were strongly stained on tumor cells of oral tumortissue but only slightly stained on endothelial cells in non-tumorous oral tissue (Fig. 1A). Furthermore, qRT-PCRshowed that transcript levels of IL-20 (Fig. 1B) and itsreceptors (Fig. 1C–E) in oral tumor tissue were significantlyhigher than in nontumorous oral tissue samples. Theirexpression levels also correlated well with the stages of theoral cancer (Fig. 1B–E). The expression levels of IL-20 andreceptor subunits were the highest in stage IV oral tumor

tissue. These data suggested that IL-20 is involved in thepathogenesis of oral cancer.

IL-20 increased cell proliferation, migration, and ROSproduction in oral cancer cellsTo study the effects of IL-20 on oral cancer cells in vitro,

the expression of IL-20 and its receptors in OC-3 and OEC-M1 cells was analyzed. OC-3 cells were established from anOSCC of a nonsmoking long-term areca (betel nut) chewer(31), whereas OEC-M1 is a cell line derived from gingivalepidermal carcinoma of a Taiwanese patient (32). RT-PCRand immunocytochemical staining showed that IL-20, IL-20R1, IL-20R2, and IL-22R1 were all expressed on OC-3and OEC-M1 cells (Fig. 2A and B). In addition, ELISA wasused to detect the endogenous IL-20 protein secreted in theconditioned media of OC-3 (373.1 pg/mL) and OEC-M1

Figure 1. Higher expression of IL-20 and its receptors in oral cancer. Surgical biopsy samples from 40 patients with oral cancer were collected. Oraltumor andnontumorous tissue from40patientswas analyzed. A, IHCstaining showed that IL-20 and its receptorswere strongly stained in 4different stages oforal tumor samples but only slightly stained on endothelial cells in nontumorous oral tissue samples (stage I, n ¼ 10; stage II, n ¼ 10; stage III, n ¼ 10;and stage IV, n ¼ 10). B–E, the mRNA of the oral tissues was isolated for qRT-PCR analysis using IL-20-, IL-20R1-, IL-20R2-, and IL-22R1-specificprimers. Quantification analysis of mRNA was normalized with hGAPDH as the housekeeping control gene. Differences between the oral tumor andnontumorous oral tissue are statistically significant. �, P < 0.05; ��, P < 0.01 versus nontumorous oral tissue controls. All experiments were done 3 times withsimilar results. Data are from a representative experiment.






(214.8 pg/mL) cells. This indicated that IL-20 might targetcancer cells using a paracrine or autocrinemechanism. A 3H-thymidine incorporation assay was used to analyze theproliferation of oral cancer cells treated with IL-20. IL-20dose dependently increased OC-3 and OEC-M1 cell pro-liferation after 72 hours of treatment (P < 0.05 comparedwith the untreated controls; Fig. 2C). To confirm thespecificity of IL-20–induced proliferation of OC-3 andOEC-M1 cells, anti–IL-20 mAb 7E was used to neutralize

the IL-20–induced proliferation of OC-3 and OEC-M1cells. 7E inhibited IL-20–induced cell proliferation (Fig.2C). To determine whether IL-20 affected cell migration inOC-3 andOEC-M1 cells, a Boyden chamber assay was usedto evaluate the migration ability (Fig. 2D). IL-20 signifi-cantly promoted OC-3 and OEC-M1 cell migration (P <0.05 compared with the untreated controls), which wasinhibited by 7E (Fig. 2D). Because oxidative stress is vitalin the malignant transformation of oral cancer, we also

Figure 2. IL-20 increased proliferation, migration, and ROS production in oral cancer cells. A, the expression of IL-20 and its receptors in OC-3 and OEC-M1cells was analyzed using RT-PCR with specific primers. B, IHC staining showed that IL-20 and its receptors, IL-20R1, IL-20R2, and IL-22R1, were expressedin OC-3 andOEC-M1 cells. C, OC-3 (3� 104) and OEC-M1 (4� 104) cells were cultured for 72 hours in 1% FBSwith or without IL-20 protein (50–200 ng/mL).Cell proliferation was determined using a 3H-thymidine incorporation assay. 7E against IL-20 (2 mg/mL) was used to specifically neutralize theproliferative activity of IL-20. �, P < 0.05 versus untreated controls. #, P < 0.05 versus treatment with IL-20 alone. Values are mean � SD of triplicateexperiments. D, OC-3 (2 � 104) and OEC-M1 (3 � 104) cells were incubated for 6 hours in medium containing 0.1% FBS with IL-20 (100–200 ng/mL),7E (2 mg/mL), or IL-20 plus 7E. Medium alone was used as a negative control. 7E against IL-20 (2 mg/mL) was used to inhibit the activity of IL-20. Cellmigration was evaluated using a modified Boyden chamber assay. �, P < 0.05 versus untreated controls (medium alone). #, P < 0.05 versus treatmentwith IL-20 alone. Values are mean � SD of triplicate experiments. E, OC-3 and OEC-M1 cells were treated with IL-20 for the indicated concentrations inserum-freemedium for 30minutes, andROSgenerationwasmeasuredusing flowcytometry. �,P<0.05 versusuntreatedcontrols. #,P<0.05 versus treatmentwith IL-20 alone. Values are mean � SD of triplicate experiments.

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analyzed the effect of IL-20 on ROS generation inOC-3 andOEC-M1 cells. IL-20 significantly and dose dependentlyincreased intracellular ROS production in OC-3 and OEC-M1 cells (P < 0.05 compared with the untreated controls),which was inhibited by 7E (Fig. 2E). These results suggestedthat IL-20 increased proliferation, migration, and ROSproduction in oral cancer cells, and was critical in theprogression of oral tumor growth.

IL-20 upregulated TNF-a, IL-1b, MCP-1, CCR4, andCXCR4 in oral cancer cellsThe molecular pathways of cancer-related inflammation

were revealed, which allowed us to identify new targetmolecules that might lead to improved diagnosis and treat-ment. Therefore, we investigated whether IL-20 is involvedin tumor-associated inflammation.We incubatedOC-3 andOEC-M1 cells with IL-20 and used qRT-PCR to analyze theexpression of proinflammatory cytokines, chemokines, andchemokine receptors. The TNF-a, IL-1b, and MCP-1

expression significantly increased in IL-20–treated OC-3(Fig. 3A–C) and OEC-M1 cells (data not shown); however,the expression of other inflammatory mediators, namely, IL-6, IL-8, matrixmetalloproteinase-2 (MMP-2),MMP-7, andMMP-9, was not affected (data not shown). IL-20 treatmentsignificantly induced CCR4 and CXCR4 expression on thesurface of OC-3 cells (Fig. 3D–E). Although flow-cyto-metric analysis showed that IL-20 increased CCR4 andCXCR4 expression on the surface of OEC-M1 cells, theyshowed no significant difference when the data were ana-lyzed using ANOVA.To confirm that IL-20 specifically induced their expres-

sion, the OC-3 cells were treated with IL-20 plus 7E. Flow-cytometric analysis showed that 7E had significantly neu-tralized CCR4 and CXCR4 expression in cotreated OC-3cells (Fig. 3D–E). In addition, we found that IL-20 expres-sion was significantly correlated with CXCR4 expression inthe oral tumor tissue of patients with stage IV cancer (datanot shown). To clarify the mechanism that IL-20 uses to

Figure 3. IL-20 upregulated TNF-a, IL-1b, MCP-1, CCR4, CXCR4, and activated intracellular signaling in oral cancer cells. A–C, OC-3 cells were treatedwith IL-20 (200 ng/mL) for the indicated times, and the expression levels of TNF-a, IL-1b, and MCP-1 were analyzed using qRT-PCR with specificprimers. �, P < 0.05 versus untreated controls. All experiments were done 3 times with similar results. Data are from a representative experiment. D–E, OC-3cells were treated with IL-20 in the indicated concentrations in DMEM for 24 hours. Cells were dissociated into a single-cell suspension and harvestedfor flow cytometry using anti-CCR4 and anti-CXCR4monoclonal antibody. �,P < 0.05 versus untreated controls. #,P < 0.05 versus treatment with IL-20 alone.Values are mean � SD of triplicate experiments. F, OC-3 cells (5 � 105) were incubated with IL-20 (200 ng/mL) for the indicated time periods, and then celllysates were collected and analyzed using immunoblotting with specific antibodies against phospho-JNK, phospho-STAT3, phospho-ERK, and phospho-AKT. a-Tubulin antibody was used as an internal control. Results are representative of 3 independent experiments. MFI, mean fluorescence intensity.






promote tumor progression, the signal molecules of JNK,STAT3, ERK, and AKT were assessed in IL-20–stimulatedOC-3 cells. Western blotting showed that IL-20 promotedthe activation of JNK, STAT3, ERK, and AKT (Fig. 3F).These results indicated that IL-20 induced inflammatorymediators in oral cancer, triggered proliferation-associatedsignals, and was a promoting factor for tumor growth.

IL-20 promoted colony formation ofOC-3 andOEC-M1cellsNeoplastically transformed cells show reduced require-

ments for extracellular growth-promoting factors, and theyare not restricted by cell–cell contact. Anchorage-indepen-dent growth is one of the hallmarks of the malignanttransformation of cells. Therefore, we used a soft agarcolony-formation assay to monitor the ability of IL-20 inthe anchorage-independent growth of OC-3 and OEC-M1cells. IL-20 significantly stimulated OC-3 and OEC-M1cells to form more colonies than the controls formed, whichwas inhibited by 7E (Fig. 4A and B). OC-3 cells cotreatedwith IL-20 and 7E formed significantly fewer and smaller(Fig. 4C) colonies in soft agar than did cells treated with IL-20 alone. These results suggest a significant role for IL-20 inthe malignant transformation of oral cancer cells.

7E suppressed in vivo tumor growth and reducedinflammation within the tumor microenvironmentIL-20 was highly expressed in oral cancer tissue, and it

increased oral tumor growth and inflammation in vitro. 7Ehas shown specificity and neutralization activity in vitro andin vivo (24, 28, 33). Therefore, we analyzed whether 7Ereduced tumor growth in vivo. OC-3 cells were injected intothe left mammary fat pads of SCID mice. One day later, the

micewere injected (s.c.) with PBS, 7E (5mg/kg), ormIgG (5mg/kg; n¼ 6 per group) every 3 days for 50 days. Fifty daysafter they had been injected with OC-3 cells, all the micewere killed and their tumors were isolated. Tumors weresignificantly smaller (Fig. 5A) and weighed significantly less(Fig. 5B) in the 7E-treated group than in the control mIgG-and PBS-treated groups. H&E staining showed thatimmune cell infiltration was reduced in the tumor mass ofthe 7E treated-group compared with the mIgG-treatedcontrol group (Fig. 5C). Tumor tissue from each groupwas then isolated and homogenized to extract RNA. qRT-PCR assays showed that IL-20 levels in the tumor were lowerin the 7E-treated group than in the mIgG-treated group(Fig. 5D). In addition, the expression of the inflammationmediators TNF-a, IL-1b, and MCP-1 was significantlylower in the 7E-treated group than in the mIgG-treatedgroup (Fig. 5E–G). Furthermore, we used OEC-M1 cellsto confirm the effect of 7E in tumor-bearing SCID miceand obtained similar results (Supplementary Fig. S1).These results indicated that 7E suppressed tumor growthin vivo by reducing IL-20 expression, decreasing otherproinflammatory cytokines, and inhibiting immune cellinfiltration.

DiscussionWe found that both IL-20 and its receptors were highly

expressed in primary, nodal-involved, and distantly meta-static oral tumor tissue. IL-20 expression was correlated withthe tumor stages in patients with oral cancer. All IL-20receptor subunits were expressed in our clinical specimens,which suggested that both autocrine and paracrine effects ofIL-20 on oral tumors and contributed to the pathogenesis oforal cancer.

Figure 4. IL-20 promoted colonyformation in vitro. A and B,OC-3 (2,500/well) and OEC-M1(3,500/well) cells were incubatedwith IL-20 for 10 days, and then acolony-formation assay was done.The culture medium was changedevery 2 days. IL-20 significantlyincreased the colony-formationability of oral cancer cells. 7E(2 mg/mL) was used to inhibit theactivity of IL-20. �, P < 0.05 versusuntreated controls. #, P < 0.05versus treatment with IL-20 alone.Values are mean � SD of triplicateexperiments. C, IL-20 alsoincreased the size of colonies ina soft agar assay. Representativephotos are shown for each group.

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IL-20 directly affected oral cancer cell proliferation byactivating proliferation-associated signals, such as JNK,ERK, and STAT3, and migration by activating migra-tion-associated signals, such as JNK and ERK. IL-20 alsoprovided a microenvironment for tumor growth and metas-tasis by inducing ROS and proinflammatory cytokines,which are essential for the malignant process from normalepithelium to invasive oral cancer (34). ROS may increasethe inflammatory responses mediated by activating signalingmolecules, such as ERK and JNK, and transcription factors,such as NF-kB. NF-kB regulates many proteins involved intumor initiation, tumor expansion, and metastasis promo-tion inmany types of cancers, including oral cancer (35, 36).A variety of cytokines, such as IL-1, IL-6, and TNF-a, as

well as chemokines, such as IL-8, CXCL5,CXCL6,CXCL7,CCL5, and CCL20, were upregulated in progressive oralcancer cells (6, 37, 38) and in patients with oral cancer (13).The effects of CCL and CXCL chemokines are mediated viatheir CCR and CXCR receptors. IL-20–treated OC-3 andOEC-M1 cells increased TNF-a and IL-1b production,

which are potent proinflammatory cytokines for the growth,metastasis, and immune evasion of oral tumors. We alsofound that IL-20 induced MCP-1 and CCR4 expression inOC-3 cells. MCP-1 (39, 40) and CCR4 (41, 42) have beenassociated with several cancers. Therefore, IL-20 may pro-mote leukocyte trafficking, which augments inflammation,tumor cell infiltration, andmetastasis in oral tumors, by sucha novel, undefined mechanism. Several studies (43, 44) haveassociated CXCR4 and oral cancer. IL-20 induced moreCXCR4 expression on the surface of IL-20–treated OC-3cells than of the untreated control group. The involvementof CXCR4 signaling in OSCC was mediated by the activa-tion of both the ERK and the AKT/PKB pathways (45).Therefore, IL-20–induced activation of ERK and AKT maycross-link to amplify the CXCR4 signaling for the chemo-taxis of oral cancer cells. 7E treatment to inhibit IL-20–induced CXCR4 expression may be a potent antimetastatictherapy against lymph node and distantmetastases in cases ofCXCR4-related OSCC. IL-20 increased the anchorage-independent growth of OC-3 and OEC-M1 cells, which

Figure 5. Anti–IL-20mAb treatment suppressed tumor growth in vivo. OC-3 cells (2�106)were injected (s.c.) into themammary fat padsof SCIDmice.Onedaylater, the mice were injected (s.c.) with PBS, 7E (5 mg/kg), andmIgG (5 mg/kg; n¼ 6 per group) every 3 days for 50 days. A, tumor size wasmeasured using avernier caliper. �,P<0.05 versusmIgGcontrols. ��,P<0.01 versusmIgGcontrols. B,micewere killed 50days after they had been injectedwithOC-3 cells, andtheir tumors were collected and weighed. �, P < 0.05 versus mIgG controls. C, tumor sections from each group were stained with H&E (magnification:�200).Representative photos are shown for each group. D–G, tumor tissue samples from each group (n ¼ 6) were isolated at the end of the experiment. Theexpression of IL-20, TNF-a, IL-1b, andMCP-1 in the tumor tissuewasanalyzedusing qRT-PCRwith specificprimers. �,P<0.05 versusmIgGcontrols. Resultsare from a representative experiment. All experiments were done 3 times. Values are means � SEM.






confirmed that IL-20 was potently involved in themalignanttransformation of oral cells.IL-20 was upregulated in the tumor tissue of patients with

oral cancer. In our mouse tumor model using OC-3 andOEC-M1 cells, we confirmed that IL-20 was highlyexpressed in the tumor mass, which is consistent with ourclinical findings. IL-20 is a hypoxia-inducible gene and thehypoxia response element (HRE) has been identified in IL-20 promoter (29). Areca (betel) nut extract modulates asignaling cascade that induces HIF-1a expression in oralcancer cells (46). HIF-1a is an independent prognosticmarker in OSCC (30). Therefore, one possible mechanismupregulating IL-20 in the tumor may be the response of oralcancer cells to hypoxia. HIF-1a binds to the HRE on IL-20promoter and activates IL-20 expression. HPV infection is aknown risk factor in the development of oral cancer (1, 5).Whether HPV regulates IL-20 expression in the pathogen-esis of oral cancer is not well understood. Additional inves-tigations of the regulation between IL-20 andHPV infectionare required.7E suppressed OC-3 and OEC-M1 tumor growth and

tumor weight in vivo. The inhibition of tumor growth andtumor size in 7E-treated mice correlated with lower IL-20expression, which indicated that IL-20 was involved in theprogression of oral cancer by regulating tumor cell prolifer-ation in vivo.We hypothesize a working model of IL-20 in oral cancer

progression. Oral cancer cells or infiltrated immune cellssecrete a large amount of IL-20, which promotes tumorcell proliferation by phosphorylating ERK, JNK, AKT,and STAT3 in an autocrine/paracrine manner. The over-expressed IL-20 stimulates ROS production in tumorcells. IL-20–induced production of proinflammatory cyto-kines and ROS by tumor cells amplify local inflammationwithin the tumor microenvironment. Eventually, all ofthese tumor-promoting effects contribute to the prolifer-ation and survival of malignant cells, angiogenesis, metas-tasis, and the impairment of immune responses. In addi-tion, the chemoattractant activity of IL-20 furtherincreases immune cell infiltration, which results in IL-20 overexpression, which, in turn, forms a positive loop to

oral cancer progression. 7E suppresses IL-20–inducedtumor growth and local inflammation in vitro and in vivo,which supports our hypothesis.We previously showed that IL-20 was induced in response

to hypoxia; promoted angiogenesis in endothelial cells;induced IL-6, IL-8, and MCP-1 expression in several typesof cells; and was a chemoattractant for neutrophils. In thepresent study, we showed that IL-20 increased the prolifer-ation and migration of oral cancer cells. IL-20 also inducedthe expression of TNF-a, IL-1b, and MCP-1, as well as theproduction of CCR4 and CXCR4, on oral cancer cells.These properties of IL-20 provide evidence that IL-20 isinvolved inmany phases of tumor progression and nurtures amicroenvironment for tumor cells.In summary, our findings indicate that IL-20 is involved

in oral cancer progression. IL-20 not only directly increasedthe proliferation, anchorage-independent growth, andmigration of cancer cells but also generated a microenvi-ronment that facilitated tumor progression by inducing theproduction of ROS and of several proinflammatory cyto-kines and chemokine receptors. We conclude that IL-20 is anovel target for pharmacologic intervention for inhibitingthe tumor growth and metastasis of oral cancer.

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

Authors' ContributionsConception and design: Y.-H. Hsu, M.-S. ChangDevelopment of methodology: Y.-H. HsuAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): Y.-H. Hsu, D.-B. ShiehAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): Y.-H. Hsu, D.-B. Shieh, C.-H. ChanWriting, review, and/or revision of the manuscript: Y.-H. Hsu, C.-C. Wei, M.-S.ChangAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): Y.-H. Hsu, C.-H. ChanStudy supervision: M.-S. Chang

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 2, 2012; revised August 14, 2012; accepted August 27, 2012;published OnlineFirst September 21, 2012.

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2012;10:1430-1439. Published OnlineFirst September 21, 2012.Mol Cancer Res Yu-Hsiang Hsu, Chi-Chen Wei, Dar-Bin Shieh, et al. Cancer and Suppresses Tumor Growth

IL-20 Monoclonal Antibody Alleviates Inflammation in Oral−Anti

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