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  • Human Cancer Biology

    Brain-Derived Neurotrophic Factor Promotes Tumorigenesis via Inductionof Neovascularization: Implication in Hepatocellular Carcinoma

    Chi-Tat Lam1, Zhen-Fan Yang1,2, Chi-Keung Lau1, Ka-Ho Tam1, Sheung-Tat Fan1,2, and Ronnie T.P. Poon1,2

    AbstractPurpose: Brain-derived neurotrophic factor (BDNF) has emerged as a novel angiogenic factor, and yet

    its impact on tumorigenesis is unclear. This study aimed at investigating the roles of BDNF in angiogenesis

    and tumor development.

    Experimental Design: BDNF was overexpressed in a mouse endothelial cell (EC) line by stable

    transfection, and angiogenic properties of the transfectants were assessed. Microarray analysis was

    employed to explore the molecular pathways. The impact of modulating BDNF levels in two mouse

    EC lines on tumorigenic potential of a transformed mouse liver cell line was evaluated by an in vivo

    cotransplantation model. BDNF and tropomyosin receptor kinase B (TrkB) protein levels were determined

    in 50 pairs of human hepatocellular carcinoma (HCC) tissues by Western blotting and immunohisto-

    chemistry. Survival analysis was carried out to determine their clinical significance.

    Results: Overexpression of BDNF could promote EC proliferation, migration, invasion, and survival.

    Microarray and molecular studies showed that RhoA, caspase-9, caspase-3, growth arrest specific 6, and

    VEGF could mediate BDNF/TrkB-induced angiogenesis. The cotransplantation experiment showed that

    high BDNF-expressing ECs could facilitate tumor angiogenesis and growth, whereas knockdown of BDNF

    by short hairpin RNAs impaired such effects. Furthermore, examination on human HCC tissues revealed

    upregulation of BDNF and TrkB protein levels in 46.0% and 33.3% of the cases studied, respectively.

    Immunohistochemistry disclosed strong BDNF reactivity in both tumor and endothelial cells. High TrkB

    expression was associated with shorter overall survival.

    Conclusions: BDNF/TrkB systemwas crucial for tumor angiogenesis and growth, whichmay represent a

    potential target for antiangiogenic therapy in HCC. Clin Cancer Res; 17(10); 312333. 2011 AACR.

    Introduction

    Antiangiogenic therapy is emerging as a therapeuticoption for cancer patients because of relatively low toxicity,low risk of drug resistance (1), and high target selectivity(2). Recent insights into the molecular mechanisms ofangiogenesis have led to identification of novel therapeutictargets, and brain-derived neurotrophic factor (BDNF)seems to be one of the candidates (35).BDNF belongs to a class of growth factors called neu-

    rotrophins, which have well-documented functions inneural development and are linked to neoplasia (4, 612). The expression of BDNF and its receptor, tropomyo-

    sin receptor kinase B (TrkB), has been reported in tumorcells of several human cancers (9, 13, 14). Intriguingly,overexpression of TrkB and BDNF is often associated withaggressive phenotype and poor prognosis of the disease(13), implying the oncogenic characteristics of BDNF/TrkB signaling cascades. Indeed, it has been shown thatBDNF/TrkB could promote cell proliferation and survival,and it induces metastasis by suppressing anoikis in var-ious cell types (8, 14, 15).

    Several lines of evidence indicate that BDNF may play arole in angiogenesis. BDNF deficiency leads to an abnor-mal cardiac vessel system in knockout mice, whereasBDNF overexpression in mouse gestational heartsincreases capillary density (3). BDNF also promotesangiogenic behaviors of rat brain endothelial cells (EC)in vitro (16). Furthermore, BDNF is capable of recruitingTrkB ECs and bone marrowderived hematopoieticprogenitor cells in an ischemic mouse model (17). Thesefindings support BDNF as a potential player in angiogen-esis. However, its detailed roles in angiogenesis andtumor development remain unclear and warrant furtherstudies.

    It is known that microenvironment plays critical roles intumorigenesis (18). Tumor microenvironment comprisingextracellular matrix, ECs, and stromal cells interacts with

    Authors' Affiliations: 1Department of Surgery and 2State Key Laboratoryfor Liver Research, The University of Hong Kong, Pokfulam, Hong Kong,China

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

    Corresponding Author: Ronnie T. Poon, Department of Surgery, TheUniversity of Hong Kong, QueenMary Hospital, 102 Pokfulam Road, HongKong, China. Phone: 852-2255-3641; Fax: 852-2817-5475. E-mail:poontp@hkucc.hku.hk

    doi: 10.1158/1078-0432.CCR-10-2802

    2011 American Association for Cancer Research.

    ClinicalCancer

    Research

    www.aacrjournals.org 3123

    Research. on August 29, 2018. 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

    Published OnlineFirst March 18, 2011; DOI: 10.1158/1078-0432.CCR-10-2802

    http://clincancerres.aacrjournals.org/

  • tumor cells by secreting soluble factors and by providingcellcell and cellmatrix interactions (19, 20). Here, wehypothesize that overexpressing BDNF in ECs may pro-mote angiogenesis and provide a favorable microenviron-ment for tumor growth. To validate this hypothesis, weconducted in vitro and in vivo assays after modulating BDNFlevels in ECs. In addition, human hepatocellular carcinoma(HCC) tissues were examined for BDNF/TrkB expressionand its implication in hepatocarcinogenesis was evaluated.

    Materials and Methods

    Cell culture and reagentsTwo mouse EC lines, MILE SVEN 1 (MS1) and SVEC4-

    10EE2 (SVEC4), and a transformed mouse liver cell line,BNL 1ME A.7R.1 (BNL), were purchased from AmericanType Culture Collection. Human umbilical vein endothe-lial cells (HUVEC) were obtained from Cascade Biologics.MS1 and BNL cells were maintained in Dulbeccos mod-ified Eagles medium (DMEM) supplemented with 10%FBS, and 100 units/mL penicillin and 100 mg/mL strepto-mycin. SVEC4 cells were cultured in DMEM with 10%horse serum. HUVECs were grown in Medium 200 sup-plemented with low serum growth supplement (CascadeBiologics). All cells were grown in a humidified 5% CO2atmosphere at 37C.

    Recombinant human BDNF and Trk inhibitor, K252a,were obtained from Calbiochem. Warfarin was purchasedfrom SigmaAldrich. Recombinant mouse Gas6 wasobtained from R&D Systems.

    Patients and sample collectionTumor and corresponding nontumorous tissues were

    collected from HCC patients undergone hepatectomy at

    Queen Mary Hospital, Hong Kong, between 2003 and2008. Fifty cases were randomly recruited in the currentstudy. All patients had a diagnosis of primary HCC, andnone of them had received treatment before surgery.Pathologic diagnosis was based on the histologic exam-ination of tumor specimens by experienced pathologists.For the total of 50 patients, 34 had cirrhotic livers and 16had noncirrhotic livers (13 chronic hepatitis and 3noncirrhotic). Six normal livers (from liver donors; 2men and 4 women; aged 5062) were included as con-trols. All tissues were obtained from consenting patientsand approved by the Institutional Review Board of theUniversity of Hong Kong. Tissues were immediatelysnap-frozen in liquid nitrogen after surgical resectionand stored at 80C prior to analysis. Parallel sectionswere formalin-fixed and paraffin-embedded for histolo-gic and immunohistochemical studies. The clinical datafor the patients are summarized in SupplementaryTable S1.

    Reverse transcription-PCR and quantitative PCRTotal RNAwas extracted from cells using RNeasyMini Kit

    (QIAGEN) according to the user manual. TRIzol reagentwas used to extract total RNA frommouse tissues followingthe manufacturers instructions (Invitrogen). cDNA wassynthesized from total RNA by using ImProm-II ReverseTranscription System (Promega). The primer sequencesand PCR conditions are summarized in SupplementaryTable S2.

    Quantitative PCR (qPCR) was carried out on a 7900HTFast Real-Time PCR system (Applied Biosystems), usingSYBRGreen PCR reagents. Relative quantificationwas doneusing DDCt method, normalizing to eukaryotic 18S rRNA.Dissociation curves were generated to evaluate PCR pro-duct specificity and purity.

    Construct preparation and stable transfectionTotal RNA was purified from adult BALB/c mouse brain,

    and cDNAwas synthesized as described above. After reversetranscription using oligo(dT) primers, cDNA template wasamplified by PCR using primers (Supplementary Table S2)designed to flank BDNF coding sequence (CDS; GenBankno. NM_001048139). PCR products were cloned intopGEM-T Easy Vector (Promega) and then subcloned intopcDNA3.1/Hygro() vector (Invitrogen) at NotI site. Theconstructs were verified by restriction enzyme digestionand sequencing using T7 primer (50-GTAATACGACTCAC-TATAGGGC-30). BDNF construct [pcDNA3.1()-BDNF]was transfected into MS1 cells by using FuGENE 6 Trans-fection Reagent (Roche) following the manufacturers pro-tocol. Clones were selected in medium furthersupplemented with 400 mg/mL Hygromycin B (Invitrogen)and verified by measuring BDNF at mRNA and proteinlevels, using reverse transcription-PCR (RT-PCR), Westernblotting, and ELISA, respectively.

    Preparation of short hairpin RNA (shRNA) constructstargeting against BDNF was described in SupplementaryMaterials and Methods. Transfections of empty vector

    Translational Relevance

    Brain-derived neurotrophic factor (BDNF) is a poten-tial angiogenic factor. In this study, we showed thatoverexpression of BDNF confers angiogenic propertieson endothelial cells (EC). By establishing an in vivocotransplantation model in nude mice, we showed thathigh BDNF-expressing ECs could promote tumorgrowth, whereas its knockdown by shRNAs impairedthe tumor-promoting effect. These data suggested acritical role of BDNF/tropomyosin recept

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