the activation of mek/erk signaling pathway by bone...

Signaling and Regulation

The Activation of MEK/ERK Signaling Pathway by BoneMorphogenetic Protein 4 to Increase HepatocellularCarcinoma Cell Proliferation and Migration

Chiang-Yen Chiu1, Kung-Kai Kuo2,3, Tzu-Lei Kuo1, King-The Lee2,3, and Kuang-Hung Cheng1,4

AbstractHepatocellular carcinoma (HCC) is one of the most common visceral malignancies worldwide, with a very high

incidence and poor prognosis. Bonemorphogenesis protein 4 (BMP4), which belongs to the TGF-b superfamily ofproteins, is a multifunctional cytokine, which exerts its biologic effects through SMAD- and non-SMAD-dependent pathways, and is also known to be involved in human carcinogenesis. However, the effects of the BMP4signaling in liver carcinogenesis are not yet clearly defined. Here, we first show that BMP4 and its receptor,BMPR1A, are overexpressed in amajority of primaryHCCs and that it promotes the growth andmigration ofHCCcell lines in vitro. We also establish that BMP4 can induce HCC cyclin-dependent kinase (CDK)1 and cyclin B1upregulation to accelerate cell-cycle progression. Our study indicates that the induction ofHCC cell proliferation isindependent of the SMADsignaling pathway, as Smad4 knockdown ofHCC cell lines still leads to the upregulationof CDK1 and cyclin B1 expression after BMP4 treatment. Using mitogen-activated protein/extracellular signal-regulated kinase (MEK) selective inhibitors, the induction of CDK1, cyclin B1 mRNA and protein were shown tobe dependent on the activation of MEK/extracellular signal-regulated kinase (ERK) signaling. In vivo xenograftstudies confirmed that the BMPR1A-knockdown cells were significantly less tumorigenic than the control groups.Our findings show that the upregulation of BMP4 andBMPR1A inHCCpromotes the proliferation andmetastasisof HCC cells and that CDK1 and cyclin B1 are important SMAD-independent molecular targets in BMP4signaling pathways, during the HCC tumorigenesis. It is proposed that BMP4 signaling pathways may havepotential as new therapeutic targets in HCC treatment. Mol Cancer Res; 10(3); 415–27. �2012 AACR.

IntroductionHepatic cancer, particularly hepatocellular carcinoma

(HCC), is one of the most common tumors worldwide andthe third most common cause of cancer-related deaths.Diagnosis of advanced stage of HCC is a devastating expe-rience for both patients and family. More than 80% ofHCCcases originate in developing countries (1, 2). Chronicinfections with hepatitis B virus or hepatitis C virus areresponsible for themajority ofHCC cases in those countries,especially in Asia (3, 4). Other risk factors include prolonged

dietary exposure to aflatoxin and cirrhosis associated withgenetic liver diseases (5, 6). Although much is known aboutthe biochemical and morphologic features of changes innormal hepatocytes which lead to HCCs in humans, his-tologic findings suggest that HCC involves a multistepcarcinogenesis process. However, the molecular pathogen-esis of HCC is still not fully understood, andmany key issuesstill remain unresolved in regard to this disease (7–9).Bone morphogenesis proteins (BMP), a large subgroup in

the TGF-b superfamily, have been shown to mediate avariety of biologic functions, which regulate cell prolifera-tion, differentiation, and the determination of cell fate (10,11). In particular, BMP signaling has been implicated in theregulation of early liver development and has been shown toplay a vital role in the determination of liver bud morpho-genesis and to promote hepatoblast migration, proliferation,and survival (12, 13). BMP4 is made up of glycosylated andsecreted cytokine, which can form a ligand-receptor complexwith type I and type II receptors, but which favors type I(BMPRIA and BMPR1B) rather than the type II receptors(BMPR2 and ACVR2B; ref. 14). BMP4, which is also partof the TGF-b family, is a vital regulatory cytokine thatfunctions throughout development in mesoderm inductionto activate downstream response genes, primarily throughthe canonical SMAD signaling pathway (15).When dimeric

Authors' Affiliations: 1Institute of Biomedical Sciences, National Sun Yat-sen University; 2Division of Hepatobiliary Surgery, Department of Surgery,3Cancer Center, Kaohsiung Medical University Chung-Ho Memorial Hos-pital; and 4National Sun Yat-sen University-Kaohsiung Medical UniversityJoint Research Center, Kaohsiung, Taiwan

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

C.-Y. Chiu and K.-K. Kuo contributed equally to this work.

Corresponding Author: Kuang-Hung Cheng, Institute of BiomedicalSciences, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan.Phone: 886-7-5252000, ext. 5817; Fax: 886-7-5250197; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-11-0293

�2012 American Association for Cancer Research.

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on May 29, 2018. © 2012 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst January 12, 2012; DOI: 10.1158/1541-7786.MCR-11-0293

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BMP4 binds to BMPR receptor complex, the type IIreceptors phosphorylate the type I receptors in their intra-cellular kinase domain and the receptor-specific SMAD1/5/8 are recruited onto the receptor complex and phosphory-lated. The phosphorylated SMAD1/5/8 then binds to acomediator, SMAD4, to convey signals to the nucleus. ThisSMAD complex translocates to the nucleus and activates orsuppresses BMP4-responsive gene expression (14–16).Recent findings have revealed that TGF-b and BMPs are

linked to a wide variety of clinical disorders and have beenshown to regulate proliferation, migration, or invasion ofcarcinoma cells, derived from several organs (17). Recentstudies have suggested that BMP2 is highly overexpressed inhuman lung cancer cells and stimulates tumor developmentand motility (18). The overexpression levels of BMP4, asevaluated in breast cancer and malignant melanoma cells,contribute to the promotion of cell invasion and migration(19–21). In addition, BMP9 has been reported to promoteovarian cancer cell proliferation through an autocrine mech-anism (22). In primary HCCs, BMP4 has been reported tobe upregulated in cancerous liver tissues by hypoxia (23);however, there is insufficient details of the molecularmechanisms of BMP4 prooncogenic functions, with regardto HCC.In this study, we provide significant molecular and

cellular evidence to support the assumption that theupregulation of BMP4 in HCCs promotes tumor prolif-eration and metastasis. First, we focused our attention onthe expression of BMP4 and its function during hepaticcarcinogenesis and showed that BMP4 activates the mito-gen-activated protein/extracellular signal-regulated kinase(MEK)/extracellular signal-regulated kinase (ERK) path-way, instead of the SMAD pathway, to promote theproliferation and migration of human HCC cells. Inparticular, the downstream signaling effectors cyclin-dependent kinase (CDK)1 and cyclin B1, which mediatethe increase in HCC cell growth stimulated by BMP4,were identified. These findings provide a better under-standing of the effects of BMP4 signaling in clinicallyrelevant diseases and could be crucial in the developmentof effective diagnostic and therapeutic strategies for HCC.

Materials and MethodsCell culture, tumor tissues, inhibitors, RNA isolation,and cDNA synthesisThe HCC cell lines, HepG2 and Hep3B, were provided

by K.-K. Kuo (Kaohsiung Medical University, Kaohsiung,Taiwan) and 293T cells were obtained fromDr.W.C.Hung(National Sun Yat-senUniversity, Kaohsiung, Taiwan). Thecells were cultured in Dulbecco's Modified Eagle's Medium(DMEM), and supplemented with 10% FBS, nonessentialamino acids, 100 units/mL penicillin, and 100 mg/mLstreptomycin at 37�C in a 5% CO2 incubator. Seventy-onepairs of HCC tumors and the adjacent benign livers wereavailable for the study. The tumor samples, with thematched adjacent benign tissue, were collected duringsurgical resections at the Kaohsiung Chung-Ho Hospital

Clinic, between 2008 and 2011. The surgically resectedtumor samples were immediately snap-frozen and shippedwithin 24 hours in dry ice and subsequently stored in liquidnitrogen. The study was approved by the InstitutionalReviewBoard at theKaohsiungMedicalUniversity. Sectionsfrom each specimen were examined by pathologists andgraded histologically. Inhibitors treatment, RNA isolation,and cDNA synthesis from the cell lines and tumor sampleswere carried out using previously described procedures(24, 25).

Lentivirus production and short hairpin RNA for geneknockdownAll the plasmids required for short hairpin RNA (shRNA)

lentivirus production were purchased from the NationalRNAi Core Facility, Academia Sinica, Taiwan. The pLKO.1-shRNA vectors used for knockdown of SMAD4, BMPR1Aare as follows: TRCN0000010321 and TRCN0000010322(SMAD4, NM_005359); TRCN0000194749, TRCN000-0000454 and TRCN0000000795 (BMPR1A, NM_00-4329). The pLKO.1-shEGFP control plasmid is TRCN-0000072190 (enhance GFP, EGFP). The Lipofectamine2000 reagent (Invitrogen) was used for lentiviral productionin 293T cells, with a packaging construct (pCMV-DR8.91),an envelope construct (pMD.G) and different shRNA orrescue constructs, according to the protocol on the RNAiCore website (http://rnai.genmed.sinica.edu.tw/protocols).

Western blot analysis and immunofluorescenceFor Western blot analysis, cells were harvested in RIPA

lysis buffer [150 mmol/L NaCl, 10 mmol/L Tris, pH 7.5,1% NP40, 1% deoxycholate, 0.1% SDS, protease inhib-itor cocktail (Roche)]. The proteins from total cell lysateswere resolved by the 10% Bis-Tris gradient gel (Invitro-gen), transferred to the polyvinylidene difluoride mem-brane, blocked in 5% non-fat milk in PBS/Tween-20, andblotted with the antibodies. The following primary anti-bodies were used: BMPR1A (SC-20736), Smad4 (SC-7966), Smad1/5/8 (SC-6031-R), p-Smad1/5/8 (#9511),p-p44/42 (#9101), cyclin B1 (SC-752), and CDK1 (SC-54; Santa Cruz and Cell Signal); BMP4 (NCL-BMP4lNovocastra); b-actin (AC-74; Sigma); and rabbit anti-GAPDH (Novus Biologicals). For immunofluorescenceanalysis, the staining of Ki-67 (NCL-Ki67-P; Novocastra)and p-histone3 (SC-8656; Santa Cruz) was conducted, aspreviously described (25).

Real-time quantitative PCR analysisTotal RNA, prepared from the samples, was used for

cDNA synthesis and PCR amplification was conducted, asdescribed above. The gene-specific primer pairs used in theanalysis of the indicated specific genes and the glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) gene used forstandardization to normalize the abundance of the varioustranscripts analyzed are described in Table 1. The relativeabundance of the targeted gene-specific PCR products wasnormalized to GAPDH, and the results of the DCt measure-ments were previously described in detail (25). These

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Mol Cancer Res; 10(3) March 2012 Molecular Cancer Research416




experiments were independently repeated 3 times and eachtreatment consisted of triplicate samples.

Transient transfections and luciferase reporter assaysCells of 60% confluence in 24-well plates were trans-

fected using Lipofectamine 2000 (Invitrogen). Fireflyluciferase reporter gene construct (200 ng) and 1 ng ofthe pRL-SV40 Renilla luciferase construct (for normali-zation) were cotransfected per well. The transfected cellswere allowed to grow overnight, prior to BMP4 (R&DSystems) treatments, and the cell extracts were thenprepared 24 to 48 hours after transfection, and theluciferase activity was measured using the Dual-LuciferaseReporter Assay Kit (Promega), according to the manu-facturers' protocol, using OrionII Microplate Lumin-ometer (Berthold Technologies) at 570 nm. Expressionwas calculated as the ratio of arbitrary firefly luciferaseunits, normalized to Renilla luciferase, and were describedin detail previously (26). These experiments were inde-pendently repeated 3 times and each treatment consistedof triplicate samples.

Wound-healing assayCells were grown to 90% of confluency and a wound was

introduced using a sterile Q-tip. The ability of cells tomigrate was monitored at different time points using a lightmicroscope under different conditions. Images were cap-tured using a Zeiss Axiocam digital camera to monitor thecell migration rate.

Cell proliferation assayFor cell growth assay, 2� 104 cells were seeded in 24-well

plates and incubated overnight. The cells were treated withor without BMP4 (10–30 ng/mL; R&D) and incubated for1 to 5 days. Twenty-five microliters of MTT (5 mg/mL;PROTECH) in 500 mL medium was then added andincubated for another 2 hours for reaction. The mediumwas removed and crystal was completely dissolved with 200mL dimethyl sulfoxide (Sigma). TheOD570 readingwas thendetected with a BioTek ELISA reader (Level).

Colony formation assayA total of 10 � 103 cells were grown in 60-mm tissue

culture dishes. After 14 days, the cells were washed with PBSand fixed with methanol and 0.1% crystal violet. Thecolonies were manually counted and then photographed.

In vitro migration and invasion assayFor Transwell migration assays, 1 � 105 to 2 � 105 cells

were plated in the top chamber, with the noncoated filtermembrane (6-well insert; pore size, 8 mm; BD Biosciences).Cells were plated in medium without serum or growthfactors and the bottom medium supplemented with 10%FBS. FBS was used as a chemoattractant in the lowerchamber. The cells were incubated for 24 hours and cellsthat did not migrate or invade through the pores wereremoved by a cotton swab. Cells on the lower surface ofthe membrane were counted under the microscope (100�)and stained with the crystal violet, at which point, therepresentative images were captured. The crystal violet wasfurther dissolved in 10% acetic acid, and the absorbance wasmeasured at 540 nm for quantitative analysis.

Fluorescence-activated cell-sorting analysisCells that were subjected to designated treatments were

trypsinized, harvested and collected, washed 3 times in PBS,and then resuspended in PBS. Cells were then fixed in 70%ice-cold ethanol, for a minimum of 2 hours, and wereethanol eliminated by washing 3 times in PBS. Cell pelletswere resuspended in PBS to adjust the cell concentration toabout 1� 106/mL and RNase to 20 mg/mL and propidiumiodide (PI) to 50 mg/mL was then added. Incubation wasthen carried out for 2 hours at room temperature, in the dark,for staining. Finally, the cells were analyzed by FACScan(Becton Dickinson), using the Cell Quest Program.

SCID mice and injectionSpecific pathogen-free, 8-week-old female C.B17/lcr-

SCID mice were purchased from BioLASCO Taiwan Co.,Ltd, for the in vivo tumorigenicity study. The animals weremaintained in the animal center at the Department ofMedical Research, Kaohsiung Medical University Hospital,and treated according to the institutional guidelines for thecare and use of experimental animals. They were housedunder controlled lighting (14 hours of light, 10 hours ofdarkness) at temperatures of 21�C to 22�C and providedwith water and NIH-31 laboratory mouse chow ad libitum.The mice were injected subcutaneously into the left and

Table 1. Comparison of clinicopathologicfeatures and the status of BMP4 mRNAexpression in 71 patients with HCC

Increased BMP4mRNA expres-sion in HCC

No Yes Pa

GenderFemale 7 13 0.792Male 17 35

Histologic gradeG1 3 4 0.444G2 18 37G3 2 1

Tumor stagepT1 15 24 0.515pT2 6 16pT3 þ pT4 2 7

Microscopic peritumoral satellites� 15 14 0.044þ 11 31

aCategorized parameters were compared using Fisher'sexact test.

BMP4 Increases HCC Cell Proliferation and Migration

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right flank with 1� 106 cells in 0.1mL and raised during thefollowing 2 to 3 months. The mice were then monitored fortumor volume, overall health, and total body weight. Thesize of the tumor was determined by caliper measurement ofthe subcutaneous tumor mass. Tumor volume was calcu-lated according to the formula 4/3pr12r2 (r1 < r2). Eachexperimental group contained more than 3 mice. All micewere killed at the end of 3 months, and the tumor volumesand weight were then measured.

Mice surgery, necropsy, histopathology, andimmunohistochemistryTissue samples were fixed in 10%buffered formalin for 12

hours, followed by a wash with PBS, a transfer to 70%ethanol, and then embedded in paraffin, sectioned, andstained with hematoxylin and eosin (H&E). Immunohis-tochemical (IHC) analysis for BMP4 and BMPR1A wasconducted, as previously described (25).

Statistical analysisData are presented as mean � SEM. Student t test (2-

tailed) was used to compare the 2 groups (P < 0.05 wasconsidered significant), unless otherwise indicated (c2 test).

ResultsBMP4 and BMPR1A are upregulated, whereas BMPR1Bis downregulated in HCCWefirst investigated the relativemRNA expression level of

BMP4 in 71 pairs of human HCC specimens, using real-time quantitative PCR (qPCR) analysis, and found thatmRNA levels of BMP4 were significantly upregulated inmore than 60% of HCC specimens compared with thoseof corresponding noncancerous liver tissues (Fig. 1A).All tumor diagnosis, and further subclassification, wereconfirmed by light microscopic examination of the H&E-stained sections by pathologists at Kaohsiung MedicalUniversity Hospital (Table 1). To confirm the results ofreal-time qPCR, BMP4 protein expression in HCC in situwas further evaluated using IHC staining, with a specificantibody against BMP4. The results also reveal that BMP4protein is overexpressed in the epithelial cells of HCCspecimens compared with the corresponding noncancerouslivers [Fig. 1B, (iii) and (v)].The type I receptors of BMPs, BMPR1A (ALK3, BRK1)

and BMPR1B (ALK6, BRK2), have been identified as beinginvolved in conducting the BMP4 signaling to the cell. Wefurther evaluated the expression pattern of BMPR1A andBMPR1B in HCC specimens using real-time qPCR anal-ysis, and our results showed that upregulation of BMPR1Awas observed in 30 of the 71 (43%) examined HCC speci-mens; however, the relative expression level of BMPR1Bwasdecreased in 71 humanHCC specimens, in comparisonwiththat of corresponding noncancerous livers (Fig. 1A, bottomand Supplementary Fig. S1). Additional IHC analysis con-firmed the upregulation of BMPRIA protein in humanHCC specimens with a specific antibody against BMPR1A,and the IHC sections revealed that the immunosignal of

BMPR1Awas highly detectable in manyHCC specimens insitu but mildly detectable in the corresponding noncancer-ous livers [Fig. 1B (iv) and (vi)].

BMP4 induced CDK1 and cyclin B1 expression topromote HCC proliferationMany studies, including our reports, have shown that

BMP4 overexpression occurs in many cancers, includingHCC, and may be associated with tumor stages and poorprognosis (25). However, the exact molecular mechanismfor BMP4-induced HCC tumor promotion is not yet fullyunderstood.Human HCC cells, HepG2 and Hep3B, were treated

with recombinant BMP4 protein (10–30 ng/mL) to test theeffects of BMP4 on HCC cells. Using a light microscope, itwas observed that HCC cell populations showed an increasein absolute numbers, during 10 ng/mL BMP4 treatment for48 hours. The growth rate was increased after BMP4treatment compared with controls during a period of 5 daysof treatment for cell proliferation analysis (Fig. 2A and B).The expression of cell-cycle-associated genes that may beinvolved in BMP4-induced HCC cell growth was thenscreened, using real-time qPCR analysis. Western blotanalysis was used to evaluate and confirm the proteinexpression of targets (Fig. 2C).The results indicate that the expression levels of cyclin

B1 and CDK1 were increased in both HepG2 and Hep3Bcells, after BMP4 treatment (Fig. 2C). Cyclin B1 andCDK1 specifically control the G2–M phase transition ofthe cell cycle and are key components in the control ofcell-cycle progression. Several studies have shown thathigh levels of cyclin B1 promote cell-cycle progressionand division. We then conducted immunostaining anal-ysis with the anti-p-histone3 antibody to analyze themitotic index in HepG2 and BMP4-treated HepG2 cells,as the histone, H3, is phosphorylated at Ser10 by activeCDK1 during cell mitosis. p-Histone3-stained cells werephotographed, and their numbers estimated by countingthe number of p-histone–positive signals by using afluorescence microscope, as p-H3–stained positive cellscan present as mitotic cells. An increase in the number ofp-histone3þ cells was observed in BMP4-treated HepG2cells (Fig. 3A). An additional study, using Ki-67 immu-nofluorescence staining, supports the findings of thep-histone3 assays (data not shown). The cell-cycle pro-gression was also confirmed, using multiple parameters,via flow cytometric analysis. Our results confirm anincrease in the number of cells in S-phase and a decreasein the percentage of viable G1 phase in the cell cycle, afterrecombinant BMP4 treatment (Fig. 3B).Further analysis of the protein expression patterns of

cyclin B1 and CDK1 was undertaken. To determine thepatterns of cyclin B1 and CDK1 expression at differenttimes during the cell cycle, the cells were tracked andcollected for up to 24 hours after serum-starved (asyn-chronous) treatment. The representative pattern of cyclinB1 and CDK1 protein expression was analyzed by West-ern blot analysis, as shown in Fig. 3C. Our results show

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that BMP4 induces the earlier expression of cyclin B1 andCDK1 to promote cell-cycle progression in HCC. Thisimplies that BMP4 promotes cell division and acceleratedcell-cycle progression, by upregulation and activation ofcyclin B1 and CDK1.

Smad4 deficiency leads to increased expression of CDK1and cyclin B and enhances cell proliferation, especially inthe presence of BMP4To determine the molecular pathway involved in

BMP4-stimulated HCC cell proliferation, we first inves-tigate the effect of canonical SMAD-dependent signalingon BMP4-induced HCC cell proliferation. Intact BMP4-induced SMAD signaling in HCC cells was first examined.We then conducted Smad-binding element-driven report-er plasmid (SBE4-Luc) assays on the HCC model cell linesto determine whether SMAD-dependent signaling is acti-vated by treatment with BMP4. The results show thatluciferase activity was significantly increased, by 2- to 3-fold, in HCC cells after stimulation with recombinantBMP4 (10 ng/mL) for 18 hours. To further confirm thatthe SMAD signaling pathway was intact and could

respond to BMP4 treatment, we conducted Western blotanalysis for detecting the status of phospho-Smad1/5/8proteins in HCC cell lines to examine the BMP4-inducedserine/threonine kinase receptors to phosphorylatedSmad1/5/8 response, which lead to intracellular activatedSmad1/5/8 binding to Smad4. The results confirm thattreatment with BMP4 increases the level of phosphory-lation of Smad1/5/8 in HCC cells, which indicates theintact functioning of the BMP4/SMAD signaling pathwayin our HCC cell lines (Supplementary Fig. S2).To evaluate whether the status of BMP4/SMAD-medi-

ated signaling has an effect on BMP4-induced HCC cellproliferation using established shRNA techniques, we suc-cessfully generated stable SMAD4-knockdown HepG2clones for comparison with wild-type parental cells. Theeffects of SMAD4 shRNAs were verified by Western blotanalysis, which showed that SMAD4 protein was suppressedby 80% in Smad4 shRNA HepG2 stable clones (Fig. 4A).The results showed that there were increased levels ofexpression of cyclin B1 and CDK1 upon SMAD4 knock-down, as verified with real-time qPCR and Western blotanalysis of those HepG2 cells [Fig. 4B (i)]. Similar

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Figure 1. The increased expression of BMP4 and BMPR1A in patients with HCC. A, real-time qPCR analysis of mRNA expression in liver tumor tissues andnormal controls, BMP4 (top), and BMPR1A (bottom). B, H&E staining in liver tissues. i, normal tissue (N); ii, tumor tissue (T). Representative IHC staining innormal liver tissues and HCC. iii and v, BMP4; iv and vi, BMPR1A (magnification, 200�).






observations were confirmed in experiments that depletedSMAD4, using different SMAD4 shRNAs that corre-sponded to a different coding sequence of the SMAD4gene (data not shown). In further tests, we added recombi-nant BMP4 protein to SMAD4-knockdownHepG2 clones.It was observed that the expression of cyclin B1 and CDK1were still upregulated in those SMAD4-knockdowncells compared with those SMAD4-knockdown groupsnot subjected to treatment with recombinant BMP4[Fig. 4B (ii)].Next, we examined the cell growth rates of HepG2

SMAD4-knockdown clones, and the corresponding mockcontrol, with or without recombinant BMP4 treatment, bycell proliferation assay during the 5 days of treatment. Thedata are consistent with the molecular studies, which clearlyindicate that the BMP4/SMAD-dependent signaling cas-cade is not involved in the BMP4 stimulation ofHCC tumorproliferation (Fig. 4C, top, middle, and bottom).

Inhibition of MEK/ERK signaling pathway efficientlydecreases the action of BMP4, which induced theincreased expression of CDK1 and cyclin B1 in HCCTGF-b and BMP signaling are composed of conical

SMAD-dependent pathways and SMAD-independent path-ways, including those involving MEK/ERK, c-jun-NH2-kinase (JNK), and p38 mitogen-activated protein kinase(MAPK) pathways. To investigate the effect of the MEK/ERK signaling pathway on BMP4-induced HCC cell pro-liferation, we used the selective inhibitor, PD98059, toinhibit MEK/ERK signaling transduction in HCC cells.We first showed that the activation of p42/44 (ERK1/2) wasdetected, using Western blot analysis for phospho-p42/44(p-ERK1/2). The results show that the expression levels ofphospho-p42/44 (ERK1/2) in cells treated with recombi-nant BMP4were higher than those for the controls, as shownin Fig. 5A, which indicates that BMP4 can stimulate thephosphorylation of p42/44 (ERK1/2) in HCC cells.

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Figure 2. BMP4 promotes HCC cells proliferation and induces cyclin B1 and CDK1 upregulation. A, the related cell number of HepG2 already had differencebetween 2 study groups, with or without BMP4 (10 ng/mL) treatment. The optical micrographs depicting the difference of cell confluence (100�).B, cell proliferation assays of HepG2 and Hep3B cell lines (P < 0.05). C, the relative mRNA expression of downstream cell-cycle–associated genes inresponse to BMP4 (10 ng/mL) treatment. The expression of cyclin B1 (CCNB) and CDK1 were significantly increased in mRNA and protein levels of bothHepG2 (left) and Hep3B (right) cells (P < 0.05).

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We then added the selective MEK/ERK inhibitor,PD98059, to cells combined with BMP4 treatment. Theresults of real-time qPCR analysis imply that the selectiveMEK/ERK inhibitor, PD98059, inhibited the level of theBMP4-induced overexpression of cyclin B1 and CDK1 inHepG2 and Hep3B cells [Fig. 5B and C (i) and (ii)]. Theprotein expression of cyclin B1 and CDK1 was furtherdetermined by Western blot analysis [Fig. 5C (iv)]. We alsoexamined the influence of the p38MAPK pathway inhibitoron BMP4-induced HCC proliferation. Using p38 MAPKinhibitor, SB203580, we aimed to determine whether thep38 MAPK pathway was involved in BMP4-induced HCCcell-cycle progression and cell proliferation. Our data sug-gested that the p38MAPK pathway was not required for theBMP4-induced upregulation of CDK1 and cylcinB1 inHCCs (Supplementary Fig. S3).

The results indicate an important role for the MEK/ERKpathway in BMP4-induced upregulation of cyclin B1 andCDK1 in HCCs and increases in HCC cell proliferation.The inhibition of MEK/REK, but not of the p38 MAPKpathway, strongly impaired the BMP4-induced upregula-tion of cyclin B1 and CDK1 and inhibited HCC cellproliferation.

BMPR1A-knockdown HCC cells are significantly lesstumorigenic in vitro and in vivoTo study the effects of BMP4 signaling inHCCon in vitro

tumor formation, we conducted a colony formation assay,which is considered to be a stringent assay for the measure-ment of cellular transformation and tumorigenic ability invitro. First, we produced BMPR1A-knockdownHepG2 cellclones, using shRNA knockdown techniques, to examine

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Figure 3. BMP4 promotes cell-cycle progression in HepG2 cells. A, top, immunofluorescent staining for p-histone3 after exposure to different doses of BMP4.FBS (20%) serves as a positive control, and DNA was visualized with DAPI staining. Bottom, quantitative analysis of p-histone3–positive signal inBMP4-treated and control groups. B, flow cytometric analysis of HepG2 cells with or without BMP4 treatment. The percentage of S-phase in cell cyclewas increase and G1 phase was decreased after BMP4 treatment for 4 hours. C, Western blot analysis for cyclin B1 and CDK1 protein expression afterBMP4 treatment at different treatment time points. C, control; DAPI, 40,6-diamidino-2-phenylindole.






their effect on tumorigenicity in HepG2 cells in vitro. Theknockdown efficiency was confirmed by Western blot anal-ysis (Fig. 6A). The BMPR1A-knockdown HepG2 cellsshowed a significant reduction in the number of coloniesformed on the Petri dish compared with scrambled shRNA-EGFP–transfected controls, confirming the requirement ofactive BMP4 signaling for HCC growth (P < 0.01; Supple-mentary Fig. S4).To further investigate the effect of BMP4 signaling on

HCC carcinogenesis in vivo, BMPR1A-knockdownHepG2cells were injected into the flanks of 2 severe combinedimmunodeficient (SCID) mice subcutaneously. Lentiviralvector bearing shRNA (shEGFP) were used as controls toinvestigate the overall effect of BMP4 signaling on HCCprogression. As expected, BMPR1A-knockdown HepG2cells showed less tumorigenicity in vivo compared with the

control groups. Our results showed that tumor size andweight were clearly reduced compared with those of thecontrols (Fig. 6B, left and right). The Western blot analysisfrom those tumor samples verified that BMPR1A proteinexpression was reduced in the knockdown tumor samplescompared with the controls (Fig. 6C). Our data imply thatBMP4 signaling through BMPR1A increases HCC tumor-igenesis and should be considered as a potential therapeutictarget for HCC.

BMP4 type IA receptor knockdown inhibits the MEK/ERK signaling pathway and attenuates BMP4-inducedHCC cell migrationA recent study has reported that hypoxia can induce

BMP4 to promote the migration of HCC cells. It confirmedthe inhibitory effect of cell migration of HCC cells treated

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Figure 4. Smad4 deficiency leads to increase expression of CDK1 and cylinB1 and enhancesHCCcell proliferation. A,Western blot analysis of SMAD4 proteinlevels in different stable SMAD4-knockdown HepG2 clones. B, top, Smad4 suppresses CDK1 and cyclin B1 protein expression in HepG2 cells. Bottom,real-time qPCR assay for CDK1 and cyclin B1 mRNA expression in SMAD4-knockdown HepG2 stable clones and control cells with or without BMP4treatment. C, top, cell growth assay inSMAD4-knockdownHepG2stable clones andcontrol cellswithoutBMP4 treatment.Middle, cell growthcurve assay forSMAD4-knockdown cells and control cells with BMP4 treatment. Bottom, cell growth assay for shSMAD4-transfected HCC cells with or without BMP4treatment (P < 0.05). C, control.

Chiu et al.





with BMP4 siRNA compared with cells treated with controlsiRNA (23). We also observed that BMP4 signaling stimu-lates HCC cell migration, possibly through the MEK/ERKsignaling cascade.

To investigate the molecular mechanisms of BMP4-induced activation of HCC cell migration, BMPR1A-knockdown HepG2 clones were chosen to examine theextent of cell migration by wound-healing assays. We

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Figure 5. Inhibition of MEK-ERK signaling pathway attenuates BMP4-induced increased expression of CDK1 and cyclin B1. A, Western blot analysis forp-p42/44 (p-ERK1/2) to confirm the MEK/ERK signaling cascades. B, using selective inhibitors, SB203580 (p38; 10 mg/mL) and PD98059 (MEK/ERK;10mg/mL), to identify the effective pathways afterBMP4 (30 ng/mL) treatment.Real-timeqPCRanalysis for detecting the expressionofCDK1mRNA inHepG2cells after treatment with different inhibitors. C, the PD98059 inhibits the BMP4-mediated CDK1 and cyclin B1 expression. i and iii, HepG2; ii and iv,Hep3B cells. C, control.

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Figure 6. BMPR1A-knockdown HepG2 cells are significantly less tumorigenic in vivo xenograft studies. A, Western blot analysis confirmed the efficiency ofBMPR1A-knockdown in HepG2 cells. Three shRNA-BMPR1A HepG2 stable clones were analyzed for BMPR1A protein expression and selected theknockdown clone no. 3 to inject into SCID mice (n¼ 3) subcutaneously for in vivo tumor formation study. B, top, gross tumor formation of shRNA-BMPR1Aknockdown and control shEGFPHepG2 cells. Bottom left, average tumor weights of shRNA-BMPR1A groupswere significantly less than the control groups.Bottom right, average tumor volumes of shRNA-BMPR1A groups were smaller than the control (P < 0.01). C, control. C, the decreased BMPR1A proteinexpression was detected in knockdown of BMPR1A in xenograft tumors by Western blot analysis.






observed that the time taken for BMPR1A-knockdownHepG2 cells tomigrate into the cell free areas, and complete-ly close the wound, was longer than 48 hours, whereas themigration rate of HepG2 shEGFP cells was faster, regardlessof the presence of BMP4 (Fig. 7B). The SMAD4 knock-down was also found to accelerate the motility of HCC cells(data not shown).Using theMEK pathway selective inhibitor, PD98059, to

block MEK/ERK signaling transduction in HepG2 andHep3B cells, our data showed that selective MEK/ERKinhibitor efficiently blocked BMP4-triggered cell migrationfor 72 hours compared with that of control cells (Fig. 7A, leftand right). It is hypothesized that BMPR1A knockdownmay diminish the BMP4-induced MEK/ERK pathway andattenuate the effect of cell migration, triggered by BMP4 inHCC cells, and that the BMP4-induced MEK/ERK path-way might be involved in the migratory and invasive prop-erties of HCC (Fig. 7C). However, a detailed understandingof BMPR1A action requires identification of molecularalterations and downstream genes regulated by BMPR1Areceptor.

DiscussionThe upregulation of BMP family members has been

found in various human malignancies, including pancre-atic, skin, and liver cancer (21, 23, 27). Several studies, ofvarious cancer models, have indicated the importance of

the activation of the BMP signaling pathway in tumortissues during carcinogenesis, through autocrine and para-crine mechanisms, for the modulation of tumor cellgrowth, invasion, and metastasis (28–30). This studyshowed that BMP4 and BMPR1A were overexpressed inthe majority of primary HCC specimens. Although therewas no overexpression of BMP4 protein in the case ofevery HCC, we observed that BMP4-overexpressedpatients accounted for a high proportion (60%) of ourpatients with HCC and that BMPR1A was overexpressedin more than 40% of patients, regardless of upregulationof their BMP4 levels. Immunohistochemistry reveals thatthe upregulation of BMP4 and BMPR1A in HCCs isprimarily localized in the cytoplasmic compartment oftumor cells, in primary human HCCs. Our results alsoindicated that the upregulation of BMP4 protein maycorrelate with HCC tumor stages and microscopic peri-tumoral satellites, which are recognized as a risk factor fordistant dissemination (Supplementary Table S2). There-fore, BMP4 and BMPR1A are ideal prognostic markers forHCC, as some cases of HCC progression and aggressionare associated with this cytokine, BMP4, or its specificreceptor, BMPR1A. In the case of BMPR1B expression,however, our results suggest that there is downregulatedexpression of BMPR1B in HCCS.In general, BMPR1A and BMPR1B are similar in

structure and are preferentially bound by BMP2 andBMP4, with differing affinities. Several studies also

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Figure 7. BMP4 signaling through MEK/ERK pathway to promote HepG2 cell migration. A, cells were treated with or without MEK kinase inhibitor, 20 mmol/LPD98059, 30 minutes priors to introduction of the cell scratch and then follow indicated different BMP4 treatment. Left, HepG2 cells. Right, Hep3B cells.B, shBMPR1A and shEGFP (mock)-transfected HepG2 cells were treated with or without 10 ng/mL BMP4 and were grown to confluency and then awound was made with Q-tips. The images of wound-healing assays were measured for the degree of wound healing by using an optical scale under alight microscope at the indicated times. Images show representative examples of 3 independent experiments. C, BMP4 signaling cascade induces HCCcell proliferation and migration by MEK/ERK-dependent pathway.

Chiu et al.





suggest that BMPR1A and BMPR1B may have distinctfunctions in embryonic development. The loss ofBMPR1A leads to embryonic lethality (31, 32), but it isstill not possible to distinguish between unique or redun-dant functions of BMPR1A and BMPR1B in the laterstages of human development. Germ line mutations ofBMPR1A have been associated with juvenile polyposis inhumans and produce a significantly higher risk of gastro-intestinal cancer (33). BMPR1B homozygous null miceare viable; however, mutants exhibit skeletal defects (34),and BMPR1B is epigenetically silenced in some patient-derived tumor-initiating glioblastoma cell lines (35). Arecent study indicated that lower BMPR1B expression wasobserved in breast cancer tissues with poor prognosis, andthe decreased expression of BMPR1B resulted in increasedcell proliferation in breast cancer cells in vitro, whichsuggested that BMPR1B mediated a repressive effect onbreast cancer cells (36). The role of the downregulation ofmRNA of BMPR1B in liver cancer cells, as determined byreal time-qPCR, is still unclear, and presents an oppor-tunity for future research.The role of BMP4 in tumorigenesis and cell-cycle

progression in HCCs was unclear, prior to our studies.To determine whether the upregulation of BMP4 proteinplays a critical role that favors tumor malignancy and apropensity for metastasis of HCC, we selected HCC celllines model systems to investigate both the molecular andcellular alterations of HCCs in response to an increase inBMP4 levels. Out results indicated that, as with otherBMP family members, BMP4 can function as an activatorin cell-cycle progression in epithelial cancer cells and maybe essential for the aggressive metastasis of tumor cells. Wefurther identified that a function of BMP4 signaling caninduce cyclin B1 and CDK1 overexpression in HCC cells.The complex kinase of cyclin B1 and CDK1 is animportant mediator of the cell-cycle regulator, whichaccelerates the progress of mitosis (37–39). It wasobserved, through Ki-67 staining, that BMP4 triggeredthe synthesis of DNA, which is required for cell division,and promoted the phosphorylation of ser10 residue ofhistone3 known as a marker for mitotic progression (40).This study shows that BMP4 accelerates the growth ofHCC cells by the upregulation of cyclin B1 and CDK1 topromote cell-cycle progression and cell division. UsingBMPR1A shRNA, we also confirmed that the HepG2cells significantly reduced the tumorigenicity in vitro, bycolony formation assays and in vivo SCID mouse xeno-graft models. The migratory ability of HepG2 cells wasalso decreased by the BMPR1A knockdown in HepG2cells, as verified by wound-healing assays. The overexpres-sion of constitutively active BMPR1A (ALK-3 QD) recep-tor enhances HepG2 cells proliferation and migration, aswell as exhibiting an increased cyclin B1 and CDK1protein expression (Supplementary Fig. S5). It is proposedthat the enforced BMP4 may contribute to progressionand malignancy of HCC.BMP4 belongs to the TGF-b superfamily. It is com-

mon knowledge that BMP4 regulates gene expression not

only through the classical SMAD-dependent signalingpathway but also by the activation of many SMAD-independent signaling pathways. We determined thepredominant pathway that mediates BMP4-inducedHCC cell proliferation by knockdown of Smad4 proteinexpression to reduce the SMAD-dependent pathway inHepG2 cells and then confirmed that BMP4 inducedphosphorylation of Smad1/5/8, and subsequently activat-ed Smad4 downstream transcriptional activation in aSMAD reporter activity assay. Next, we used the selectiveMEK/ERK signaling pathway inhibitor, PD98059, toattenuate the BMP4-activated MEK/ERK pathway. Onthe basis of these studies, we found that further upregula-tion of the expression of cyclin B1 and CDK1 could resultfrom Smad4 deficiency in HepG2 cells, whereas inhibi-tion of the MEK/ERK signaling pathway efficientlyattenuates BMP4-induced upregulation and maintainedbasal levels of cyclin B1 and CDK1 in HepG2 cells.MEK/ERK signaling is believed to play an important rolein the regulation of cell proliferation and its signalingpathway is activated by different growth factors, includingBMP4 cytokines (41, 42). Growth factor–induced MEK/ERK cascade is considered to be an inducer for theregulation of multiple phosphorylating proteins and theirtarget proteins and to promote the interaction betweenretinoblastoma (Rb) protein and E2F-1 and to affecttranscriptional regulation of target genes involved incell-cycle control (43–45). E2F family–regulated genesare required for cell-cycle progression, depending on theactivation status of MEK/ERK. The E2F-regulating genesinclude CDK1, Cdc25A, and cyclin B1, which are asso-ciated with cell-cycle progression (46, 47). Therefore, ourfindings suggest that BMP4 induces the increased expres-sion of cyclin B1 and CDK1 to accelerate cell-cycleprogression through the MEK/ERK pathway but notthrough the SMAD-dependent pathway in HCCs (Fig.7C). It was also noted that the BMP4-induced phospho-level of Smad1/5/8, at 24 hours after BMP4 treatment,was lower than that at 1 hour after treatment. It ispresumed that there was cross-talk between the 2 path-ways in BMP4 activation, the MEK/ERK signaling path-way and SMAD-dependent pathway, during the 24 hoursof the study. Recent studies have indicated that Smad1 istargeted to proteasome for degradation in response toBMP type I receptor activation and that p-Smad1 targetedfor degradation to the centrosome is associated withMEK/ERK activation (48, 49). This may explain howthe MEK/ERK pathway overcomes the SMAD-depen-dent signaling to become the predominant pathway dur-ing BMP4 activation in liver carcinogenesis. Our study ofthe BMP4-induced MEK/ERK pathway in HCC is con-sistent with the results of our previous work, whichdescribed the TGF-b1–mediated activation of MEK/ERKpathways, combined with Smad4 deficiency, to promotecolorectal cancer malignancy (26, 50). We also deter-mined that the MEK/ERK signaling pathway has apotential role in regulating the proliferation and migra-tion of HCC cells.






In conclusion, our findings further support the notion thata BMP4!BMPR1A! SMAD1/5/8 signaling axis acts as atumor suppressor in HCC cells (16, 25, 26), and that theBMP4-induced MEK/ERK signaling pathway facilitatesproliferation and migration of liver cancer cells and theupregulation of BMP4, as an aggressive enhancer for hepa-tocarcinogenesis, by stimulating the overexpression of cyclinB1 andCDK1proteins inHCCcells. The results of the studysuggest that the effects of BMP4 signaling, which contributeto HCC, are worthy of further investigation. They also showthat the BMP4-inducedMEK/ERK cascade inHCC tumorsis an ideal therapeutic target for HCC treatment.

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

AcknowledgmentsThe authors thank Drs. Sam Thiagalingam, W.C. Hung, and Jeffrey Wrana for

generously providing cell lines and reagents.

Grant SupportThese studies were supported in part by grants (NSC 98-2314-B-037-027-

MY3 to K.-K. Kuo) and (NSC 98-2320-B-110-003-MY3 to K.-H. Cheng) fromthe National Science Council, Taiwan ROC, Department of Health, ExecutiveYuan, ROC (DOH100-TD-C-111-002 to K.-T. Lee and K.-K. Kuo), and grant ofNational Sun Yat-Sen University–Kaohsiung Medical University Joint ResearchCenter.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received June 21, 2011; revisedDecember 19, 2011; acceptedDecember 27, 2011;published OnlineFirst January 12, 2012.

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2012;10:415-427. Published OnlineFirst January 12, 2012.Mol Cancer Res Chiang-Yen Chiu, Kung-Kai Kuo, Tzu-Lei Kuo, et al. Proliferation and Migration

CellMorphogenetic Protein 4 to Increase Hepatocellular Carcinoma The Activation of MEK/ERK Signaling Pathway by Bone

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