IL-4 and IL-17A Cooperatively Promote Hydrogen Peroxide

IL-4 and IL-17A Cooperatively Promote Hydrogen Peroxide Production, Oxidative DNA Damage, and Upregulation of Dual Oxidase 2 in Human Colon and Pancreatic This information is current as Cancer Cells of September 26, 2021. Yongzhong Wu, Mariam M. Konaté, Jiamo Lu, Hala Makhlouf, Rodrigo Chuaqui, Smitha Antony, Jennifer L. Meitzler, Michael J. Difilippantonio, Han Liu, Agnes Juhasz,

Guojian Jiang, Iris Dahan, Krishnendu Roy and James H. Downloaded from Doroshow J Immunol published online 23 September 2019 http://www.jimmunol.org/content/early/2019/09/20/jimmun ol.1800469 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 23, 2019, doi:10.4049/jimmunol.1800469 The Journal of Immunology

IL-4 and IL-17A Cooperatively Promote Hydrogen Peroxide Production, Oxidative DNA Damage, and Upregulation of Dual Oxidase 2 in Human Colon and Pancreatic Cancer Cells

Yongzhong Wu,* Mariam M. Konate´,† Jiamo Lu,* Hala Makhlouf,† Rodrigo Chuaqui,† Smitha Antony,† Jennifer L. Meitzler,* Michael J. Diﬁlippantonio,† Han Liu,† Agnes Juhasz,* Guojian Jiang,* Iris Dahan,† Krishnendu Roy,† and James H. Doroshow*,†

Dual oxidase 2 (DUOX2) generates H2O2 that plays a critical role in both host defense and chronic inflammation. Previously, we demonstrated that the proinflammatory mediators IFN-g and LPS enhance expression of DUOX2 and its maturation factor DUOXA2 through STAT1- and NF-kB‒mediated signaling in human pancreatic cancer cells. Using a panel of colon and pancre- Downloaded from atic cancer cell lines, we now report the induction of DUOX2/DUOXA2 mRNA and protein expression by the TH2 cytokine IL-4. IL-4 activated STAT6 signaling that, when silenced, significantly decreased induction of DUOX2. Furthermore, the TH17 cytokine IL-17A combined synergistically with IL-4 to increase DUOX2 expression in both colon and pancreatic cancer cells mediated, at least in part, by signaling through NF-kB. The upregulation of DUOX2 was associated with a significant increase in the production

of extracellular H2O2 and DNA damage—as indicated by the accumulation of 8-oxo-dG and gH2AX—which was suppressed by the NADPH oxidase inhibitor diphenylene iodonium and a DUOX2-speciﬁc small interfering RNA. The clinical relevance of these experiments is suggested by immunohistochemical, microarray, and quantitative RT-PCR studies of human colon and pancreatic http://www.jimmunol.org/ tumors demonstrating signiﬁcantly higher DUOX2, IL-4R, and IL-17RA expression in tumors than in adjacent normal tissues; in pancreatic adenocarcinoma, increased DUOX2 expression is adversely associated with overall patient survival. These data suggest

a functional association between DUOX2-mediated H2O2 production and induced DNA damage in gastrointestinal malignancies. The Journal of Immunology, 2019, 203: 000–000.

ecent studies suggest that reactive oxygen species (ROS) of activation (7–9). DUOX2 is a membrane-localized glycopro- play an important role as mediators of inflammation- tein composed of six transmembrane helices bearing a cytosolic R related malignancies by enhancing tumor cell prolifera- C-terminal FAD/NADPH binding domain, two cytosolic EF-hands by guest on September 26, 2021 tion and angiogenesis (1–3). ROS are produced in human tumors for calcium binding, and an extracellular N-terminal peroxidase-like by a variety of mechanisms, including by members of the mi- domain. In the presence of its cognate maturation factor DUOXA2, tochondrial electron transport chain (4) and the NADPH oxidase these structural components regulate the transfer of electrons from (NOX) gene family (5, 6). NADPH to molecular oxygen to generate H2O2 (7, 10–12), which is The seven NOX family members share certain structural homol- important for the synthesis of thyroid hormone (13) and plays an ogies while retaining distinct tissue specificities and mechanisms important host defense role against pathogens in the airway and gastrointestinal mucosal epithelia (10, 14, 15). *Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Increasing evidence suggests an etiologic role for DUOX2 in Institute, National Institutes of Health, Bethesda, MD 20892; and †Division of Cancer gastrointestinal malignancies; it is overexpressed in premalignant Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 chronic inflammatory states of the colon (16, 17), adenomatous large intestinal polyps (18), and in chronic pancreatitis (19) as well as ORCIDs: 0000-0003-1393-0998 (H.M.); 0000-0002-4676-7034 (M.J.D.); 0000- 0003-0541-0714 (K.R.). early stages of pancreatic ductal adenocarcinoma (PDAC) (20). The Received for publication March 27, 2018. Accepted for publication August 27, 2019. proinflammatory microenvironment of these diseases, including This work was supported by the Division of Cancer Treatment and Diagnosis and the overexpression of TH17 and TH2 cytokines (IL-17A and IL-4/IL-13) Center for Cancer Research, National Cancer Institute, National Institutes of Health. and their corresponding receptors, has been demonstrated to sig- The content of this publication does not necessarily reflect the views or policies of the nificantly alter epithelial tumor cell proliferation, survival, and Department of Health and Human Services, nor does mention of trade names, com- mercial products, or organizations imply endorsement by the U.S. Government. metastatic potential (21–24). Furthermore, prior studies from our Address correspondence and reprint requests to Dr. James H. Doroshow, Division of group demonstrated that LPS and the TH1 cytokine IFN-g stimulate Cancer Treatment and Diagnosis, National Cancer Institute, Building 31, Room 3A- DUOX2 expression and activity in pancreatic cancer cells by acti- 44, 31 Center Drive, National Institutes of Health, Bethesda, MD 20892. E-mail vating signaling through NF-kB and STAT1 (19, 25, 26). address: [email protected] In the present work, we found significant upregulation of The online version of this article contains supplemental material. DUOX2 and DUOXA2 in surgically resected colon cancer spec- Abbreviations used in this article: DPI, diphenylene iodonium; gH2AX, histone H2AX phosphorylation at serine 139; IHC, immunohistochemical, immunohistochemistry; imens compared with adjacent normal colonic epithelium and NAC, N-acetyl- L-cysteine; NOX, NADPH oxidase; 8-oxo-dG, 8-oxo-7,8-dihydro- have shown that DUOX2 expression is significantly associated 29-deoxyguanosine; PDAC, pancreatic ductal adenocarcinoma; PEG-catalase, with poor prognosis in pancreatic cancer clinical samples from catalase/polyethylene glycol; ROS, reactive oxygen species; siRNA, small interfering RNA; TCGA, The Cancer Genome Atlas; WCE, whole-cell extract. The Cancer Genome Atlas (TCGA). To understand the potential This article is distributed under The American Association of Immunologists, Inc., role of DUOX2 in adenocarcinomas of the pancreas and colon, we Reuse Terms and Conditions for Author Choice articles. examined the mechanisms of proinflammatory cytokine-related

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800469 2 IL-4 AND IL-17A ENHANCE DUOX2-RELATED DNA DAMAGE

ROS production by DUOX2 in human pancreatic and colonic tumors (31). RNA expression levels for 11 oxidases or related proteins cancer cells. We report in this study that IL-4—alone or in com- were expressed for the 23 colon adenocarcinoma samples relative to the bination with IL-17A—strongly induces DUOX2/DUOXA2 mRNA pool of five normal colon mucosae from noncancer patients (31). Unsu- pervised clustering analysis with BRB-ArrayTools (32) was used to gen- and DUOX protein expression, leading to significantly increased erate a heat map. extracellular H2O2 accumulation and ROS-related DNA dam- For other comparative analyses between normal and malignant tissues, age. DUOX2 upregulation by IL-4 occurred as a consequence of primary human colon and pancreatic cancers and adjacent nonmalignant STAT6 activation and translocation to the nucleus, suggesting a tissue samples were acquired from the National Cancer Institute–sponsored Cooperative Human Tissue Network (Eastern, Mid-Western, and Mid- role for STAT6 in the transcriptional regulation of DUOX2 fol- Atlantic Divisions) in compliance with the Office of Human Subjects lowing proinflammatory stimulation. Enhanced expression of Research at the National Institutes of Health, Bethesda, MD. Specimens DUOX2 by IL-17A appears to be a consequence of NF-kB– were selected without regard to age, race/ancestry, or sex and were ob- related signal transduction. These results suggest that DUOX2 and tained from patients who had not received chemotherapy or irradiation associated ROS play a role in inflammation-related gastrointestinal treatment prior to surgical intervention. Tumors were preserved by snap freezing in liquid nitrogen within 60 min of surgery. RT-PCR methods are malignancies. described in the corresponding section below. Materials and Methods Immunohistochemistry and tissue microarray Reagents and Abs Immunohistochemical (IHC) staining was performed on 1203 formalin- fixed, paraffin-embedded tissue microarray blocks from US Biomax. Human rIFN-g (285-IF), IL-17A (317-ILB), IL-4 (204-IL), and IL-13 These included the following: colon disease tissue arrays no. BC05002, no. (213-ILB) were purchased from R&D Systems (Minneapolis, MN). All

BC051110, and no. CO809a; small intestine tissue array no. SM2081; breast Downloaded from cytokines were dissolved in 0.1% BSA in sterilized PBS. Actinomycin D invasive ductal carcinoma tissue array no. BC08118; breast cancer tissue (A9415), cycloheximide (C7968), and catalase/polyethylene glycol (PEG- array no. BR1505b; lung cancer tissue array no. LC2085b; midadvanced- catalase; lyophilized powder, 40,000 U/mg, C4963-2MG) were from stage ovary cancer tissue array no. OV8010; prostate cancer tissue array no. Sigma-Aldrich (St. Louis, MO); and ionomycin (calcium salt, 407952-1MG) PR2085b; and a tissue array of gastritis with intestinal metaplasia and and LPS (437627) were from Calbiochem (MilliporeSigma, Billerica, MA). gastric carcinoma no. IC00011. The tissue microarrays were deparafﬁnized Anti-human DUOX Ab, which reacts with both DUOX1 and DUOX2, was in alcohol and rehydrated with graded alcohol prior to Ag retrieval. A previously developed by Creative Biolabs (Port Jefferson Station, NY) and validated DUOX Ab developed in mouse and described previously (26) was

characterized by our laboratory (26). Anti-STAT6 (catalog no. 611290) http://www.jimmunol.org/ used. Slides were then counterstained with hematoxylin, dehydrated, and and anti–p-STAT6 (catalog no. 611566) used for Western blot analysis Y641 coverslipped. All samples were processed in parallel with a no-primary-Ab were from BD Transduction Laboratories (San Diego, CA). Abs against control to evaluate possible artifactual nonspecific staining from the sec- human lamin A/C (no. 2032), p-histone H2AX (no. 2577S), p-STAT1 Y701 ondary Ab. Isotype control staining was prepared with normal rabbit IgG (no. 9167), STAT3 (79D7, no. 4904), p-STAT3 (no. 9145), and NF-kB Y705 (Cell Signaling Technology, Danvers, MA) at a comparable concentration (p65) (no. 8242) were purchased from Cell Signaling Technology to the primary Ab. The verification of staining performance was con- (Beverly, MA). STAT1 p84/p91 (sc-346), STAT3 (C-20, sc-482), goat firmed on a series of cancer tissue samples. In addition, a series of normal, anti-rabbit IgG/HRP (sc-2004), and goat anti-mouse IgG/HRP (sc-2005) nontumor tissues were evaluated to establish immunoreactivity and assay were from Santa Cruz Biotechnology (Dallas, TX). All human primer specificity. Evaluation and comparison of staining on sections exposed to and probe sets, b-actin (Hs99999903_m1), DUOX2 (Hs00204187_m1), the primary and secondary Abs were compared with negative control DUOXA2 (Hs01595310_g1), STAT6 primer (Hs00598625_m1), IL-17RA sections that were not exposed to the primary Ab. Each slide was digi- primer (Hs01064648_m1), and IL-4Ra (Hs00166237_m1), were from by guest on September 26, 2021 tally imaged using an Aperio ScanScope. Tissues were scored as posi- Life Technologies (Carlsbad, CA). Negative Control No. 1 siRNA tively stained only if they exhibited a staining pattern with the primary (AM4635) and human DUOX2 siRNA (S27012) were obtained from Ab that was significantly different from that found when omitting the Applied Biosystems (Carlsbad, CA). ON-TARGETplus Human STAT6 primary Ab. Those that did not demonstrate a significant difference be- siRNA SMARTpool (L-006690-00-0005) was purchased from GE tween primary and omitting primary staining were graded as 0+ no stain Healthcare Dharmacon (Lafayette, CO). and 0% cells stained. Tissues that demonstrated a significant difference Data mining and bioinformatic analyses of TCGA datasets between the two conditions were graded as follows. The assay was interpreted with a scoring system of 0+, 1+, 2+, and 3+ for staining Level 3–normalized (RNASeq V2) gene expression datasets were gener- intensity, corresponding to negative, weak, moderate, and strong DUOX ated by TCGA Research Network (http://cancergenome.nih.gov) and staining, respectively. The percentage of stained tumor/lesion cells downloaded from the cBioPortal for Cancer Genomics (27, 28). Overall, (distribution) was estimated for each patient, and 0 to ,10% was DUOX2 mRNA expression levels in 9121 samples from 30 studies were considered negative. retrieved and log2 transformed. For the survival analysis of patients with pancreatic cancer, only primary solid tumors with measured DUOX2 ex- Cell culture pression and known survival data (n = 177) were included in the analysis. The samples were stratified into low- and high-DUOX2 expression groups Human colon cancer cell lines T84 (CCL-248) and LS513 (CRL-2134) and using Cutoff Finder to determine the optimal expression cutoff point for human pancreatic cancer cell lines BxPC-3 (CRL-1687), AsPC-1 (CRL- statistical significance by log-rank test (29). DUOX2, IL-4R, IL-17RA, 1682), and CFPAC-1 (CRL-1918) were obtained from American Type and IL-17A gene expression levels in primary solid tumors of the pan- Culture Collection (Manassas, VA). T84 cells were cultured in a 1:1 mixture creas (n = 178) and colon (n = 380) from TCGA were retrieved using of Ham’s F12 medium and DMEM with 2.5 mM L-glutamine and 5% FBS; cBioPortal. LS513, BxPC-3, and AsPC-1 cells were cultured in RPMI 1640 medium (SH30255.01; GE Healthcare HyClone, Logan, UT) with 1.0% sodium Patients and sample collection pyruvate and 10% FBS (100-106; Gemini Bio-Products, Sacramento, CA). CFPAC-1 cells were cultured in IMDM with 10% FBS. The identity of The tissue samples analyzed for this study were from patients diagnosed each cell line was confirmed by the Genetic Resources Core Facility at with colon adenocarcinoma and treated by the Department of General Johns Hopkins University (Baltimore, MD). To establish starvation con- Surgery, University Medicine Gottingen (Gottingen, Germany). These data ditions before each experiment, cells were cultured overnight in the same were previously described by Camps et al. (30, 31), and the complete gene medium without FBS. Starvation conditions were used because DUOX2 expression dataset was previously deposited into the National Center for induction by different cytokines is stronger after serum starvation, as noted Biotechnology Information Gene Expression Omnibus database, accession previously (25). In all cases, cells were cultured in a humidified incubator number GSE10402 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? at 37˚C in an atmosphere of 5% CO2 in air. acc=GSE10402). As described in Camps et al. (30), a subset of samples was selected based on also having oligonucleotide array data, and then by Quantitative RT-PCR applying a 90th interpercentile range procedure to equalize the spread of Cy3 measurement per array (in log2 scale). The resulting subset contained For analysis of human tissues, samples ranging in size from 200 to 28 samples: 23 primary colon tumors and 5 normal colon mucosae sam- 750 mg were homogenized on ice, and RNA was isolated using the ples. RNA extraction, quantification, quality assessment, expression RNeasy Plus Universal Mini Kit (catalog no. 73404; QIAGEN, Germantown, microarrays, and data analysis were previously reported for the 23 primary MD) according to the manufacturer’s protocol. Two micrograms of total The Journal of Immunology 3

RNA isolated from each specimen was used for cDNA synthesis in a Detection of 8-oxo-7,8-dihydro-29-deoxyguanosine 20 ml reaction system with the following cycles: 25˚C for 5 min, 42˚C by immunofluorescence for 50 min, and 75˚C for 5 min. After the reaction was complete, the synthesized cDNA was diluted with H2Oto100ml prior to quantita- Immunofluorescence staining was performed following the manufacturer’s tive RT-PCR. For the analysis of human tumor and adjacent normal protocol (Trevigen, Gaithersburg, MD) with some modifications described surgical specimens, b-actin and 18S rRNA were used as housekeeping as follows. A total of 6 3 104 AsPC-1 cells were seeded onto four-well control genes for colon cancer and pancreatic cancer patient samples, glass chamber slides, then treated with either solvent or IL-4 (50 ng/ml) respectively. plus IL-17A (50 ng/ml) for 48 h. The primary Abs were incubated For in vitro measurements, total RNA was extracted from 1 3 106 cells overnight at 37˚C on a humidified chamber with dilution 1:2000 in 13 using the RNeasy Mini Kit (catalog no. 74104; QIAGEN, Valencia, CA) PBS containing 1% BSA and 0.01% Tween 20. The secondary Abs were following the manufacturer’s instructions. Two micrograms of total RNA incubated using a dilution of 1:400 in 13 PBS containing 1% BSA for were then used for cDNA synthesis, along with SuperScript III Reverse 1 h at room temperature in the dark. Slides were mounted using VEC- Transcriptase (18080-044) and random primers (48190-011), both from TASHIELD Antifade Mounting Medium (catalog no. H-1200; Vector Life Technologies, in a 20 ml reaction. The cDNA synthesis steps consisted Laboratories, Burlingame, CA) with DAPI for counterstaining of nu- of cycles of 25˚C for 10 min, 42˚C for 50 min, and 75˚C for 10 min. The clei. The Abs for immunofluorescence staining of 8-oxo-7,8-dihydro-29- synthesized cDNA was diluted to 100 ml with diethylpyrocarbonate-treated deoxyguanosine (8-oxo-dG) were a mouse monoclonal 8-oxo-dG Ab, H2O, and quantitative PCR was conducted in 384-well plates in a 20-ml catalog no. 4354-MC-050, from Trevigen and a goat anti-mouse sec- volume consisting of 2 ml diluted cDNA, 1 ml primers, 7 mlH2O, and ondary Ab conjugated with Alexa Fluor 488, catalog no. A-11001, from 10 ml TaqMan Universal PCR Master Mix (4364340; Life Technologies). Thermo Fisher Scientific. Fluorescence images were captured using a The PCR was performed using the default cycling conditions (50˚C for confocal microscope (Zeiss LSM 780); ZEN Blue version 2.3 software 2 min and 95˚C for 10 min and 40 cycles of 95˚C for 15 s and 60˚C for was used for image quantitation. 10 min) with the ABI PRISM 7900HT Sequence Detection System (Ap- plied Biosystems, Foster City, CA). Triplicate samples were used for the Statistical analysis Downloaded from quantitative RT-PCR, and the mean values were calculated. The data in Data represent the mean 6 SD from three or more independent experi- all figures represent three independent experiments. Relative gene ex- ments. Statistical differences between the mean values of samples were pression was calculated from the ratio of the target gene expression to assessed using the two-tailed Student t test or the Wilcoxon signed-rank the internal reference gene (b-actin or 18S rRNA) expression based on test. Statistical significance was defined as *p , 0.05, **p , 0.01, and the cycle threshold values. ***p , 0.001. For the analysis of human tumor surgical specimens, NOX gene expression levels were measured by quantitative RT-PCR with Power SYBR Green technology (Applied Biosystems). For each RT-PCR, 300 ng cDNA Results http://www.jimmunol.org/ was used. PCR was done with the default variables of the Applied Bio- DUOX2 is highly expressed in specific human tumors systems’ Prism 7000 sequence detector with a total reaction volume of 25 ml. Primers were obtained from Operon Technologies (Huntsville, AL). To assess the potential importance of DUOX2 expression in tu- Each sample was analyzed in triplicate, and each data point was calculated morigenesis, we examined the relative expression levels of DUOX2 as the median of the three measured cycle threshold values. across a broad range of human malignancies using human tumor Western analysis datasets from TCGA (Fig. 1A). DUOX2 mRNA expression varied widely in human tumors, with thyroid, pancreas, and colorectal To prepare extracts, cells were lysed in 13 RIPA Lysis Buffer (catalog no. 20-188; MilliporeSigma) supplemented with a phosphatase inhibitor cancers exhibiting the highest levels. These results are consis- tablet (04-906-837001) and a protease inhibitor tablet (11-836-153001), tent with our recent report demonstrating significantly increased by guest on September 26, 2021 both from Sigma-Aldrich, to generate whole-cell extracts (WCEs). Nu- DUOX2 expression in patients with early stage PDAC (20). The clear extracts were additionally prepared using the NE-PER Nuclear and relative mRNA expression levels of members of the NOX gene Cytoplasmic Extraction Kit (78833; Thermo Fisher Scientific, Rockford, IL). The protein concentrations of both types of extracts were measured family (NOX1/4/5, DUOX1/2, and their accessory proteins) were using the Pierce BCA Protein Assay Kit (23227; Thermo Fisher Scientific). also examined in surgical samples from 23 colon cancer patients The extracts were then combined with an equal volume 23 SDS Protein relative to normal colon mucosae from five noncancer patients. As Gel Loading Solution (351-082-661; Quality Biological, Gaithersburg, shown in Fig. 1B, NOX1 and DUOX2 were both highly expressed MD). Next, unless otherwise indicated, 50 mgofWCEor20mgof in colon cancers. Some tumors had either increased expression nuclear extract was electrophoretically separated on a 4–20% Tris- Glycine Gel (EC6028; Life Technologies) and transferred to nitrocel- of NOX1 or DUOX2 (versus normal colonic mucosa), whereas lulose membranes using an iBlot Transfer Stack (IB 3010-01; Life other colon cancers demonstrated concurrent overexpression of Technologies). The membranes were blocked in 5% nonfat milk in 13 both genes. TBST (TBS with 0.1% Tween 20) buffer for 1 h at room temperature To confirm these findings, we evaluated DUOX protein ex- and then incubated overnight with the indicated primary Abs in TBST. After three washes in TBST, the membranes were incubated with the pression by IHC staining of tissue microarrays from normal tissues, appropriate HRP-conjugated secondary Abs for 1 h at room tempera- as well as benign and malignant tumors. Generally, DUOX ex- ture on a shaker. SuperSignal West Pico Luminol/Enhancer Solution pression was observed in epithelial cells, often with a cytoplasmic (1856136; Thermo Fisher Scientific) was then applied to visualize the pattern. The most significant overexpression relative to normal proteins of interest. tissue was identified in gastrointestinal tumors such as colonic , Amplex red assay to detect extracellular H2O2 adenomas and adenocarcinomas (***p 0.001; Table I), with DUOX located mainly in the cytoplasm with increasing intensity Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (catalog no. at the brush border of the malignant glands (Fig. 1Ca); in normal A22188; Life Technologies) was employed to detect extracellular H2O2 release. Cells were washed twice with 13 PBS, trypsinized, and colon mucosa, DUOX protein was detected in neuroendocrine counted. Next, for BxPC-3 and AsPC-1 cells, 20 ml cell suspension cells (Fig. 1Cb, inset) and at the tips of villi. DUOX protein ex- 4 containing 2 3 10 live cells in 13 Krebs–Ringer phosphate glucose pression was also increased in small intestinal adenocarcinomas buffer was mixed with 100 ml solution containing 50 mMAmplexRed (***p , 0.001; Table I); it was localized in the cytoplasm and the and 0.1 U/ml HRP in Krebs–Ringer phosphate glucose buffer with 1 mM ionomycin and incubated at 37˚C for the indicated times. The fluores- brush border and apical membrane of malignant cells (Fig. 1Cc); cence of the oxidized 10-acetyl-3,7-dihydroxyphenoxazine was then normal small intestinal mucosa was mostly negative for DUOX measured at excitation and emission wavelengths of 530 and 590 nm, (Fig. 1Cd). High levels of DUOX staining were also observed but respectively, using a SpectraMax Multi-Mode Microplate Reader (Mo- less frequently in prostate adenocarcinomas (**p , 0.01 versus lecular Devices, Sunnyvale, CA), and the amount of extracellular H O 2 2 normal prostate; Table I); staining was principally cytoplasmic was determined using a standard curve from 0 to 2 mMH2O2.Each value in the figures is the mean value for triplicate or quadruplicate and at the luminal surface of malignant cells (Fig. 1Ce), whereas experiments. adjacent nonmalignant tissue showed mild focal DUOX staining 4 IL-4 AND IL-17A ENHANCE DUOX2-RELATED DNA DAMAGE Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. Expression of DUOX2 in human cancers. (A) Boxplot representations of DUOX2 mRNA expression levels in human tumor samples from TCGA datasets sorted by median expression of DUOX2. Open circles represent individual tumor samples; numbers of samples per primary tumor site are shown in parentheses. Data were retrieved from the cBioPortal for Cancer Genomics. ACC, adrenocortical carcinoma; AML, acute myeloid leukemia; ccRCC, clear cell kidney carcinoma; chRCC, chromophobe renal cell carcinoma; DLBC, diffuse large B cell lymphoma; PCPG, pheochromocytoma and paraganglioma; pRCC, papillary kidney carcinoma. (B) The mRNA expression level of 11 oxidases (rows) in 23 colon cancer patients (columns) was expressed relative to their expression level in a pool of five normal colon mucosae from noncancer patients. An unsupervised clustering heat map and dendrograms were generated from these data. Green indicates decreased gene expression in a patient’s colon tumor compared with normal; black indicates that there was no change between tumor and normal; red indicates increased expression in the colon tumor compared with normal colon mucosae. (Ca–h) Representative images of DUOX protein expression examined by IHC in multitumor and normal tissue microarrays. All images were taken at digital magnification 53.(D) Kaplan–Meier overall survival curves of patients with pancreatic adenocarcinoma (n = 177; TCGA) stratified by DUOX2 mRNA levels. High-DUOX2 mRNA expression is associated with decreased overall survival. *p , 0.05.

(Fig. 1Cf). DUOX protein was also signiﬁcantly overexpressed in (Fig. 1Ch). Finally, we found that 65% (n = 32) of cases with some invasive ductal carcinomas of the breast (**p , 0.01; chronic gastritis and 35% (n = 8) of those with gastric carcinomas Fig. 1Cg, Table I), compared with normal ducts and breast tissues demonstrated moderate or intense (2+ and 3+) DUOX epithelial The Journal of Immunology 5

Table I. Expression levels of DUOX protein in human malignancies

High Unstained, Low Expressers Cell Positive (%) Unscored Expressers 2+ and 3+ p Organ Pathologic Diagnosis 0+ (No. [%]) 1+ (No. [%]) (No. [%]) Valuea 0–9 10–24 25–49 $50 Colon Colon adenocarcinoma 7 (5) 84 (56) 59 (39) 0.000 *** 55 585 Colon adenoma 0 (0) 19 (40) 29 (60) 0.000 *** 10 693 Inﬂammatory bowel disease 0 (0) 16 (84) 3 (16) 0.301 NS 0 5 21 74 Colon tissue adjacent to 0 (0) 25 (89) 3 (11) — — 3 18 18 61 cancer Breast Breast invasive ductal 65 (30) 134 (62) 18 (8) 0.003 ** 30 1 5 64 carcinoma Breast invasive lobular 6 (60) 4 (40) 0 (0) 1.000 NS 60 10 0 30 carcinoma Normal breast tissue 1 (25) 3 (75) 0 (0) — — 25 0 0 75 Stomach Gastric carcinoma 8 (35) 7 (30) 8 (35) 0.007 ** 61 26 4 9 Gastritis 6 (12) 11 (22) 32 (65) — — 51 22 8 18 Lung Lung adenocarcinoma 37 (54) 21 (31) 10 (15) 0.181 NS 54 7 6 32 Lung squamous cell 34 (46) 23 (31) 17 (23) 0.569 NS 46 3 4 47 carcinoma

Lung small cell carcinoma 22 (100) 0 (0) 0 (0) 1.000 NS 100 0 0 0 Downloaded from Lung tissue adjacent to 16 (80) 2 (10) 2 (10) — — 80 0 0 20 cancer Prostate Prostate adenocarcinoma 46 (25) 120 (66) 15 (8) 0.001 ** 25 6 9 59 Normal prostateb 0 (0) 7 (88) 1 (13) — — 0 0 0 100 Prostate tissue adjacent to 1 (8) 11 (92) 0 (0) — — 8 0 0 92 cancerb Small intestine Small intestine 12 (14) 56 (64) 19 (22) 0.001 *** 26 1 10 62 adenocarcinoma http://www.jimmunol.org/ Small intestine metastatic 8 (42) 10 (53) 1 (5) 0.701 NS 53 5 11 31 adenocarcinoma to lymph node Small intestine chronic 0 (0) 32 (97) 1 (3) 0.214 NS 27 12 9 52 inﬂammation (not speciﬁed) Small intestine adjacent to 3 (16) 13 (68) 3 (16) 0.018 * 42 32 0 26 cancer Normal small intestine 2 (13) 13 (81) 1 (6) — — 31 12 19 38

Tissues were scored as unstained/unscored (0+), low-DUOX expressers (1+), or high-DUOX expressers (2+ and 3+). Percentage of tissues per score is shown in parentheses. by guest on September 26, 2021 The p values reflect comparisons between the proportion of low and high DUOX expression in malignant tissues and normal tissue samples of the same histology.The dashes (—) shown in the columns for the p values reflect the fact that no comparisons were performed amongst normal tissue samples themselves. aThe x2 statistical test was used to determine significant differences between the proportions of low (1+ staining) and high expressers (2+ and 3+) between malignant tissue and matched or adjacent normal tissue. bMost of the samples from normal prostate and normal prostate adjacent to cancer showed very faint staining for DUOX in the majority of the epithelial cells (.50% of cells). ***p , 0.001, **p , 0.01, *p , 0.05. staining (Table I). Recently, we reported similar results in patients colon and pancreatic cancer cell lines for the RNA expression of with chronic pancreatitis (19). These data suggest that chronic both chains of the type II IL-4R and found measurable RNA ex- inflammation, as well as frank malignancy, may be associated with pression of IL-4Ra and IL-13Ra1 by quantitative RT-PCR in enhanced DUOX2 expression in multiple organs. AsPC-1 and BxPC-3 pancreatic cancer cells and in LS513 and To interrogate possible associations between DUOX2 and T84 colon cancer cell lines (Supplemental Fig. 1). To characterize patient survival, we performed a Kaplan–Meier analysis and Cox whether inflammatory cytokines modulate DUOX2 expression regression of next-generation sequencing data from the TCGA in tumor cells, BxPC-3 pancreatic cancer cells were exposed to pancreatic cancer cohort. The dataset was stratified into two 25 ng/ml IFN-g, IL-4, or IL-13 for 24 h. In BxPC-3 cells, IFN-g groups representing high- and low-DUOX2 expression, with the increased DUOX protein expression and the phosphorylation of DUOX2 expression cutoff optimized by log-rank analysis (29). STAT1 at Tyr701 (p-STAT1Y701), consistent with our previous We discovered a significant association between high-DUOX2 report (25). In contrast, IL-4 and IL-13 increased DUOX protein mRNA expression and worse overall survival (Fig. 1D; hazard in concert with p-STAT6Y641, but not p-STAT1Y701 (Fig. 2A). At ratio = 1.66 and *p , 0.05). These results support the hypothesis the RNA level, the IL-4‒mediated upregulation was specific for that DUOX2 expression could contribute to the development or DUOX2, and to a lesser extent, DUOX1; the expression levels of outcome of gastrointestinal malignancies known to occur in the NOX1/2/4/5 were unchanged by IL-4 exposure for 24 h in BxPC-3 setting of inflammatory stress (1, 33). cells (Supplemental Fig. 2A). IL-4 and IL-13 increase DUOX2 expression in BxPC-3 IL-4 upregulates DUOX2/DUOXA2 expression in a time- and pancreatic cancer cells via the type II IL-4R concentration-dependent manner, promotes the production of IL-4 activates IL-4Ra, which, in turn, heterodimerizes with H2O2, and activates the DNA damage response in BxPC-3 cells IL-13Ra to form the type II IL-4R (34). Several reports indicate A time course experiment with IL-4 (20 ng/ml) in BxPC-3 cells that the type II IL-4R is upregulated and activated in various ep- revealed that after 3 h, IL-4 significantly increased DUOX2 and ithelial tumors, including malignant glioma, ovarian, lung, breast, DUOXA2 RNA expression (**p , 0.01 versus 0 h), with a pro- pancreas, and colon carcinomas (35, 36). We therefore screened gressive increase from 3 to 24 h (Fig. 2B, Supplemental Fig. 3A). 6 IL-4 AND IL-17A ENHANCE DUOX2-RELATED DNA DAMAGE Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. IL-4 and IL-17 induce DUOX2 expression in BxPC-3 pancreatic cancer cells. (A) Western analysis of WCEs from BxPC-3 cells treated with 25 ng/ml IFN-g, IL-4, or IL-13 in serum-free medium for 24 h. b-Actin served as the loading control. (B) BxPC-3 cells grown in serum-free medium were treated with IL-4 (20 ng/ml) for different durations (0, 1, 3, 6, 12, and 24 h) without inhibitor or pretreated with actinomycin D (100 ng/ml; †) or cycloheximide (1 mg/ml; ‡) for 30 min before 12 h incubation with IL-4 (20 ng/ml). Upper panel, relative DUOX2 mRNA expression normalized to b-actin determined by quantitative RT-PCR. Lower panel, Western analysis of 50 mg BxPC-3 WCEs. (C) Concentration response for IL-4–induced DUOX2 expression in BxPC-3 cells treated with IL-4 at different concentrations (0–50 ng/ml) for 24 h. Upper panel, relative DUOX2 mRNA expression normalized to b-actin was evaluated using quantitative RT-PCR. Lower panel, Western analysis of 50 mg BxPC-3 WCEs. (D) Amplex Red assay for extracellular H2O2 production by BxPC-3 cells treated with solvent or IL-4 (50 ng/ml) for 24 h. (E) Western analysis of 50 mg WCEs from BxPC-3 cells treated for 24 h with solvent, IL-4 (25 ng/ml), or IL-17A (25 ng/ml); 6 2 h preincubation with PEG-catalase (2000 U/ml). Data represent mean 6 SD for at least three independent experiments. **p , 0.01, ***p , 0.001 versus nontreated cells.

The effect of 12 h IL-4 treatment was mitigated by 30-min pre- p-STAT1Y701 or p-STAT3Y705)at1h,whichwassustainedupto treatment with either the transcription initiation inhibitor actino- 24 h (Fig. 2B). mycin D (100 ng/ml) or the protein synthesis inhibitor cycloheximide Upregulation of DUOX2/DUOXA2 mRNA and protein levels (1 mg/ml; ***p , 0.001). Western analysis demonstrated similar were associated with enhanced histone H2AX phosphorylation results for DUOX protein induction following IL-4 treatment at serine 139 (gH2AX), a marker of DNA double-strand breaks, (Fig. 2B, lower panel). Analysis of the kinetics of activation of 12–24 h after IL-4 treatment. A concentration-response evaluation the IL-4R–mediated signaling pathway revealed that IL-4 se- of 24 h IL-4 exposure demonstrated that 1 ng/ml IL-4 was suffi- lectively induced maximal phosphorylation of STAT6 (but not cient to significantly increase both DUOX2 and DUOXA2 mRNA The Journal of Immunology 7 and DUOX protein expression in BxPC-3 cells, and to activate STAT6-specific siRNA also inhibited STAT6 protein expression, p-STAT6Y641 (Fig. 2C, Supplemental Fig. 3B); maximal induction but had no effect on the expression of total STAT1 and STAT3 of DUOX protein expression and the p-STAT6Y641 signal occurred (Fig. 3D). Furthermore, silencing STAT6 also inhibited IL-4– with 10 ng/ml IL-4 (Fig. 2C, lower panel). Moreover, BxPC-3 induced DUOX protein expression in BxPC-3 cells. These results cells treated with IL-4 produced significantly more H2O2 over suggest that IL-4–mediated DUOX2/DUOXA2 gene regulation time compared with solvent-treated cells (***p , 0.001 at occurs via STAT6 signaling in BxPC-3 pancreatic cancer cells. 15–180 min) as measured by Amplex Red assay (Fig. 2D). The IL-4–enhanced STAT6 nuclear translocation in BxPC-3 cells addition of membrane permeable PEG-catalase to the reaction system decreased extracellular H2O2 accumulation from both To investigate the nuclear distribution of STAT6 following treat- solvent-treated and IL-4‒exposed BxPC-3 cells below the limit ment with IL-4 (20 ng/ml), nuclear extracts of BxPC-3 cells treated of detection (data not shown). for different durations (0, 1, 24, and 48 h) were prepared for We also assessed the expression level of the receptor for the TH17 Western analysis. Lamin A/C served as a loading control. After 1 h proinflammatory cytokine IL-17A, IL-17RA, and found that it was exposure to IL-4, nuclear translocation of STAT6 was observed expressed in our panel of cell lines (Supplemental Fig. 1, dark (Fig. 3E). After 24 and 48 h, nuclear STAT6 signal persisted, al- gray bars). In BxPC-3 cells, treatment with IL-17A alone (25 ng/ml) beit to a lesser extent compared with 1 h of treatment. Three- increased the expression of DUOX protein as well as gH2AX, kilobase segments of human DUOX2 promoter were scanned to analogous to treatment with IL-4 (Fig. 2E). However, this ef- identify a potential STAT6 binding site matching the canonical fect was independent of p-STAT6Y641 activation. The enhanced STAT6 binding motif 59-TTCN(3–4)GAA-39 (37), in which the gH2AX signal produced by exposure to IL-4 or IL-17A, was underlined letters correspond to conserved palindromes and N Downloaded from attenuated by pretreatment of these cells with 2000 U/ml PEG- denotes any of the 4 nt. We identified such a site in the human catalase (Fig. 2E). The catalase treatment, however, did not pre- DUOX2 promoter region with sequence 59-TTCACTGAA-39, vent cytokine-enhanced DUOX protein expression. localized at around –4594 bp from the DUOX2 transcription start site. Activated STAT6 signaling is essential for IL-4‒induced DUOX2 expression in BxPC-3 cells IL-4 and IL-17A synergize to upregulate DUOX2 expression http://www.jimmunol.org/ To define the role of STAT6 in the regulation of IL-4–mediated and potentiate DNA damage in AsPC-1 pancreatic cancer cells DUOX2 expression further, we performed RNA interference ex- To explore the effects of IL-4 and IL-17A on DUOX2 expression in periments in the BxPC-3 pancreatic cancer cell line. Transfecting a different model of human pancreatic cancer, AsPC-1 cells were BxPC-3 cells with human STAT6-specific small interfering RNA treated for 1 or 24 h with IL-4 (50 ng/ml) or IL-17A (50 ng/ml) (siRNA) resulted in 75% inhibition of STAT6 mRNA expression alone or in combination. The stimulatory effects of IL-4 and compared with endogenous levels (Fig. 3A). STAT6 knockdown IL-17A on DUOX protein expression, p-STAT6Y641, and gH2AX was accompanied by significant attenuation of IL-4‒induced described above for BxPC-3 cells were confirmed in AsPC-1 cells DUOX2 (Fig. 3B; ***p , 0.001 versus control siRNA with IL-4) (Fig. 4). Whereas 50 ng/ml IL-4 alone or with IL-17A (50 ng/ml) and DUOXA2 mRNA expression (Fig. 3C; ***p , 0.001 versus induced a robust p-STAT6Y641 signal as early as 1 h, cytokine- by guest on September 26, 2021 control siRNA with IL-4). Western analysis demonstrated that induced upregulation of DUOX protein and production of gH2AX

FIGURE 3. STAT6 signaling is essential for IL-4R‒ mediated induction of DUOX2 in BxPC-3 cells. (A–C) Control siRNA or STAT6-specific siRNA were transiently transfected into BxPC-3 cells; 24 h following transfection, cells were incubated in serum-free medium with or without IL-4 (20 ng/ml) for another 24 h; RNAthenwasextractedandsubjectedtoquantita- tive RT-PCR. Relative STAT6 (A), DUOX2 (B), and DUOXA2 (C) mRNA levels were normalized to b-actin. Data represent mean 6 SD for at least three independent experiments. ***p , 0.001. (D) WCEs from BxPC-3 cells transiently transfected with control siRNA or STAT6-specific siRNA were analyzed by Western blot using specific Abs. Representative results are shown from three independent experiments. (E) Nuclear extracts from BxPC-3 cells treated with IL-4 (20 ng/ml) for different durations in triplicate were evaluated by Western analysis; nuclear envelope marker lamin A/C served as the loading control. 8 IL-4 AND IL-17A ENHANCE DUOX2-RELATED DNA DAMAGE occurred later and could be detected at 24 h (Fig. 4A). As dem- support a role for STAT6 signaling in the cytokine-mediated onstrated for BxPC-3 cells, RNA interference experiments con- overexpression of DUOX2 in pancreatic cancer cells. firmed that the STAT6/siRNA not only significantly decreased To corroborate the association of DUOX2-derived H2O2 with STAT6 mRNA (***p , 0.001; Fig. 4B, left) and protein (Fig. 4C) DNA damage in this model, AsPC-1 cells were preincubated with expression by more than 50%, but also diminished IL-4 plus the flavin dehydrogenase (and NOX) enzyme inhibitor diphenylene IL-17A‒mediated upregulation of DUOX2 mRNA (***p , 0.001; iodonium (DPI) or the reduced thiol N-acetyl-L-cysteine (NAC). Fig. 4B, right) and DUOX protein (Fig. 4C). These results Both DPI and NAC decreased the IL-4 plus IL-17A–mediated Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. IL-4 and IL-17A synergize to upregulate DUOX2 and potentiate DNA damage in AsPC-1 human pancreatic cancer cells. (A) Western analysis of 50 mg WCEs from AsPC-1 treated with IL-4 (50 ng/ml) and/or IL-17A (50 ng/ml) for 1 or 24 h using specific Abs, as indicated. (B) Control siRNA or STAT6-specific siRNA were transiently transfected into AsPC-1 cells. Twenty-four hours following transfection, cells were incubated in serum- free medium with or without IL-4 (50 ng/ml) plus IL-17A (50 ng/ml) for another 24 h; RNA was then extracted and subjected to quantitative RT-PCR for quantitation of STAT6 (left) and DUOX2 (right) RNA expression levels normalized to b-actin. ***p , 0.001. (C) Transient transfection with specific STAT6/siRNA decreased STAT6 and IL-4 plus IL-17A–induced DUOX protein expression in these cells, as demonstrated by Western analysis. (D) Pretreatment with flavin dehydrogenase and NOX inhibitor DPI (1 mM) and reduced thiol NAC (10 mM) for 30-min-attenuated, cytokine-mediated DNA damage at 24 h in AsPC-1 cells. (E) Transient transfection with specific DUOX2/siRNA attenuated IL-4/IL-17A–mediated DUOX protein expression and DNA damage response (i.e., gH2AX signal) as measured by Western analysis in AsPC-1 cells. Data shown for (A) through (E) are representative of at least three independent replicates. (F) Amplex Red assay for extracellular H2O2 levels in AsPC-1 cells treated with solvent or IL-4 (50 ng/ml) plus IL-17A (50 ng/ml) for 24 h. H2O2 concentrations were calculated by interpolation from a standard curve, using 0–2 mMH2O2. Data represent mean 6 SD for three independent experiments. ***p , 0.001 between the two conditions compared. IO, ionomycin. (G) Representative immunofluorescence images (left) and quantitation (right) showing induction of the oxidative DNA base marker 8-oxo-dG in green and DAPI nuclear counterstaining in blue. AsPC-1 cells were treated for 48 h with solvent (top panels) or with IL-4 (50 ng/ml) plus IL-17A (50 ng/ml; lower panels). Data represent the mean 6 SD from five randomly selected fields for each condition. The confocal images with a pixel size of 0.130 mm were collected with a 633 objective using a 23 optical zoom. The Journal of Immunology 9

activation of gH2AX but had no effect on DUOX or p-STAT6Y641 Western analysis using whole-cell lysates from CFPAC-1 cells protein levels (Fig. 4D). The role of DUOX2 in the induction of treated with LPS or IL-17A for different durations revealed that DNA damage was also evaluated by addition of a DUOX2-specific although neither cytokine affected the expression of the p65 subunit siRNA to AsPC-1 cells; in these experiments, DUOX2-specific of NF-kB, both induced robust DUOX protein expression after 24 h siRNA abrogated DUOX protein expression and activation of exposure (Fig. 5C, lane 4 for LPS; lane 7 for IL-17A). We then gH2AX following IL-4 plus IL-17A exposure (Fig. 4E, lane 4 compared NF-kB signal transduction in CFPAC-1 cells exposed to compared with lane 2). However, the DUOX2 siRNA had no in- LPS or IL-17A. Nuclear extracts prepared from CFPAC-1 cells hibitory effect on total STAT6 protein expression or Y641 phos- treated with LPS or IL-17A for 1 or 24 h were analyzed (Fig. 5D); phorylation (Fig. 4E). To evaluate the involvement of DUOX2 in 1 h exposure to either LPS (1 mg/ml; lane 2) or IL-17A (50 ng/ml; the activation of DNA damage repair, we examined whether IL-4 lane 3) resulted in an increase in the nuclear translocation of and IL-17A enhanced the enzymatic activity of DUOX2 in AsPC- p65 compared with solvent-treated cells (lane 1). At 24 h, the en- 1 cells. In this cell line, the combination of IL-4 with IL-17A and hanced nuclear localization of p65 was maintained in LPS-treated the calcium ionophore ionomycin significantly enhanced the cells, but not in IL-17A‒treated cells, revealing ligand-dependent production of H2O2 compared with cells that had not been ex- differences in the kinetics of NF-kB signaling activation. posed to the cytokine combination (***p , 0.001; Fig. 4F). IL-4 alone and combined with IL-17A upregulate DUOX2 in Finally, to evaluate the potential mutagenic effect of cytokine- human colon cancer cells enhanced DUOX2 expression (38, 39), we measured the production of the promutagenic oxidized DNA base 8-oxo-dG in AsPC-1 Because DUOX2 is overexpressed in surgically resected tumors cells following a 48-h exposure to the combination of IL-4 and from a substantial fraction of colorectal cancer patients (Fig. 1A, Downloaded from IL-17A (Fig. 4G). Treatment with the cytokine combination sub- 1B, Table I), we evaluated the effects of IL-4 and IL-17A on stantially increased 8-oxo-dG levels in AsPC-1 cells (p=0.053). DUOX2 mRNA and protein expression in colon cancer lines. For T84 colon cancer cells (Fig. 6A), 24 h exposure to IL-4 (50 ng/ml) k ‒ Role of NF- B in IL-17A mediated upregulation of DUOX2 significantly upregulated both DUOX2 and DUOXA2 mRNA expression in pancreatic cancer cells expression (***p , 0.001 versus control). Western analysis con-

IL-17A regulates immune function through its cognate receptor firmed the increase in DUOX protein levels and the activation of http://www.jimmunol.org/ IL-17RA and downstream activation of MAPK and NF-kB sig- p-STAT6Y641 following IL-4 exposure (Fig. 6B). Furthermore, the naling (40, 41). To understand the molecular mechanism under- combination of IL-4 and IL-17A significantly enhanced the in- lying IL-17A–mediated DUOX2 regulation in human pancreatic duction of DUOX2 and DUOXA2 mRNA (***p , 0.001 versus cancer cells, the potential of IL-17A alone or in combination with control and IL-4 alone) and increased DUOX protein expression in IFN-g/IL-4 to enhance DUOX2 mRNA expression was compared this colon cancer model (Fig. 6A, 6B). The effect of the IL-4 and with that of LPS (Fig. 5). The latter has been previously shown to IL-17A combination on NOX expression in T84 cells was ob- activate NF-kB signaling and induce DUOX2 expression either served for DUOX2, and to a much lesser extent DUOX1 and alone or in combination with IFN-g in CFPAC-1 pancreatic cancer NOX1; these cytokines did not affect the mRNA expression of cells (19). As demonstrated in Fig. 5A, LPS, IFN-g, and IL-17A other NOX family enzymes (Supplemental Fig. 2B). Likewise, in by guest on September 26, 2021 independently induce DUOX2 mRNA expression (***p , 0.001 another colon cancer cell line that expresses NOX1 (Supplemental versus solvent treated). The combination of either LPS or IL-17A Fig. 4A), LS513, IL-4 alone (24 h, 25 ng/ml) significantly upreg- with IFN-g increased DUOX2 mRNA expression .8-fold com- ulated DUOX2 mRNA (***p , 0.001; Supplemental Fig. 4B), as pared with LPS or IL-17A alone and .20-fold compared with IFN- well as DUOX protein and p-STAT6Y641 (Supplemental Fig. 4C). g alone in CFPAC-1 cells (Fig. 5A; ***p , 0.001 versus either The addition of IL-17A to IL-4 further enhanced the DUOX2 RNA cytokine alone). However, the cotreatment of LPS or IL-17A with and protein expression over IL-4 treatment alone (Supplemental IL-4 did not result in a further increase in DUOX2 mRNA ex- Fig. 4B, 4C). In T84 cells, the upregulation of DUOX protein pression compared with LPS/IL-17A exposure alone (Fig. 5B). by IL-4 and IL-17A treatment was accompanied by a DNA

FIGURE 5. IL-17A activates NF-kB and induces DUOX2 expression in CFPAC-1 human pancreatic cancer cells. (A and B) CFPAC-1 cells were grown in serum-free medium and treated with LPS (1 mg/ml) or IL-17A (25 ng/ml) alone or in combination with IFN-g (25 ng/ml) (A) or IL-4 (25 ng/ml) (B) for 24 h, as indicated on the x-axis. DUOX2 RNA expression normalized to b-actin was analyzed by quantitative RT-PCR. Data represent the mean of three independent experiments. ***p , 0.001 for single cytokine or combinations versus solvent-treated cells, unless spec- iﬁed otherwise. (C) Western analysis of 50 mg cell lysates from CFPAC-1 cells treated with LPS (1 mg/ml) or IL-17A (25 ng/ml) for 0.25, 1, or 24 h. (D) Western analysis of 20 mg of nuclear extract from CFPAC-1 cells treated with LPS (1 mg/ml) or IL-17A (25 ng/ml) for 1 or 24 h. NF-kB subunit p65 nuclear translocation was compared between LPS- and IL-17A–treated cells. Lamin A/C served as the loading control. Experi- ments shown in (B)and(C) are representative of three independent studies. 10 IL-4 AND IL-17A ENHANCE DUOX2-RELATED DNA DAMAGE

addition to IL-4 plus IL-17A‒induced DUOX2 upregulation (Fig. 6C [right], Fig. 6D). Relationship between the expression of DUOX2 and mediators of proinﬂammatory cytokine signaling in human pancreatic and colon cancers To assess the clinical relevance of IL-4‒mediated upregulation of DUOX2 in pancreatic and colon cancer cells, we measured the expression of DUOX2 and IL-4R in 13 surgically resected PDACs and their adjacent normal pancreatic tissues, and in 19 surgically resected colon adenocarcinomas and associated normal colon (42) (Figs. 7, 8). In the pancreatic cancer cohort, the expression of DUOX2, IL-4R, and IL-17RA was signiﬁcantly higher in tumors compared with adjacent normal tissues (*p , 0.05, **p , 0.01; Fig. 7A–C). In the colon cancer resection specimens, DUOX2 (but not IL-4R) was strongly overexpressed in tumor samples compared with normal tissue (***p , 0.001; Fig. 8A, 8B). DUOXA2 expression levels in these samples followed the same pattern as

DUOX2 (data not shown). Downloaded from In an evaluation of tumor expression proﬁles from the TCGA, we found signiﬁcant positive correlations between the expression of DUOX2 and the expression of IL-4R in large pancreatic (n = 178; Pearson r = 0.37; ***p , 0.001) and colon (n = 380; Pearson r = 0.20; ***p , 0.001) cancer cohorts (Figs. 7D, 8C, respec-

tively). Additionally, in the TCGA pancreatic cancer dataset, http://www.jimmunol.org/ DUOX2 and IL-17RA expression were also significantly corre- lated (Pearson r = 0.29; ***p , 0.001; Fig. 7E); the expression of the IL-17RA ligand IL-17A was found to be significantly corre- lated with the expression of DUOX2 in TCGA colorectal cancer cohort (Pearson r = 0.41; ***p , 0.001; Fig. 8D). These results support a relationship between proinflammatory signaling via IL-4 and IL-17A, and DUOX2 in human pancreatic and colon cancers.

Discussion by guest on September 26, 2021 DUOX2, which plays an important role in gastrointestinal and respiratory host defense (10, 13–15), is upregulated during chronic inflammation in humans and in multiple cancers (Fig. 1A) (18, 25, 26, 43, 44). Using our human DUOX mAb (26), we recently demonstrated that DUOX is highly expressed in most early stage PDACs as well as pancreatic intraepithelial neoplasms (20). We report in this study that evaluation of over 1200 tumors and normal FIGURE 6. IL-4 alone and combined with IL-17A upregulate DUOX2 through STAT6 signaling in T84 human colon cancer cells. (A) T84 cells samples by IHC revealed that, in addition to PDAC, DUOX is most grown in serum-free medium were treated with solvent IL-4 (50 ng/ml), frequently overexpressed in adenocarcinomas of the large and small IL-17A (50 ng/ml), or both in combination for 24 h. RNA expression levels bowel (Fig. 1C, Table I). These results are supported by a bio- of DUOX2 (dark gray bars) and DUOXA2 (light gray bars) normalized to informatic examination of expression levels of DUOX2 mRNA b-actin were analyzed by quantitative RT-PCR. (B) Western analysis of across the TCGA (Fig. 1A), as well as our two newly reported 50 mg WCEs from T84 cells grown in serum-free medium, treated for 24 h datasets from surgical samples comparing tumor to adjacent normal with solvent IL-4 (50 ng/ml) and/or IL-17A (50 ng/ml) using specific Abs tissue levels of DUOX2 expression by expression array profiling C for DUOX, p-STAT6Y641, STAT6, and b-actin. ( ) Control siRNA or (Fig. 1B) or by quantitative RT-PCR (Figs. 7A–C, 8A, 8B). STAT6-specific siRNA were transiently transfected into T84 cells; 24 h Because we found a significant association between high-DUOX2 following transfection, cells were incubated in serum-free medium with or mRNA expression and poor prognosis for patients with PDAC without IL-4 (50 ng/ml) alone or IL-4 (50 ng/ml) plus IL-17A (50 ng/ml) for another 24 h; RNA then was extracted and subjected to quantitative (Fig. 1D), and because genetically engineered mouse models have RT-PCR for quantitation of STAT6 (left) and DUOX2 (right) mRNA ex- established an intimate link between chronic inflammation be- pression levels normalized to b-actin. (D) Transfection with specific cause of IL-4 and IL-17A and pancreatic and bowel carcinogen- STAT6-siRNA decreased STAT6 and IL-4 plus IL-17A-induced DUOX esis (45–49) as well as a role for DUOX2 in the pathogenesis of protein expression in T84 cells, as demonstrated by Western analysis. IBD (50), we initiated the current studies to examine potential Data represent mean 6 SD for at least three independent experiments. mechanistic interactions between these proinflammatory cyto- ***p , 0.001 between the two conditions compared. kines, NOX isoforms, and reactive oxygen production. In a recent study, we reported that IL-4 exposure induces NOX1 expression in damage response in the form of an increase in gH2AX ex- HT-29 and WiDr colon cancer cells (42). Our demonstration in the pression (Fig. 6B), as described above in pancreatic cancer cell present work that IL-4 can also increase DUOX2 expression in lines BxPC-3 and AsPC-1. Moreover, in this model of colon colorectal cancer lines previously known to express only NOX1, cancer as well, STAT6-specific siRNA significantly inhibited including T84 (Fig. 6A) and LS513 cells (Supplemental Fig. 4), both STAT6 RNA (Fig. 6C, left) and protein (Fig. 6D), in is consistent with the data in Fig. 1B demonstrating that surgically The Journal of Immunology 11

FIGURE 7. Relationships between expression of DUOX2andcomponentsoftheIL-4andIL-17sig- naling pathways in human pancreatic cancer. (A–C) Thirteen pairs of human pancreatic cancer surgical specimens and adjacent normal tissues were analyzed by quantitative RT-PCR for the expression of DUOX2 (A), IL-4R (B), and IL-17RA (C), with b-actin as control. The Wilcoxon signed-rank test was used to evaluate the difference in expression between normal and tumor samples. (D and E) DUOX2, IL-4R, and IL-17RA log2 mRNA levels were retrieved from the TCGA for 178 primary pancreatic cancer specimens. Pearson and Spearman correlation coefﬁcients (r)and level of signiﬁcance p using a two-tailed t test for DUOX2 versus IL-4R (D) and DUOX2 versus IL-17RA (E)areshown.*p , 0.05, **p , 0.01, ***p , 0.001 between the two conditions compared. Downloaded from

resected colorectal tumors can express either NOX1 or DUOX2 or (Fig. 5). Interactions and cross-talk between STAT6 and NF-kB both at high levels compared with normal colonic mucosa. have been studied by other groups. For instance, Shen et al. (53) http://www.jimmunol.org/ In PDAC and colon cancer cells, IL-4 significantly increased provided the proof of concept for the functional interaction of DUOX2 mRNA and DUOX protein expression through a STAT6- STAT6 and NF-kB using HEK293 and I.29m B lymphoma cells dependent mechanism (Figs. 2–4, 6, Supplemental Fig. 4). Kinetic and an IL-4–inducible reporter gene containing cognate binding analysis of IL-4–mediated signaling revealed that STAT6 activa- sites for both STAT6 and NF-kB. Upon stimulation with IL-4, the tion occurred first (after 1 h in BxPC-3 cells), suggesting that transcription factors cooperatively activated the transcription of this step is upstream of the transcriptional regulation of DUOX2 the IL-4 response gene (53). Correspondingly, the DUOX2 pro- (Fig. 2B). IL-4 promotes the nuclear translocation of STAT6 moter contains canonical binding motifs for both STAT6 and (Fig. 3E); the IL-4–mediated DUOX2 response can be silenced NF-kB. We have previously demonstrated that the p65 component by STAT6 siRNA (Fig. 3A–C) supporting this thesis. of the NF-kB complex binds the DUOX2 promoter about 22700 bp by guest on September 26, 2021 IL-17 is often described as an potentiator of inflammatory re- from the transcription start site (19). sponse because it promotes the production of cytokines such as Cytokine-enhanced ROS formation and resulting DNA dam- IL-6, chemokines such as CCL5, and other inflammatory mediators age play an important role in chronic inflammation-associated and, as such, has been shown to synergize with other cytokines to gastrointestinal malignant transformation (19, 54–56). Produc- increase inflammation (51, 52). Addition of IL-17A to IL-4 further tion of H2O2 by IL-4/IL-17A-enhanced DUOX2 expression led increased DUOX2 overexpression in pancreatic (Fig. 4A, 4B) and to DNA double-strand breaks in BxPC-3, AsPC-1, and T84 cells colon cancer (Fig. 6A, 6B) cells, which may be NF-kB related (Figs. 2, 4, 6), as well as the production of the oxidized DNA

FIGURE 8. Relationships between expression of DUOX2 and components of the IL-4 and IL-17 signaling pathways in human colon cancer. (A and B) Nineteen pairs of human colon cancer surgical specimens and adjacent normal tissues were analyzed by quantitative RT-PCR for the expression of DUOX2 (A) and IL-4R (B), with b-actin as control. The Wilcoxon signed-rank test was used to evaluate the difference in expression between normal and tumor samples. (C and D) DUOX2, IL-4R, and IL-17A log2 mRNA levels were retrieved from the TCGA for 380 primary colorectal cancers. Pearson and Spearman correlation coefficients (r) and level of significance p using a two- tailed t test for DUOX2 versus IL-4R (C), and DUOX2 versus IL-17A (D) are shown. *p , 0.05, **p , 0.01, ***p , 0.001 between the two conditions compared. 12 IL-4 AND IL-17A ENHANCE DUOX2-RELATED DNA DAMAGE base 8-oxo-dG in the AsPC-1 line. Oxidant DNA damage could 7. Bedard, K., and K. H. Krause. 2007. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87: 245–313. help to explain at least part of the genetic instability that ac- 8. Brandes, R. P., N. Weissmann, and K. Schro¨der. 2014. Nox family NADPH companies chronic pancreatic and bowel inflammation. In all oxidases: molecular mechanisms of activation. Free Radic. Biol. Med. 76: 208– three cell lines, IL-4 and IL-17A increased gH2AX in concert 226. 9. Nauseef, W. M. 2008. Biological roles for the NOX family NADPH oxidases. with DUOX protein; this effect was mitigated by PEG-catalase J. Biol. Chem. 283: 16961–16965. in BxPC-3 cells (Fig. 2E) and by the flavoprotein and NOX 10. De Deken, X., B. Corvilain, J. E. Dumont, and F. Miot. 2014. Roles of DUOX- inhibitor DPI and the reduced thiol NAC in AsPC-1 cells mediated hydrogen peroxide in metabolism, host defense, and signaling. Anti- oxid. Redox Signal. 20: 2776–2793. (Fig. 4D). These results suggest that DUOX2-associated ROS 11. Morand, S., T. Ueyama, S. Tsujibe, N. Saito, A. Korzeniowska, and T. L. Leto. play a role in the induction of DNA damage following cytokine 2009. Duox maturation factors form cell surface complexes with Duox affecting the specificity of reactive oxygen species generation. FASEB J. 23: 1205–1218. treatment and that an increase in DUOX2 activity could pro- 12. Grasberger, H., and S. Refetoff. 2006. Identification of the maturation factor for mote malignant transformation in response to inflammatory dual oxidase. Evolution of an eukaryotic operon equivalent. J. Biol. Chem. 281: stimuli. 18269–18272. 13. Caillou, B., C. Dupuy, L. Lacroix, M. Nocera, M. Talbot, R. Ohayon, D. De`me, Binding of IL-4 to the IL-4R initiates a signaling cascade that J. M. Bidart, M. Schlumberger, and A. Virion. 2001. Expression of reduced activates STAT6 (57, 58). Engagement of IL-17 with IL-17RA nicotinamide adenine dinucleotide phosphate oxidase (ThoX, LNOX, Duox) mediates NF-kB and MAPK activation, leading to production of genes and proteins in human thyroid tissues. J. Clin. Endocrinol. Metab. 86: 3351–3358. proinflammatory cytokines and chemokines, and subsequent re- 14. Geiszt, M., J. Witta, J. Baffi, K. Lekstrom, and T. L. Leto. 2003. Dual oxidases cruitment of myeloid cells to the inflamed tissues (21, 41). There- represent novel hydrogen peroxide sources supporting mucosal surface host defense. FASEB J. 17: 1502–1504. fore, we posit that the combination of TH2andTH17 cytokines 15. Leto, T. L., and M. Geiszt. 2006. Role of Nox family NADPH oxidases in host synergistically enhance oxidative stress by activating distinct reg- defense. Antioxid. Redox Signal. 8: 1549–1561. Downloaded from ulatory pathways to increase DUOX2 expression in pancreatic and 16. Sommer, F., and F. Ba¨ckhed. 2015. The gut microbiota engages different signaling pathways to induce Duox2 expression in the ileum and colon epithelium. colon cancer. Our observations of the significant correlation be- Mucosal Immunol. 8: 372–379. tween the mRNA expression of DUOX2 and IL-4R in TCGA 17. MacFie, T. S., R. Poulsom, A. Parker, G. Warnes, T. Boitsova, A. Nijhuis, pancreatic and colorectal cancer cohorts, as well as the correlation N. Suraweera, A. Poehlmann, J. Szary, R. Feakins, et al. 2014. DUOX2 and DUOXA2 form the predominant enzyme system capable of producing the re- of DUOX2 expression with IL-17RA in pancreatic tumors and active oxygen species H2O2 in active ulcerative colitis and are modulated by

IL-17A in colorectal tumors, provide support for a pathogenetic 5-aminosalicylic acid. Inﬂamm. Bowel Dis. 20: 514–524. http://www.jimmunol.org/ ‒ 18. Kita, H., Y. Hikichi, K. Hikami, K. Tsuneyama, Z. G. Cui, H. Osawa, role of IL-4 and/or IL-17 mediated upregulation of DUOX2 in H. Ohnishi, H. Mutoh, H. Hoshino, C. L. Bowlus, et al. 2006. Differential gene those cancers (Figs. 7, 8). expression between ﬂat adenoma and normal mucosa in the colon in a micro- In summary, we have shown that IL-4 alone, or combined with array analysis. J. Gastroenterol. 41: 1053–1063. 19. Wu, Y., J. Lu, S. Antony, A. Juhasz, H. Liu, G. Jiang, J. L. Meitzler, IL-17A, strongly induces DUOX2 and its cognate maturation factor M. Hollingshead, D. C. Haines, D. Butcher, et al. 2013. Activation of TLR4 is DUOXA2, leading to long-lasting H2O2 production and DNA required for the synergistic induction of dual oxidase 2 and dual oxidase A2 by damage in human pancreatic and colon tumor cells. These studies, IFN-g and lipopolysaccharide in human pancreatic cancer cell lines. J. Immunol. 190: 1859–1872. in concert with mRNA and IHC data demonstrating high-level 20. Wu, Y., J. L. Meitzler, S. Antony, A. Juhasz, J. Lu, G. Jiang, H. Liu, DUOX protein expression in colon and pancreatic cancer and M. Hollingshead, D. C. Haines, D. Butcher, et al. 2016. Dual oxidase 2 and

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SUPPLEMENTAL FIGURE S1. Endogenous expression of cytokine receptors in colon and

pancreatic cancer cell lines. Quantitative RT-PCR analysis of relative IL4Rα (black bars), IL13Rα

(light grey bars), and IL17RA (dark grey bars) expression normalized to β-actin in human pancreatic cancer cell lines AsPC-1 and BxPC-3, and in colon cancer cell lines LS513 and T84, respectively. Data represent mean ± SD for at least 3 independent experiments.

SUPPLEMENTAL FIGURE S2. RNA expression of NOX family enzymes in human cancer

cell lines. (A-B) Expression of 6 NADPH oxidase isoforms relative to β-actin. Cell were grown

for 24 h in serum-free conditions with solvent or IL-4 (25 ng/ml) for BxPC-3 (A), and with solvent or 50 ng/ml IL-4 and IL-17A for T84 cells (B). Data represent mean ± SD for at least 3 independent

experiments. *p<0.05, **p<0.01, ***p<0.001 between the 2 conditions compared.

SUPPLEMENTAL FIGURE S3. Time- and concentration-dependent overexpression of

DUOXA2 mRNA by IL-4 in BxPC-3 cells. (A) BxPC-3 cells grown in serum free medium were treated with IL-4 (20 ng/ml) for different durations (0, 1, 3, 6, 12, 24 h) without inhibitor, or pretreated with actinomycin D (100 ng/ml, †) or cychloheximide (1 µg/ml, ‡) for 30 minutes before

12 h incubation with IL-4 (20 ng/ml). Relative DUOXA2 mRNA expression normalized to β- actin was determined by quantitative RT-PCR. (B) Concentration response of IL-4-induced

DUOXA2 expression in BxPC-3 cells treated with IL-4 at different concentrations (0-50 ng/ml) for 24 h. Relative DUOXA2 mRNA expression normalized to β-actin was evaluated using quantitative RT-PCR. **p<0.01, ***p<0.001 between the 2 conditions compared for three independent experiments. Data represent mean ± SD for 3 independent experiments.

SUPPLEMENTAL FIGURE S4. IL-4 with or without IL-17A upregulates DUOX2 mRNA and

DUOX protein in LS513 cells. (A-C) LS513 cells grown in serum-free medium were treated with

IL-4 (25 ng/ml) and IL-17A (25 ng/ml) alone or in combination for 24 h. NOX1 (A) and DUOX2

(B) mRNA, and NOX1 and DUOX protein expression (C) were determined using quantitative RT-

PCR and Western analysis, respectively. *p<0.05, **p<0.01, and ***p<0.001 between the 2

conditions; data represent mean ± SD for 3 independent experiments.