Interleukin-32 Increases Human Gastric Cancer Cell Invasion Associated with Tumor Progression and Metastasis

Published OnlineFirst March 6, 2014; DOI: 10.1158/1078-0432.CCR-13-1221

Clinical Cancer Human Cancer Biology Research

Chung-Ying Tsai1, Chia-Siu Wang4, Ming-Ming Tsai2, Hsiang-Cheng Chi1, Wan-Li Cheng3, Yi-Hsin Tseng1, Cheng-Yi Chen1, Crystal D. Lin6, Jun-I. Wu5, Lu-Hai Wang5, and Kwang-Huei Lin1

Abstract Purpose: The proinflammatory cytokine interleukin-32 (IL-32) is a novel tumor marker highly expressed in various human carcinomas, including gastric cancer. However, its effects on prognosis of patients with gastric cancer and cancer metastasis are virtually unknown at present. The main aim of this study was to explore the clinical significance of IL-32 in gastric cancer and further elucidate the molecular mechanisms underlying IL-32–mediated migration and invasion. Experimental Design: Gastric cancer cells with ectopic expression or silencing of IL-32 were examined to identify downstream molecules and establish their effects on cell motility, invasion, and lung metastasis in vivo. Results: IL-32 was significantly upregulated in gastric cancer and positively correlated with aggressiveness of cancer and poor prognosis. Ectopic expression of IL-32 induced elongated morphology and increased cell migration and invasion via induction of IL-8, VEGF, matrix metalloproteinase 2 (MMP2), and MMP9 expression via phosphor-AKT/phospho-glycogen synthase kinase 3b/active b-catenin as well as hypoxia- inducible factor 1a (HIF-1a) signaling pathways. Conversely, depletion of IL-32 in gastric cancer cells reversed these effects and decreased lung colonization in vivo. Examination of gene expression datasets in oncomine and staining of gastric cancer specimens demonstrated the clinical significance of IL-32 and its downstream molecules by providing information on their coexpression patterns. Conclusions: IL-32 contributes to gastric cancer progression by increasing the metastatic potential resulting from AKT, b-catenin, and HIF-1a activation. Our results clearly suggest that IL-32 is an important mediator for gastric cancer metastasis and independent prognostic predictor of gastric cancer. Clin Cancer Res; 20(9); 2276–88. 2014 AACR.

Introduction nantly unknown at present. The tumor–node–metastasis Gastric cancer is the fourth most common cancer type (TNM) staging system is the only tool routinely used for worldwide and ranks second in terms of global cancer- predicting prognosis in the clinical setting. Currently, related mortality (1). In Taiwan, gastric cancer was surgery is the major known cure for the disease. Therefore, recorded as the sixth leading cause of cancer-related deaths elucidation of the molecular mechanisms involved in in 2010 (2). The prognosis of gastric cancer is poor, and gastric cancer and identification of valuable prognostic the key players in molecular pathogenesis are predomi- markers as well as novel therapeutic strategies is of essential clinical value (3). Increasing evidence has confirmed the hypothesis that Authors' Affiliations: 1Department of Biochemistry, College of Medicine; several pro- and anti-inflammatory cytokines promote 2Department of Nursing, Chang-Gung University of Science and Technol- tumor progression and affect the host antitumor response 3 ogy; Department of Gastroenterology and Hepatology, Chang Gung (4). Inflammatory tumor microenvironments play a pivotal Memorial Hospital, Taoyuan; 4Department of General Surgery, Chang Gung Memorial Hospital at Chiayi; 5National Health Research Institute, role, not only in tumor development but also metastasis. Zhunan, Miaoli, Taiwan, Republic of China; and 6Pre-med Program, Pacific Cytokines released in response to infection affect tumor Union College, Angwin, California development in different ways. IL-8 produced by gastric Note: Supplementary data for this article are available at Clinical Cancer cancer cells contributes to sustained angiogenesis and Research Online (http://clincancerres.aacrjournals.org/). tumor invasion and metastasis via either autocrine or para- C.-Y. Tsai and C.-S. Wang contributed equally to this article. crine mechanisms (5). VEGF promotes breast cancer cell Corresponding Author: Kwang-Huei Lin, Department of Biochemistry, invasion through an autocrine pathway (6). Recent studies Chang-Gung University, 259 Wen-hwa 1 Road, Taoyuan, Taiwan. Phone: have revealed higher expression of IL-32, a novel pro- 886-3-211-8263; Fax: 886-3-211-8263; E-mail: [email protected] inflammatory cytokine, in human stomach (7, 8), lung doi: 10.1158/1078-0432.CCR-13-1221 (9), pancreas (10), liver (11), and esophagus (12) cancer 2014 American Association for Cancer Research. tissues, compared with normal tissue or serum.

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them were subjected to gastric resection that included total Translational Relevance gastrectomy in 35 patients and subtotal gastrectomy in 85. Metastasis is the major cause of death in patients with The pathologic examination and further biological studies gastric cancer and involves a multistep process, includ- were carried out under informed consents of patients. This ing invasion of surrounding tissue. Although interleu- study protocol was approved by the Medical Ethics and kin-32 (IL-32) has been reported as a gastric cancer Human Clinical Trial Committee of the CGMH (IRB NO. tumor marker, the role of IL-32 in gastric cancer pro- 95-0472B). gression and metastasis is unknown. This study shows Postoperatively, the patients with stage 2 to 3 diseases that IL-32 contributes to gastric cancer progression by received adjuvant chemotherapy with 5-fluorouracil–based increasing the metastatic potential, resulting in worse regimens, and those with stage 4 diseases were treated with patient survival. Silencing of IL-32 revealed decreased palliative chemotherapy of various regimens as indicated. cell invasion ability and lung colonization in vivo by All patients were followed up regularly in the outpatient downregulation of IL-8 and VEGF expression via phos- service of our hospital, of every 3 months in the first 2 years, pho-AKT/active b-catenin signaling pathways. These every 6 months between the 3rd and 5th years and then once results demonstrate a novel function and regulation of a year thereafter. IL-32 in gastric cancer and imply that it might be advan- tageous to target IL-32 for cancer therapy. New thera- Clinicopathologic studies peutic strategies directed at blocking IL-32 signaling or Resected specimens were examined pathologically using function may effectively be applied to prevention of the criteria of the 6th edition of the American Joint Com- gastric cancer metastasis. mittee on Cancer (pTNM) classification system and the Japanese General Rules for gastric cancer Study (18). Clin- icopathologic parameters included patient age and gender, tumor location, and size, gross (Borrmann) tumor type, IL-32, originally designated natural killer cell transcript 4 wall invasion, resection margin, histologic type, lymph- (NK4) in humans, was found to be absent in rodents. The node metastasis, vascular invasion, lymphatic invasion, and recently described cytokine is mainly produced by T cells, perineural invasion. After discharge, all patients had peri- NK cells, epithelial cells, and monocytes after stimulation odic follow-up visits at the outpatient department of CGMH with IL-2, IL-18, or IFN-g (13). The IL-32 gene is located on until death or the beginning of preparation of this article. human chromosome 16p13.3, and has a full length of 705 bp. Moreover, the gene is organized into 8 exons and has 6 Tumor samples splice variants, IL-32a,IL-32b,IL-32g,IL-32d, IL-32e, and IL- Fresh samples of tumor tissue and adjacent noncancerous 32z, among which the g isoform exhibits the highest cytokine mucosa were harvested immediately following gastric resec- producing activity (14). IL-32b is the major secreted isoform, tion. Samples were dissected from resected specimens by a whereas the a isoform is located intracellularly (15). How- pathologist at CGMH, and immediately snap-frozen in ever, the functionaldifferences between these isoforms are yet individual vials using liquid nitrogen. Frozen specimens to be established. The cytokine induction activity of IL-32 is were stored at 70 C in a tumor bank until use. activated via partial cleavage by proteinase-3 (16). Although a receptor for IL-32 has not been described, it is defined as Real-time quantitative reverse transcription PCR a proinflammatory cytokine, in view of its stimulation of Quantitative reverse transcription PCR (qRT-PCR) was TNF-a,IL-1b, IL-6, and IL-8 production, and activation of the performed using SYBR green as described previously (19). NF-kB and the p38 mitogen-activated protein kinase path- Fluorescence emitted by SYBR green was detected using the ways (17). Overexpression of IL-32 in stomach cancer tissue ABI PRISM 7500 sequence detection system (Applied Bio- has been reported (7). However, the specific role of this systems). The following primers were used: human IL-32 0 cytokine in gastric cancer has received little attention, and qPCR (forward primer, 5 -TGAGGATAAAAATG ACATCCC- 0 0 its clinicopathologic significance and underlying molecular TAAAGA-3 , and reverse primer, 5 -TGTGTCAGCTCAAG- 0 mechanisms have rarely been studied to date. Therefore, we TGT AGCTTTC-3 ); human IL-8 qPCR (forward primer, 0 0 0 focused on the clinical and functional significance, as well as 5 -CTGGCCGTGGCTCTC TTG-3 , and reverse primer, 5 - 0 the signaling pathway of IL-32 in gastric cancer. CCTTGGCAAAACTGCACCTT-3 ); human 18s rRNA qPCR (forward primer, 50-CGAGCCGCCTGGATACC-30,and reverse primer, 50-CCTCAGTT CCGAAAACCAACAA-30). Materials and Methods Subjects Immunoblot analysis After obtaining informed consent, 120 patients (67 males Total cell lysates from tumors and adjacent noncancerous and 53 females; median age, 66 years; range 28–86 years) mucosa or cell lines were extracted by lysis buffer (150 mM diagnosed patholgically with gastric cancer at the Chang NaCl/50 mM Tris, pH 7.8/1% Triton X-100/1 mM EDTA, 1 Gung Memorial Hospital (CGMH) from 2000 to 2005 were mM phenylmethylsulfonylfluoride) and quantified by Brad- enrolled in the study. None of the patients had received ford method. Equal amounts of protein were electrophor- chemotherapy or radiotherapy before operation and all of esed through a 10% Tris-SDS gel and transferred onto a

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polyvinylidene difluoride membrane. The membranes were 30-gauge needle into the tail vein of 4 to 6 weeks old male blocked overnight with PBS containing 0.1% Tween 20 in severe combined immunodeficiency (SCID; C.B17/Icr- 5% skimmed milk at 4 C, and subsequently immunoblotted scid) mice. Five weeks later, mice were sacrificed by CO2 with primary antibody listed in Supplementary Table S1. and the lung was removed, fixed with 3.7% formaldehyde After further washing, the membrane was incubated for 1 and sectioned. Paraffin sections were stained with hema- hour with a horseradish peroxidase-conjugated, affinity- toxylin and eosin (H&E; Sigma). Lung metastasis was quan- purified anti-rabbit antibody (1:5,000 dilution in TBS; Santa tified by counting the total tissue area per lung section (A1) Cruz Biotechnology Inc.). Immune complexes were visual- and metastasis present in the same area (A2). The metastatic ized via chemiluminescence using an ECL Detection Kit index was calculated by the ratio A2/A1. (Amersham Inc.). Statistical analysis Immunohistochemistry The Mann–Whitney U test or Fisher exact test was used for Formalin-fixed and paraffin-embedded tissues were between-group comparisons, where appropriate, and the examined with immunohistochemistry (IHC) using prima- correlation between the results obtained with the 2 different ry antibody listed in Supplementary Table S2 and the analyses analyzed with Spearman test. Follow-up studies of avidin–biotin complex method according to previously patients were performed until the time of writing or patient described procedures (20). The staining intensity was grad- death. The cancer-specific survival outcome was evaluated ed as absent (0), weak (1þ), medium (2þ), or strong (3þ). by applying the Kaplan–Meier method for all patients, The histoscore (Q) was calculated by multiplying the per- except those who died from surgical complications. The centage (P) of positive cells by the intensity (I), according to log-rank test was used to compare the prognostic signifi- the formula: Q ¼ P1 I1 þ P2 I2 þ ... þ Pn In. cance of individual variables on survival. Cox’s proportional hazards model was applied in a multivariate analysis to RNA interference identify independent predictors of survival. A P-value of The lentiviral pLKO, control shLuc, and shRNA were <0.05 was considered statistically significant. obtained from the National RNAi Core Facility at the Institute of Molecular Biology/Genomic Research Center, Results Academia Sinica. Target information is listed in Supplemen- High expression of IL-32 in gastric cancer is correlated tary Table S3. Recombinant lentivirus carrying shRNA was with tumor progression and poor prognosis produced by cotransfecting 293FT cells with a mixture of In our previous study (21), differential gene expression plasmid DNA consisting of pMD-G (VSV-G envelope), patterns from genome-wide microarray analyses revealed pCMV-cR8.91 (Gag/Pol/Rev), and pLKO/1-shRNA vectors upregulation of several inflammation-associated genes in using TurboFect reagent (Fermentas) according to the man- gastric cancer tissues, compared with adjacent nontumor- ufacturer’s recommendations. Virus-containing culture sup- ous mucosa tissues. CXCL1 (22), SLIP (23), and SPARC (21) ernatants were collected 2 days after transfection and used have been verified and studied for clinical significances in to infect AGS and TSGH9201 cells in combination with 10 our setting. Furthermore, we focused on a novel pro-inflam- mg/mL polybrene (Sigma-Aldrich). Stable cell lines were matory gene, IL-32, as a potential tumor marker for gastric selected by culturing cells in 2 mg/mL puromycin (Calbio- cancer development. To verify the enhanced expression of chem) for 1 week. Western blot analysis or qPCR was used IL-32 in gastric cancer, IHC (n ¼ 120) and real-time qPCR to determine the effects of gene expression knockdown. (n ¼ 66) were performed on clinical specimens. Among the 120 patients analyzed using IHC, the mean score in tumor Cell lines and culture tissues was 107.2 4.8, which was significantly greater than TSGH 9201 human gastric cancer cell line (BCRC 60146) that (32.33 3.98) in the matching adjacent mucosa. IL-32 was obtained from the Bioresource Collection and Research was consistently detected in the majority of tumor regions, Center of Taiwan. Human gastric carcinoma cell line AGS whereas levels were reduced in nontumor areas (Supple- was obtained from American Type Culture Collection (CRL- mentary Fig. S1A). Four pairs of representative cases from 1739). The cell line was authenticated by assessments of IHC analysis are shown in Supplementary Fig. S1B. Strong short tandem repeat loci following database comparison. staining of IL-32 was mostly detected in the cytosol of gastric All cell lines were cultured in RPMI 1640 medium (Invitro- tumor cells. In contrast, absent or weak staining for IL-32 gen), supplemented with 10% FBS (Invitrogen), 100 IU/mL was observed in nontumor gastric epithelial cells. Staining penicillin G, and 100 mg/mL streptomycin sulfate (Sigma- was more intense in the advanced stages (Supplementary Aldrich) at 37C in a humidified atmosphere containing 5% Fig. S1B, right), relative to early stages of the disease (Sup- CO2. All kinase inhibitors used in cell culture are listed in plementary Fig. S1B, left). Data from qPCR experiments Supplementary Table S4. revealed high IL-32 expression (1.5-fold) in 48 of 66 (74%) gastric cancer tissues, compared with paired non- In vivo tumor metastasis model cancerous tissues (P < 0.001; Supplementary Fig. S1C). TSGH9201 cells with or without silencing IL-32 were Because the IL-32 isoforms cannot be distinguished harvested and washed twice with PBS. Cells (1 using qPCR and IHC, we further analyzed expression of 106/150 mL per mouse) were injected intravenously via a IL-32 protein in paired specimens via Western blotting

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(Supplementary Fig. S1D). As expected, IL-32 expression metastasis, lymph node metastasis, peritoneal seeding, vas- in tumor areas was higher than that in matched noncan- cular invasion, lymphatic invasion, perineural invasion, cerous adjacent mucosa in 3 representative patients. gross type, and tumor size. Distant metastasis [P < 0.001; Notably, all IL-32 isoforms, including a, b,andg,were HR ¼ 3.325; 95% confidence interval (CI), 1.838–6.014], expressed in the tumor, with the b isoform as the dom- higher IL-32 expression (P ¼ 0.006; HR ¼ 2.340; 95% CI, inant variant, as reported previously (15). Similar results 1.277–4.290), serosal invasion (P ¼ 0.014; HR ¼ 2.893; were obtained from a RT-PCR assay (data not shown). 95% CI, 1.239–6.752), and histologic type (P ¼ 0.005; HR To determine the association between IL-32 expression ¼ 2.918; 95% CI, 1.387–6.141) significantly emerged as and tumor characteristics, the IHC score for IL-32 was independent prognostic markers for gastric cancer in a analyzed using clinicopathologic parameters. Table 1 lists stepwise forward-conditional multivariate regression mod- the characteristics of study patients (n ¼ 120). Tumor size el (Table 1). These data indicate IL-32 as well as 3 pathologic (maximum diameter) was on average 5.6 cm (median, 5.2 parameters (distant metastasis, serosal invasion, and histo- cm; range, 0.3–20 cm). Tumors were located in the prox- logic type) displayed an independent prognostic value for 5- imal third of the stomach in 24 cases (20%), middle third in year cumulative survival. Our results support a strong asso- 29 (24.1%), distal third in 61 (50.8%), and the whole ciation between IL-32 expression and gastric cancer pro- stomach in 6 (5%). Histologic tumor types were intestinal gression, and support the utility of IL-32 as a useful prog- (n ¼ 42, 35%) and diffuse (n ¼ 78, 65%). As defined by the nosis marker for gastric cancer. depth of wall invasion, early gastric cancer (T1) was diagnosed in 24 (20%) cases (mucosal in 10 and submucosal in Ectopic expression of IL-32 in TSGH9201 cells 14), whereas advanced cancers (T2; muscle proper and promotes migration and invasion subserosa) was diagnosed in 18 (15%) cases, and serosa Clinicopathologic data indicate that IL-32 is involved in (T3) in 59 (49.1%), and invasion to adjacent organs (T4) in cell invasiveness. To confirm whether IL-32 is associated 19 (15.8%) cases. Lymph node metastasis was diagnosed in with invasiveness of gastric cancer cell lines, we established 87 cases (72.5%). During surgery, peritoneal seeding was stable IL-32–expressing sublines. Because all IL-32 isoforms demonstrated in 22 (18.3%) cases, and liver metastasis was are expressed in clinical specimens, the largest g variant that identified in 2 (1.6%) patients. Pathologic staging was can be spliced into IL-32b and IL-32a (24) was introduced distributed as follows: I, 30 cases, 25%; II, 10 cases, in TSGH9201 cells (Supplementary Fig. S2A). All cells 8.3%; III, 47 cases, 39.1%; and IV, 33 cases, 27.5%. overexpressing IL-32 displayed protrusions at the leading Interestingly, higher levels of IL-32 were detected in T3 and edge and elongated spindle-like morphology, compared T4 groups where the serosal surface of the gastric wall was with control cells (Supplementary Fig. S2B). Based on these invaded by cancer, compared with T1 and T2 groups where morphologic characteristics, we propose that IL-32 no invasion was evident (P ¼ 0.016; Table 1 and Fig. 1A). In enhances cell motility (25). Indeed, IL-32–overexpressing addition, IL-32 was significantly increased in patients with cells exhibited significantly higher migration (Supplemen- metastasis to the lymph nodes (P ¼ 0.005; Table 1 and Fig. tary Fig. S2C) and invasive (Fig. 2A) abilities toward a serum 1B) and those at the more advanced pathologic stages (III and gradient, as well as wound healing ability (Supplementary IV) of gastric cancer, compared with those in the earlier Fig. S2D), relative to control cells. Similar findings were pathologic stages (I and II; P ¼ 0.011; Table 1 and Fig. 1C). consistently obtained in doxycycline-inducible IL-32–over- Among the 120 patients, 65 died: 56 died because of expressing cells (Supplementary Fig. S3A), whereby cell gastric cancer progression; 2 died from surgical complica- migration ability was increased following induction of IL- tions; and 7 died because of noncancerous causes. Median 32 with doxycycline (Supplementary Fig. S3B). Our results follow-up duration for survivors (n ¼ 55) was 83.1 months clearly indicate that IL-32 is involved in gastric cancer cell (range, 64–137 months). Overall cumulative 5-year surviv- migration and invasion. al rate for the 120 patients was 53.4%. After dichotomiza- tion based on the median IL-32 expression level, the IL-32- IL-32 accelerates cell invasion through IL-8, VEGF, high group (n ¼ 73) presented significantly shorter cumu- MMP9, and MMP2 in an AKT-dependent manner lative survival, compared with the IL-32-low group (n ¼ 47) In general, cancer cells utilize an autocrine mechanism using univariate analysis (Table 1 and Fig. 1D; 66.4% vs. by releasing several important factors to enhance prolifer- 40.4%, log-rank P ¼ 0.0045). Other factors that predicted ation and metastasis (26). Accordingly, we investigated outcome significantly for cumulative survival in univariate whether IL-32 induces secretory factors to facilitate gastric analysis included tumor location, gross type, tumor size, cancer cell invasion. Following the addition of concentrat- histologic type, depth of invasion, lymph node metastasis, ed conditioned medium to the lower Boyden chamber as a distant metastasis, pathologic stage, liver metastasis, peri- chemoattractant, IL-32 and the induced secretory proteins toneal seeding, vascular invasion, lymphatic invasion, and significantly enhanced the invasion ability of parental perineural invasion (Table 1). Cox regression analysis (mul- TSGH9201 cells (Supplementary Fig. S2E). To further tivariate) was performed to determine the prognostic poten- define the chemoattractant in the IL-32–induced secre- tial of IL-32 independently for gastric cancer in relation to tome, several well-known invasion-associated factors, such the significant clinicopathologic parameters in univariate as IL-8, VEGF, MMP2, and MMP9, were determined in analysis, including histologic type, serosal invasion, distant conditioned medium. As expected, ectopic expression of

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Table 1. Clinicopathologic correlations of IL-32 expression and 5-year survival rate in patients with gastric cancer

Univariate analysis Multivariate analysis Parameters n Mean SEa Pb 5-y S.R.c Log-rank Pd HR 95% CI P Age (y) <65 58 108.7 6.9 0.876 45.1 0.2329 65 62 107.0 6.6 56.1 Gender Male 67 109.0 6.7 0.880 52.0 0.7656 Female 53 106.4 6.7 48.8 Location Upper third 24 110.0 8.9 0.985 52.2 0.0005 Middle third 29 106.6 10.5 54.5 Lower third 61 106.7 8.8 53.3 Whole 6 112.7 24.6 0.0 Gross type Localized 46 99.7 8.4 0.095 77.4 <0.0001 Inﬁltrative 74 112.9 5.6 33.4 Size <5 cm 65 102.4 6.8 0.187 72.3 <0.0001 5 cm 55 114.3 6.5 24.6 Histologic type 2.918 1.387–6.141 0.005 Intestinal 42 101.3 8.8 0.657 77.5 <0.0001 Diffuse 78 108.7 5.6 35.4 Depth of invasion (pT) T1 24 83.8 12.5 0.033 91.5 <0.0001 T2 18 108.6 12.5 68.9 T3 59 115.3 6.3 39.8 T4 19 114.2 9.6 11.1 Serosal invasion 2.893 1.239–6.752 0.014 No (T1, T2) 42 93.5 9.0 0.016 82.4 <0.0001 Yes (T3, T4) 78 115.6 5.3 32.7 Lymph node status (pN) N0 33 88.5 10.5 0.024 87.4 <0.0001 N1 43 115.4 7.4 48.2 N2 23 106.5 10.9 43.0 N3 21 124.3 7.3 5.0 Lymph node metastasis No (N0) 33 88.5 10.5 0.005 87.4 <0.0001 Yes (N1, N2, N3) 87 115.2 5.0 36.1 Distant metastasis (pM) 3.325 1.838–6.014 <0.001 No 91 106.9 5.8 0.631 64.1 <0.0001 Yes 29 110.7 7.4 5.6 Pathologic stage (pStage) I 30 84.3 11.2 0.007 92.7 <0.0001 II 10 116.5 11.0 80.0 III 47 121.6 7.5 47.6 IV 33 107.0 6.9 4.8 Stages I, II 40 92.4 9.1 0.011 89.4 <0.0001 Stages III, IV 80 115.6 5.3 30.4 Liver metastasis No 118 108.0 4.8 0.853 51.5 0.0135 Yes 2 100.0 50.0 0.0 (Continued on the following page)

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Table 1. Clinicopathologic correlations of IL-32 expression and 5-year survival rate in patients with gastric cancer (Cont'd )

Univariate analysis Multivariate analysis Parameters n Mean SEa Pb 5-y S.R.c Log-rank Pd HR 95% CI P Peritoneal seeding No 98 107.1 5.5 0.688 60.4 Yes 22 110.9 8.3 0.0 <0.0001 Vascular invasion No 95 107.1 5.6 0.654 59.6 <0.0001 Yes 25 110.8 7.6 12.2 Lymphatic invasion No 48 101.4 9.0 0.123 76.2 <0.0001 Yes 72 112.2 5.1 32.6 Perineural invasion No 76 106.8 6.3 0.682 63.7 0.0002 Yes 44 109.6 6.9 28.4 IL-32 (IHC score) 2.34 1.277–4.29 0.006 <100 (median) 47 56.4 3.5 <0.001 66.4 0.0045 100 73 141.0 4.1 40.4

NOTE: HRs, 95% CI, and P values were generated using a Cox proportional hazard analysis. Boldface indicates statistical signiﬁcance. aIHC scores. bMann–Whitney test. cFive-year survival rate. dLog-rank test.

IL-32 was accompanied by induction of IL-8, VEGF, MMP2, GSK3b, resulting in nuclear translocation and activation of and MMP9 expression, both at the RNA (data not shown) b-catenin (27). Several studies have reported that b-catenin and protein levels (Fig. 2B), as well as zymographic activity of regulates IL-8, VEGF, MMP2, and MMP9 expression (28, MMP2 and MMP9 (Supplementary Fig. S2F). Next, we inves- 29), and acts as a major pathway frequently altered in tigated the mechanism by which IL-32 regulates invasion- human gastric cancer (30). Accordingly, we hypothesized associated factors. To elucidate the specific signaling pathway that b-catenin operates in IL-32-phospho-AKT signaling. underlying IL-32–mediated expression of IL-8, IL-32–over- Interestingly, expression of active b-catenin was dramati- expressing cells were treated with inhibitors for the following cally increased in the nuclei of IL-32–overexpressing cells in pathways: phosphoinositide 3-kinase (PI3K; LY294002), which p-AKT was activated as well as the intermediate extracellular signal–regulated kinase (ERK; U0126), NF-kB regulator, phospho-GSK-3b (Fig. 3A). This finding was (Bay 11-7082), and TGF-b (SB431542). LY294002, which further confirmed using doxycycline-inducible IL-32–over- markedly inhibits PI3K downstream phosphorylated AKT (p- expressing cells in which active nuclear b-catenin was AKT) expression (Fig. 2C), suppressed IL-32–induced IL-8 increased via inducing IL-32 production over a short period mRNA (Supplementary Fig. S4) and protein (Fig. 2C) expres- of time, compared with empty vector control cells (Supple- sion to a significant extent. In addition, suppression of p-AKT mentary Fig. S4A). To provide evidence of the mechanistic activity led to decreased VEGF, MMP2, and MMP9 expression role of b-catenin on the regulation of IL-32/p-AKT–induced (Fig. 2C) and inhibition of the invasive ability of IL-32– IL-8, VEGF, MMP2, and MMP9, we transduced vector- or IL- overexpressing cells (Fig. 2D). Similar results were observed 32–overexpressing TSGH cells with short hairpin RNAs by using MK-2206, a highly selective non-ATP competitive (shRNA) targeting b-catenin (shb-catenin), IL-8 (shIL8), or allosteric Akt inhibitor, to block AKT activity (Supplementary luciferase control (shLuc), using the lentiviral delivery sys- Fig. S5). Our results collectively indicate that AKT activation tem. Silencing of b-catenin or IL-8 led to a dramatic decrease by IL-32 is required to induce IL-8, VEGF, MMP2, and MMP9 in IL-32–induced VEGF, MMP2, and MMP9 expression, but expression for cell invasion. had little effect on the endogenous level (Fig. 3B). Notably, IL-32–induced IL-8 was additionally decreased by silencing Active b-catenin is a downstream effector of phospho- b-catenin, whereas knockdown of IL-8 did not affect active AKT in IL-32–induced cell invasion b-catenin expression, implying that b-catenin regulates IL-8 Next, we investigated the mechanism underlying AKT- production in IL-32- overexpressing cells. LY294002 treat- dependent invasiveness in IL-32–overexpressing cells. Acti- ment suppressed active b-catenin expression (Fig. 2C), vated AKT is capable of phosphorylating and inactivating whereas, in contrast, p-AKT expression was not affected by

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AB 300 300

Figure 1. IL-32 overexpression in 200 200 human gastric cancer is correlated with prognosis of patients. A, scatter plot of IL-32 IHC scores according to depth of wall invasion 100 100 ( , P ¼ 0.016, T1–T2 vs. T3–T4). B, scatter plot of IL-32 IHC scores according to lymph node IL-32 expression (IHC score) IL-32 expression IL-32 expression (IHC score) IL-32 expression metastasis ( , P ¼ 0.005, N0 vs. 0 – T1 T2 T3 T4 0 N1 3). C, scatter plot of IL-32 IHC N0 N1 N2 N3 Depth of wall invasion (pT) scores according to pathologic ¼ – Lymph node metastasis (pN) stage ( , P 0.011, stage I II vs. CD stage III–IV). D, Kaplan–Meier 300 survival curves of 2 groups of 1.0 patients with gastric cancer Lower expression (n = 47) deﬁned by an IL-32 expression level cutoff value of 100 (the 0.8 median), established on the basis 200 of IHC scores. The 5-year survival n 0.6 Higher expression ( = 73) rate of the lower expression groups (n ¼ 47) was signiﬁcantly better 0.4 than that of the higher expression 100 groups (n ¼ 73; 66.4% vs. 40.4%; 0.2 log-rank P ¼ 0.0045). Cumulative survival Cumulative Cutoff point = 100 (IL-32 IHC score) Log-rank P = 0.0045 IL-32 expression (IHC score) IL-32 expression 0.0 0 0122436486072 IIIIII IV Postoperative months Pathological stage (pStage)

introducing either b-catenin or IL-8 shRNA (Fig. 3B). The accompanied by decreased cell invasion, compared with data indicate that b-catenin and IL-8 act as downstream shLuc control, in both gastric cancer cell lines (Fig. 4A and targets of p-AKT in the IL-32 signaling pathway and induce B). Consistent with the molecular changes in IL-32–over- VEGF, MMP2, and MMP9 production. To determine wheth- expressing cells, IL-32 silencing resulted in decreased er active b-catenin and IL-8 are required for IL-32–induced expression of p-AKT, active b-catenin, IL-8, and VEGF (Fig. cell invasiveness, we performed an invasion assay with 4C). We additionally re-expressed a siRNA-resistant form b-catenin or IL-8 knockdown cells. Consistent with their of IL-32 by introducing a silent mutation in the siRNA well-established role in cancer cell invasion, depletion of target region into IL-32 knockdown cells. Immunoblot b-catenin and IL-8 significantly suppressed IL-32–induced analyses confirmed that expression of IL-32 is restored to matrix invasion ability, but had little effect on invasiveness the control cell level (Supplementary Fig. S6A). As of control cells (Fig. 3C). Interestingly, elongated IL-32– expected, re-expression of IL-32 was sufficient to restore overexpressing cells was partly restored to a round shape the invasive properties of TSGH IL-32 knockdown cells upon b-catenin knockdown, but not IL-8 depletion (Fig. (Fig. 4A). Next, we compared in vivo metastasis formation 3D). Taken together, these results indicate that IL-32 of TSGH shLuc control and IL-32 knockdown cells to enhances the activity of AKT, and subsequent b-catenin further establish the relationship between IL-32 expression activation results in the production of IL-8, VEGF, MMP2, levels and metastasis formation. Following injection of and MMP9 to promote cell invasion. cells into the tail vein of immunodeficient mice for 5 weeks, the number of experimental lung metastatic nodules was Silencing of IL-32 reduces the invasive ability of human examined macroscopically and microscopically for the gastric cancer cells mediated via inactivation of the occurrence of metastasis. The number decreased signifi- p-AKT/b-catenin pathway cantly in animals injected with IL-32 knockdown cells, To investigate the significance of IL-32 in gastric cancer compared with those injected with shLuc control cells (Fig. metastasis, we transduced AGS or TSGH cells with 2 inde- 4D). Our results clearly show that silencing of IL-32 pendent shRNAs targeting human IL-32. Introduction of decreases invasive activity in vitro and pulmonary meta- IL-32 siRNA resulted in reduced IL-32 expression (Supple- static potential to a substantial extent in vivo in human mentary Fig. S6A and S6B), and more importantly, was gastric cancer cells.

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Regulation of IL-32 in Gastric Cancer

Figure 2. Ectopic overexpression of IL-32 increases IL-8, VEGF, MMP2, MMP9 expression as well as TSGH9201 cell invasion in a phospho-AKT–dependent manner. Three IL-32–overexpressing sublines (IL-32-pool, IL-32-1, and IL-32-2) and 2 control lines (v1 and v2) were established using parental TSGH9201 cells. A, quantification of serum-induced transwell invasion (4 105 cells) by IL-32– overexpressing and control cells. The top panel shows representative microscopic images of transwells. Magnification, 40. The scale bar represents 500 mm (mean SEM; 3 independent experiments) , P < 0.05; , P < 0.01. B, IL-8, VEGF, MMP2, and MMP9 protein expression in the supernatant of IL-32–overexpressing and control cells. Fascin1 was used as a loading control. C, V1 and IL-32-2 cells were treated with vehicle (dimethyl sulfoxide) or LY294002 (LY) for 48 hours. Expression of the indicated molecules was detected via Western blotting. b-Actin and fascin1 were used as loading controls of cytosolic and secreted proteins, respectively. D, after treatment, cells were analyzed with the invasion assay, presented as a histogram. The top panel shows representative microscopic images of transwells. Magnification, 40. The scale bar represents 500 mm. , P < 0.01. One-way ANOVA with Tukey multiple comparison tests (mean SEM; 3 independent experiments).

IL-32, CTTNB1 (b-catenin), IL-8, VEGF-A, MMP2, and mens (Fig. 5A and Supplementary Fig. S7). However, this MMP9 are coexpressed in human gastric malignancies finding remains to be experimentally confirmed. Further To determine the clinical significance of the IL-32/b-cate- immunohistochemical analysis for IL-32 and IL-8 in 85 nin/IL-8/VEGF/MMP2/MMP9 axis, we examined whether cases of gastric cancer disclosed a significantly moderate the observed correlation between IL-32 and these down- correlation between IL-32 and IL-8 (Fig. 5B and C; Spear- stream effectors could be extended to human gastric cancer. man r ¼ 0.562, P < 0.001). These results strongly suggest Using oncomine, expression values for IL-32, CTTNB1, IL-8, that the regulation of CTTNB1 (b-catenin), IL-8, VEGF-A, VEGF-A, MMP2, and MMP9 were regained from the pub- MMP2, and MMP9 by IL-32 occurs not only in gastric cancer lished Cho gastric microarray dataset (GEO accession: cell lines in vitro, but also human gastric tumors in vivo. GSE13861; ref. 31). Spearman analysis performed on the plotted mRNA expression values revealed a significantly Discussion moderate correlation between expression of IL-32 and its Gastric cancer is the second most common cause of downstream effectors in 65 gastric adenocarcinoma speci- cancer-induced death worldwide. The prognosis in the late

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Regulation of IL-32 in Gastric Cancer

stage of gastric cancer remains extremely poor, and median exchange factor, Tiam1, is upregulated in IL-32–overexpres- survival rarely approaches 1 year, mainly because of metas- sing cells. Furthermore, IL-32–induced cell migration was tasis (32). No effective therapeutic targets have been iden- abolished by a selective Rac1/Tiam1 inhibitor, NSC23766 tified to date. The most important objective of gastric cancer (Supplementary Fig. S8). However, activation of the Wnt/ research is to elucidate the molecular mechanism(s) under- b-catenin pathway has also been shown to trigger changes in lying metastasis. epithelial cell morphology (e.g., epithelial-mesenchymal IL-32 was overexpressed in gastric cancer, whereas expres- transition) and activate migration and invasion (36). sion was significantly low in nontumor mucosa, as described Accordingly, downregulation of b-catenin in IL-32–overex- previously (7). IL-32 expression in tumors from the Cho pressing cells partly reversed IL-32–induced cell elongation. gastric microarray dataset was higher than that in nontumor Together, these results give possible molecular mechanisms specimens, in keeping with our data. The mechanism under- by which IL-32 induce cell migration. lying IL-32 overexpression in gastric cancer is unclear at Data from this study indicate that IL-32 triggers cell inva- present. It has been reported that chromosome 16p13.3 sion partially by inducing IL-8, VEGF, MMP2, and MMP9 region, which contains the IL-32 gene is frequently amplified expression, resulting in degradation of the extracellular in breast and small intestine cancer (33, 34). The above matrix. These results are in line with previous findings show- findings provide implications of IL-32 dysregulation in the ing that IL-32 is associated with acquisition of an invasive and mechanism of gastric cancer development, but more exper- metastasis phenotype in lung cancer mediated via coexpres- imental evidence is needed to confirm this hypothesis. In our sion of IL-8 and VEGF (9). Furthermore, IL-8 and VEGF are analyses, Kaplan–Meier survival analysis showed that higher well defined pro-angiogenic and pro-metastatic factors in IL-32 expression is significantly associated with poor prog- numerous human cancers. Ectopic expression of IL-32 is nosis of patients with gastric cancer. IL-32 was additionally accompanied by hypoxia-inducible factors 1a (HIF-1a)acti- identified as an independent prognostic marker in multivar- vation, one of the most powerful regulators of angiogenesis iate analysis in parallel with the known clinical and patho- (Supplementary Fig. S9). Furthermore, depletion of HIF-1a logic factors for gastric cancer. Further analysis of the predic- decreased IL-32–induced cell invasion and IL-8, VEGF, tive potential of IL-32 expression supported its utility as a MMP9, MMP2 expression (Supplementary Fig. S9B and predictor to identify aggressive gastric cancer, consistent with S9C). However, the expression of IL-32–induced phospho- both Kaplan–Meier and Cox regression analyses. This study AKT and active b-catenin did not change by HIF-1a depletion provides evidence that IL-32 is a useful independent biomark- (Supplementary Fig. S9B). In addition, the expression of IL- er for prognosis. Shortly before the publication of this article, 32–induced HIF-1a also did not change by b-catenin or IL-8 a study also found higher IL-32 expression is correlated with depletion (Supplementary Fig. S9D). Notably, both active poor postoperative outcomes of patients with gastric cancer b-catenin and HIF-1a were inhibited whereas AKT was inac- (8). Although that study only focused on clinical significance, tivated by MK-2206 (Supplementary Fig. S5A). Therefore, the findings lend support to our claim of the importance of both b-catenin and HIF-1a involve in IL-32–induced cell the regulation and function of IL-32 in gastric cancer. invasion under phospho-AKT activation. Other investigators Ectopic expression of the largest g variant of IL-32, which have demonstrated an involvement of IL-32 in the regulation can be spliced into IL-32b and IL-32a in gastric cancer cells, of IL-8 in endothelial cells, indicative of a role in modulation leads to spindle-like morphology and increased cell migra- of endothelial function (37). The results collectively support tion ability. Analogous results were obtained upon ectopic an important role of IL-32 in angiogenesis in gastric cancer. expression of the b variant of IL-32, which generated both b To our knowledge, this study has shown for the first time and a isoforms without the g isoform (data not shown). that IL-32 stimulates activation of AKT and b-catenin as well These results imply that all IL-32 isoforms may have similar as HIF-1a to induce IL-8, VEGF, MMP2, and MMP9 secre- functions in cell migration. A more recent study showed that tion for gastric cancer metastasis (Fig. 5D). Clearly, p-AKT, IL-32 contains an RGD motif that potentially binds to and activated b-catenin as well as HIF-1a are upstream regula- activates integrins and focal adhesion kinase (FAK) essential tors of IL-8 expression. However, we cannot exclude the for cell anchorage and migration, consistent with our find- possibility of cross-talk signaling between IL-8, VEGF, ings (35). Previous studies have identified Rac1 as an impor- MMP2, and MMP9. Previous studies have reported suppres- tant downstream component of integrin/FAK signaling (30). sion of IL-1b, TNF-a, and INF-g–induced IL-32a expression Accordingly, we hypothesize that IL-32 induces integrin and via inhibition of the PI3K/AKT pathway in myofibroblasts Rac1 activation to enhance cell migration ability. Our experi- (38). Gastric cancer cells may use the positive feedback loop ments showed that the Rac1-specific guanine nucleotide to activate the PI3K/AKT pathway for cancer progression.

Figure 3. IL-32 induces IL-8, VEGF-A, MMP2, and MMP9 expression via b-catenin activation. A, expression of indicated molecules in IL-32–overexpressing and control cells was detected via Western blotting. B, expression of indicated proteins in v1 and IL-32-2 cells transfected with the gene silencers, shLuciferase (shLuc), b-catenin shRNA (shb-catenin), or IL-8 shRNA (shIL8), was detected using Western blotting. C, invasion assay of indicated cells was performed. Magnification, 100. Scale bar represents 200 mm. Quantified results are presented as a histogram in the right panel. One-way ANOVA with Tukey multiple comparison tests (mean SEM; 3 independent experiments). , P < 0.01. D, quantification of cells displaying elongated morphology. Elongation is presented as a percentage (mean SEM; 3 independent experiments, n > 400) of cells with a length of more than 3 times the width. , P < 0.05. Left: representative phase-contrast images. The arrows indicate the presence of a spindle shape. Magnification, 400. Scale bar represents 50 mm.

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Figure 4. Depletion of IL-32 in gastric cancer cell lines reduces invasion ability in vitro and in vivo. A, invasion properties of the indicated cells (6 105) were analyzed. Magnification, 100. Scale bar represents 200 mm. Quantified results are presented as a histogram in the right panel. One-way ANOVA with Tukey multiple comparison tests (mean SEM; 3 independent experiments). , P < 0.05; , P < 0.001. B, the invasive properties of indicated cells were examined. Magnification, 100. Scale bar represents 200 mm. Quantified results are presented as a histogram in the right panel. One-way ANOVA with Tukey multiple comparison tests (mean SEM; 3 independent experiments). , P < 0.001. C, expression of indicated molecules in control or IL-32 knockdown TSGH cells was detected via Western blotting. D, top: H&E staining of metastatic lesions upon intravenous administration of shLuc-1, KD-IL-32-1, and KD-IL-32-2 cells (1 106) into SCID mice. Microscopic images of the lung tumor nodules indicated with arrowheads. Magnification, 100. Scale bar represents 200 mm. Bottom: lung sections of tumor-bearing mice were microscopically analyzed, and metastases per histologic section counted. N ¼ 4; , P < 0.05; , P < 0.01; , P < 0.001.

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Regulation of IL-32 in Gastric Cancer

Figure 5. IL-32 is correlated with IL-8, VEGF-A, MMP2, MMP9, and CTNNB1 in human gastric cancer. A, heat map of expression profiles for IL-32, IL-8, VEGF-A, MMP2, MMP9, and CTNNB1 (b-catenin) of 65 human gastric adenocarcinomas from an oncomine microarray dataset (as indicated in the figure). Spearman rank correlation coefficient of individual genes with IL-32 shown in the left. B, representative positive and negative staining of IL-32 and IL-8 in human gastric cancer tissue sections. Magnification, 200. Scale bar represents 100 mm. C, IL-32 and IL-8 IHC scores were plotted on the basis of expression values. Spearman rank correlation coefficients between IHC scores of IL-8 (y-axis) and IL-32 (x-axis) in 85 clinical specimens were analyzed. Spearman r ¼ 0.562, P < 0.001. D, proposed model representing the prometastatic effect of IL-32 in the pathogenesis of gastric cancer.

In summary, significant overexpression of IL-32 was Writing, review, and/or revision of the manuscript: C.-Y. Tsai, C.-S. Wang, K.-H. Lin observed in gastric cancer, suggesting a critical role in Administrative, technical, or material support (i.e., reporting or orga- development and progression to malignancy. IL-32 may nizing data, constructing databases): H.-C. Chi, Y.-H. Tseng, K.-H. Lin be a useful independent prognostic tumor marker to pre- Study supervision: C.-Y. Tsai, K.-H. Lin dict survival and metastasis of gastric cancer patients. Our Acknowledgments findings collectively provide evidence that IL-32 functions The authors thank Dr. Y. Liang for immunohistochemistry and pathologic as a potential therapeutic target and support its utility as a comments. useful independent prognostic marker of patient survival. Grant Support Disclosure of Potential Conflicts of Interest This work was supported by grants from Chang-Gung University, No potential conflicts of interest were disclosed. Taoyuan, Taiwan (CMRPG6B0011, NMRP 170441), and the National Sci- ence Council of the Republic of China (NSC 92-2314-B-182-077; 97-2314-B- Authors' Contributions 182-009-MY2; 100-2314-B-182A-074). Conception and design: C.-Y. Tsai, C.-S. Wang, Y.-H. Tseng, K.-H. Lin The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Development of methodology: M.-M. Tsai, H.-C. Chi, J.-I. Wu, L.-H. Wang advertisement Acquisition of data (provided animals, acquired and managed patients, in accordance with 18 U.S.C. Section 1734 solely to indicate provided facilities, etc.): C.-S. Wang, C.-Y. Chen, C.D. Lin this fact. Analysis and interpretation of data (e.g., statistical analysis, biosta- tistics, computational analysis): C.-S. Wang, M.-M. Tsai, W.-L. Cheng, Received May 9, 2013; revised December 19, 2013; accepted January 25, C.-Y. Chen, K.-H. Lin 2014; published OnlineFirst March 6, 2014.

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2288 Clin Cancer Res; 20(9) May 1, 2014 Clinical Cancer Research

Interleukin-32 Increases Human Gastric Cancer Cell Invasion Associated with Tumor Progression and Metastasis

Chung-Ying Tsai, Chia-Siu Wang, Ming-Ming Tsai, et al.

Clin Cancer Res 2014;20:2276-2288. Published OnlineFirst March 6, 2014.

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