MPO Promoter Polymorphism rs2333227 Enhances Malignant Phenotypes … · Population and Prevention Science MPO Promoter Polymorphism rs2333227 Enhances Malignant Phenotypes of Colorectal

Population and Prevention Science

MPO Promoter Polymorphism rs2333227Enhances Malignant Phenotypes ofColorectal Cancer by Altering the BindingAffinity of AP-2aQingtao Meng1, Shenshen Wu1, Yajie Wang1, Jin Xu2, Hao Sun1, Runze Lu1, Na Gao3,Hongbao Yang4, Xiaobo Li1, Boping Tang5, Michael Aschner6, and Rui Chen1,7

Abstract

Myeloperoxidase (MPO) promoter SNPs rs2243828 (�764T>C)and rs2333227 (G-463A) program malignant phenotypes byregulating MPO transcriptional activity. In this study, we enrolleda total of 1,175 controls and 1,078 patients with colorectal cancerwith comprehensive clinical and survival information to assesswhether these SNPs could affect the susceptibility and developmentof colorectal cancer. TheMPO rs2333227 TT genotype significantlyincreased the risk of colorectal cancer and decreased the overallsurvival time of patients. Colorectal cancer cells with the rs2333227TT genotype exhibited enhanced proliferation, migration, and inva-

sion capacity in vitro and in vivo. Mechanistically, we found thatMPO SNP rs2333227C to Tmutation altered the binding affinity ofthe transcription factors AP-2a to the rs2333227 mutation region,sequentially enhancing expression levels of MPO and activatingfurther IL23A–MMP9 axis–mediated oncogenic signaling. Takentogether, our findings indicate that MPO SNP rs2333227 serves asa marker of enhanced risk for development of colorectal cancer.

Significance: MPO polymorphisms are a guide for high riskand poor prognosis in patients colorectal cancer.Cancer Res; 78(10);2760–9. �2018 AACR.

IntroductionGlobally, colorectal cancer is the third most common form of

cancer and the second leading cause of cancer-related death in thewestern world, with a lifetime risk in the United States of approx-imately 7% (1). Several factors are routinely used as prognosticmarkers for predictive prognosis, including depth of tumor inva-sion, nodal status, and distant metastasis (2). The 5-year relativesurvival rate ranges from 90% in colorectal cancer patients withstage y to 10%in colorectal cancer patientswith stage IV (3).Otherenvironmental factors thought to influence colorectal cancer

prognosis include lifestyle (4) and the tumor immunologicmicroenvironment (5).

Myeloperoxidase (MPO) is a lysosomal enzyme that is pres-ent in neutrophils, monocytes, and tissue macrophages. Itprotects against microbial infections (6) by generating variousfree radicals and reactive oxidants. Published data provideevidence of high circulating levels of MPO in patients withheart failure or coronary artery diseases (7–9). The SNPsrs2333227 (G-463A) and rs2243828 (T-764C), located in the50 upstream region of the MPO gene, are functional SNPs, asreported in the Carotene and Retinol Efficacy Trial (CARET)cohort (10) and other studies on rectal cancer (11). It is wellknown that the MPO rs2333227 polymorphism is associatedwith increased risk for disease, including breast cancer (12),prostate cancer (13, 14), and peripheral arterial disease (15).However, an association between MPO variants and colorectalcancer risk has yet to be reported.

In the current study, we investigated the association betweenMPO gene polymorphisms and the risk of colorectal cancer in aChinese population. Experiments were performed to assess therole of SNPs rs2333227 in promoting colorectal cancer.

Materials and PatientsStudy subjects

This case–control study included 1,277 patients with colorectalcancer and 1,175 cancer-free controls. Newly diagnosed andhistologically confirmed colorectal cancer patients were recruitedbetween January 2007 and October 2011 the Jiangsu province(China). Controls were randomly selected from patients attend-ing the same hospital for physical examination. The recruitedpatients had no history of previous cancer in other organs, or

1Key Laboratory of Environmental Medicine Engineering, Ministry of Education,School of Public Health, Southeast University, Nanjing, China. 2Department ofMaternal, Child and Adolescent Health, School of Public Health, Nanjing MedicalUniversity, Nanjing, China. 3Institute ofBioinformatics, HeinrichHeineUniversity,D€usseldorf, Germany. 4Center for New Drug Safety Evaluation and Research,China Pharmaceutical University, Nanjing, China. 5Jiangsu Key Laboratory forBioresources of Saline Soils, Jiangsu Synthetic Innovation Center for CoastalBioagriculture, YanchengTeachersUniversity, Yancheng, China. 6Department ofMolecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York.7Institute for Chemical Carcinogenesis, Guangzhou Medical University, Guangz-hou, China.

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

Q. Meng and S. Wu contributed equally to this article.

Correspondence Author: Rui Chen, Southeast University, 87 Dingjiaqiao, GulouDistrict, Nanjing 210009, China. Phone: 868-327-2560; Fax: 8625-8332-4322; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-2538

�2018 American Association for Cancer Research.

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preoperative chemotherapy or radiotherapy, and the controlswere genetically unrelated to the colorectal cancer patients. Thepathologic stage of colorectal cancer was assessed using the SixthEdition of the American Joint Committee on Cancer (AJCC)Cancer Staging Manual. The time of death or the last follow-up(in June 2016) of each patient was confirmed by a trained clinicalspecialist. Themedian survival time (MST) was 75.0months, andthemaximum follow-up timewas 112.7months. Of the recruitedpatients, 1,078 patients who completed the follow-up wereenrolled in the study, with a response rate of 84.4%. Writteninformed consent was obtained from each enrolled subject beforerecruitment. The study was conducted in accordance with Decla-ration ofHelsinki and approved by the Institutional ReviewBoardof Southeast University (Nanjing, China).

DNA extraction and genotype analysisBecause of lack of blood samples, the genomic DNA of the

colorectal cancer patients was extracted from paraffin-embeddedtissues using the EZNA Tissue DNA Kit (Omega Bio-Tek). For thecontrols, the genomic DNA was extracted from venous bloodusing the RelaxGene Blood DNA Kit (Tiangen Biotech). Theselected polymorphisms were genotyped using TaqMan allelicdiscrimination assays with the Quant Studio 6 Flex system(Applied Biosystems). The loading wells without DNA were usedas negative controls. Ten percent of the samples were randomlyselected for confirmation, and the results were 100% consistentwith previous genotyping results.

Tissue microarray construction and IHCThe colorectal cancer tissue microarrays (TMA) were created by

the National Engineering Center for Biochip (Shanghai, China).Duplicate 1.0-mmdiameter cores of tissue from each samplewerepunched from paraffin tumor blocks and adjacent colorectaltissues. The IHC assay procedure used has been described previ-ously (16). In brief, the specimens were incubated overnight withrabbit polyclonal anti-MPO antibody (BOSTER) and anti-MMP9antibody (Abcam) at 4�C. Each specimen was stained with 3, 30-diaminobenzidine (DAB; Zhongshan Biotech). The evaluation ofMPO and MMP9 expression was blindly conducted by twoindependent pathologists. The specimens were categorized usinga semiquantitative immunoreactivity score (17). Briefly, theintensity of immunostaining was deemed as 0 to 3 (0, negative;1, weak; 2, moderate; 3, strong), and the percentage of immuno-reactive cells was regarded as 1 (0%–25%), 2 (26%–50%), 3(51%–75%), and 4 (76%–100%). The immunoreactivity score(IRS) was calculated by the intensity multiply the percentage. Theoptimal cut-off value for theMPOandMMP9 IRSwas obtained bythe receiver operating characteristic (ROC) analysis. Briefly, thearea under the curve (AUC) at different cut-off values of MPO IRSfor survival time from 1 to 9 years was calculated, respectively.Under this condition, samples with IRS 0–4 and IRS 6–12 weredefinedas lowandhigh expression forMPO, respectively,whereassamples with IRS 0–6 and IRS 8–12 were defined as low and highexpression for MMP9, respectively.

Cell cultureThe human colorectal cancer cell lines (SW620 and SW480)

were purchased from the Shanghai Institute of Biochemistry andCell Biology, Chinese Academy of Sciences (Shanghai, China) in2016 and maintained in DMEM (HyClone) at 37�C under 5%CO2. Themediumwas supplementedwith 10%FBS (Sigma), 100

U/mL penicillin, and 100 mg/mL streptomycin (HyClone). Thecells were periodically tested and validated to be free of myco-plasma. These two cell lines were tested by short tandem repeatanalysis and used within 6 months. The cells were grown for nomore than 25 passages for any experiments.

Chromatin immunoprecipitation assaysChromatin immunoprecipitation (ChIP) experiments were

performed using the ChIP-ITTM magnetic Chromatin Immuno-precipitation Kit (cat. no. 53008, Active Motif) according to themanufacturer's instructions. Briefly, samples were fixed in form-aldehyde for 10 minutes at room temperature. Then, sampleswere neutralized by treated with glycine stop-fix solution for 5minutes at room temperature. The isolated nuclei and chromatinwere incubatedwith antibody against AP-2a, or control IgGat 4�Covernight. After sequential ChIP, quantitative PCRwas performedto determine the precipitated genomic DNA using the QuantStudio 6 Flex system (Applied Biosystems, Life Technologies),followed by human MPO rs2333227 (G-463A) promoterprimers identifying the AP-2a–binding sequence: forward: 50-GCCTCTAGCCACATCATCAA -30; reverse: 50- AATTTAGCACTAC-CAGCCCA-30.

RNA extraction and real-time RT-PCR analysisTotal RNA was isolated with TRIzol reagent (Invitrogen) from

frozen tissues and colorectal cancer cells. Quantitative PCR reac-tions were performed using SYBR Green Real-Time PCR Kits(Toyobo). Primers were 50-GACAAATACCGCACCATC-30 (for-ward) and 50-AAGCCGTCCTCATACTCC-30 (reverse) for MPO;50-ATCCGCAAAGACCTGT-30 and 50-GGGTGTAACGCAACTA-AG-30 for b-actin.

Western blot analysisTotal proteinswere extracted fromcolorectal cancer tumor tissues

with RIPA lysis buffer. Immunoblot assays were used to detect theMPO protein level by using primary antibodies human MPO(1:1,000 dilution, BOSTER Biological Technology) and b-actin(1:10,000 dilution). Semiquantitative analysis of MPO proteinlevels was performed with Image Lab 3.0 software (BIO-RAD).

CRISPR/Cas9–mediated genome engineeringThe CRISPR/Cas9 expression vectors expressing Cas9, puromy-

cin-resistant gene, and sgRNA were constructed (SupplementaryFig. S1A) based on a pX330 vector. The sequences of sgRNAtargeting the rs2333227 mutation region are shown in Supple-mentary Fig. S1A. One day before the transfection, the cells wereseeded in 6-well plates (2� 105 cells/well). Colorectal cancer cellswere cotransfected with single-stranded DNA donor construct(0.1 mg/mL) and CRISPR/Cas9 expression plasmids (1 mg/mL)using Lipofectamine 2000 according to the manufacturer's pro-tocol (Invitrogen). After 24 hours, the mediumwas replaced withcomplete medium (2 mL) containing puromycin (0.6 mg/mL;VWR Pty Ltd.), and then every 2 days until day 14. Surviving cellswere plated in a 96-well plate (1 cells/well) for single cell–derivedcolonies. Cells were then harvested for DNA sequencing(rs2333227, Supplementary Fig. S1B).

Analysis of colorectal cancer cell colony formation, migration,and invasion

For colony formation ability assays, 500 cells were seeded in100-mm culture dishes. We added DMEM (HyClone) containing

MPO Polymorphism and Colorectal Cancer Development

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10% FBS (Sigma) and placed the dishes at 37�C with 5% CO2 inan incubator, allowing the colonies to grow for 10 days. Cellcolonies were then stained and imaged according to our previousstudy (18). Transwell (Corning) assays were performed to detectthe effect of different MPO rs2333227 colorectal cancer cellgenotypes on colorectal cancer cell migration and invasion. Cells(5 � 104) were added to transwell chamber with a membranepore size of 8 mm (Corning) in triplicate. The cells (5� 104) wereadded to the 8-mm pore insert in serum-free medium. The poreinserts were coated with Matrigel (Cell Biolabs). As a chemoat-tractant, DMEM containing 10% FBS was added to the lowerchambers. After 24 hours, cells were fixed and stained with crystalviolet (1%) in PBS/ethanol (85%/15%). Cell migration wasdetermined in an analogous fashion, but in the absence ofMatrigel in the transwell chambers.

In vivo nude mice flank tumor experimentsAll animal studies were performed with the approval of the

Committee on Animal Care and Use of Southeast University. Atotal of 36 nude mice (6 mice for each group) were injectedsubcutaneously with 5 � 106 luciferase-expressing stably trans-formed colorectal cancer cells (SW620 and SW480) with CC, CT,and TT genotype, respectively. Two weeks later, the nude micewere euthanized and tumor, lung, and liver tissues were removed,and luciferase activity was determined with a luminometer (Sir-ius, Berthold Detection Systems).

ELISAThe MPO and IL23A levels of the colorectal cancer patients'

serum and colorectal cancer cell culture supernatant weredetected by ELISA (Abcam) according to the manufacturer'sinstructions.

Statistical analysisThe genotype frequencies of the controls were analyzed for

deviations from the Hardy–Weinberg equilibrium (HWE) usingthe goodness-of-fit c2 test. t test (for continuous variables) and c2

test (for categorical variables) were used to evaluate the differ-ences in demographic characteristics between the colorectal can-cer patients and controls and the genotype results of the SNPs. Theassociation between different genotypes and colorectal cancer riskwere estimated using the logistic regression models with adjust-ment for age, gender, smoking, and drinking status. The Kaplan–Meier method and log-rank test were used to analyze the overallsurvival in different subgroups classified by demographiccharacteristics, genotype, and clinical information. Univariate ormultivariate Cox regression analysis was used to analyze HRs and95% confidence interval (CI), adjusted for age, sex, drinking orsmoking status, tumor site (colon or rectum), grade (low orintermediate/high differentiated), and clinical stage (I to IV).Stepwise Cox regression analysis was conducted to assess predic-tive factors of colorectal cancer survival, with a significance level ofP < 0.05 for entering and P > 0.10 for removing the variablesadjusted in Cox regression analysis. The mediation model wasused to explore whether the association betweenMPO rs2333227C>T polymorphism and colorectal cancer patients' survival wasmediated byMPOprotein level. Briefly, thefirst assumption of themediationmodelwas applied to evaluate the association betweenrs2333227 CT/TT genotypes and colorectal cancer patients' sur-vival. The second assumption of the mediation model wasapplied to assess the association between rs2333227 CT/TT gen-otypes andMPOprotein levels. For the third assumption, the Coxregression analysis was performed to test the association betweenlevels ofMPOprotein and colorectal cancer patients' survival. Thestatistical significance criterion was set with a two-sided P < 0.05.All the statistical analyses were performed using SAS version 9.1.3(SAS Institute).

ResultsSubject characteristics

The detailed information on the subjects has been shownpreviously (19). Briefly, the age and sex distribution of colorectalcancer patients and controls were similar. More smokers and

Table 1. Association between the MPO polymorphisms and risk of colorectal cancer

Cases ControlsSNPs Genotype n (%) n (%) P Adjusted OR (95% CI)a

rs2243828 AA 714 (66.2) 787 (67) 0.3981 1.00 (ref)AG 308 (28.6) 341 (29) 0.99 (0.82–1.19)GG 56 (5.2) 47 (4) 1.33 (0.89–1.99)HWE 0.0034 0.1924Ptrend 0.4212

rs2333227 CC 746 (69.2) 875 (74.5) 0.0066 1.00 (ref)CT 300 (27.8) 281 (23.9) 1.27 (1.05–1.53)TT 32 (3) 19 (1.6) 2.00 (1.12–3.56)CC 746 (69.2) 875 (74.5) 0.0054 1.00 (ref)CT/TT 332 (30.8) 300 (25.5) 1.31 (1.09–1.58)T allele 0.1688 0.1357 0.002HWE 0.7824 0.5097Ptrend 0.002

aAdjusted for age, gender, smoking, and drinking status.

Table 2. Association between MPO rs2333227 polymorphisms and overall survival of the colorectal cancer patients

Genotype Patients (n) Deaths (n) MST Log-rank P Adjusted HRa (95% CI)

CC 746 427 79.5 0.0026 1.00 (ref)CT 300 194 59.4 1.18 (0.99–1.40)TT 32 23 58.4 1.28 (0.84–1.96)CT/TT 332 217 59.3 0.0006 1.19 (1.01–1.40)aAdjusted for age, gender, smoke and drink status, location, grade, and TNM.

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Figure 1.

MPO mediated the relationship between rs2333227 C>T polymorphism and colorectal cancer patients' survival. A, Schematic of the study. B, MPO rs2333227genotypes and colorectal cancer survival in a dominant genetic model in all patients. C, Protein expression of MPO obtained by IHC assay in human colorectal cancerand adjacent tissues TMA. D, The distribution of MPO protein expression of human colorectal cancer and adjacent tissues TMA in patients carrying threers2333227 genotypes. P values were calculated with theWilcoxon test. E,MPO rs2333227 genotypes and protein expression of MPO in human colorectal cancer andadjacent tissues TMA. �� , P < 0.001; NS, no significance. F, Schematic of mediation model for rs2333227 C>T polymorphism, MPO protein level, and patientswith colorectal cancer survival. G, MPO rs2333227 genotypes and colorectal cancer survival in a dominant genetic model (log-rank P < 0.001). H, Therelationship between MPO protein expression and patients with colorectal cancer survival (log-rank P < 0.001). Kaplan-Meier analysis was calculated with1,005 patients with colorectal cancer.






drinkers were observed in colorectal cancer patients than thecontrols (P¼ 0.0116 and 0.0347 for smoking and drinking status,respectively).

Association between MPO polymorphism and risk ofcolorectal cancer

The genotype distribution of MPO rs2243828 and rs2333227among the controls was consistent with HWE (P ¼ 0.1924 forrs2243828, P ¼ 0.5097 for rs2333227). As shown in Table 1,frequencies of MPO rs2243828 genotypes were not significantlydifferent between the colorectal cancer patients and controls (P¼0.3981). However, the frequency distributions of rs2333227genotypes were significantly different between colorectal cancerpatients and controls (P ¼ 0.0066). When the rs2333227 CCgenotype was used as a reference, the heterozygous CT and TTgenotypes imparted increased risk of colorectal cancer (adjustedOR ¼ 1.27; 95% CI, 1.05–1.53 for the CT genotype; 2.00, 1.12–3.56 for the TT genotype), and the increased risk did not changesubstantially in a dominant genetic model (adjusted OR ¼ 1.31;95% CI, 1.09–1.58).

The stratification analysis revealed that rs2333227 CT/TTimparted a higher risk for colorectal cancer, especially in sub-groups of age (>56 years), female, colon cancer, and tumor–node—metastasis (TNM) stages (III–IV; adjusted OR ¼ 1.42;95% CI, 1.09–1.85 for age > 56 years; 1.53, 1.15–2.04 forfemale; 1.64, 1.30–2.08 for patients with colon cancer; 1.44,

1.11–1.87 for patients with TNM stage III tumors; 2.49, 1.70–3.66 for patients with TNM stage IV tumors), compared withcolorectal cancer patients with rs2333227 CC genotype (Sup-plementary Table S1).

Association between rs2333227 and colorectal cancer survivalAs shown in Table 2 and Fig. 1A and B, Kaplan–Meier

survival curves were used to assess the association betweencolorectal cancer survival and rs2333227 genotype. In theoverall model, the MST of colorectal cancer patients with MPOrs2333227 CC genotype was 79.5 months, and 59.4 and 58.4months for the CT and TT genotypes, respectively (log-rank P ¼0.0026). Compared with colorectal cancer patients withrs2333227 CC genotype, those with rs2333227 CT/TT had ashortened survival time in the dominant model (MST ¼ 79.5and 59.3 months, respectively, log-rank P ¼ 0.0006). Althoughprevious studies noted that SNP rs2243828 was positivelyassociated with other cancers (8, 16), we found that the SNPrs2243828 does not contribute to the clinical occurrence ofcolorectal cancer.

In the stratification analysis (Supplementary Table S2), wefound that the CT/TT genotypes were significantly associated witha decreased survival time in the subgroup of age >56 years, femalegender, never drink, and smoke (adjusted HR ¼ 1.33; 95% CI,1.05–1.67; adjusted HR ¼ 1.61; 95% CI, 1.24–2.10, adjustedHR ¼ 1.24; 95% CI, 1.01–1.53; adjusted HR ¼ 1.25; 95%

Figure 2.

AP-2a promoted tumor MPO transcription by binding to the rs2333227 T genotype in colorectal cancer patients. A–C, The binding affinity between AP-2aand the indicated MPO rs2333227 genotypes were determined by ChIP assay in colorectal cancer. Human peripheral white blood (A), SW620 cells (B), andSW480 cells (C). D, MPO mRNA levels in colorectal cancer tissues expressing CC or CT/TT genotype were detected by qPCR analysis. E, MPO protein levels incolorectal cancer tissues expressing CC, CT, or TT genotype were detected by immunoblotting. �� , P < 0.001.

Meng et al.





CI, 1.01–1.54, respectively). We also found a similar result in thesubgroups of intermediate/high grade and TNM stage III (adjust-ed HR ¼ 1.25; 95% CI, 1.02–1.54; adjusted HR ¼ 1.29; 95% CI,1.01–1.65, respectively).

We then performed a Cox stepwise regression analysis toevaluate the effects of selected demographic, clinical information,and the rs2333227 variant on colorectal cancer survival. As aresult, four variables (age, drinking status, TNM stage, andrs2333227 dominant model) were included in the model (P <0.0001 for age, P < 0.0001 for drinking status, P < 0.0001 for TNMstage, and P ¼ 0.013 for rs2333227 dominant model; Supple-mentary Table S3).

MPO rs2333227 genotype and MPO expression in colorectalcancer

To further determine whether the MPO rs2333227 polymor-phism can affect its protein expression, TMAs from each geno-type were collected for IHC analysis. IHC assay revealed that

MPO protein expression levels were upregulated in the cancertissue of colorectal cancer patients as well as patients with theMPO rs2333227 CT/TT genotype (Fig. 1C–E).

Next, we used the mediation model to explore whetherMPO mediated the relationship between rs2333227 C>T poly-morphism and colorectal cancer patients' survival. We foundthat the levels of MPO protein were significantly upregulatedin the patients carrying the CT/TT genotypes comparedwith those with the CC genotype (by the logistic regressionanalysis; adjusted OR ¼ 2.74; 95% CI, 2.00–3.75; Fig. 1Eand F), thus supporting the second assumption of the medi-ation model. Moreover, the first assumption of the mediationmodel showed the CT/TT genotypes significantly decreasedthe colorectal cancer patients' survival time when comparedwith those with the CC genotype (adjusted HR ¼ 1.21;95% CI, 1.01–1.44; Fig. 1F and G). Furthermore, Cox regres-sion analysis indicated that high MPO expressions was asso-ciated with shorter survival time (adjusted HR ¼ 3.36; 95% CI,

Figure 3.

MPO rs2333227 T genotype promotes in vitro and in vivo cell proliferation, migration, and invasion of human colorectal cancer cell lines. A and B, Representation ofcolony formation images and colony formation counting are shown [SW620 (A) and SW480 (B)]. C and D, Transwell assay performed to determine themigration and invasion of cell lines SW620 (C) and SW480 (D). E and F, Flank tumor, liver, and lung metastatic burden in nude mice (n ¼ 6 for each group)injected with colorectal cancer cell lines with rs2333227 CC, CT, and TT genotypes, measured by luciferase activity. � , P < 0.05; ��, P < 0.01; �� , P < 0.001 .






2.77–4.07; Fig. 1F–H), confirming the third assumption ofthe mediation model. Moreover, the relationship betweenrs2333227 C>T polymorphism and colorectal cancer patients'survival became insignificant once the MPO was included inthe model (adjusted HR ¼ 0.89; 95% CI, 0.74–1.06; Fig. 1F).

MPO rs2333227 T genotype recruited AP-2a to bind onthe -463 region

According to the JASPAR CORE database andGene Regulation,the MPO rs2333227 SNP site, located at the �463 promoter, iscontained in the AP-2a–binding motif. Accordingly, we hypoth-esized that the MPO rs2333227 variant might affect the bindingaffinity of AP-2a to the MPO rs2333227 mutation region. Thus,the efficacy binding affinity of AP-2a to the -463 site was assessedby ChIP analysis using colorectal cancer human peripheral whiteblood. Results showed that the rs2333227 CT/TT genotype pro-moted AP-2a binding to theMPO promoter, but the CC genotype

did not in colorectal cancer human peripheral white blood(Fig. 2A). Similar results were also confirmed in SW620 andSW480 cells with the indicated genotypes (Fig. 2B and C; threeseparate colonies with rs2333227 CT/TT genotype). To furtherdetermine whether theMPO rs2333227 polymorphism can affectMPO transcriptional activity, the MPO mRNA level of colorectalcancer patients was determined. Results showed that individualswith mutant CT/TT genotypes at the rs2333227 had significantlyhigherMPO levels than thosewith the CC genotype (Fig. 2D). TheMPO protein expression levels of colorectal cancer patients withthe CT/TT genotype were significantly higher than those with theCC genotype (Fig. 2E). Moreover, the serum MPO levels ofcolorectal cancer patients with the CT/TT genotype were statisti-cally significantly higher than those with the CC genotype (Sup-plementary Table S4). Thus, we posit that higher AP-2a–bindingefficiency is driving increased MPO protein expression in thers2333227 genotypes.

Figure 4.

IL23A and MMP9 mediated the relationship between MPO and development of colorectal cancer. A, Protein interaction network for genes regulated by MPOanalyzed by STRING (version 10.0). B, Correlation analysis between mRNA expression levels of MPO and IL23A by qRT-PCR. C, Correlation analysisbetween mRNA expression levels of MPO and MMP9 by qRT-PCR. D, Protein expression of MMP9 obtained by IHC assay in human colorectal cancerand adjacent tissues TMA. E, MPO protein expressions of human colorectal cancer and adjacent tissues TMA in patients carrying three rs2333227genotypes were determined. P values were calculated with the Wilcoxon test. F, MPO rs2333227 genotypes and protein expression of MMP9 inhuman colorectal cancer and adjacent tissues TMA. ��, P < 0.001; NS, no significance. G, The relationship between MPO and MMP9 protein expressionand colorectal cancer patients' survival (log-rank P < 0.001). Kaplan–Meier analysis was calculated in a sample of 915 patients with colorectal cancer.H, Time-dependent ROC analysis for the clinical risk score (TNM, grade), MPO, MMP9, or the combined MPO, MMP9, and the clinical risk score(TNM, grade).

Meng et al.





MPO rs2333227 TT genotype promotes MPO expression andcolorectal cancer cell proliferation, invasion, and migration

The SNPs rs2333227 (G-463A) and rs2243828 (T-764C),located in the 50 upstream region of MPO gene, are functionalSNPs, as reported in various diseases (10, 11).

We hypothesized that the MPO-mutant rs2333227 genotypemight alter colorectal cancer cell invasion and migration. Ourresults indicated that colony formation in both SW620 andSW480 cells were markedly enhanced by the rs2333227 CT/TTgenotype compared with the CC genotype (Fig. 3A and B). TheMPO rs2333227 TT genotype significantly promoted the migra-tion and invasion of SW480 and SW620 colorectal cancer celllines (Fig. 3C and D).

Next, we injected the colorectal cancer cell lines (SW480 andSW620 with rs2333227 CC, CT, and TT genotype) into thedorsal flanks of nude mice. The results showed that the growthrate of xenograft colorectal cancer cells was significantly higherwith the rs2333227 CT/TT genotype than CC genotype (Fig. 3Eand F).

IL23A andMMP9mediated the relationship between MPO anddevelopment of colorectal cancer

To further explore the potential mechanism underlying thebiological effects of MPO in colorectal cancer development, weconstructed a protein interaction network for genes regulated byMPO using STRING version 10.0 (search tool for the retrieval ofinteracting genes; Fig. 4A). The correlations betweenmRNA levelsof MPO and key components in the predicted network werevalidated in colorectal cancer tissues (Fig. 4B; Supplementary Fig.S2A–S2I). Among all the components, IL23A transcriptionallevels exhibited the strongest correlation with MPO, especiallyin patients carrying with CT/TT genotype (r ¼ 0.8560 for CT/TTand r ¼ 0.5362 for CC, respectively; Fig. 4B). Given that IL23upregulates the matrix metalloproteinase 9 (MMP9) expression(20), we next evaluated themRNA and protein levels of MMP9 incolorectal cancer patients. We found a significantly positive cor-relation between MPO and MMP9 mRNA expression levels (r ¼0.5163, P <0.001, Fig. 4C). Moreover, the correlation in patientswith CT/TT genotypes was stronger than with the CC genotype(r ¼ 0.7689 and r ¼ 0.1563, respectively), corroborating the CT/TT genotypes might be a higher risk factors for colorectal cancermetastasis (Fig. 4C). Similar to our finding in MPO, MMP9 wereupregulated in colorectal cancer patients' cancer tissues andpatients carrying the MPO rs2333227 CT/TT genotypes (Fig.4D–F.). Furthermore, colorectal cancer cells carrying the CT/TTgenotypes also showed increased mRNA levels of MPO, IL23A,and MMP9 compared with the CC genotype (SupplementaryFig. S3A–S3F). The trend for increased MPO and IL23 proteinlevels was also consistent with the mRNA levels in the cell culturesupernatants assayed by ELISA (Supplementary Fig. S3G–S3J.).Kaplan-Meier analysis showed that both high expressions of thosetwo proteins showed worsening outcome for survival comparedwith the other groups (one high or both low; Fig. 4G.). Further-more, multivariate Cox regression analysis indicated that highMPO and MMP9 expressions were independent prognostic mar-kers for colorectal cancer (Table 3). We then performed a time-dependent ROC analysis to evaluate the prognostic efficacy ofMPO and MMP9 expressions on colorectal cancer patients. Wefound that the combination of the clinical variables, MPO, andMMP9 risk score contributed more than either one of these alone(Fig. 4H). Ta

ble

3.MultivariateCoxregressionan

alysisofMPO

andMMP9expressionan

dselected

clinical

patho

logic

variab

lesin

colorectal

cancer

survival

MPOa

MMP9a

Combined

a,b

Combined

a,c

Variables

Adjusted

HR(95%

CI)

PAdjusted

HR(95%

CI)

PAdjusted

HR(95%

CI)

PAdjusted

HR(95%

CI)

P

Age(>56

vs.�

56)

1.41(1.18

–1.67)

0.0001

1.47(1.24–1.75)

<0.0001

1.39(1.17

–1.65)

0.0002

1.52(1.24–1.85)

<0.0001

Gen

der

(fem

ales

vs.m

ales)

0.86(0.71–1.0

3)0.0975

0.82(0.68–0

.98)

0.028

40.80(0.67–

0.96)

0.019

0.89(0.72–

1.10)

0.2861

Smoke

(eve

rvs.n

ever)

1.04(0.83–

1.30)

0.753

80.94(0.76–1.18

)0.5959

0.97(0.78–1.21)

0.772

81.11(0.85–

1.45)

0.450

3Drink

(eve

rvs.n

ever)

1.16(0.91–1.4

6)

0.2312

1.19(0.94–1.50)

0.14

101.17(0.93–

1.47)

0.18

191.0

5(0.79–1.39)

0.732

7Lo

cation(rectum

vs.colon)

0.89(0.75–

1.06)

0.2060

0.90(0.76–1.07)

0.230

70.86(0.72–

1.03)

0.0935

0.95(0.78–1.17

)0.6402

Grade(intermed

iate/highvs.low)

0.95(0.80–1.14

)0.5813

0.93(0.78–1.12

)0.4515

0.98(0.82–

1.17)

0.832

0.94(0.76–1.16

)0.5652

TNM(3/4

vs.1/2)

5.17

(4.24–6

.29)

<0.0001

6.10

(5.04–7

.39)

<0.0001

5.63(4.64–6

.84)

<0.0001

5.71

(4.55–

7.18)

<0.0001

Protein

(highvs.low)

3.48(2.89–4

.21)

<0.0001

2.23

(1.88–2.64)

<0.0001

2.91(2.40–3

.53)

<0.0001

4.04(3.25–

5.03)

<0.0001

aMultivariateCoxregressionan

alyses

wereperform

edwith915

subjectsbecau

seofloss

ofsample

duringMPO

andMMP9an

tigen

retrieval.

bMultivariateCoxregressionan

alyses

werecalculated

withfollo

wingproteinsexpressionstatus

(one

highvs.b

oth

low).

c MultivariateCoxregressionan

alyses

werecalculated

withfollo

wingproteinsexpressionstatus

(both

highvs.b

oth

low).






DiscussionIn the current study, we explored the role of promoter MPO

SNPs in susceptibility and development of colorectal cancer. Byconducting a hospital-based case–control study, we found thatsubjects carrying rs2333227 CT/TT genotypes had a significantlyhigher risk for colorectal cancer and poorer survival outcomeswhen compared with the CC genotype. With in vivo and in vitroassays,wedemonstrated that transcription factor AP-2apresentedstronger binding affinitywith rs2333227CT/TT genotype thanCCgenotype, thus upregulating MPO expressions, and ultimatelypromoting colorectal cancer proliferation and metastasis. There-fore, our results verified the critical role ofMPO functional variant(rs2333227) in colorectal cancer.

MPO catalyzesoxidants generation, thus contributing to the riskof cancer (21). It has been reported thatMPO rs2333227 T allelealters the susceptibility of breast (12) and prostate cancer (14).However, few studies were paid attention to the role of MPOrs2333227 in colorectal cancer. In our cohort, we demonstratedthat rs2333227 TT genotype was a risk factor for colorectal cancerand associated with worse clinical features, suggesting thatdifferent clinical features of colorectal cancer might be regulatedby different molecular mechanisms. Interestingly, we found thatage, drinking status, TNM stage, and rs2333227 dominant modelwereindependentfactorsaffectingcolorectalcancerinourstepwiseCox regression analysis. Taken together, our results have verifiedthe oncogenic role of rs2333227 TT genotype in colorectal cancer.

Transcription factor AP-2a promotes the expressions of targetgenes (such as DEK; ref. 22) through directly binding to thepromoter region. By conducting the ChIP assay, we demonstratedthat AP-2a preferred to binding with MPO rs2333227 CT/TTgenotype. As colorectal cancer tissues, serum and cells carryingCT/TT genotype exhibited much higher MPO protein levels, wespeculated that AP-2a was responsible for rs2333227 CT/TTgenotype–induced higher transcriptional activity. As few reportswere focused on AP-2a/rs2333227 T allele axis, further researchesare still needed to confirm our findings.

It has been reported that MPO plays an important role incellular processes, such as DNA damage, oxidative stress (23),and innate immune response (24). Its major function is tomediate hypochlorous acid (HOCl) generation (25). However,HOCl and other oxidants derived byMPO are considered impor-tant mediators of oxidative damage to cellular biomolecules (26)in that they may damage the structure of host tissues (27).Moreover, HOCl can decrease the adhesiveness of extracellularmatrix proteins and affect endothelial function (11), destabilizingthe tissue environment surrounding endothelial cells (28). Thissuggests that overexpressed MPO in human tumors may beassociated with enhanced cell invasion andmigration. Moreover,we found that MPO protein expression levels were upregulated inthe cancer tissue of colorectal cancer patients, which corroboratesthat MPO could be an oncogene in colorectal cancer.

To further ascertain the downregulators of MPO that driveaggressive phenotype, protein interaction network were con-structed (29, 30). Among the 10 candidates, IL23A showed thestrongest correlation with MPO levels, especially in patientscarrying TT genotype. Interestingly, Lan and colleagues showedthat IL23A was upregulated in the colorectal cancer tissues (31).Previous studies have showed that IL23A promotes tumor inci-dence and growth by targetingMMP9 (20). In our study, we againdemonstrated that MMP9 was not only the downregulator ofIL23A, but also synergistically predicted colorectal cancer patients'prognosis with MPO. Taken together, our results indicated thatMPO/IL23A/MMP9 axis might promote colorectal cancer pro-gression and metastasis.

In summary, our results revealed that functional rs2333227 TTgenotype upregulatedMPO expressions in colorectal cancer, thusactivating IL23A/MMP9 signaling–medicated tumor growth anddevelopment. However, further studies are still required to con-firm our findings.

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

Authors' ContributionsConception and design: M. Aschner, R. ChenDevelopment of methodology: R. ChenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Wang, H. Sun, R. Lu, X. LiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Q. Meng, S. Wu, J. Xu, N. Gao, M. AschnerWriting, review, and/or revision of the manuscript:Q. Meng, S. Wu, X. Li, M.Aschner, R. ChenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. Yang, B. Tang, R. ChenStudy supervision: R. Chen

AcknowledgmentsThisworkwasfinancially supported byNationalNatural Science Foundation

of China (81472938 and 91643109 to R. Chen; 81703261 to J. Xu), theNational Key Research andDevelopment Program of China (2017YFC0211600and 2017YFC0211603 to R. Chen), the Natural Science Foundation of JiangsuProvince (BK20151418 to X. Li; BK20171060 to J. Xu), the Fund of theDistinguished Professor of Jiangsu Province to R. Chen, the Fund of theDistinguished Talents of Jiangsu Province to R. Chen (BK20150021), the Sixtalent peaks project in JiangsuProvince toR.Chen(2016-WSN-002), theNaturalScience Foundation of Jiangsu Higher Education Institutions to J. Xu(17KJB330002), the Fund of the Post-graduate Innovative Talents to Q. Meng(KYZZ16_0137), the Fundamental Research Funds for the Central UniversitiestoR.Chen andX. Li, and theNational Institute of Environmental health Sciences(NIEHS; R01 ES10563, R01 ES07331, and R01 ES020852 to M. Aschner).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 25, 2017; revised December 13, 2017; accepted March 9,2018; published first March 14, 2018.

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2018;78:2760-2769. Published OnlineFirst March 14, 2018.Cancer Res Qingtao Meng, Shenshen Wu, Yajie Wang, et al.

αAP-2Phenotypes of Colorectal Cancer by Altering the Binding Affinity of

Promoter Polymorphism rs2333227 Enhances MalignantMPO

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