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Molecular and Cellular Pathobiology

Mycoplasma Hyorhinis Infection PromotesNF-kB–Dependent Migration of Gastric Cancer Cells

Hongying Duan1, Ling Chen1, Like Qu1, Hua Yang1, Sonya Wei Song1, Yong Han1, Meihua Ye2,Wanyuan Chen2, Xianglei He2, and Chengchao Shou1

AbstractChronic infection of Mycoplasma hyorhinis (M. hyorhinis) has been postulated to be associated with several

types of cancer, but its effect on patients' survival and host factors mediating its infection remain unclear. Herein,we demonstrated that M. hyorhinis p37 protein expression in gastric cancer tissues predicts poor survival andassociateswithmetastasis.M. hyorhinis infectsmammalian cells andpromotes gastric cancer cell invasiveness viaits membrane protein p37. Synthesized peptide corresponding to the N-terminus of p37 prevents M. hyorhinisinfection. Host AnnexinA2 (ANXA2) interactswith theN-terminus of p37. In addition, EGFR forms a complexwithp37 andANXA2, and is required forM. hyorhinis–induced phosphorylation andmembrane recruitment of ANXA2.M. hyorhinis infection is inhibited by siRNA-mediated knockdown of ANXA2 or EGFR, but is enhanced byexpression of ectopic ANXA2 or EGFR. Downstream of ANXA2 and EGFR, the NF-kB pathway is activated andmediatesM. hyorhinis–driven cellmigration. In conclusion, our study unveils the effect ofM. hyorhinis infection ongastric cancer survival and uncovers the mechanisms by which M. hyorhinis infects mammalian cells andpromotes cancer cell migration. Cancer Res; 74(20); 5782–94. �2014 AACR.

IntroductionIt has been reported thatmore than 16% of new cancer cases

worldwide are attributable to infection (1). The associationbetween Helicobacter pylori and gastric cancer suggests thattumor formation could be initiated by persistent infection ofthe pathogenic microbes (2). Several other infectious organ-isms had been investigated for their roles in tumor develop-ment, including mycoplasmas (3, 4).

To date, at least 16 mycoplasma species have been isolatedfrom human (3). Mycoplasma hyorhinis (M. hyorhinis), firstisolated from the respiratory tracts of pigs (5), can causepolyserositis and arthritis in piglets (6). In addition,M. hyorhi-nis is one of common contaminants of mammalian cells incultures, which affects the host metabolic pathways (7, 8).Several studies, including ours, have revealed an associationbetween M. hyorhinis infection and malignancy (9–15). By the

ELISA method, M. hyorhinis lipoprotein p37 was shown to bepositive in sera of 36% men with benign prostatic hyperplasiaand 52% with prostate cancer (10). We used a monoclonalantibody (mAb) PD4 against p37 to examine M. hyorhinisinfection and found that the positive rate of M. hyorhinis ingastric cancer was higher than those in chronic superficialgastritis and gastric ulcer (12). However, the prevalence ofM. hyorhinis infection in the general population is unknown.In addition, the effect of M. hyorhinis infection on patients'survival remains unclear.

M. hyorhinis could induce cell transformation (13) andincreased the invasiveness of gastric cancer cells (11). It alsopromoted melanoma cell invasiveness (14). The percentage ofCD133þ cells in human colorectal cancer cell lines was influ-enced byM. hyorhinis infection (15), indicating thatM. hyorhi-nis affects the stemness of cancer cells.

Early studies demonstrated p37 as a major component ofthe high-affinity transport system of M. hyorhinis (16, 17),whereas biochemical characterization of this protein is stillinsufficient. Full-length p37 has a signal peptide of 23 aminoacids (16, 17), which was found to be peculiar because of fourphenylalanine residues and an untypical cleavage site (17).Following the cleavage site, the C-S-N motif fits the con-sensus sequence of bacterial lipoprotein and was thought toplay a role in the membrane recruitment of M. hyorhinis(16, 17). p37 shares partial homology to the hemagglutininprotein of influenza A, a sialic acid–binding protein criticalfor viral entry (18), but p37 has no homology to mammalianproteins. Proinvasive function of p37 has been documented(18, 19), but the role of p37 in M. hyorhinis infection andmechanisms underlying M. hyorhinis– and p37-inducedmalignant phenotypes are unknown.

1Key Laboratory of Carcinogenesis and Translational Research (Ministry ofEducation), Department of Biochemistry and Molecular Biology, PekingUniversity Cancer Hospital and Institute, Beijing, China. 2Department ofPathology, Zhejiang Provincial People's Hospital, Hangzhou, Zhejiang,China.

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

Corresponding Authors: Chengchao Shou, Department of BiochemistryandMolecular Biology, Peking University Cancer Hospital and Institute, 52Fucheng Road, Beijing 100142, China. Phone: 0086-10-88196766; Fax:0086-10-88122437; E-mail: [email protected]; and Xianglei He,Department of Pathology, Zhejiang Provincial People's Hospital, Hang-zhou, Zhejiang 310014, China, Phone: 0086-571-85893833; Fax: 0086-571-85893288, E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-0650

�2014 American Association for Cancer Research.

CancerResearch

Cancer Res; 74(20) October 15, 20145782

on September 30, 2020. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst August 18, 2014; DOI: 10.1158/0008-5472.CAN-14-0650

http://cancerres.aacrjournals.org/

A D

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Gender Male 253 105 (41.5) 0.610 1

Female 86 33 (38.4) 1.139

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59 178 67 (37.6) 0.208 1

>59 160 71 (44.4) 1.322

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1353.0)5.83(472914

>4 147 64 (43.5) 1.230

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I/II 154 58 (37.7) 0.521 1

III/IV 185 76 (41.1) 4.937

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p 1 0.952 (40.9)54132T1 and pT2

pT3 207 84 (40.6) 0.986

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1 0.831 (41.6)42101WD and MD

96 (40.3)238 PD 0.950

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1 0.974 54 (40.6)133 Negative

84 (40.8)206 Positive 1.007

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1 0.074 39 (33.1)118 Negative

64 (43.8)146 Positive 1.581

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Negative 1 0.021 (35.5)75211

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85 (44.3)192 Positive 1.409

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1 0.029 113 (38.4)294 Negative

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qPCR (−) qPCR (+)n = 41n = 47

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Figure 1. M. hyorhinis infection predicts metastasis and poor survival for patients with gastric cancer. A, representative immunohistochemical staining ofp37 protein by PD4 mAb in human gastric tissues. Original magnification, �400. B, representative immunofluorescence staining of p37 protein by PD4 mAbin human gastric tissues. Sections were counterstained by DAPI. C, comparison of IHC and qPCR results for cohort 2 by the c2 test. D, correlations ofM. hyorhinis infectionwith clinicopathologic factors in cohort 1. E, Kaplan–Meier estimation of overall survival for patients with gastric cancer (cohort 1) by thelog-rank test.

Mycoplasma Hyorhinis Infection and Carcinogenesis

www.aacrjournals.org Cancer Res; 74(20) October 15, 2014 5783




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Figure 2. p37 is required forM. hyorhinis infection ofmammalian cells. A,M. hyorhinis binds toMGC803 and AGS cells in a dose-dependent manner. The cellswere exposed to 103, 104, 105 CCU/mL live or heat-activated M. hyorhinis for 24 h, followed by Cell ELISA with PD4 antibody. B, p37 binds to cancer,immortalized, and primary cells in a dose-dependentmanner. The cells were treated with indicated concentration of GST-p37 for 24 h, followed byCell ELISAwith anti-GST antibody. GST and GST-ricin A chain were used as controls. C, qPCR analysis of M. hyorhinis infection. 5 mg/mL anti-p37 polyclonal orpreimmune IgG was coincubated with 105 CCU/mL live M. hyorhinis for 2 h before adding to cells for another 24 h. Alternatively, 105 CCU/mL live or heat-activated M. hyorhinis was used to treat cells for 24 hours; qPCR was performed to amplify p37 DNA. (Continued on the following page.)

Duan et al.

Cancer Res; 74(20) October 15, 2014 Cancer Research5784




In this study, we demonstrated thatM. hyorhinis infection ingastric cancer tissues predicts metastasis and poor survival.We further uncovered the roles of p37 as well as host AnnexinA2 (ANXA2) and EGFR inM. hyorhinis infection and infection-induced cell migration.

Materials and MethodsSpecimen and immunohistochemistryThe studywith clinical sampleswas approvedby theMedical

Ethic Committee of Zhejiang Provincial People's Hospital. Twocohorts of formalin-fixed and paraffin-embedded (FFPE) gas-tric cancer tissues were obtained from archives of the Depart-ment of Pathology, Zhejiang Provincial People's Hospital(Hangzhou, Zhejiang, China): 339 collected during 1998 to2004 (cohort 1), 88 collected during 2013 to 2014 (cohort 2).Written informed consents were obtained from all patientsbefore operation. Specimens were diagnosed histopathologi-cally and staged according to the TNM International UnionagainstCancer classification system.Data onpatients of cohort1, who had survived until the end of follow-up period, werecensored at the date of last contact and their clinicopathologiccharacteristics were summarized in Fig. 1D. The tissue micro-array was constructed as described previously (20). Immuno-histochemistry (IHC) was performed to detect p37 in bothcohorts with PD4 antibody. The degree of immunostainingwas reviewed and scored independently by two pathologist(W. Chen and X. He) based on the intensity of staining: 0 (nostaining), 1 (weak staining, light yellow), 2 (moderate staining,yellow brown), and 3 (strong staining, brown). Moderate andstrong stainingswere defined asM. hyorhinis–positive, whereasno and weak stainings were defined as negative.

DNA extraction and detection of M. hyorhinis DNA byquantitative PCRDNA was extracted from FFPE tissues (cohort 2) with the

QIAamp DNA FFPE Tissue Kit from Qiagen. Alternatively,DNA was isolated from cells by the standard protocol. Quan-titative PCR (qPCR) was performed with 30 ng (cell) or 100 ng(tissue) DNA using the StepOne system ABI and SYBR GreenReal-Time PCR 2� premixKit (Takara). The reaction programsand p37-specific primers (forward: 50-TATCTCATTGACCTT-GACTAAC-30, reverse: 50-ATTTTCGCCAATAGCATTTG-30)were reported previously (21). Using DNA from M. hyorhi-nis–infected and –uninfected AGS cells as references, clinicalsamples with any recorded threshold cycle number (Ct) valuewere scored as positive. To compareM. hyorhinisDNA levels incells, GAPDH (forward primer: 50-TGAAGGTCGGAGTCAAC-GG-30, reverse primer: 50-CCTGGAAGATGGTGATGGG-30)was amplified as control and the data were analyzed usingthe 2�DDCt method. Primers were synthesized by Sangon.

Cell cultureGastric cancer cell AGS was from the ATCC. Gastric cancer

cell MGC803 and immortalized human gastric epithelia GES-1were kept in Peking University Cancer Hospital and Institute(Beijing, China). Primary human fetal colon fibroblasts CC-18Co was gifted by Drs. Dajun Deng and Baozhen Zhang(Peking University Cancer Hospital and Institute). Humanumbilical vein endothelial cell (HUVEC) was gifted by Dr.Chuanke Zhao (Peking University Cancer Hospital and Insti-tute). Primary mouse embryonic epithelia (MEF) and primarymouse gastric epithelia (PMGE)were gifted byTingMa (PekingUniversity Cancer Hospital and Institute). PMGE was isolatedas previously reported (22). AGS, MGC803, and GES-1 cellswere cultured in RPMI-1640 medium. MEF was cultured inDMEM. HUVEC, CC-18Co, and PMGE were cultured in EBM-2medium. Media were supplemented with 5% to 10% FBS plusantibiotics from Invitrogen. Primary cells were used at passage3 to 5, whereas cell lines were never used beyond passage 20.The mycoplasma test was performed biweekly by qPCR ampli-fication of M. hyorhinis p37, Hochest 33258 staining, and PCRamplification of mycoplasma genome with a kit from HDBiosciences.

M. hyorhinis propagation and infectionM. hyorhinis (ATCC 17981) was grown for 72 hours at 37�C in

a modified Hayflick medium supplemented with 20% heat-inactivated FBS (23). The mycoplasmas were serially passagedfor three times and harvested at the mid-exponential phase ofgrowth by centrifugation for 20minutes at 12,000� g and storedat �80�C. Titer of M. hyorhinis was quantified as color changeunits (CCU) per milliliter (24). Cells at 80% confluence wereserum starved for 24 hours before addition of 105 CCU/mL M.hyorhinis, which was equal to 0.33 to 1 multiplicity of infection.Heat-inactivation of M. hyorhinis was reported previously (25).

Antibodies, reagents, and plasmidsCommercial antibodies were listed in Supplementary

Materials and Methods. Anti-p37 mAb PD4 was generatedand characterized previously (11, 12, 26). Polyclonal anti-p37 antibody was prepared by immunizing rabbit withGST-p37 fusion protein following the standard protocol.Bay 11–7082 was from Sigma-Aldrich. GST-ricin A chainwas gifted by Dr. Xianping Wang (Institute of Biophysics,Chinese Academy of Sciences, Beijing, China). p37-2-23,ANXA2-2-26, and random control peptides were synthe-sized by SBS Bio. pcDNA3.0-ANXA2 was cloned in ourlaboratory. Deletion of N-terminus of ANXA2 was per-formed by PCR. ANXA2 cDNAs (full-length and truncated)were cloned into pET-28a-c and the recombinant proteinswere prepared. pcDNA3.1-EGFR was gifted by Dr. ZhijieChang (Tsinghua University, Beijing, China).

(Continued.) D, cell ELISA analysis of M. hyorhinis infection. Cells were treated as in C with 1, 2, and 5 mg/mL anti-p37 polyclonal antibody or IgG. E,immunofluorescence analysis ofM. hyorhinis infection. AGS cells grown on coverslips were treated as in C with 5 mg/mL anti-p37 antibody or IgG, then werefixed, immunostained with PD4, and counterstained with DAPI. F, flow-cytometry analysis ofM. hyorhinis infection. Gastric cancer cells and primary mousegastric epithelia were treated as in Cwith 5 mg/mL anti-p37 antibody or IgG. Cells were fixed and immunostainedwith PD4. PD4-positive rate (%) was shown.G, heat-inactivation (left two) or anti-p37 (right two) inhibitsM. hyorhinis–induced cell migration. Cells were treated as in C, followed bymigration assay. Data,mean � SD from three to four experiments with triplicate for each sample; �, P < 0.05; ��, P < 0.01; ��, P < 0.001; n.s., no significance.






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Duan et al.





ELISA and solid-phase–binding assayOf note, 96-well plates were seeded with cells (1� 104/well)

or were immobilized with polypeptide for 24 hours, then cellELISA or solid-phase binding was performed as describedpreviously (27).

Flow cytometryCells (1 � 106/10-cm plate) were infected with M. hyorhinis

for 24 hours, harvested, fixed in 2% paraformaldehyde, stainedwith PD4 (2 mg/mL) at 4�C overnight and FITC-conjugatedsecondary antibody for 30 minutes, then were subjected toflow cytometry with BD FACSAria.

Identification of p37-binding proteinsMGC803 cells were homogenized in lysis buffer (50 mmol/L

Tris-HCl pH 8.0, 150 mmol/L NaCl, 1% Triton X-100, 0.5mmol/L DTT, 1 mmol/L PMSF, and 1x complete proteaseinhibitors) for 20 minutes. After centrifugation, supernatantswere incubatedwith 2mg purifiedGST-p37 orGST (glutathioneS–transferase) protein plus 20 mL glutathione sepharosebeads (GE Healthcare) at 4�C overnight. The precipitates wereresolved by SDS-PAGE and stained by Coomassie brilliantblue G-250. Protein bands precipitated by GST-p37 were sub-jected to MALDI-TOF mass spectrometry analysis by theCentral Laboratory of Peking University Cancer Hospital andInstitute.

Cell migration assayCell migration assay was carried out as previously

reported (11).

Immunofluorescence stainingCells grown on the coverslips were fixed with 4% para-

formaldehyde for 30 minutes at 4�C, followed by permeabi-lization with 0.1% Triton X-100 in PBS for 5 minutes andblocked with 5% BSA at room temperature for 1 hour.Antibodies were then applied to the cells overnight at 4�C,followed by probing with secondary antibodies, counter-staining with DAPI, and mounting in 50% glycerol/PBS. Toobserve membrane localization of FITC-p37-2-23 peptide,cells were coincubated with peptide plus DiIC18(3)-DS(Beyotime) for 10 minutes. A Leica SP5 confocal system(Leica) was used to observe the localization of indicatedproteins or peptides. Digital images were processed withAdobe Photoshop CS (Adobe Systems) by adjusting thelinear image intensity display range.

siRNA and plasmids transfectionCells were plated in 6-well plates at 3� 105 cells per well and

transfected with siRNAs plus siRNA mate (both from Gene-

Pharma). The target sequences of siRNAs were listed in Sup-plementary Materials andMethods. Plasmids were transfectedinto cells with Lipofectamine 2000 (Invitrogen).

Statistical analysisData analysis was performed using SPSS 13.0 (SPSS, Inc.). A

standard c2 test was performed to assess the associationbetween p37 and clinicopathologic characteristics. Survivalcurves were estimated using the Kaplan–Meier method andcompared with the log-rank test. A two-tailed Student t testwas used to determine the significance of differences. Valuesrepresent mean � SD. A P value of less than 0.05 was consid-ered statistically significant.

ResultsM. hyorhinis infection predicts metastasis and poorsurvival in patients with gastric cancer

To determine the association betweenM. hyorhinis infectionand clinicopathologic parameters in gastric cancer, we exam-ined the p37 protein expression in two cohorts of samples byIHC. p37 exhibited punctuate distribution in the cytoplasm,especially in the compartment adjacent to cell membrane(Fig. 1A and Supplementary Fig. S1A). Immunofluorescencestaining was also performed in a subset of samples to bettervisualize p37 in tissues (Fig. 1B and Supplementary Fig. S1B). Ofnote, 39.5% (134/339, cohort 1) and 44.3% (39/88, cohort 2)cases were positive for M. hyorhinis in IHC. To validate thereliability of IHC technique, we performed qPCR with DNAextracted from cohort 2 and found a positive rate of 46.5% (41/88). In addition, 31 of 41 of qPCR-positive samples were IHC-positive, whereas 39 of 47 of qPCR-negative samples were IHC-negative (Fig. 1C). Comparison of the results of two techniquesconfirmed the reliability of IHC (Fig. 1C), in which specificitywas 83.0% and the sensitivity was 75.6%. As for cohort 1,although p37 expression exhibited no correlation with gender,age, tumor size, clinical stage, differentiation, or lymph nodemetastasis, significant correlations with blood vessel invasionand metastasis were observed (Fig. 1D). The Kaplan–Meierplotting of cohort 1 showed that patients positive forM. hyorhinis had poorer survival than those negative forM. hyorhinis (Fig. 1E), suggesting that M. hyorhinis infectionmight act as an unfavorable prognostic factor for patients withgastric cancer.

p37 is essential for M. hyorhinis infection and infection-induced cell migration

M. hyorhinis could infect human gastric cancer cells andpromoted cell invasion (11), and purified p37 alone wassufficient to promote cancer cell invasiveness (18, 19).

Figure 3. N-terminal peptide of p37 preventsM. hyorhinis infection. A, N-terminus of p37 binds to gastric cancer cells in a time- and dose-dependent manner.The cells were treated with increasing amount of GST-p37-2-23 or GST-p37-D2-23 for 24 hours, or 300 pmol/L GST-p37-2-23 or GST-p37-D2-23 for 1,6, 12, and 24 hours, followed by Cell ELISA. B, immunofluorescence stainings of FITC-conjugated N-terminal peptide of p37 (green) and DiIC18(3)-DS(red) in AGS cells. FITC-control peptide was used as negative control. C, N-terminal peptide of p37 inhibitsM. hyorhinis infection. p37-2-23 or control peptidewas added in cell medium 1 hour before M. hyorhinis exposure for 32 hours. The infected cells were subjected to qPCR. Data, mean � SD from threeexperiments with triplicate for each sample; �, P < 0.05; ��, P < 0.001; n.s., no significance.






However, the role of p37 in M. hyorhinis infection remainedunclear. We found that live, but not heat-inactivated,M. hyorhinis bound to gastric cancer cells MGC803 and AGSin a time- and dose-dependent manner in the cell ELISA(Fig. 2A and Supplementary Fig. S2A). Purified GST-p37 boundto gastric cancer cells MGC803 and AGS, immortalized gastricepithelia GES-1, and primary cells MEF, PMGE, and CC-18Co;however, GST or GST-ricin A chain, which has similar iso-electric point to p37, had low binding activity (Fig. 2B andSupplementary Fig. S2B), indicating that GST moiety orelectrostatic interaction plays minimal role in the binding ofGST-p37 to cells.M. hyorhinis could infect both cancerous andnoncancerous cells, as detected by qPCR amplification of p37(Fig. 2C), but heat inactivation abolished this potential (Fig.2C). A polyclonal anti-p37 antibody blocked the binding ofM.hyorhinis to gastric cancer cells in qPCR, cell ELISA, andimmunofluorescence staining assays (Fig. 2C–E). Similarly,M. hyorhinis infection of GES-1 and HUVEC cells was blockedby this antibody (Fig. 2C). Results of flow cytometry furthervalidated M. hyorhinis infection and inhibition by anti-p37 inboth cancer cells and primary cells (Fig. 2F and Supplemen-tary Fig. S2C). M. hyorhinis promoted gastric cancer cellmigration in a dose-dependent fashion (Fig. 2G), whereasheat inactivation or anti-p37 decreasedM. hyorhinis–promot-ed cell migration (Fig. 2G). These results indicate that infec-tion of mammalian cells by M. hyorhinis is p37 dependent.

N-terminal peptide of p37 prevents M. hyorhinisinfection

p37 was reported to localize on the surface of mammaliancells (16), but the mechanism is elusive. Its N-terminalregion (amino acids 2–23) was predicted to be hydrophobic(16), and has limited homology to proteins of other myco-plasma species (Supplementary Fig. S3). We found thatGST-p37-2-23 bound to MGC803 and AGS cells, but GST-p37-D2-23 had no binding (Fig. 3A). The immunofluores-cence assays revealed the colocalization between FITC-p37-2-23 and the membrane indicator DiIC18 (3)-DS (Fig. 3B). Inaddition, application of a synthesized p37-2-23 peptideblocked the M. hyorhinis infection of AGS, MGC803, GES-1, and PMGE cells, as evaluated by qPCR (Fig. 3C). Theseresults suggest that p37-mediated M. hyorhinis infectionrequires its N-terminal region.

p37 interacts with ANXA2To identify the host-binding partner(s) of p37, we per-

formed pull-down assay and identified a 36-kD protein pre-cipitated by GST-p37 from MGC803 cell lysates, which wascharacterized as ANXA2 (Fig. 4A). Coimmunoprecipitationassay confirmed the binding of p37 to endogenous ANXA2,but not to annexin family members ANXA1 or ANXA4(Fig. 4B). The immunofluorescence assay further showed thecolocalization of p37 and ANXA2 in M. hyorhinis–infectedAGS cells (Fig. 4C). Using pull-down assays, we found that p37that lacked its N-terminal region failed to precipitate ANXA2,whereas this region alone was sufficient to interact withANXA2 (Fig. 4D), suggesting that the N-terminal region ofp37 mediates p37-ANXA2 interaction.

ANXA2 belongs to the annexin family of calcium-bindingproteins (28). The unique N-terminal domains of annexinsdetermine their distinct functions (28). TheN-terminal domainof ANXA2 (amino acids 2–26) is conserved across vertebratesand mediates its interactions with other proteins (28). Wefound that His-ANXA2 lacking this domain failed to be pre-cipitated by GST-p37 (Fig. 4D). Furthermore, the results ofsolid-phase–binding assay indicated that the N-terminalregion of p37 directly interacted with the N-terminal domainof ANXA2 (Fig. 4E), and p37-2-23 peptide blocked interactionbetween GST-p37 and His-ANXA2 (Fig. 4F).

ANXA2 is essential for M. hyorhinis infection and p37binding to host cells

Next, we found thatM. hyorhinis infection was blocked by ananti-ANXA2 antibody in the immunofluorescence stainingassay and qPCR assay (Fig. 5A and B). Binding of GST-p37 toMGC803 and AGS cells was also reduced by anti-ANXA2antibody (Supplementary Fig. S4A and S4B). Anti-ANXA2antibody antagonized M. hyorhinis– and GST-p37–promotedcell migration (Fig. 5C and Supplementary Fig. S4C). In addi-tion, when ANXA2 was knocked down by siRNAs targeting thenoncoding regions (Fig. 5D), infection of M. hyorhinis andbindings of GST-p37 to cells were lowered (Fig. 5E, Supple-mentary Fig. S4D and S4E). Conversely, expression of ectopicANXA2 potentiated M. hyorhinis infection (Fig. 5F). Moreover,knockdown of ANXA2 inhibited M. hyorhinis–promoted cellmigration (Fig. 5G). These results indicate that ANXA2 is a hostreceptor mediating M. hyorhinis infection.

EGFR facilitates M. hyorhinis infectionTyr23 phosphorylation of ANXA2 is associated with its

membrane localization under stress conditions (29). UponM. hyorhinis infection, Tyr23 phosphorylation of ANXA2 wasupregulated, whereas total ANXA2was unaltered (Fig. 6A). Themembrane ANXA2 was increased and cytosolic ANXA2 wasdecreased when the cells were exposed to M. hyorhinis (Sup-plementary Fig. S5A). Treatment with GST-p37 could induceANXA2 phosphorylation (Supplementary Fig. S5B), suggestingthat p37 is critical for ANXA2 phosphorylation upon M. hyor-hinis infection.

EGFR plays a role in regulating ANXA2 phosphorylation andlocalization (30, 31). By coimmunoprecipitation assay, wefound that p37 interacted with both ANXA2 and EGFR(Fig. 6B). Colocalization of these proteins was observed inM. hyorhinis–infected cells (Fig. 6C). M. hyorhinis infectionpotentiated EGFR–ANXA2 association and EGFR preferential-ly interacted with phosphorylated ANXA2 (Fig. 6D). WhenEGFR was knocked down (Fig. 6E), M. hyorhinis–inducedANXA2 Tyr23 phosphorylation and membrane localization ofANXA2 were decreased (Fig. 6F and Supplementary Fig. S5C).M. hyorhinis infection was decreased by knockdown of EGFR,but was increased by expression of ectopic EGFR (Fig. 6G).Furthermore,M. hyorhinis–promoted migration was inhibitedby knockdown of EGFR (Fig. 6H). Therefore, EGFR couldfacilitate M. hyorhinis infection likely by enhancing ANXA2phosphorylation.

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Figure 4. ANXA2 and p37 interact through their N-terminal regions. A, GST pull-down combinedwithMALDI-TOF spectrometry analysis identifiedAXNA2 as aGST-p37–binding protein (arrow). Sequences of six peptides identified byMALDI-TOFwere shown. B, coimmunoprecipitation assays to validate ANXA2-p37interaction in M. hyorhinis–infected MGC803 cells. HC, IgG heavy chain. C, localizations of p37 (green) and ANXA2 (red) in M. hyorhinis–infected AGScells. Colocalization was shown bymerged signals (yellow). D, left, N-terminal region of p37mediates its interaction with ANXA2. Right, N-terminal domain ofANXA2 mediates its interaction with p37. Purified recombinant proteins were coincubated as indicated. The complexes were precipitated byglutathione beads, followed by Western blot analysis with antibodies against ANXA2 and GST. E, N-terminal region of p37 interacts with N-terminaldomain of ANXA2. N-terminal peptide (ANXA2-2-26) or control peptide was immobilized to the 96-well plate. GST-fusion proteins were coincubated asindicated and binding assay was performed with anti-GST. F, N-terminal region of p37 blocks p37-ANXA2 interaction. His-ANXA2 was immobilized on the96-well plate. Then 10 or 30 mmol/L synthesized p37-2-23 was added, followed by GST-p37 incubation. Control peptide was used as negative control. A andD, red arrowhead, the proteolytic degradation of GST-p37, potentially due to cleavage site in the N-terminal region of p37 (shown in Supplementary Fig. S3).Data, mean � SD from three experiments with triplicate for each sample; ��, P < 0.01; ��, P < 0.001; n.s., no significance.






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Figure 5. ANXA2mediatesM. hyorhinis infection. A, immunofluorescence analysis of anti-ANXA2 effect onM. hyorhinis infection. Anti-ANXA2 or IgG (5 mg/mL)was added in cell medium for 1 hour, followed by M. hyorhinis exposure for 24 hours. Cells were fixed and immunostained with PD4. B, qPCR analysisof anti-ANXA2’s effect on M. hyorhinis infection. Cells were treated as in A and DNA was subjected to qPCR. C, anti-ANXA2 antibody inhibitsM. hyorhinis–induced cell migration. D, knockdown efficiency of ANXA2 after transient transfection of 50 nmol/L indicated siRNAs for 48 hours.E, ANXA2 knockdown reduces M. hyorhinis infection. Twenty-four hours after transfection with indicated siRNAs, cells were serum starved for 24 hours,followed byM. hyorhinis infection for another 24 hours. Then DNA was subjected to qPCR. F, expression of ectopic ANXA2 promotesM. hyorhinis infection.MGC803 cells were transfected with 50 nmol/L siRNAs for 24 hours, followed by transfection with indicated plasmids (1–2 mg) for 24 hours, serumstarvation for 24 hours, and M. hyorhinis infection for 24 hours. Expression of endogenous ANXA2 and ectopic myc-ANXA2 were analyzed. M. hyorhinisinfection was examined by qPCR. Arrowhead, nonspecific bands. G, ANXA2 knockdown abolishes M. hyorhinis–induced cell migration. Cells were treatedas in E, followedbymigration assay. Data,mean�SD from two to three experimentswith triplicate for each sample; �,P < 0.05; ��,P <0.01; ��,P < 0.001; n.s.,no significance.

Duan et al.





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Figure 6. EGFR facilitatesM. hyorhinis infection. A, ANXA2 phosphorylation inM. hyorhinis–infected cells. B, p37 interacts with both ANXA2 and EGFR inM. hyorhinis–infected MGC803 cells. C, localizations of p37 (green), ANXA2 (red), and EGFR (blue) inM. hyorhinis–infected cells. Colocalization of threeproteins was shown in merge (white). D,M. hyorhinis infection increases ANXA2–EGFR interaction. E, validation of knockdown efficiency of EGFR aftertransfection with 50 nmol/L indicated siRNAs for 48 hours. Of note, #1–#3, targeting coding regions; #4–#6, targeting noncoding regions. F, EGFRknockdown abolishes M. hyorhinis–induced ANXA2 phosphorylation. AGS cells were transfected with siRNAs for 24 hours, followed by serumstarvation for 24 hours andM. hyorhinis infection for 24 hours. G, EGFR knockdown decreases M. hyorhinis infection, whereas ectopic EGFR increasesM. hyorhinis infection. MGC803 cells were transfected with 50 nmol/L siRNAs for 24 hours, followed by transfection with indicated plasmids (2 mg)for 24 hours, serum starvation for 24 hours, and M. hyorhinis infection for 24 hours. H, EGFR knockdown abolishes M. hyorhinis–induced cellmigration. Cells were treated as in G, followed by migration assay. Data, mean � SD from three experiments with triplicate for each sample; ��, P < 0.01;��, P < 0.001.






M. hyorhinis infection activates NF-kB signaling topromote cell migration

To explore the mechanisms ofM. hyorhinis–promoted inva-siveness, we performed microarray analysis and the NF-kBpathway was predicted to be activated in the infected cells(Supplementary Fig. S6A and S6B). Quantitative RT-PCR anal-ysis showed increased expression in 5 of 6 of NF-kB targetgenes (IKBA, COX2, MMP1, PRDM1, SOCS2, MAP2K1) byM. hyorhinis infection (Fig. 7A). Consistently, S536 phosphor-ylation of NF-kB p65 was upregulated and p65 tended to beaccumulated in the nuclei of M. hyorhinis–infected cells, sup-porting the activation of NF-kB (Fig. 7B and C). Paralleled withenhanced p65 phosphorylation, EGFR phosphorylation waselevated by M. hyorhinis infection (Fig. 7B). When ANXA2 orEGFR was knocked by siRNA, M. hyorhinis–induced Ser536phosphorylation of p65 was attenuated (Fig. 7D), implying thatNF-kB signaling is downstream of ANXA2 and EGFR in thecontext of M. hyorhinis infection. When cells were pretreatedwith the NF-kB signaling inhibitor Bay 11–7082,M. hyorhinis–and p37-induced cell migrations were significantly inhibited(Fig. 7E), suggesting that activated NF-kB is responsible forM. hyorhinis–induced cell invasiveness.

DiscussionIn this study, we revealed that M. hyorhinis infection corre-

lated withmetastasis and poor survival of patients with gastriccancer in cohort 1, confirming the previous proposal thatM. hyorhinis could be a risk factor for gastric cancer (9–15)We used both IHC and qPCR techniques to detectM. hyorhinisinfection in FFPE gastric cancer tissues. By using qPCR andfreshly isolated DNA samples, M. hominis infection wasshown to be associated with prostate cancer (32). However,extracting mycoplasma DNA from formalin-fixed tissues ischallenging. There would be a quick decline in the success ofPCR for large fragment after storage in FFPE after 1 to 2 years(33). Moreover, formalin can introduce damages to AT-richregions of DNA (34), whereas M. hyorhinis genome has an A-Tcontent of more than 74% (35). For these reasons, it is hard tovalidate the IHC results by qPCR for samples of cohort 1, whichwere collected 10 to 16 years ago. This issue was resolved byusing cohort 2, which was collected for less than 1 year.Statistical analysis of M. hyorhinis–positive rate and clinico-pathologic factors indicated that results calculated from IHCof cohort 1 were consistent with those from qPCR of cohort 2and IHC of cohort 2. By this approach, we indirectly validated

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Figure7. M. hyorhinis–activated NF-kB signaling contributes to cell migration. A, qRT-PCR analysis of six NF-kB p65 downstream genes inM. hyorhinis–infected cells. B, phosphorylations of EGFR and p65 were induced by M. hyorhinis infection. C, distribution of p65 in cytoplasma and nuclearfractionations before and after M. hyorhinis infection. Qualities of extracts were validated with antibodies against b-tubulin and Histone 2B. D,knockdown of EGFR or ANXA2 abolishes M. hyorhinis-induced p65 phosphorylation. Cells were transfected with 50 nmol/L indicated siRNAs for24 hours, serum starved for 24 hours, and M. hyorhinis infection for 24 hours. E, inhibition of NF-kB by Bay 11-7082 (2 mmol/L) abolishes M. hyorhinis(left)– and GST-p37 (right)–induced cell migration. Data, mean � SD from three experiments with triplicate for each sample; �, P < 0.05; ��, P < 0.01;��, P < 0.001.

Duan et al.





the results of IHC-based survival prediction. However, wecould not exclude the effects of recent mycoplasma coloniza-tion and/or antibiotics treatment on the results of IHC andqPCR. It should be pointed out that we previously foundM. hyorhinis infection correlates with clinical stage and differ-entiation of tumor (12), which was not supported by thepresent study. The inconsistence could be resulted from dif-ference in sample size and/or source of samples. Thus, multi-centric studies using multiple techniques and more samplesare required to further verify the association betweenM. hyorhinis infection and gastric cancer.We found M. hyorhinis could infect both primary cells and

cancerous cells, whereas results of qPCR and flow cytometrysuggest that primary cells are less vulnerable to be infectedby this microbe. We further identified ANXA2 as a hostfactor mediating M. hyorhinis infection. ANXA2 plays mul-tiple roles in cancer development (28, 36, 37). Recent studiesdisclosed the relationship between ANXA2 and microbalinfection. ANXA2 contributes to HPV16 infection (31), andis also involved in the formation of hepatitis C virus (HCV)replication complex on the lipid raft (38). Our findingsdemonstrate that ANXA2 contributes to M. hyorhinis infec-tion through its interaction with p37 at their N-termini.Potential inhibitors targeting the binding interface betweenp37 and ANXA2 may provide therapeutic opportunities tocombat M. hyorhinis infection.ANXA2 phosphorylation at Tyr23 is associated with its cell

surface translocation (29). In this study, Tyr23 phosphory-lation was found to be enhanced and the ANXA2 on cellsurface was increased upon M. hyorhinis infection in anEGFR-dependent manner. It is noteworthy that EGFR playssome roles in microbial infection. EGFR is a cofactor forHCV entry, whereas kinase inhibitors targeting EGFR reduceHCV infection (39). EGFR and HER2 function together asreceptors for C. albicans (40). It is also a receptor for HCMV(41). Alternatively, EGFR signaling mediates infection-induced malignant phenotype. For example, Helicobacterpylori upregulates EGFR in AGS cells and promotes migra-tion by activating the EGFR–PI3K pathway (42, 43). Ourpresent results again underscore the essential role of EGFR

in microbial infection. We noticed an increased ANXA2–EGFR interaction and EGFR preferentially interacts withphosphorylated ANXA2 in M. hyorhinis–infected cells, themechanisms underlying these alterations need to beexplored in future studies.

In addition, we showed that M. hyorhinis activates NF-kBsignaling, which is required for M. hyorhinis–induced gastriccancer cell migration. Considering the critical role of the NF-kB pathway in inflammation-related carcinogenesis (44), ourresults provide another potential approach for counteract-ing M. hyorhinis-related malignant phenotypes.

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

Authors' ContributionsConception and design: H. Duan, L. Qu, X. He, C. ShouDevelopment of methodology: H. Duan, L. Chen, L. QuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Duan, M. Ye, X. HeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Duan, L. Chen, L. Qu, S.W. Song, Y. Han, M. Ye,X. HeWriting, review, and/or revision of the manuscript: H. Duan, L. Qu,S.W. Song, M. Ye, X. He, C. ShouAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. Chen, H. Yang, W. Chen, C. ShouStudy supervision: L. Qu, W. Chen, C. Shou

AcknowledgmentsThe authors thank Dr. Dajun Deng, Dr. Bin Dong, Dr. Caiyun Liu, Ting Ma,

Lin Meng, Dr. Zhihua Tian, Lixin Wang, Dr. Baozhen Zhang, Dr. ChuankeZhao (all from Peking University Cancer Hospital and Institute), Dr. ZhijieChang (Tsinghua University), and Dr. Xianping Wang (Institute of Biophysics,Chinese Academy of Sciences) for providing critical reagents or technicalassistance. Transcript profiling: Genechip accession number is GSE52638.

Grant SupportThis work was supported by National Natural Science Foundation of

China (no. 91029713) and National Basic Research Program of China (no.2010CB529303).

The costs of publication of this article were defrayed in part 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 March 3, 2014; revised July 4, 2014; accepted July 28, 2014;published OnlineFirst August 18, 2014.

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Duan et al.




2014;74:5782-5794. Published OnlineFirst August 18, 2014.Cancer Res Hongying Duan, Ling Chen, Like Qu, et al. Migration of Gastric Cancer Cells

Dependent−Bκ Infection Promotes NF-Mycoplasma Hyorhinis

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