Myeloid Differentiation Factor 88 Promotes Growth and...

Human Cancer Biology

Myeloid Differentiation Factor 88 Promotes Growth andMetastasis of Human Hepatocellular Carcinoma

Beibei Liang1,2, Rui Chen2, Tao Wang2, Lei Cao1,2, Yingying Liu2, Fan Yin2, Minhui Zhu2, Xiaoyu Fan2,Yingchao Liang2, Lu Zhang2, Yajun Guo1,2,3, and Jian Zhao2,3

AbstractPurpose: To investigate the expression of myeloid differentiation factor 88 (MyD88) in hepatocellular

carcinoma (HCC) and its prognostic value in patients with HCC.

Experimental Design: Expression of MyD88 was detected by immunohistochemistry in surgical HCC

specimens (n ¼ 110). The correlation of MyD88 expression to clinicopathologic characteristics was

analyzed. The involvement of MyD88 in tumor growth and invasion was investigated.

Results: The expression of MyD88 was significantly higher in HCC tumors than that in adjacent

nontumor tissues. Particularly, high expression of MyD88 was found in HCCs with late tumor stage

(P¼ 0.029). Patients with highMyD88 staining revealed a higher recurrence rate (65% vs. 40%; P¼ 0.008).

Kaplan–Meier analysis showed that recurrence-free survival (RFS; P ¼ 0.011) and overall survival (OS;

P¼ 0.022) were significantly worse among patients with highMyD88 staining. Univariate andmultivariate

analyses revealed that MyD88 was an independent predictor for OS and RFS. Ectopic expression of MyD88

promoted HCC cell proliferation and invasion in vitro. Suppression of MyD88 expression with lentivirus

encoding short hairpin RNA reduced tumor growth and invasion, as well as lung metastasis. Finally,

silencing of MyD88 inhibited the activation of NF-kB and AKT in HCC cells, whereas forced expression of

MyD88 was able to enhance the activation of NF-kB and p38/extracellular signal–regulated kinase without

Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) signaling.

Conclusion: Elevated expression ofMyD88may promote tumor growth andmetastasis via both TLR/IL-

1R–dependent and –independent signaling and may serve as a biomarker for prognosis of patients with

HCC. Clin Cancer Res; 19(11); 2905–16. �2013 AACR.

IntroductionInvasion andmetastasis are the leading causes of death in

patients with cancer. Inflammation is considered to be themost important environmental factor contributing totumor progression by promoting proliferation, antiapop-tosis, invasion, and angiogenesis (1–3). The inflammatoryresponse can be initiated by several types of pattern-recog-nition receptors (PRR), the Toll-like receptors (TLR) are thewell-characterized PRR (4). The interleukin (IL)-1 receptors(IL-1R) share a common Toll/IL-1 receptor (TIR) motif in

their cytoplasmic domain with TLRs. TIR domain-contain-ing adaptor proteins are required to bridge the TLR/IL-1Rreceptors to the intracellular molecules and transmit cellu-lar signaling. The first such adaptor molecule to be discov-ered ismyeloid differentiation factor 88 (MyD88; refs. 5–7).By interaction with TIR domain of TIR, MyD88 recruits IL-1R–associated kinase (IRAK) and TNF receptor–associatedfactor-6 (TRAF6), leading to activation of NF-kB and mito-gen-activated protein kinases (MAPK; ref. 8).

There is increasing evidence forMyD88playing an impor-tant role in carcinogenesis. Mice lacking MyD88 formedfewer tumors than wild-type (WT) mice in diethylnitrosa-mine (DEN)-induced hepatocarcinogenesis or azoxy-methane (AOM)-induced intestinal tumorigenesis (9,10). In DEN-induced hepatocarcinogenesis, MyD88 wasfound critical for the production of IL-6 in Kupffer cells (9).The contribution of MyD88 to inflammation-associatedtumorigenesis was further confirmed in 7,12-dimethyl-benz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol13-acetate (TPA)–induced skin papilloma (11). In additionto inducing proinflammatory response, recent evidenceshowed that MyD88 may act intrinsically in epithelial cellsto promote carcinogenesis by noninflammatory functions.In APCMIN mice, MyD88-dependent signaling was foundrequired for positive regulators of tumor progression

Authors' Affiliations: 1School of Pharmacy, Shanghai Jiao Tong Univer-sity; 2International Joint Cancer Research Institute, The Second MilitaryMedical University; and 3National Engineering Research Center of Anti-body Medicine, State Key Laboratory of Antibody Medicine and TargetingTherapy, Shanghai, PR China

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

B. Liang, R. Chen, and T. Wang contributed equally to this work.

Corresponding Author: Jian Zhao, International Cancer Research Insti-tute, The Second Military Medical University, 800 Xiang Yin Road, NewBuilding 10–11th Floor, Shanghai 200433, PR China. Phone: 86-21-81870807; Fax: 86-21-81870801; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-1245

�2013 American Association for Cancer Research.

ClinicalCancer

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derived from epithelial cells, such as matrix metalloprotei-nase 7 (MMP7) and COX-2 (10). In addition, in methyl-cholanthrene (MCA)-induced fibrosarcoma, a model thathas not been classically defined as having a significantinflammatory origin, fewer MyD88�/� mice developedfibrosarcoma than WT control. MyD88 might act intrinsi-cally to facilitate fibroblast and epithelial cell to transfor-mation (11). The direct evidence of intrinsic MyD88 in theregulation of carcinogenesis by noninflammatory responsecame from a RAS-dependent skin carcinogenesis model(12). MyD88 was found to play a cell-autonomous role inthe regulation of cell-cycle checkpoint and proliferation viaits interaction with activated extracellular signal–regulatedkinase (ERK). Thus, MyD88 may have multiple facets intumorigenesis, via both proinflammatory and noninflam-matory responses.

Abnormal expression of MyD88 has been previouslyreported in various types of cancer, which is related totumor development and chemoresistance. High expres-sion of MyD88 was found in stomach, colon, and lungprimary human cancer tissues, as well as in papillomasdeveloped from DMBA/TPA–treated mice (12). In colo-rectal cancer (CRC), high expression of MyD88 wasfrequently detected in CRC with liver metastasis andsignificantly related to poor prognosis of patients withcancer (13). Upregulation of MyD88 was also found inovarian cancer cells and in ovarian cancer tissues (14, 15).SKOV3, a cell line obtained from the ascites of a patientwith advanced, metastatic ovarian cancer, expresses highlevel of MyD88 (15). Patients with ovarian cancer whosetumors did not express MyD88 had a statistically signif-icant improved progression-free interval compared withpatients whose tumors expressed MyD88 (14). Moreover,high expression of MyD88 conferred ovarian cancer cellsresistance to chemotherapy (14, 16). Patients whosetumor was MyD88-positive had a poor response to pac-

litaxel chemotherapy (16). Recently, RNA interferencescreening revealed that somatically acquired MyD88mutations in activated B-cell–like (ABC) subtype of dif-fuse large B-cell lymphoma (DLBCL) activated NF-kB andJAK–STAT3 signaling to promote cell survival (17).

The development of human hepatocellular carcinoma(HCC) is closely associated with chronic inflammation.Evidences have shown that TLR/IL-1R signaling plays animportant role in hepatocarcinogenesis (18, 19). Recentevidence showed that TLR4 single-nucleotide polymorph-ismsmight be associatedwith the development ofHCC (20,21). However, little is known about the expression ofMyD88 in human HCCs and its correlation with tumordevelopment. In this study, we analyze the expression ofMyD88 in 110 cases of HCCs and evaluate its correlationwith clinicopathologic characteristics. The effects of hepaticMyD88 on cell survival, proliferation, and invasion areassessed in vitro as well as in vivo.

Materials and MethodsPatient samples

In all, 110 primary HCC samples with adjacent nontu-mors liver tissues were obtained from patients who hadundergone curative hepatic resection between 2003 and2006 at Guangxi Cancer Hospital (Nanning, Guangxi, PRChina). Patient’s consent and approval from Guangxi Can-cer Hospital Ethics Committee were obtained to use theseclinicalmaterials for researchpurposes. The entering criteriaof all patients were described as previously reported (22,23). Curative resectionwas defined as complete resection ofall tumor nodules and the cut surface being free of cancer byhistologic examination (23, 24). The clinicopathologiccharacteristics of the patients are summarized in Supple-mentary Table S1.

Patient follow-upwas completed onMarch 15, 2011. Themedian follow-up period was 41 months (range, 1–79months). All patients were monitored postoperatively byserum a-fetoprotein (AFP), abdomi-nal ultrasonography,and chest radiograph every 1 to 6months depending on thepostoperative time as previously described (25, 26). Ifrecurrence was suspected, the patients were detected bycomputed tomography and/or MRI. Recurrences were con-firmed on the basis of typical imaging appearances incomputed tomography scans and/or MRI and an elevatedAFP level. Most patients died from intrahepatic recurrence,distal metastasis, or complicated cirrhosis.

Immunohistochemical stainingThe expression of MyD88 was analyzed with EnVision

system (Changdao Biotech, Shanghai, China) in formalin-fixed, paraffin-embedded sections of primary tumors. Brief-ly, the slides were dewaxed, hydrated and washed, and theendogenous peroxidase activity was quenched. After micro-wave antigen retrieval, slides were blocked and then incu-batedwith the antibody againstMyD88 (T3223; Epitomics)overnight at 4�C. Subsequently, sections were rinsed andincubated with the working solution of horseradish perox-idase–labeled goat anti-rabbit for 60 minutes at 37�C. After

Translational RelevanceTumor metastasis is the leading cause for the death in

patients with hepatocellular carcinoma (HCC) aftercurative resection. Therefore, it is urgent to reveal themechanism underlying HCC metastasis. Here, we showthat the expression of myeloid differentiation factor 88(MyD88), an adaptor molecule for Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) signaling, is en-hanced in HCCs. Patients with high MyD88 stainingreveal ahigher recurrence rate andpoorer recurrence-freesurvival and overall survival. MyD88 may promotetumor metastasis through regulation of apoptosis andexpression of inflammation factors and other tumormodifiers such asmatrixmetalloproteinase 7 andCOX2,via both TLR/IL-1R–dependent and –independent sig-naling. Our results suggest that MyD88 may be a can-didate prognostic factor for HCC and a valuable targetfor therapy.

Liang et al.

Clin Cancer Res; 19(11) June 1, 2013 Clinical Cancer Research2906



rinsing for 3 times, staining was visualized using the per-oxide substrate solution diaminobenzidine. Counter-stained by hematoxylin, the slides were dehydrated ingraded alcohol and mounted. Negative controls were pre-pared in the absence of primary antibody.Evaluation of immunostaining was independently con-

ducted by 2 experienced pathologists. The expression ofMyD88 was scored according to the signal intensity anddistribution. Briefly, a mean percentage of high tumorcells was determined in at least 5 areas at �400 magni-fication and assigned to 1 of 5 following categories: 0,<5%; 1, 5%–25%; 2, 25%–50%; 3, 50%–75%; and 4,>75%. The intensity of immunostaining was scored asfollows: 1, weak; 2, moderate; and 3, intense. For tumorsthat showed heterogeneous staining, the predominantpattern was taken into account for scoring. The percent-age of high tumor cells and the staining intensity weremultiplied to produce a weighted score for each case.Tissues with immunohistochemical scoring 2 or less wereconsidered as low, 3 to 12 as high.

Cells and plasmids, siRNA, lentivirusPLC/PRF/5, HepG2, and Hep3B were purchased from

American Type Culture Collection (ATCC). MHCC97-Land HCC-LM3 were obtained from the Liver Cancer Insti-tute, Zhong Shan Hospital, Fudan University (Shanghai,PR China). HL7702, Huh-7, and SMCC-7721 wereobtained from Cell Bank of Shanghai Institutes for Bio-logical Sciences, Chinese Academy of Sciences (Shanghai,PR China). All these cell lines were cultured in Dulbecco’smodified Eagle’s medium (Gibco) supplemented with10% (v/v) FBS (Hyclone) at 37�C in a humidified incu-bator containing 5%CO2. IL-1a and IL-1bwere purchasedfrom Perprotech.MyD88 cDNA was amplified by PCR and subcloned into

pcDNA3.0 vectors (Invitrogen), designated as pMyD88.siRNA-targeting MyD88 (siMyD88) was generated by Gen-ePharma. Lentiviral plasmid vectors encoding short hairpinRNAs (shRNA)–targeting MyD88 or scramble shRNA weregenerated and designated as shMyD88 and shNon, respec-tively. Further details are available in the SupplementaryMaterial.

Real-time PCR and Western blottingIsolationof total cellular RNAwas carriedout byusing the

NucleoSpin RNAII (740955; MACHEREY-NAGEL), andfirst-strand cDNA was generated using the PrimeScript RTreagent kit (DRR037A; Takara). The cDNA sample was thenmeasured by real-time PCR (RT-PCR) with an AppliedBiosystems 7500 Real-Time PCR System as recommendedby the manufacturer. Relative mRNA levels of differenttarget genes were calculated depended on the Ct values,corrected for b-actin expression, according to the equation:

2�DDCt [DDCt ¼ DCt (treatment) � DCt (control), DCt ¼ Ct

(target genes) � Ct (b-actin)].Total cell lysate was prepared as described before (27).

Proteins at the same amount were separated by SDS-PAGEand transferred onto polyvinylidene difluoride (PVDF)

membranes. After probing with primary and secondaryantibody, antigen–antibody complex was visualized byenhanced chemiluminescence’s reagents Supersignal(Pierce). The primers and antibodies used in this study arelisted in the Supplementary Tables S4 and S5.

Luciferase reporter assays and electrophoreticmobility shift assay

HCC (3 � 104) cells were plated in 48-well plates andtransfected with full NF-kB–driven luciferase constructtogether with the pRL-TK in triplicate by FuGENE HDTransfection Reagent (Roche). We harvested cells 48 hoursafter transfection and conducted the luciferase assays usingthe Dual Luciferase Reporter Assay System (Promega).Luciferase activities were calculated as fold induction com-paredwith that in pGL3.0. All bar diagrams are shown as themean � SD.

The nuclear extracts from cells were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit(Pierce). Detection of DNA–protein binding by electropho-retic mobility shift assay (EMSA) was done using LightShiftchemiluminescent EMSA kit (Pierce). Double-stranded gelshift probes corresponding to the human consensus NF-kBsequences2 (50-AGTTGAGGGGACTTTCCCAGGC-30) wereend-labeled with biotin.

Plate colony formation, cell invasion, and migrationassay

Forty-eight hours after transfection, cells were dispersedinto single-cell suspension, which was prepared and inoc-ulated in 100-mmdishes with a density of 5� 103 cells andmaintained for 12 days. Afterward, the colonies werestained with 1% crystal violet for 30 seconds after fixationwith 4% paraformaldehyhe for 5 minutes, and the colonieswere counted. Each experiment was repeated in triplicate.The results presented are averages from 3 independentexperiments.

Ability of cell invasion and migration was evaluated bythe Cell Invasion Assay Kit (Millipore) and Transwell Per-meable Support (Corning) according to the manufacturer’sdirectory. Five 200-multiple microscopic fields were ran-domly selected to calculate the total count of the invaded ormigrated cells. The relative number of cells having pene-trated the ECM or basement membrane was used to denotethe invasion ormigration ability of the cells. All assays wereconducted 3 times.

Detection of apoptosisApoptotic cells were analyzed by the ApoDETECT

Annexin V–FITC Kit (Invitrogen) in vitro. In situ apoptosisassay was conducted with the Fluorescein FragEL DNAFragmentation Dectection Kit (QIA39-1EA; Merck). Theformalin-fixed paraffin sections were deparaffinized andincubated with terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) reaction mix-ture. Apoptotic cells carrying DNA labeled with fluoresceinisothiocyanate (FITC)–dUTP were observed under fluores-cence microscope (Olympus).

MyD88 Promotes Growth and Metastasis in HCC

www.aacrjournals.org Clin Cancer Res; 19(11) June 1, 2013 2907



Animal studiesMale athymicBALB/c nudemicewere purchased from the

Shanghai Experimental Animal Center of Chinese Academ-ic of Sciences (Shanghai, PRChina) andweremaintained inspecific pathogen-free conditions. Animal care and exper-imental protocols were conducted in accordance withthe guidelines of Shanghai Medical Experimental AnimalCare Commission. For in vivo treatment, HCC-LM3 cells (5� 106) infected with lentivirus encoding shMyD88 [at amultiplicity of infection (MOI) of 50] were implantedsubcutaneously into theflankofnudemice (6 ineachgroup,maleBALB/cnu/nu, 4–6weeks), andHCC-LM3cells treatedwith lentivirus encoding shNonwere used asmock control.Tumor growth was monitored with tumor volume, whichwas calculated as described before (27). The mice weresacrificed 6 weeks later, and the lungs were removed. Con-secutive sectionsweremade for every tissueblockof the lungand stained with hematoxylin and –eosin (H&E). Theincidence and classification of lung metastasis were calcu-lated and evaluated independently by 2 pathologists.

Statistic analysisAll statistical analyseswere carried out using SPSS 16.0 for

Windows software. The x2 test was used to compare qual-itative variables; quantitative variables were analyzed bytwo-tailed Student t test and Wilcoxon rank sum test.Clinical variables included age, gender, hepatitis B virus(HBV) active status, AFP, cirrhosis, tumor number, vascularinvasion, tumor size, and stage. Tumor stage was deter-mined according to the American Joint Committee onCancer (AJCC) classification system. Kaplan–Meier analysiswas used to determine the survival data. Overall survival(OS) was defined as the interval between surgery and death

or between surgery and the last observation point. Forsurviving patients, the data were censored at the last fol-low-up. Recurrence-free survival (RFS) was defined as theinterval between the date of surgery and the date of diag-nosis of any type of relapse (intrahepatic recurrence andextrahepatic metastasis). Difference in survival betweengroups was evaluated by the log-rank test. Univariate andmultivariate analyses were based on the Cox proportionalhazards regression model. Data were presented as themean � SEM. All statistical tests were two-sided, and P <0.05 was considered statistically significant.

ResultsEnhanced expression of MyD88 is associated with poorprognosis in HCC patients

The expression of MyD88 was examined in 110 hepato-carcinomas and adjacent nontumor tissues using anti-human MyD88 antibody (Fig. 1A). Immunostaining ofMyD88 was mainly detected in the cytoplasm of hepaticcells. Most of the stroma cells were negative staining,although sporadic positive staining on these cells was alsoobserved (Fig. 1A and Supplementary Fig. S1A–S1C). Highstaining of MyD88 could be observed in 57 of 110 (51.8%)cases of HCCs, whereas MyD88 showed high staining inonly 10 of 110 (9.1%) cases of adjacent nontumor tissues.Statistical analysis revealed that the immunostaining scoresin tumor tissues [score value:median, 9 (range, 2–12)] weresignificantly higher than that in adjacent nontumor tissues[score value: median, 6 (range, 2–12); P < 0.001]. Consid-ering the relationship between MyD88 expression levelsand inflammation, CD68 was stained in MyD88-high andMyD88-low groups to detect the levels of inflammation.The average amounts of CD68þ cells in intratumor or in

Figure 1. MyD88 expression inhuman HCC tissues. A,representative images ofMyD88 expression in HCCtissues examined byimmunohistochemistry. MyD88was low detected in adjacentnormal liver cells (a and b), whereasit was weakly (c and d) or highly (eand f) detected in HCC cells. T,tumor tissue; NT, matched normaltissue. B and C, Kaplan–Meieranalysis of survival in patients withHCC. Kaplan–Meier survivalcurves for patients with HCCaccording to levels of MyD88expression were shown in B (RFS)and C (OS). Green, patients withhigh MyD88 expression. Blue,patients with low MyD88expression.

Liang et al.




peritumor were similar with no significant differencebetween HCC tissues with high or low MyD88 expression(Supplementary Fig. S1).Statistical analysis showed that MyD88 expression was

not significantly correlated with age, gender, hepatitis Bsurface antigen (HbsAg), serum AFP level, cirrhosis, vascu-lar invasion, and tumor number (Table 1). However, itsexpression level was found to be significantly higher inHCCs with AJCC stage III–IV than HCCs with stage I–II(P ¼ 0.029). Notably, patients with high MyD88 stainingrevealed a higher recurrence rate after surgical resection(65% vs. 40%; P ¼ 0.008; Table 1).The potential association between MyD88 expression

level and RFS or OS was retrospectively evaluated.Kaplan–Meier analysis showed that RFS (P ¼ 0.011; Fig.

1B) and OS (P ¼ 0.022; Fig. 1C) were significantly worseamong patients with high MyD88 staining. Patients inMyD88-high group had less median cancer-free survivalthan patients in MyD88-low group (14 vs. 38months). TheOS estimates showed that patients in MyD88-high grouphad less median (32 vs. 42 months) and lower OS (37.5%vs. 66.7%) than patients in MyD88-low group. Consistent-ly, the 1-, 3-, and 5-yearOS and RFS after surgery weremuchworse for patients with MyD88-high than patients withMyD88-low. RFS and OS (in brackets) rates at 1, 3, and 5years posthepatectomy were 74% (83%), 55% (66.5%),and 48% (54%) in MyD88-low expression group and were55% (77%), 31.5% (43.8%), and 26% (23.9%) in MyD88-high expression group (Supplementary Table S2). In uni-variate analysis, vascular invasion and MyD88 expressionstatus were prognositic factors for RFS and OS (Table 2).Multivariate analysis revealed thatMyD88 expression statuswas defined as an independent prognostic for both RFS (P¼0.027) and OS (P ¼ 0.026). MyD88-high expressionpatients were about 1.8 times more likely to suffer fromrelapse than MyD88-low expression patients (Table 2).Thus, increased expression of MyD88 may serve as a prog-nostic indicator for patients with HCC.

Enhanced MyD88 promotes proliferation and survivalof HCC cells

The mRNA level of MyD88 was markedly higher in 5HCC cells compared with HL7702, a normal hepatic cell(Fig. 2A). Moreover, the level of MyD88 in HL7702 wasmarkedly elevated by the stimulation of lipopolysaccharide(LPS), IL-1a, or IL-1b (Supplementary Fig. S2A). We thenused lentivirus-encoding shRNA to knockdown MyD88 inHepG2 with high level of MyD88 and HCC-LM3 withmedium level of MyD88, or pcDNA3.0 vector encodingMyD88 cDNA to overexpression MyD88 in MyD88-lowHL7702 and MyD88 medium HCC-LM3 (SupplementaryFig. S2B and S2C). Plate colony formation assay showedthat knockdown of MyD88 greatly inhibited cell prolifer-ation in HCC-LM3 and HepG2 cells, whereas overexpres-sion of MyD88 markedly enhanced cell proliferation inHL7702 and HCC-LM3 cells (Fig. 2B). The effect of MyD88on the cell growth was further confirmed by in vivo assay inHCC-LM3 xenografts. As shown in Fig. 2C, depletion ofMyD88 in HCC-LM3 cells dramatically inhibited tumorgrowth in nude mice. The tumor volumes developed byMyD88-deficient HCC-LM3 cells were about half of thatdeveloped by control cells.

Evading apoptosis has been regarded as oneof the cellularmechanisms contributing to the development of cancer(28).We then investigatewhether enhancedMyD88 expres-sion in HCC leads to resistance to apoptotic stimulation.The percentage of apoptotic cell greatly increased withdepletion of MyD88 under the stimulation of serum star-vation, whereas the apoptotic rate dropped dramaticallywith the overexpression of MyD88 in HCC-LM3 cells (Fig.2D). Apoptosiswas further examinedby in situTUNEL assayon HCC-LM3 xenografted tumor tissues. More apoptoticnuclei, seen as green color excited under fluorescence

Table 1. The associations ofMyD88 expressionwith clinicopathologic characteristics in 110patients with HCC

MyD88 expression

FeatureLow(n ¼ 53)

High(n ¼ 57) P

Gender 0.305Male 45 52Female 8 5

Age, y 0.488<50 31 37�50 22 20

HbsAg 0.227Positive 47 55Negative 6 2

AFP, ng/mL 0.138�400 26 20>400 27 37

Cirrhosis 0.670� 10 9þ 43 48

Tumor size, cm 0.790<5 18 18�5 35 39

Vascular invasion 0.148No 45 42Yes 8 15

Tumor number 0.495Single 51 52Multiple 2 5

AJCC stage 0.029I–II 36 27III–IV 17 30

Recurrence 0.008� 32 20þ 21 37

NOTE: P values are two-tailed and based on the Pearson x2

test.





microscope, were detected in MyD88-deficient HCC-LM3xenografts than that in control xenografts (Fig. 2E). Inconsistencewith in vitro analysis,more apoptotic cells couldbe observed in MyD88-low HCC tissues than that inMyD88-high tissues (Supplementary Fig. S2D). Thus,MyD88 may contribute to tumor progression through reg-ulation of apoptosis.

Enhanced MyD88 promotes invasion andmetastasis inHCC cells

We further investigated the role of MyD88 in tumormetastasis, which has been implicated from clinical data.HepG2 and HCC-LM3 cells migrated slower and had lessability to invade through Matrigel when MyD88 wasknocked down (Fig. 3A and B). In contrast, HL7702 andHCC-LM3 cells migrated faster and had more invasiveability when MyD88 was overexpressed (Fig. 3C and D).HCC-LM3 cell has a high degree of pulmonary metastasisafter subcutaneous injection (29), we therefore examinedthe effects of MyD88 on tumor metastasis in HCC-LM3xenografts in vivo. Six weeks after transplantation, none ofthe mice developed lung metastasis when MyD88 wassilenced. In contrast, 6 of 7 control mice developed lungmetastasis (Fig. 3E and Supplementary Table S3). Thesedata suggest that enhancedMyD88may promote themotileand invasive abilities of HCC cells.

Enhanced MyD88 promotes activation of NF-kB, PI3K/AKT, and p38/ERK in HCC cells

To explore the role ofMyD88 in both TLR-dependent and-independent pathway, several genes that relate to tumorprogression including MMP7, COX2, osteopontin (OPN),and proinflammatory cytokines such as IL-6, IL-1b, andTNF-awere analyzed with the presence or absence of IL-1b.

As expected, silence of MyD88 dramatically inhibited IL-1b–induced expression of these tumor modifiers in HCC-LM3 cells (Supplementary Fig. S3A and S3B). Interestingly,knockdown of MyD88 alone was sufficient to inhibitendogenous expression of MMP7, IL-1b, IL-6, and TNF-a.Moreover, forced expression of MyD88 was able to induceexpression of these tumormodifiers to the level comparablewith that induced by IL-1b (Supplementary Fig. S3C andS3D). The alterations of IL-1b, IL-6, and TNF-a were con-firmed by ELISA assay (Supplementary Fig. S3E).

NF-kB is one of the main downstream signaling compo-nents of TLR/MyD88 signaling (8) and is constitutivelyactivated in HCC (30). We then investigate whetherenhanced expression of MyD88 leads to activation of NF-kB in HCC cells. As expected, knockdown of MyD88 greatlyattenuated IL-1b–induced NF-kB activation in HL7702,HepG2, and HCC-LM3 (Fig. 4A). Notably, depletion ofMyD88 was able to significantly inhibit intrinsic NF-kBactivity in MyD88-high expression cells HCC-LM3 andHepG2, whereas overexpression of MyD88 alone was ableto enhance NF-kB transcriptional activity in these cells (Fig.4A). The requirementofMyD88forNF-kBactivation inTLR/IL-1R–independentmanner was further confirmed byDNA-binding activity analysis (Fig. 4B). Silencing of MyD88attenuated NF-kB DNA-binding activity in MyD88-highHepG2 cells (Fig. 4B, left), whereas overexpression ofMyD88 enhanced NF-kB DNA-binding activity in HL7702cells (Fig. 4B, right). Moreover, depletion of MyD88 inhib-ited phosphorylation of IkBa and its degradation indepen-dent of IL-1b stimulation, whereas forced expression ofMyD88 enhanced phosphorylation of IkBa and its degra-dation in HCC-LM3 cells (Fig. 4C). These data suggest thatenhanced expression of MyD88 is able to activate NF-kB viaTLR/IL-1R–independent signaling in HCC cells.

Table 2. Univariate and multivariate analyses of factors associated with disease-free survival and OS

RFS OS

Variables HR (95% CI) P value HR (95% CI) P value

Univariate analysesGender (female vs. male) 3.151 (0.985–10.08) 0.053 1.036 (0.442–2.428) 0.936Age, y (<50 vs. �50) 0.791 (0.460–1.359) 0.396 0.589 (0.322–1.075) 0.085HbsAg (negative vs. positive) 1.184 (0.428–3.275) 0.745 2.790 (0.677–11.492) 0.155AFP, ng/mL (�400 vs. >400) 1.144 (0.673–1.944) 0.620 1.020 (0.588–1.770) 0.943Cirrhosis (no vs. yes) 2.149 (0.922–5.013) 0.077 1.186 (0.559–2.518) 0.657Tumor size, cm (<5 vs. �5) 1.191 (0.677–2.097) 0.544 1.106 (0.615–1.989) 0.737Tumor number (single vs. multiple) 1.138(0.412–3.143) 0.804 0.797 (0.247–2.571) 0.704AJCC stage (I–II vs. III–IV) 0.580 (0.335–1.004) 0.052 0.619 (0.351–1.094) 0.099Vascular invasion (no vs. yes) 2.243 (1.277–3.939) 0.005 1.855(1.040–3.306) 0.036MyD88 (low vs. high) 1.959 (1.145–3.353) 0.014 1.913 (1.082–3.380) 0.026

Multivariate analysesVascular invasion (no vs. yes) 2.084 (1.184–3.669) 0.011MyD88 (low vs. high) 1.841 (1.072–3.161) 0.027 1.913 (1.082–3.380) 0.026

NOTE: Multivariate analysis, Cox proportional hazards regression model. Variables were adopted for their prognostic significance byunivariate analysis and no obvious correlation between each other.

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Phosphoinositide 3-kinase (PI3K) and AKT has beenimplicated in TLR/IL-1R signaling pathway, and may reg-ulate NF-kB activation in both TLR/IL-1R–dependent and–independent signaling (31–33). Here, we found thatdownregulation of MyD88 greatly inhibited the intrinsicactivation of AKT in HCC-LM3 cells independent of IL-1bstimulation, overexpression of MyD88 or stimulation withIL-1b did not cause further activation of AKT in HCC-LM3cells (Fig. 4DandE). Treatment ofHCC-LM3cellswith PI3Kinhibitor LY294002 significantly inhibitedMyD88-induced

NF-kB activation, indicating that MyD88-induced NF-kBactivation is, at least in part, via PI3K/AKT (SupplementaryFig. S3F).

TLR/IL-1R–MyD88 signaling also leads to activation ofMAPKs such as c-jun-NH2-kinase (JNK), p38, and ERK (8).Here, we founddownregulation ofMyD88 inhibited IL-1b–induced p38 phosphorylation, whereas overexpression ofMyD88 alone was able to enhance p38 phosphorylation tothe level similar to the one stimulated by IL-1b inHCC-LM3cells (Fig. 4D and E). It has been reported thatMyD88 binds

Figure 2. MyD88 promotes proliferation and survival of HCC cells. A, expression of MyD88 mRNA in HCC cell lines compared with that in normal livercell line HL7702 by RT-PCR. Expression levels were normalized for b-actin. B, downregulation of endogenous MyD88 in HepG2 and HCC-LM3 inhibits cellgrowth (left), whereas ectopic expression of MyD88 promotes HL7702 and HCC-LM3 proliferation as analyzed by colony assay (right). HCC-LM3 or HepG2cells were infected with lentivirus encoding shMyD88 or shNon, whereas HL7702 or HCC-LM3 cells were transfected with pMyD88 or pcDNA3.0. C,downregulation of MyD88 in HCC-LM3 cells inhibits tumorigenicity in nude mice. The tumor volume of each group was scored every 5 days. D,downregulation of endogenous MyD88 in HCC-LM3 promotes cell apoptosis as analyzed by Annexin V–FITC/PI (propidium iodide) double staining (top),whereas ectopic expression of MyD88 inhibits serum-starvation–induced apoptosis (bottom). HCC cells were transfected with siNon and siMyD88, orpcDNA3.0 and pMyD88 for 48 hours, and then subjected to serum starvation for 24 hours. The early apoptotic cells (Annexin Vþ/PI�) were quantified. E,downregulation of endogenous MyD88 in HCC-LM3 promotes cell apoptosis as determined by in situ TUNEL apoptosis analysis in xenografted tumorsection. The percentage of apoptotic cells was calculated by counting green-stained nuclei (the apoptotic nuclei) versus blue-stained nuclei (the total nuclei)from 6 randomly chosen fields in each section. Data represented the mean � SD �, P < 0.05; ��, P < 0.01. DAPI, 40,6-diamidino-2-phenylindole.





to ERK and prevents its inactivation by its phosphatase,MKP3, thereby amplifying the activation of the canonicalRAS pathway in a TLR/IL-1R–independent manner (12).Indeed, herewe foundoverexpressionofMyD88was able toenhance ERK activation without IL-1b stimulation (Fig. 4Dand E). These results suggest that enhanced MyD88 maylead to activation of p38/ERK via TLR/IL-1R–independentsignaling. Treatment of HCC-LM3 cells with p38 inhibitorSB2035 andERK inhibitorU0126 greatly inhibitedMyD88-induced NF-kB activation (Supplementary Fig. S3F). More-over, U0126 inhibited the migration in MyD88-highexpression cells HepG2 and HCC-LM3, and HCC-LM3

overexpressing MyD88 (Supplementary Fig. S4A andS4B). These data indicate that MyD88-induced NF-kB acti-vation and invasion also involves ERK activation.

Taken together, these findings suggest that MyD88 maypromote tumor progression through TLR/IL-1R signalingindependent activation of NF-kB via PI3K/AKT and p38/ERK.

DiscussionThe development of HCC is closely associated with

chronic inflammation caused by viral infection, alcoholconsumption, or hepatic metabolic disorders. Evidences

Figure 3. MyD88 promotes tumormetastasis in vitro and in vivo. Aand B, downregulation ofendogenous MyD88 in HepG2 andHCC-LM3 inhibits cell migration(A) and invasion (B) as analyzed bycell migration assays and cellinvasion assays. C and D, ectopicexpression of MyD88 stimulatesHL7702 and HCC-LM3 migration(C) and invasion (D). For the cellinvasion assay, HCC cells wereincubated for 72 hours; for themigration assay, cells wereincubated for 48 hours. Datashown are the mean � SD(�, P < 0.05; ��, P < 0.01). E,downregulation of endogenousMyD88 in HCC-LM3 inhibits in vivometastasis as determined bycalculating the number of lungmetastatic foci in each group ofmice under microscope.Representative lung tissuesections (H&E stain; magnification,�400) from each group are shown(bars, 400 mm). Arrows indicatemetastatic tumors.

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have suggested that TLR/IL-1R signaling plays an importantrole in various live disease and HCC (34). Recent evidenceshave indicated the involvement of MyD88 in the develop-

ment of HCC. In DEN-induced inflammation-associatedliver cancer model, loss of TLR4 or MyD88 decreased DEN-induced tumor development including the incidence, size,

Figure 4. MyD88 activates NF-kB, PI3K/Akt, and p38/ERK in HCC cells. A, ectopic expression of endogenous MyD88 in HL7702, HepG2, andHCC-LM3 increases NF-kB transcriptional activities with and without IL-1b (10 ng/mL) stimulation for 24 hours (top), whereas downregulation of MyD88decreases NF-kB transcriptional activities (bottom). NF-kB activities are analyzed by the luciferase activities 48 hours after transfection. Data are indicated asthe number of folds (n-fold) over that of pGL3.0, and shown as the mean � SD (�, P < 0.05; ��, P < 0.01). B, downregulation of endogenous MyD88 inHepG2 decreases NF-kB DNA-binding activities (left), whereas ectopic expression of MyD88 in HL7702 increases NF-kB DNA-binding activities (right) asanalyzed by EMSA. NS, nonspecific signal. C–E, Western blot analysis of the expression of p-IKB-a, total IKB-a, MyD88, p-Akt, total Akt, p-p38, total p38,p-Erk1/2, total Erk1/2, p-JNK, and total JNK in HCC-LM3 infected with shNon or shMyD88, and transfected with pcDNA3.0 or pMyD88, in the presence orabsence of IL-1b (10 ng/mL). The cells were collected 24 hours after treatment and then analyzed by Western blotting. Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was used as an internal control for analysis in C–E.





and number of tumor (9, 18). Recently, HBx protein wasfound to stimulate MyD88 expression in hepatocytes tostimulate IL-6 expression (35). Collectively, these dataindicate a strong contribution of TLR/MyD88 signaling tohepatocarcinogenesis. In current study, we showed thatMyD88 was frequently upregulated in HCCs, which wasclosely related with the worse stage of tumor and the higherrecurrent rate in patients with HCC. Moreover, the survivalanalysis revealed that patients with high expression ofMyD88 hadworse RFS andOS. Univariate andmultivariateanalyses revealed thatMyD88was a significant predictor forOS and RFS. In addition, in an external cohort’s dataavailable in public database [National Center for Biotech-nology Information (NCBI)’s Gene Expression Omnibusaccession # GSE14520], the expression of MyD88 is signif-icantly higher in 10 cases of HCC with portal vein tumorthrombi (PT) than that in metastasis-free HCCs (PN) at thetime of surgery (PN/PT is 0.5731; P ¼ 0.0009693; ref. 36).Our findings and previous observations strongly implicatethat elevated MyD88 is involved in the tumor progressionandmay serve as a prognostic factor for patients with HCC.

Although Kupffer cells are considered the primary cells inliver to respond to TLR/IL-1R signaling, recent studiesprovide evidence of TLR/IL-1R signaling in hepatic nonim-mune cell populations, including hepatocytes, biliary epi-thelial cells, endothelial cells, and hepatic stellate cells (34).Even though hepatocytes express very low levels of TLR2,TLR3, TLR4, and TLR5 and their responses are fairly weakin vivo (37), recent evidence suggests that TLR/MyD88signaling is involved in the biologic or pathologic processesin hepatocytes. HBx stimulated IL-6 expression in hepato-cytes via aMyD88-dependentmanner (35). LPS suppressedglucose production in hepatocytes through the TLR4/MyD88/NF-kB pathway (38). In Plasmodium-infected hepa-tocytes, MyD88 was required for NF-kB activation andinducible nitric oxide synthase expression (39). Here, weshowed that elevated hepatic MyD88 was involved in thegrowth and metastasis of HCC cells.

ElevatedMyD88 expression has been found in parenchy-mal cells in various types of cancer (13–15, 35, 40), eventhough themechanisms forMyD88upregulation are largelyunknown. Here, we found that the expression of MyD88could be upregulated by the stimulation of LPS, IL-1a, andIL-1b. The contribution of MyD88 to tumor progression ismainly linked to its antiapoptosis ability (14, 40). Here, wehave shown that besides the ability to evade apoptoticstimulation, elevated expression of MyD88 conferred HCCcells with enhanced abilities of migration and invasion.Furthermore, several metastasis-related genes such asMMP7, COX2, OPN, and inflammation factors such asIL-6, IL-1b, and TNF-a were regulated in a MyD88-depen-dent manner. Anti-IL-6 antibody was able to attenuateMyD88-induced enhanced migration ability in HCC-LM3cells (Supplementary Fig. S4C and S4D). Our results revealthe importance of hepatic MyD88 in the regulation oftumor microenvironment and tumor progression.

Notably, the effects ofMyD88 on tumor progressionmaybe via both TLR/IL-1R–dependent and –independent sig-

naling. On one hand, MyD88 was upregulated by thestimulation of LPS and IL-1 and was required for IL-1b–induced upregulation of tumor modifiers such as MMP7,COX2, IL-6, and TNF-a, and the activation of NF-kB andp38. On the other hand, overexpression of MyD88 alonewas sufficient to regulate the ability to evade apoptoticstimulation,migrate and invade throughmatrix, the expres-sion of tumor modifiers, and the activation of NF-kB andp38/ERK in HCC cells. Although HCC cells expressed lowlevel of TLRs as detected by PCR, most of HCC cellsexpressed high level of IL-1R compared with hepatic cellHL7702 (Supplementary Fig. S5A and S5B). In the light ofthis, IL-1 is a major factor in the tumor microenvironment,thus enhanced MyD88 may promote tumor metastasis viaIL-1R/MyD88 signaling in vivo.

However, the role of MyD88 in TLR/IL-1R–independentsignaling during tumor development still needs to be elu-cidated. Previous studies have suggested that MyD88 maybe involved in TLR/IL-1R–independent signaling. The upre-gulation of MMP7 in dysplastic epithelium was perturbedby MyD88 deficiency rather than by TLR2/TLR4 doubledeficiency in APCMIN mice (9, 10). In RAS-mediated tumordevelopment, MyD88 mutant that cannot interact withERK, but not mutant that cannot interact with IRAK, lostthe ability to regulate cell transformation, indicating therole of MyD88 in TLR/IL-1R–independent signaling (12).Interaction of MyD88 with p-ERK has been suggested tocause ERK activation and make contribution to RAS-medi-ated cell transformation. We assessed the potential linkbetween MyD88 mRNA expression and RAS pathway acti-vation by testing RAS target gene signatures available inMolecular Signature Database. InMyD88 gene set, there is a"BILD_HRAS_ONCOGENIC_SIGNATURE," which indi-cates that expression of MyD88 may be increased in theactivation status of RAS oncogenic pathways (41). Here, wefound ERK was phosphorylated when MyD88 was over-expressed inHCC-LM3 cells (Fig. 4E), however, knockdownMyD88 did not attenuate ERK activation (Fig. 4D). Whenwe examined the RAS–MEK–ERK activation in HCC cells,we could not find a correlation between MyD88 expressionand RAS pathway activation in HCC cells we tested (Sup-plementary Fig. S5C). Therefore, it would be interesting toextend this analysis in more HCC tissue samples. Never-theless, ERK inhibitor U0126 inhibited cell migration inMyD88-high HCC cells HepG2 andHCC-LM3, as well as inHCC-LM3 overexpressingMyD88 (Supplementary Fig. S4Aand 4B). These indicate that ERK activation contributes toMyD88-mediated HCC development via TLR/IL-1R–inde-pendent signaling.

Our data suggested thatMyD88may regulate TLR/IL-1R–independent signaling through activation of PI3K/AKT/NF-kB in HCC. Here, we found depletion of MyD88 greatlyattenuated NF-kB transcriptional activity, DNA-bindingability, and AKT phosphorylation in HCC cells. PI3K/AKTsignaling has been suggested as one of the regulators forNF-kB activation induced by TNF, IL-1, and LPS (31–33). AKThas been shown to induce activation of NF-kB throughinhibitor of IkB kinase a (IKKa) phosphorylation and

Liang et al.




liberation of IkBa or p65 phosphorylation independent ofIkBa degradation (31, 32, 42, 43). MyD88 has a bindingmotif YXXM for p85, a regulatory unit of PI3K. MyD88 hasbeen shown to be associated with p85 by coimmunopre-cipitation in LPS stimulated RAW 264.7 cells (44). LTA-andStaphylococcus aureus–induced cPLA and COX-2 expressionare mediated through the formation of a TLR2/MyD88/PI3K/Rac1 complex in human tracheal smoothmuscle cells(45). The association ofMyD88with p85 is also observed inthe context of TLR5 signaling in intestinal epithelial cellsand is required for TLR5-induced phosphorylation of Akt,NF-kB activation, and IL-8 production (46, 47). However,whetherMyD88 and p85 interaction participates in TLR/IL-1R–independent signaling during HCC progression stillneeds further investigation.Here, we show that enhanced MyD88 expression is a

strong indicator for more aggressive tumors and poorerclinical outcome in HCC. Elevated MyD88 may promoteHCC metastasis through activation of NF-kB, PI3K/AKT,and p38/ERK, regulation of apoptosis and tumor micro-environment through both TLR/IL-1R–dependent and-independent signaling. Moreover, high expression ofMyD88 may serve as a prognostic factor and a therapeutictarget for HCC.

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

Authors' ContributionsConception and design: J. ZhaoDevelopment of methodology: R. Chen, T. WangAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): B. Liang, R. Chen, T. WangWriting, review, and/or revision of the manuscript: B. Liang, J. ZhaoAdministrative, technical, or material support (i.e., reporting or orga-nizingdata, constructingdatabases): L. Cao, Y. Liu, F. Yin,M. Zhu, X. Fan,Y. Liang, L. ZhangStudy supervision: Y. Guo, J. Zhao

Grant SupportThis work is supported in part by grants from Ministry of Science and

Technology of China "973" and "863" programs (2010CB945600 and2011CB966200), National Nature Science Foundation of China, State KeyProject for Infection Disease (2012ZX10002011-009) and New Drug Devel-opment and Programs of Shanghai Subject Chief Scientists, MunicipalCommission of Education and Municipal Commission of Science andTechnology.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 18, 2012; revised February 27, 2013; accepted March 12,2013; published OnlineFirst April 2, 2013.

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