Tumor and Stem Cell Biology

ANGPTL1 Interacts with Integrin a1b1 to SuppressHCC Angiogenesis and Metastasis by InhibitingJAK2/STAT3 SignalingQian Yan1,2, Lingxi Jiang1,2, Ming Liu1,2,3,4, Dandan Yu1,2, Yu Zhang1,2, Yan Li1,2,5,Shuo Fang1,2, Yan Li6, Ying-Hui Zhu6, Yun-Fei Yuan6, and Xin-Yuan Guan1,2,6

Abstract

Downregulation of tumor suppressor signaling plays animportant role in the pathogenesis of hepatocellular carcino-ma (HCC). Here, we report that downregulation of the angio-poietin-like protein ANGPTL1 is associated with vascular inva-sion, tumor thrombus, metastasis, and poor prognosis inHCC. Ectopic expression of ANGPTL1 in HCC cells effectivelydecreased their in vitro and in vivo tumorigenicity, cell motility,and angiogenesis. shRNA-mediated depletion of ANGPTL1exerted opposing effects. ANGPTL1 promoted apoptosis via

inhibition of the STAT3/Bcl-2–mediated antiapoptotic path-way and decreased cell migration and invasion via down-regulation of transcription factors SNAIL and SLUG. Further-more, ANGPTL1 inhibited angiogenesis by attenuating ERKand AKT signaling and interacted with integrin a1b1 receptorto suppress the downstream FAK/Src–JAK–STAT3 signalingpathway. Taken together, these results suggest ANGPTL1 as aprognostic biomarker and novel therapeutic agent in HCC.Cancer Res; 77(21); 5831–45. �2017 AACR.

IntroductionHepatocellular carcinoma (HCC) is one of the most frequent

human malignancies and ranks as the third leading cause ofcancer-related deaths worldwide (1). A number of HCC studiesrevealed promoter DNA hypermethylation could lead tothe silencing of tumor suppressor genes (2, 3), which is pos-itively correlated with HCC development and progression (4).In a recent study, genome-wide methylation analysis (27,578CpG sites) was applied to identify differentially methylatedgenes between 71 primary HCCs and 8 nondiseased (ND)normal liver tissues (5). A total of 678 genes were significantlyhypermethylated in HCCs. Our previous RNA-sequencingresults derived from 3 pairs of HCC cases found 407 geneswere downregulated in all 3 cases (GenBank accession no.GSE33294; ref. 6). By combining these two databases, weidentified 20 genes that were overlapped and angiopoietin-like

protein 1 (ANGPTL1) is one of the significantly downregulatedgenes with promoter hypermethylation.

Structurally similar to angiopoietins, angiopoietin-like pro-teins (ANGPTL) were recently identified as a protein familyconsisting of 8 members, ANGPTL1–ANGPTL8 (7). Angiopoie-tin-like proteins were reported to be able to potently regulateangiogenesis (8–10). Some of them were also found to affectinflammation (11), lipid metabolism (12), hematopoietic stemcell behavior (13), and cancer cell invasion (14–18). Previousstudies revealed that ANGPTL1 functions as a key antiangiogenicprotein (19). The downregulated transcription level of ANGPTL1has been found in various cancers (19), Several studies havedemonstrated the inhibitory role of ANGPTL1 on tumor growthand metastasis (20, 21). However, little is known about the roleand underlying mechanism of ANGPTL1 in HCC pathogenesisand progression.

In this research, we studied expression status of ANGPTL1 inHCC and its clinical significance. Both in vitro and in vivo func-tional assays were applied to characterize the effects of ANGPTL1in cell growth, apoptosis, cell motility, and angiogenesis. We alsodemonstrated that ANGPTL1 interacts with integrin a1b1 andsuppresses its downstream signaling to promote apoptosis andrepress cancer cell invasion and angiogenesis.

Materials and MethodsHCC clinical samples and cell lines

A total of 122 primary HCC samples and their adjacent non-tumor tissues were obtained from patients who underwent hep-atectomy from HCC at Sun Yat-sen University Cancer Center(Guangzhou, China). All HCC patients gave written informedconsent on the use of clinical specimens for medical research. Thesamples used in this study were approved by the Committees forEthical Review of Research Involving Human Subjects at the SunYat-Sen University Cancer Center (Guangzhou, China). All HCC

1Department of Clinical Oncology, The University of Hong Kong, Hong Kong,China. 2State Key Laboratory for Liver Research, The University of Hong Kong,Hong Kong, China. 3Key Laboratory of Protein Modification and Degradation,School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou,China. 4Affiliated Cancer Hospital of GuangzhouMedical University, Guangzhou,China. 5Department of Biology, South University of Science and Technology ofChina, Shenzhen, China. 6State Key Laboratory of Oncology in Southern China,Sun Yat-sen University Cancer Center, Guangzhou, China.

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

Q. Yan and L. Jiang contributed equally to this article.

Corresponding Author: Xin-Yuan Guan, The University of Hong Kong, RoomL10-56, Laboratory Block, 21 Sassoon Road, Hong Kong 852, China. Phone: 852-3917-9782; Fax: 852-2816-9126; E-mail: xyguan@hku.hk

doi: 10.1158/0008-5472.CAN-17-0579

�2017 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 5831

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579

cell lines used in this study were obtained from the Institute ofVirology at the Chinese Academy of Medical Sciences (Beijing,China), and maintained in DMEM (Gibco BRL) supplementedwith 10% FBS (Gibco BRL) and 1% penicillin/streptomycin.Human umbilical vein endothelial cells (HUVEC) were main-tained in Medium 200 supplemented with low serum growthsupplement (Thermo Fisher Scientific). All cell lines used weremycoplasma free.

Antibodies and reagentsThe following antibodies were used in the immunoblot assays:

ANGPTL1 (Thermo Fisher Scientific), b-actin (Abcam), ERK1/2(ImmunoWayBiotechnology), phospho-STAT3 (Tyr705), STAT3,Bcl-xL, phospho-AKT, AKT, c-Jun, phospho-MEK1/2 (Ser217/221), phospho-p44/42 MAPK, E-cadherin, vimentin, b-catenin,SLUG, SNAIL, caspase-3, caspase-9, PARP, phospho-FAK, FAK,phosphor-Src, phospho-JAK2, c-Myc, cyclin D1, Pim2, IntegrinAntibody Sampler Kit (Cell Signaling Technology), Bcl-2, JAK2(Santa Cruz Biotechnology), Fibronectin, N-cadherin, MMP-9(Proteintech), HIF1a (GeneTex), anti-Mouse CD34 (BD Phar-mingen), and VEGFA (Boster). Recombinant protein rANGPTL1was purchased from Abnova. Integrin a1b1 inhibitor obtustatinwas obtained from Tocris Bioscience.

RNA extraction and qRT-PCRTotal RNAwas extracted andpurifiedusing TRIzol (Invitrogen),

and then cDNA was synthesized using the Transcriptor HighFidelity cDNA Synthesis Kit (Roche). SYBR Green PCR Kit(Applied Biosystems) was applied to conduct qRT-PCR. Primersequences are listed in Supplementary Table S1.

Construction of ANGPTL1 overexpression and knockdowncells

To evaluate the tumor-suppressive function of ANGPTL1, full-length ANGPTL1 cDNAwas cloned into the pLenti6/V5 Lentiviralexpression vector (Invitrogen). Blasticidin (Sigma-Aldrich) wasused to select for stably transduced cells. For ANGPTL1 knock-down assay, short hairpin RNAs (shRNA) specifically targetingANGPTL1 were cloned into the lentiviral vector PLL3.7(Addgene), followed by puromycin (Sigma-Aldrich) selection.

Bisulfite treatment and methylation analysisGenomic DNA was isolated and purified from HCC clinical

samples and cell lines by phenol-chloroform method. EpiTECTBisulfite Kit (Qiagen) was used to treat DNA.Methylation-specificPCR and bisulfite genomic sequencing were carried out usingprimers listed in Supplementary Table S1.

In vitro tumorigenicity assaysFoci formation assay was performed to determine the anchor-

age-dependent tumor cells' growth ability. Briefly, 1 � 103 cellswere seeded in a 6-well plate. Two weeks later, the survivingcolonies were fixed, stained with Crystal violet, and counted.Anchorage-independent growth was assessed by colony-formingability in soft agar.

Migration and invasion assaysCells suspended in serum-free DMEM were seeded into

wells with a 8-mm microporous filter (Becton Dickinson lab-ware). Cells were attracted by 10% FBS-containing medium.After 48 hours, cells were fixed, stained, and counted. For the

invasion assay, the microporous filter was coated with Matrigel(Becton Dickinson). Triplicate independent experiments wereperformed.

In vivo tumorigenicity assayThe in vivo tumorigenic ability of ANGPTL1 was investigated

in a xenograft mouse model. Cells were injected subcutane-ously into the left and right dorsal flank of BALB/cAnN-nu(nude) mice. Tumor formation was monitored by measuringthe tumor volume weekly. Tumor volume was calculated as0.5 � l � w2, in which l is the length and w is the width ofthe tumor.

In vivo metastasis assayCells were transplanted through intrasplenic injection into

4-week-old BALB/cAnN-nu (nude) mice, and all animal pro-cedures were approved by Committee on the Use of LiveAnimals in Teaching and Research. All of the mice were eutha-nized 6 weeks after injection. Tumor nodules that formed onthe liver surfaces were detected and counted. After the exper-iment, hematoxylin and eosin staining was performed to visu-alize the structure of the livers. IHC and TUNEL assays wereperformed to confirm the expression of ANGPTL1 and apopto-tic cells, respectively.

Microvessel countsIHC was performed on sections using anti-CD34 antibodies.

Microvessel counts were analyzed by counting cells stained withanti-CD34. The highest number of vessels was counted under a20� objective.

Immunofluorescence stainingCells were seeded onto cover slides in 6-well plates. When

reached to 60%–80% confluence the next day, cells were fixed,permeablilized, blocked, and then incubated with primary anti-bodies. Alexa Fluor dye–conjugated secondary antibodies wereadded, followed by counterstaining with 40,60-diamidino-2-phe-nylindole (Invitrogen). Imageswere acquired using laser scanningconfocal microscope.

Immunoprecipitation assaysFor immunoprecipitation assay, 5 mg of total cell lysate was

immunoprecipitated with anti-FLAG or anti-IgG Affinity gel(Biotool) at 4�C overnight. Extensive washing and immunocom-plexes denaturation steps were carried out according to themanufacturer's instructions. Denatured immunocomplexes wereanalyzed by Western blotting. About 5% of the whole lysate(Input) was used as a positive control. Western blotting analysiswas performed with the standard protocol.

Endothelial cell tube formation assayHUVECs were resuspended in Medium 200 in the presence

or absence of different concentrations of rANGPTL1. Cells(4 � 104) were seeded per well onto solidified Matrigel (BDBiosciences) in 96-well plates. The cells were incubated at37�C for 4–6 hours. Tubular networks were visualized withmicroscope.

Morphometric score assessment of IHC stainingFor IHC assays, IHC results were assessed by a semiquantitative

scoring criterion in which both the staining intensity and the

Yan et al.

Cancer Res; 77(21) November 1, 2017 Cancer Research5832

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579

percentage of positive-stained cells were recorded. A stainingindex (with values from 0 to 9) was obtained as the intensity ofstaining (negative ¼ 0, weak ¼ 1, moderate ¼ 2, or strong ¼ 3)times the proportion of immunopositive cells (0%–25% ¼ 1,25%–50% ¼ 2, >50% ¼ 3). The morphometric score in 10 pairsof HCC clinical samples were listed in Supplementary Table S2.

Statistical analysisStatistical analysis was performed using SPSS (version 16.0).

Paired Student t test was used to compare the mRNA level ofANGPTL1 inpaired nontumor and tumor tissues. An independentStudent t test was used to compare the mean value of any twopreselected groups. The association of ANGPTL1 expression withclinicopathologic parameters was analyzed with Pearson c2 test.Survival analysis was conducted by using Kaplan–Meier plots andlog-rank tests. Univariable and multivariable Cox proportionalhazard regression models were used to analyze independentprognostic factors. The data are presented as the mean � SD ofthree independent experiments. Fisher exact test was used toanalyze the association of promoter methylation and the expres-sion of ANGPTL1. A P value less than 0.05 was consideredstatistically significant.

ResultsANGPTL1 was frequently downregulated in HCC

To determine whether downregulation of ANGPTL1 was acommon event in HCC, qPCR was used to detect the expressionlevel of ANGPTL1 in 122 pairs of HCC samples. Downregula-tion of ANGPTL1 was detected in 64 of 122 (52.5%) HCCs(cut-off score is 0.2). Compared with nontumor tissue, therelative level of ANGPTL1 was significantly lower in its tumorcounterparts (paired Student t test, P < 0.0001; Fig. 1A). In aKaplan–Meier survival analysis comparing patients with dif-ferent ANGPTL1 expression levels, lower ANGPTL1 expressionwas significantly correlated with a shorter overall survival (OS;log-rank test, 10.487; P ¼ 0.001; Fig. 1B) and shorter disease-free survival time (log-rank test, 4.073; P ¼ 0.044; Fig. 1B). Toinvestigate the clinical significance of ANGPTL1 expression inHCC, clinicopathologic association study was performed in122 pairs of HCC samples. The results showed that lowerexpression of ANGPTL1 was significantly correlated with seruma-fetoprotein (AFP) level (P ¼ 0.014), vascular invasion (P ¼0.029), poor differentiation (P ¼ 0.022), metastasis (P ¼0.043), and tumor thrombus (P ¼ 0.049; Pearson c2 test,Supplementary Table S3). Multivariate Cox regression analysisfurther revealed that ANGPTL1 was an independent prognosticmarker for the OS time of HCC patients (HR, 2.048; 95%confidence interval, 1.051–3.992, P ¼ 0.035; SupplementaryTable S4).

Protein expression level of ANGPTL1 was also determined byIHC staining and Western blot analysis. We examined theANGPTL1 protein expression in 10 pairs of HCC clinical samplesby IHC analysis, and a relatively strong ANGPTL1 staining wasconsistently observed in thenontumor samples (Fig. 1C).Westernblot was also performed inHCC cell lines, and downregulation ofANGPTL1 was detected in almost all HCC cell lines comparedwith immortalized liver cell line MiHA, LO2, and primary culturehepatocytes (Fig. 1D). TO detect the secreted protein, conditionalmedia of those cell lines were collected and ELISA assay wasperformed. Consistently, the level of secreted ANGPTL1 in HCC

cell lines was less than immortalized liver cell line MiHA andLO2 (Fig. 1E).

Promoter hypermethylation of ANGPTL1To determine the effects of epigenetic regulation on

ANGPTL1 expression, two HCC cell lines PLC-8024 and Huh7with undetectable ANGPTL1 expression were treated with dif-ferent concentrations of DNA methyltransferase inhibitor5-azacytidine (5-aza-dC) and histone acetylation agent tricho-statin A (TSA). Restoration of ANGPTL1 mRNA and proteinexpression was observed when the concentration of 5-aza-dCincreased to 400 mmol/L (Fig. 1F and G). Treatment with TSA,however, did not enhance ANGPTL1 expression (Fig. 1F),indicating that DNA methylation, but not histone modifica-tion, should be responsible for ANGPTL1 downregulation inHCC. To further confirm the methylation status of ANGPTL1promoter region, bisulfite genomic sequencing (BGS) andmethylation-specific PCR (MSP) were applied to study themethylation status of ANGPTL1 promoter in clinical samples.One CpG-island at the promoter region (�2971 nt to �2732nt) of ANGPTL1 gene was predicted by a publicly availabledatabase (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi; Fig. 1H). BGS was conducted in three pairs ofmatched HCC and nontumor clinical samples with ANGPTL1downregulation in tumor sample. A higher density of methyl-ation was detected in all three HCC samples compared withtheir nontumor counterparts (Fig. 1H).

MSP using methylation- and unmethylation-specific primerswas then conducted in 50 primary HCC tumors and their pairednontumor tissue. Downregulation of ANGPTL1 was detected in30of 50of tumor tissues byqRT-PCR, comparedwith correspond-ing nontumor tissues. Interestingly, MSP study found that meth-ylated PCR product was detected in all tumor and nontumorspecimens. Unmethylated PCR product was detected in all non-tumor specimens, but only in 13of 30 (43.3%)of the tumor tissuewith lower ANGPTL1 expression, and 17 of 20 (85%) tumor withnormal ANGPTL1 expression, respectively (Fig. 1I and J). Statis-tical analysis showed ANGPTL1 expression is significantly corre-lated with promoter hypermethylation (P ¼ 0.0038, Fisher exacttest, Fig. 1J), supporting that DNA promoter methylation is themajor cause of ANGPTL1 downregulation in HCC.

Tumor-suppressive function of ANGPTL1To explore whether ANGPTL1 possesses a tumor-suppressive

function in HCC, ANGPTL1 was cloned into a lentivectorand infected HCC cell lines PLC-8024 and Huh7. Expressionof ANGPTL1 in these two cell lines was determined by qPCRand Western blot analysis (Fig. 2A and B). The tumor-suppres-sive role of ANGPTL1 was addressed by cell proliferation, fociformation, and soft agar assay. Compared with control cells,ANGPTL1-transfected cells showed impaired abilities of fociformation (Fig. 2C) and colony formation in soft agar(Fig. 2D). However, cells overexpressing ANGPTL1 exhibitedno difference in cell proliferation (data not shown). Then,parental cells BLE-7402 and QGY-7703 with relatively highexpression level of ANGPTL1 were treated with ANGPTL1-neutralizing antibody, and foci formation assay results reveal-ed that treatment with neutralizing antibody increased thenumber of foci formed significantly (Supplementary Fig.S1A and S1B). To assess the in vivo tumor-suppressive abilityof ANGPTL1, LacZ- and ANGPTL1-transfected cells were

ANGPTL1 Suppresses HCC Angiogenesis and Metastasis

www.aacrjournals.org Cancer Res; 77(21) November 1, 2017 5833

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579

Figure 1.

ANGPTL1 was frequently downregulated in HCC. A, Scatterplots of the relative expression of ANGPTL1 detected by qRT-PCR in HCC and paired adjacentnontumor tissues. Black lines, mean � SD. B, Kaplan–Meier overall survival curve (left) and disease-free survival curve (right) of HCC patientscorrelated with ANGPTL1 expression. ANGPTL1(�), patients with ANGPTL1 downregulation; ANGPTL1(þ), patients without ANGPTL1 downregulation.C, Representative image of IHC staining with an anti-ANGPTL1 antibody, showing higher expression of ANGPTL1 in nontumor tissues (magnification,�400). D,Expressions of ANGPTL1 in HCC cell lines, immortalized liver cell lines, and hepatocytes were detected by Western blot analysis. b-Actin was used as a loadingcontrol. E, Detection of secreted ANGPTL1 level in HCC cell lines and immortalized liver cell lines. F and G, Detection of ANGPTL1 mRNA and proteinexpression in PLC-8024 and Huh7 cells by qRT-PCR after demethylation (5-Aza-dC) or histone acetylation (TSA) treatment. The results are expressed as mean� SD of three independent experiments. H, A schematic diagram showing the predicted CpG island upstream of the ANGPTL1 gene. The methylation status ofCpG dinucleotides in three pairs of HCC cases was detected by BGS sequencing. The percentage of methylation at each CpG site is displayed in thepie charts. Primers for MSP were mapped. I, Representative MSP analysis of ANGPTL1 promoter in primary HCCs. M, methylation; U, unmethylation. b-Actinwas used as a loading control. Clinical samples with case number 523, 531, and 558 were patients with ANGPTL1 normal expression in HCC tissues, while 171,147, and 219 were patients with ANGPTL1 lower expression in HCC tissues, compared with nontumor tissues. J, Association study indicates that thedownregulation of ANGPTL1 was significantly associated with aberrant methylation of the ANGPTL1 gene (P < 0.01, Fisher exact test).

Yan et al.

Cancer Res; 77(21) November 1, 2017 Cancer Research5834

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579

Figure 2.

ANGPTL1 has strong tumor-suppressive function in HCC. Ectopic expression of ANGPTL1 was detected in ANGPTL1-transfected cells by qRT-PCR (A)and Western blotting (B). 18S and b-actin were used as loading control, respectively. C, Representative images of foci formation in monolayer culture.The numbers of foci were calculated and are depicted in the bar chart. Values are the mean � SD of three independent experiments. �� , P < 0.01,independent Student t test. D, Representative images of colony formation in soft agar culture. The numbers of colonies were calculated and aredepicted in the bar chart. Values are the mean � SD of three independent experiments. �� , P < 0.01, independent Student t test. E, Images of micewith tumor formed induced by injecting the indicated cells. Tumor growth curves are summarized in the line chart. The average tumor volume is expressed asthe mean � SD of 5 mice. � , P < 0.05; ��� , P < 0.001, independent Student t test. F, IHC staining of proliferative index Ki67 in xenograft tumors. The percentageof Ki67-positive cells is summarized in the bar chart. ��� , P < 0.001.

ANGPTL1 Suppresses HCC Angiogenesis and Metastasis

www.aacrjournals.org Cancer Res; 77(21) November 1, 2017 5835

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579

injected subcutaneously into the left and right dorsal flank ofnude mice (n ¼ 10), respectively. Tumors induced byANGPTL1-transfected PLC-8024 cells (tumor formation in2/10 of mice) showed significantly longer latency and smallermean tumor volume than tumors induced by LacZ-transfectedcells (tumor formation in 9/10 of mice; P < 0.01, Student ttest, Fig. 2E). A similar result was obtained when usingANGPTL1-transfected Huh7 cells in a xenograft mouse exper-iment (P < 0.05, Student t test, Fig. 2E). To confirm cellproliferative status in xenograft tumors, IHC staining of pro-liferative index Ki67 was performed. Results demonstrated thattumors induced by 8024- or Huh7-ANGPTL1 cells showed asignificantly lower percentage of Ki67-positive signals com-pared with 8024- or Huh7-LacZ cells (Fig. 2F), indicating thetumor-suppressive function of ANGPTL1.

shRNA-mediated ANGPTL1 silencing abolishes its tumor-suppressive function

To confirm whether ANGPTL1 is required for the tumor-sup-pressive phenotype of HCC cells, we silenced ANGPTL1 expres-sion with short hairpin RNA (shRNA) against ANGPTL1. TwoshRNAs (ANGPTL1-sh4, ANGPTL1-sh5) were introduced into theHCCcell lineQGY-7703and immortalized liver cell line LO2. Thesilencing effect was determined by qPCR and Western blot anal-ysis, and the result showed that both shRNAs could dramaticallydecreased the ANGPTL1 expression level compared with Vector-transfected cells (Fig. 3A and B). Functional assays revealed thatANGPTL1 silencing could increase the efficiency of foci formation(P < 0.05, Student t test, Fig. 3C) and colony formation in soft agar(P < 0.05, Student t test, Fig. 3D), compared with Vector-trans-fected cells. For in vivo xenograft experiments, 7703-shANGPTL1and 7703-Vec cells were subcutaneously injected into nude mice(n¼ 11). The average volume of tumors induced by shANGPTL1-transfected cells (tumor formation in all 11 mice) was signifi-cantly larger than that induced by Vec-transfected cells (tumorformation in 10 mice; Fig. 3E). Similar result was obtained whenusing LO2-Vec and LO2-shANGPTL1 cells in xenograft mousemodels (Fig. 3E). IHC staining of Ki67 was conducted to confirmthe proliferative status in xenografts. Tumors induced byshANGPTL1-transfected cells showed a significantly higher per-centage of Ki67-positive cells (Fig. 3F). Collectively, these dataindicate that ANGPTL1 possesses strong tumor-suppressive abil-ity both in vitro and in vivo.

ANGPTL1 promotes apoptosis induced by cisplatin inHCC cells

To explore the molecular mechanism involved in ANGPTL1-mediated HCC cell survival, we investigated the effect ofANGPTL1 on apoptosis induced by chemotherapeutic agentcisplatin. ANGPTL1- and LacZ-transfected cells were treatedwith different concentrations of cisplatin for 48 hours. TheXTT results showed that ANGPTL1 could decrease the viabilityof HCC cells (Fig. 4A). Next, we studied whether the decreasedviability of ANGPTL1-transfected cells was attributed to ahigher rate of apoptosis. After treatment with cisplatin for 24hours, the apoptotic index was measured by flow cytometry.Compared with LacZ-transfected cells, the apoptotic index ofANGPTL1-transfected cells increased dramatically (Fig. 4B andC). Conversely, knockdown of ANGPTL1 had the oppositeeffect (Fig. 4B and C). Furthermore, the typical molecularindicators of apoptosis, cleaved caspase-3, caspase-9, and PARP

increased more rapidly in ANGPTL1-transfected cells (Fig. 4D).To determine whether ANGPTL1 could affect survival of tumorcells in vivo, we performed in situ detection of apoptotic cells inxenograft tumor. The results showed that 10% of xenografttumor cells were undergoing apoptosis derived from 8024-LacZcells, whereas 50% of tumor cells were apoptotic in 8024-ANGPTL1–derived tumor (Fig. 4E and F). Results were consis-tent in ANGPTL1-silenced cells. Furthermore, cleaved caspase-3(CC3) staining was conducted in xenograft tumors, and resultsdemonstrated that ANGPTL1 could reduce CC3 expression invivo (Supplementary Fig. S2A and S2B). These results indicatethat ANGPTL1 not only restored the cellular response to apo-ptotic stimulation, but also made HCC cells more vulnerable tocisplatin-induced apoptosis.

ANGPTL1 suppresses tumor cell migration, invasion, andmetastasis

As downregulation of ANGPTL1was associated with vascularinvasion and metastasis (Supplementary Table S3), we nextinvestigated the effect of ANGPTL1 on tumor cell migration andinvasion. In vitro cell Transwell migration andMatrigel invasionassays found that the migratory and invasive abilities of 8024-ANGPTL1 and Huh7-ANGPTL1 cells were inhibited comparedwith control cells (Fig. 5A). When ANGPTL1 was silenced byshRNA in QGY-7703 and LO2 cells, the migratory and invasiveabilities were dramatically increased (Fig. 5B and C), suggestingthat ANGPTL1 could inhibit cancer cell motility. To test the invivo suppressing effect of ANGPTL1 on metastasis, 8024-LacZand 8024-ANGPTL1 cells were injected into spleen of nudemice (n ¼ 10), respectively. Liver metastatic nodules werecounted 6 weeks after injection. Liver metastatic nodules wereformed in 9 of 10 and 3 of 10 mice injected with 8024-LacZcells and 8024-ANGPTL1 cells, respectively (Fig. 5D). Thenumber of metastatic nodules formed on the surface of theliver was significantly lower in mice injected with 8024-ANGPTL1 cells (4.2 � 2.3) than in mice injected with 8024-LacZ cells (32.3 � 8.2; P < 0.05, Student t test, Fig. 5E).Histologic analysis of liver dissected from each mouse con-firmed liver metastases (Fig. 5D) and ANGPTL1 expression (Fig.5F, top). To detect the apoptotic cells in liver metastatic lesions,TUNEL assay was performed in liver tissue dissected from eachmouse. The results showed that metastatic lesions derived from8024-ANGPTL1 cells had significantly higher apoptotic indexcompared with that derived from 8024-LacZ cells (Fig. 5F,bottom).

ANGPTL1 inhibits epithelial-to-mesenchymal transitionAs epithelial-to-mesenchymal transition (EMT) is a critical

step in metastasis that involves diverse biological mechanisms,we next studied the effects of ANGPTL1 on EMT process.Western blot analysis was performed to detect the expressionlevels of EMT markers and EMT-related transcription factors.Results showed that ANGPTL1 could upregulate epithelialmarkers (E-cadherin and b-catenin) and downregulate mesen-chymal markers (N-cadherin, fibronectin, and vimentin) andtranscription factor SNAIL and SLUG in HCC cell lines(Fig. 5G). As MMP-9 is responsible for the degradation ofbasement membrane of extracellular matrix, which is impor-tant in the process of tumor invasion, we also investigatedwhether ANGPTL1 could suppress MMP-9 expression. Our datashowed that MMP-9 expression level was relatively lower in

Yan et al.

Cancer Res; 77(21) November 1, 2017 Cancer Research5836

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579

Figure 3.

Tumor-suppressive effect of ANGPTL1 can be inhibited by RNA interference. Two shRNAs targeting ANGPTL1 (ANGPTL1-sh4, ANGPTL1-sh5) could effectivelydecrease ANGPTL1 expression in mRNA (A) and protein (B) level detected by qRT-PCR and Western blotting. Scrambled shRNA (Vec) was used as negativecontrol. SilencingANGPTL1 expression could increase abilities of foci formation (C) and colony formation (D) in soft agar. The results are expressed as mean� SD ofthree independent experiments. � , P < 0.05; �� , P < 0.01, independent Student t test. E, Images of mice with tumor formed induced by injecting the indicatedcells. Tumor growth curves are summarized in the line chart. The average tumor volume is expressed as the mean � SD of 5 mice. � , P < 0.05; ��� , P < 0.001,independent Student t test. F, IHC staining of proliferative index Ki67 in xenograft tumors. The percentage of Ki67-positive cells is summarized in the bar chart.�� , P < 0.01; ��� , P < 0.001.

ANGPTL1 Suppresses HCC Angiogenesis and Metastasis

www.aacrjournals.org Cancer Res; 77(21) November 1, 2017 5837

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579

Figure 4.

ANGPTL1 promotes the cisplatin-induced apoptosis. A, Cell viability between ANGPTL1- and LacZ-transfected cells was compared by XTT assay aftertreatment with cisplatin at indicated concentration for 48 hours. Data represent the mean � SD of three independent experiments with three repeats.� , P < 0.05; ��, P < 0.01, independent Student t test. B and C, Apoptotic indexes between LacZ- and ANGPTL1-transfected cells, or Vec- andshANGPTL1-transfected cells were compared by flow cytometry with Annexin-V–fluorescein isothiocyanate double staining after treatment with cisplatinat indicated concentration for 24 hours. The apoptotic index was defined as the percentage of apoptotic cell. Results represent the mean � SD ofthree independent experiments with three repeats. �, P < 0.05; �� , P < 0.01, independent Student t test. D, The activation of caspase-9, caspase-3,PARP was compared between LacZ- and ANGPTL1-transfected PLC-8024 cells (left) or Vec- and shANGPTL1-transfected LO2 cells (right) by Westernblot analysis. Cells were incubated with indicated concentrations of cisplatin for 24 hours. b-Actin was used as a loading control. E, The in situapoptosis in xenograft tumors was determined by TUNEL assay. Apoptotic cells were stained with fluorescein isothiocyanate–labeled DNA strand breaks, andnuclei were counterstained with DAPI. F, Quantification result of the in situ apoptotic cells in xenograft tumors. The apoptotic index, defined as thepercentage of apoptotic cells, was calculated and is summarized in the bar chart.

Yan et al.

Cancer Res; 77(21) November 1, 2017 Cancer Research5838

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579

Figure 5.

ANGPTL1 suppresses tumor migration, invasion, and metastasis. A, Transwell migration and Matrigel invasion assays were performed to compare cellmotilities between LacZ- and ANGPTL1-transfected PLC-8024 (left) and Huh7 cells (right). The number of migrated or invaded cells was calculated andis depicted in the bar chart. B and C, Cell migration and invasion assays were performed to compare cell motilities between Vec-7703 and shANGPTL1-7703cells (B) or Vec-LO2 and shANGPTL1-LO2 cells (C). The number of migrated or invaded cells was calculated and is depicted in the bar chart. Thevalues indicate the mean � SD of three independent experiments (�� , P < 0.01; ��� , P < 0.001, independent Student t test). D, An in vivo experimentalmetastasis assay was performed to evaluate the effect of 8024-LacZ and 8024-ANGPTL1 cells on tumor metastasis. Representative images of liversderived from nude mice after intrasplenic injection of indicated cells (left). Representative images of hematoxylin and eosin staining of liver sectionsmentioned above (right; original magnification, �200). Tumor sections are indicated within the circles depicted. E, The numbers of metastatic nodules on thesurface of the liver are summarized. The values for the individual mice are shown above the bars; the values by group are also denoted (�� , P < 0.01;independent Student t test). F, Representative images of IHC staining with ANGPTL1 antibody on liver tissues from nude mice after intrasplenicinjection of indicated cells. Tumor section is indicated within the circle depicted (top; magnification, �200). Representative images of in situ apoptotic cells inliver tissues detected by TUNEL assay (bottom). G, The protein levels of E-cadherin, b-catenin, N-cadherin, fibronectin, vimentin, SNAIL, SLUG, and MMP-9were compared between cells transfected with LacZ and ANGPTL1, or Vec and shANGPTL1. b-Actin was used as a loading control.

ANGPTL1 Suppresses HCC Angiogenesis and Metastasis

www.aacrjournals.org Cancer Res; 77(21) November 1, 2017 5839

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579

Figure 6.

ANGPTL1 inhibits angiogenesis both in vitro and in vivo. A, Effects of ANGPTL1 on endothelial cell tube formation. Well-formed tubes were observed incontrol group, whereas recombinant ANGPTL1 inhibited tube formation of HUVECs in a dose-dependent manner. A representative tube formation imagefrom an independent field is shown. The experiment was repeated three times with similar results. B, Quantification of the extent of vascular tubeformation. Three parameters were scored in nine visual fields per well using the angiogenesis quantification platform (Wimasis Image Analysisplatform), and all the parameters were shown to be suppressed by ANGPTL1. (Continued on the following page.)

Yan et al.

Cancer Res; 77(21) November 1, 2017 Cancer Research5840

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579

ANGPTL1-transfected cells compared with control cells(Fig. 5G). When ANGPTL1 was silenced, a higher level ofMMP-9 was detected compared with control cells (Fig. 5G).These results suggest that the suppressive effect of ANGPTL1 onHCC invasion and metastasis is via the inhibition of EMTprocess.

ANGPTL1 inhibits angiogenesisAs previous studies revealed that ANGPTL1 functions as a

key antiangiogenic protein (19), we next studied whetherANGPTL1 could inhibit angiogenesis in HCC. First, vasculartube formation assay was used to test the stage of angiogene-sis in HUVECs. As expected, ANGPTL1 recombinant protein(rANGPTL1) could inhibit vascular tube formation ofHUVECs, as visualized by morphologic changes (Fig. 6A), andthe parameters to assess vascular formation were markedlysuppressed (Fig. 6B). Then the effect of ANGPTL1 on themigration and proliferation of HUVECs was analyzed. Treat-ment with rANGPTL1 significantly suppressed the migratoryability of endothelial cells in a dose-dependent manner(Fig. 6C). Compared with vehicle (PBS)-treated group, 58%of migration inhibition was observed when treated with thehighest dose (100 ng/mL) of rANGPTL1 (Fig. 6C). Also, theproliferation of HUVECs in low-serum condition was inhibitedby rANGPTL1 (Fig. 6D). To further confirm the ANGPTL1-mediated autocrine pathway, conditioned medium was usedto treat HUVECs as well as HCC cell line PLC8024. By utilizingthe ANGPTL1-neutralizing antibody, the inhibiting phenotypeof vascular tube formation by conditioned medium was recov-ered (Supplementary Fig. S3A and S3B). Also, blockingANGPTL1 activity by neutralizing antibody could abolish cells'migration reduction induced by conditioned medium (Sup-plementary Fig. S3C and S3D).

Angiogenesis-inhibiting mechanisms of ANGPTL1To further confirm the effect of ANGPTL1 on HCC angio-

genesis, the microvessel formation frequency was comparedbetween xenograft tumors induced by shANGPTL1- and emptyvector–transfected LO2 cells. The in vivo tumor growth rate oftumors induced with shANGPTL1 cells was significantly higherthan tumors induced by vector-transfected cells (P < 0.01;Fig. 3E). By IHC staining with CD34 and CD31 (both areepithelial markers), tumors induced by LO2-shANGPTL1 cellsshowed a significantly higher frequency of microvessel forma-tion compared with tumors induced by LO2-Vec cells (Fig. 6Fand G; Supplementary Fig. S2A and S2B). Furthermore, expres-sions of angiogenesis regulators HIF1a and VEGF weredecreased and increased in ANGPTL1-expressing and -silenced

cells, respectively (Fig. 6H). Taken together, these observationssuggest the inhibiting role of ANGPTL1 in the regulation ofangiogenesis in HCC.

To further explore the underlying molecular mechanism, wetested whether ANGPTL1 could affect ERK and AKT signalingpathways, both of which play important roles in angiopoietin-mediated endothelial cell behavior (22–24). Results showedthat ANGPTL1 could effectively inhibit the phosphorylation ofERK1/2 and AKT in serum-starved HUVECs (Fig. 6E), suggest-ing that ANGPTL1 inhibits endothelial cell proliferation, migra-tion, and tube formation by blocking AKT and ERK signals,which likely underlie the inhibitory activity of ANGPTL1 ontumor angiogenesis and metastasis.

ANGPTL1 interacts with integrin a1b1 and inhibitsdownstream JAK2/STAT3 signaling pathway

As ANGPTL proteins have been reported to deliver theirsignals via integrin receptor–related pathways (25–27), we nextstudied whether ANGPTL1-mediated effect is through integrinsignaling pathways. To identify candidate integrin receptors forANGPTL1, immunoprecipitation was performed to analyze theimmunoprecipitates from Flag-tagged ANGPTL1-transfectedPLC-8024 cells. Our data revealed that ANGPTL1 interactedwith the integrin a1b1 receptor (Fig. 7A). ANGPTL1 bindingwith integrin b1 was also confirmed by immunofluorescence asa result of treating PLC-8024 cells with rANGPTL1 (Fig. 7B).Furthermore, silencing of integrin a1b1 in PLC8024 cellssignificantly reduced their adhesive capability to recombinantprotein ANGPTL1, indicating the role of a1b1 as ANGPTL1receptor (Fig. 7C; Supplementary Fig. S4A). To further study theintegrin signaling related with the ANGPTL1-inhibited HCCprogression, we investigated the effects of ANGPTL1 on thedownstream signal transduction pathways. Proteins were col-lected at different time points after rANGPTL1 treatment.Results showed that apart from phosphorylated ERK and AKT,both of which are classic integrin downstream signaling (28),phosphorylated STAT3 was repressed rapidly after rANGPTL1treatment (Fig. 7D). This result was further confirmed inANGPTL1-transfected cells. ANGPTL1-transfected cells showeda lower expression level of phosphorylated STAT3, ERK, andAKT, compared with LacZ-transfected cells (Fig. 7E). In con-trast, these signaling pathways were activated in ANGPTL1-silenced cells compared with control cells (SupplementaryFig. S4B). As non-receptor tyrosine kinases including focaladhesion kinase (FAK) and Src are key signaling proteinsrecruited and activated by integrins (28), we next studied theirexpressions in ANGPTL1-transfected or -silenced cells. Our datashowed that phosphorylated FAK and Src were suppressed by

(Continued.) The results are shown as mean � SD of three independent experiments. � , P < 0.05; �� , P < 0.01, independent Student t test. C, Effects ofANGPTL1 on endothelial cell migration. Cells were seeded in the migration chambers, and the number of migrated cells in the presence or absence ofANGPTL1 was counted in three independent fields. The values indicate the mean � SD of three independent experiments (�� , P < 0.01, independent Student ttest). D, Suppression of the HUVEC growth by ANGPTL1. Recombinant protein rANGPTL1 was supplemented on days 1, 3, and 5. The number of cellswas measured by XTT assay on days 1, 3, and 6. The values indicate the mean � SD of three independent experiments. �� , P < 0.01; ��� , P < 0.001,independent Student t test. E, ANGPTL1 blocks activation of ERK1/2 and AKT kinases. HUVECs were cultured and switched into serum-free mediumwith 200 ng/mL of rANGPTL1 for indicated time periods. Cells were lysed, and protein samples were analyzed by Western blotting. F, IHC staining withANGPTL1, VEGF, and CD34 antibodies was performed in sections of xenograft tumors induced by LO2 cells transfected with empty vector or shANGPLT1(original magnification, �200). G, The frequency of microvessel formation was determined by counting microvessels stained by CD34 undermicroscope. H, The VEGF and HIF1a protein level was compared by Western blotting between LacZ- and ANGPTL1-transfected PLC-8024 and Huh7 cells,or Vec- and shANGPTL1-transfected LO2 and 7703 cells.

ANGPTL1 Suppresses HCC Angiogenesis and Metastasis

www.aacrjournals.org Cancer Res; 77(21) November 1, 2017 5841

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579

ANGPTL1 (Fig. 7F). Also, the expression of activated JAK2, acrucial molecule that phosphorylates STAT3, was reduced inANGPTL1-transfected cells and increased in ANGPTL1-silencedcells, compared with their control cells (Fig. 7F; SupplementaryFig. S4C). Phosphorylated STAT3 translocates into the nucleusto induce transcription of genes involved in cell proliferation,survival, metastasis, and angiogenesis (29). We detected theexpression levels of cell proliferation–related proteins c-Jun,cyclin D1, c-Myc, and Pim2, and survival related proteins Bcl-2and Bcl-xL in ANGPTL1-transfected or -silenced cells. Resultsshowed that ANGPTL1 could decrease the expressions of theseproliferative- or survival-related proteins, and subsequentlyinhibited HCC progression (Supplementary Fig. S4D and S4E).

To study whether integrin a1b1 is involved in the suppres-sing effect of ANGPTL1 on HCC invasion and metastasis, HCCcells were treated with an integrin a1b1 inhibitor obtustatin(30) followed by treating cells with rANGPTL1. The resultfound that the inhibition of integrin a1b1 activity in PLC-8024 cells by obtustatin could abolish rANGPTL1-mediatedreduction of cancer cell invasion (Fig. 7G and H). In addition,our data revealed that after incubation with obtustatin, thelevels of phosphorylated FAK, Src, JAK, STAT3, AKT and ERKwere not altered by rANGPTL1 treatment (Fig. 7I and J),indicating the important role of ANGPTL1/integrin a1b1 sig-naling axis in HCC progression. Taken together, our resultssuggest that ANGPTL1 exerts its tumor-suppressive function inHCCs through interaction with integrin a1b1 and inhibitingdownstream Src/JAK2/STAT3 signaling pathways, and subse-quently promotes apoptosis and suppresses HCC cell angio-genesis and metastasis (Fig. 7K).

DiscussionTumor suppressor genes (TSG) are frequently downregulated

by genetic (e.g., deletion and mutation) or epigenetic (e.g.,methylation and miRNA) mechanisms in various cancer types(31). Recently, Revill and colleagues identified 678 genes, includ-ingANGPTL1, whichwere significantly hypermethylated inHCCs(5). In this study, downregulation ofANGPTL1was detected in 64of 122 (52.5%) of primary HCCs, which was significantly corre-lated withmanymalignant phenotypes such as higher serum AFPlevel, vascular invasion, poor differentiation, metastasis, andpoorer survival rates. Multivariate analyses demonstrated thatANGPTL1 could be an independent prognostic biomarker ofpoorer HCC outcomes. Although ANGPTL1 has been proposedto inhibit tumor growth and metastasis (20, 21), the underlyingmolecular mechanisms remain unclear.

Previous studies have shown that angiopoietin-like proteinsdeliver their signals via integrin pathways. ANGPTL1 suppresseslung cancer cell motility through inhibiting integrin a1b1activity (18). ANGPTL3 interacts with integrin anb3 to promoteendothelial cells migration and adhesion ability (32). ANGPTL4helps tumors to escape anoikis and regulate the migratoryability of keratinocytes via integrin b3 and b5. Integrin down-stream signaling activation, such as FAK and ERK, AKT phos-phorylation, has significant impact on cancer cell behavior(33, 34). In this study, immunoprecipitation analysis revealedthat ANGPTL1 interacts with integrin a1b1 of HCC cells.To further explore the tumor-suppressing mechanisms ofANGPTL1, the effects of ANGPTL1 on the integrin a1b1 down-stream signal transduction pathways were investigated. Besides

the classical integrin/FAK/ERK/AKT signaling, our results alsoshowed for the first time that ANGPTL1 represses the Src/JAK/STAT3 signaling, and inhibition of integrin a1b1 activity attenu-ates ANGPTL1-mediated pathways and subsequent tumor cellbehaviors. However, whether ANGPTL1 plays a role as anantagonist for integrin a1b1 remains unclear and needs moreevidence. Previous studies demonstrated that ANGPTL1 over-expression repressed the expression of activated integrin a1b1(18). Therefore, the activity of integrin a1b1 receptor may besuppressed when bound by ANGPTL1.

STAT3 is a well-characterized oncogene that affects variousbiological processes including to promote: (i) cell proliferationand survival by regulating cyclin D1, Bcl-2 and Bcl-xl; (ii) tumorinvasion and metastasis by regulating E-cadherin, MMP-9; (iii)angiogenesis by targeting VEGF and HIF1a expression (35). Bothin vitro and in vivo functional assays demonstrated that ANGPTL1could suppress tumor cell growth, whichmight be attributed to itsproapoptotic ability. Further study showed that ANGPTL1 couldpromote apoptosis through inhibiting the STAT3 signaling, fol-lowed by suppressing antiapoptotic protein Bcl-2 and Bcl-xlexpression, thus activating the cytochrome c/caspase-9/caspase-3 pathway. The mechanism of apoptosis is complex and involvesmany pathways, in which any defect may lead to malignanttransformation, tumor metastasis, and resistance to anticancerdrugs (36). Therefore, downregulation of ANGPTL1 in HCCmight be an early and critical event that propels uncontrolledtumor cell expansion duringHCC initiation.Moreover, one of theimportant clinical problems when treating cancer with chemo-therapy is that apoptosis defects may occur in response to che-motherapeutic drugs (37). The contribution of ANGPTL1 to theincreased rate of apoptosis inHCCmay provide novel therapeuticstrategies in enhancing HCC chemosensitivity.

A previous report showed a negative correlation between theactivated STAT3 and E-cadherin in human cutaneous squamouscell carcinoma (38). E-cadherin is an important epithelial cellmarker, and decreased E-cadherin expression has been consid-ered one of the hallmarks of EMT. Previous studies demon-strated that angiopoietin-like protein 6 interacted with integrina6 and E-cadherin to drive liver metastasis of colorectal cancer,suggesting the angiopoietin-like 6/integrin a6/E-cadherin func-tions as an interconnected network that facilitates metastaticprocess (15). A similar network was found in this study, inwhich ANGPTL1 binds with integrin a1b1 of tumor cells,suppressing the expression of E-cadherin and inhibiting met-astatic process. Furthermore, our data revealed that ANGPTL1could downregulate transcription factors SNAIL and SLUGexpression, suggesting that ANGPTL1 could inhibit EMTthrough suppressing SNAIL and SLUG expression, and subse-quently decrease cell motility. Consistent with what weexpected, both in vitro and in vivo functional assays demon-strated that ANGPTL1 could decrease HCC cell migration,invasion, and metastasis. All these data suggest that ANGPTL1plays important inhibiting roles in HCC progression, whichmay serve as a novel therapeutic target for the prevention ofcancer metastasis.

Angiogenesis is also reported to be linked with STAT3 (39).As prominent transcription targets for STAT3, VEGF and HIF1aare important proangiogenic factors (40). Transfection of cellswith activated STAT3 was found to be sufficient to increaseVEGF expression and induce in vivo angiogenesis (41). Ourresults showed that silenced expression of ANGPTL1 could

Yan et al.

Cancer Res; 77(21) November 1, 2017 Cancer Research5842

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579

Figure 7.

ANGPTL1 interacts with integrin a1b1 and represses downstream JAK2/STAT3 signaling pathways. A, PLC-8024 cells were transfected with Flag-taggedANGPTL1. Cell lysateswere immunoprecipitatedwith anti-Flag andnormal IgG.Western blot analysiswas performed to detect integrinsa1, -a4, -a5, -av, -a7, -b1, -b3,-b4, and -b5 in this immunoprecipitation. B, PLC-8024 cells were treated with rANGPTL1 for 2 hours, followed by double immunofluorescence staining withANGPTL1 (red) and integrin b1 (green) antibodies. Nuclei were counterstained with DAPI (blue). C, Adhesion assay was performed to analyze the adhesive ability ofPLC8024 cells with or without integrin a1b1 silencing to 96-well plate coated with ANGPTL1 recombinant protein. D, Western blot analysis of phosphorylation ofSTAT3, MEK, ERK, and AKT in PLC-8024 cells treated with 50 ng/mL rANGPTL1 for various periods of time. E and F, Western blot analysis of phosphorylation ofSTAT3, ERK, AKT, FAK, Src, and JAK in ANGPTL1-overexpressing PLC-8024 and Huh7 cells. G, Measurement of the invasion of PLC-8024 cells incubated withobtustatin, an integrin a1b1 inhibitor, followed by rANGPTL1 treatment during the invasion process. H, The number of invaded cells was calculated and is depicted inthe bar chart. The values indicate the mean � SD of three independent experiments (�� , P < 0.01, independent Student t test; n.s., nonsignificant.). I, Western blotanalysis of phosphorylation of FAK, Src, JAK, STAT3, AKT, andERK in PLC8024 cells treatedwith 100ng/mL rANGPTL1 for indicated periods of time in the presence orabsence of obtustatin. J,Quantification analysis of theWestern blotting assaywas conducted by ImageJ software, and the valuewas calculated and is depicted in thebar chart. The values indicate the mean � SD of three independent experiments (� , P < 0.01; ��� , P < 0.001, independent Student t test; n.s., nonsignificant.).K, A schematic diagram illustrating the proposed ANGPTL1-regulated tumor-suppressive mechanism in HCC tumorigenesis.

ANGPTL1 Suppresses HCC Angiogenesis and Metastasis

www.aacrjournals.org Cancer Res; 77(21) November 1, 2017 5843

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579

increase VEGF level as well as the frequency of microvesselformation in xenograft tumors, indicating that ANGPTL1 couldinhibit angiogenesis by suppressing VEGF signaling. We alsoexamined the expression of HIF1a, a transcription factor thatactivates downstream target genes associated with angiogene-sis, tumor cell invasion, and metastasis (32). The result indi-cated that ANGPTL1 could suppress HIF1a expression in HCCcells via inhibiting STAT3 signaling pathway. Our in vitro assayfurther confirmed the inhibitory role of ANGPTL1 in endothe-lial cell tube formation, migration, and proliferation. ANGPTL1could also block activation of ERK1/2 and AKT kinases inendothelial cells, which partly underlie the ANGPTL1-mediatedinhibition on tumor angiogenesis.

In summary, we provide clinical evidence that ANGPTL1expression inversely correlates with vascular invasion andmetastasis in HCC, and is positively associated with clinicaloutcomes including overall and disease-free survival times inHCC patients. We demonstrate that ANGPTL1 promotes apo-ptosis and suppresses tumor cell angiogenesis and metastasisin vitro and in vivo by regulating the integrin a1b1/FAK-Src/JAK/STAT3 signaling axis. Molecular target therapy aiming at TSGshas been tried mainly for p53 (42). As p53 gene product isneither a cell surface protein nor a typical enzyme, the targettherapy has not been easy. In this perspective, TSGs, whoseproducts are secreted proteins and function extracellularly,have more advantages as targets for cancer therapy in termsof delivery efficiency. As a secreted tumor suppressor, ANGPTL1has more potential to be developed as a novel therapeuticstrategy for HCC treatment.

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

Authors' ContributionsConception and design: Q. Yan, L. Jiang, X.-Y. GuanDevelopment of methodology: Q. Yan, L. Jiang, M. Liu, Y. LiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Q. Yan, L. Jiang, D. Yu, Y. Zhang, S. Fang, Y. Li,Y. YuanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Q. Yan, L. Jiang, D. Yu, Y. Zhang, Y. Li, Y.-H. Zhu,X.-Y. GuanWriting, review, and/or revision of the manuscript: Q. Yan, L. Jiang,X.-Y. GuanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Q. Yan, L. Jiang, Y. Li, S. Fang, X.-Y. GuanStudy supervision: X.-Y. Guan

AcknowledgmentsThe authors thank Dr. Stephanie Ma of School of Biomedical Sciences, the

University of Hong Kong, for her generous gift of theHUVEC cell line. ProfessorX.Y. Guan is Sophie YM Chan Professor in Cancer Research.

Grant SupportThis work was supported by grants from the Hong Kong Research Grant

Council (RGC) grants, including GRF (17143716 and 767313), CollaborativeResearch Funds (C7038-14G and C7027-14G), NSFC/RGC Joint ResearchScheme (N_HKU712/12), Theme-based Research Scheme Fund (T12-704/16-R), Health and Medical Research Fund (04150826), and National NaturalScience Foundation of China (81472250).

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 March 7, 2017; revised July 15, 2017; accepted August 25, 2017;published OnlineFirst September 13, 2017.

References1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of

worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer2010;127:2893–917.

2. Matsuda Y, Ichida T, Matsuzawa J, Sugimura K, Asakura H. p16(INK4)is inactivated by extensive CpG methylation in human hepatocellularcarcinoma. Gastroenterology 1999;116:394–400.

3. Lim SO, Gu JM, Kim MS, Kim HS, Park YN, Park CK, et al. Epigeneticchanges induced by reactive oxygen species in hepatocellular carcinoma:methylation of the E-cadherin promoter. Gastroenterology 2008;135:2128–40.

4. Nishida N, Kudo M, Nagasaka T, Ikai I, Goel A. Characteristic patterns ofaltered DNA methylation predict emergence of human hepatocellularcarcinoma. Hepatology 2012;56:994–1003.

5. Revill K, Wang T, Lachenmayer A, Kojima K, Harrington A, Li J, et al.Genome-wide methylation analysis and epigenetic unmasking identifytumor suppressor genes in hepatocellular carcinoma. Gastroenterology2013;145:1424–35.

6. Chen L, Li Y, Lin CH, Chan TH, Chow RK, Song Y, et al. Recoding RNAediting of AZIN1 predisposes to hepatocellular carcinoma. Nat Med2013;19:209–16.

7. Santulli G. Angiopoietin-like proteins: a comprehensive look. FrontEndocrinol 2014;5:4.

8. Fidler IJ. The pathogenesis of cancer metastasis: the 'seed and soil' hypoth-esis revisited. Nat Rev Cancer 2003;3:453–8.

9. Oike Y, Yasunaga K, Suda T. Angiopoietin-related/angiopoietin-like pro-teins regulate angiogenesis. Int J Hematol 2004;80:21–8.

10. Le Jan S, Amy C, Cazes A, Monnot C, Lamande N, Favier J, et al.Angiopoietin-like 4 is a proangiogenic factor produced during ische-

mia and in conventional renal cell carcinoma. Am J Pathol 2003;162:1521–8.

11. Tabata M, Kadomatsu T, Fukuhara S, Miyata K, Ito Y, Endo M, et al.Angiopoietin-like protein 2 promotes chronic adipose tissue inflam-mation and obesity-related systemic insulin resistance. Cell Metab2009;10:178–88.

12. Kersten S. Regulation of lipid metabolism via angiopoietin-like proteins.Biochem Soc Trans 2005;33:1059–62.

13. Zhang CC, Kaba M, Ge G, Xie K, Tong W, Hug C, et al. Angiopoietin-likeproteins stimulate ex vivo expansion of hematopoietic stem cells. Nat Med2006;12:240–5.

14. Galaup A, Cazes A, Le Jan S, Philippe J, Connault E, Le Coz E, et al.Angiopoietin-like 4 prevents metastasis through inhibition of vascularpermeability and tumor cell motility and invasiveness. Proc Natl AcadSci U S A 2006;103:18721–6.

15. Marchio S, Soster M, Cardaci S, Muratore A, Bartolini A, Barone V, et al. Acomplex of alpha6 integrin and E-cadherin drives liver metastasis ofcolorectal cancer cells through hepatic angiopoietin-like 6. EMBO MolMed 2012;4:1156–75.

16. Sato R, Yamasaki M, Hirai K, Matsubara T, Nomura T, Sato F, et al.Angiopoietin-like protein 2 induces androgen-independent andmalignantbehavior in human prostate cancer cells. Oncol Rep 2015;33:58–66.

17. Parri M, Pietrovito L, Grandi A, Campagnoli S, De Camilli E, Bianchini F,et al. Angiopoietin-like 7, a novel pro-angiogenetic factor over-expressedin cancer. Angiogenesis 2014;17:881–96.

18. Kuo TC, Tan CT, Chang YW, Hong CC, Lee WJ, Chen MW, et al. Angio-poietin-like protein 1 suppresses SLUG to inhibit cancer cell motility.J Clin Invest 2013;123:1082–95.

Yan et al.

Cancer Res; 77(21) November 1, 2017 Cancer Research5844

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579

19. Dhanabal M, LaRochelle WJ, Jeffers M, Herrmann J, Rastelli L, McDonaldWF, et al. Angioarrestin: an antiangiogenic protein with tumor-inhibitingproperties. Cancer Res 2002;62:3834–41.

20. Sasaki H, Moriyama S, Sekimura A, Mizuno K, Yukiue H, Konishi A, et al.Angioarrestin mRNA expression in early-stage lung cancers. Eur J SurgOncol 2003;29:649–53.

21. Xu Y, Liu YJ, Yu Q. Angiopoietin-3 inhibits pulmonary metastasis byinhibiting tumor angiogenesis. Cancer Res 2004;64:6119–26.

22. Papapetropoulos A, Fulton D, Mahboubi K, Kalb RG, O'Connor DS, Li F,et al. Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survi-vin pathway. J Biol Chem 2000;275:9102–5.

23. Kim I, KimHG, So JN, Kim JH, KwakHJ, Koh GY. Angiopoietin-1 regulatesendothelial cell survival through the phosphatidylinositol 30-Kinase/Aktsignal transduction pathway. Circ Res 2000;86:24–9.

24. Kim I, RyuYS,KwakHJ, AhnSY,Oh JL, YancopoulosGD, et al. EphB ligand,ephrinB2, suppresses the VEGF- and angiopoietin 1-induced Ras/mitogen-activated protein kinase pathway in venous endothelial cells. FASEB J2002;16:1126–8.

25. CamenischG, PisabarroMT, ShermanD, Kowalski J, NagelM,Hass P, et al.ANGPTL3 stimulates endothelial cell adhesion and migration via integrinalpha vbeta 3 and induces blood vessel formation in vivo. J Biol Chem2002;277:17281–90.

26. Huang RL, Teo Z, Chong HC, Zhu P, Tan MJ, Tan CK, et al. ANGPTL4modulates vascular junction integrity by integrin signaling and disruptionof intercellular VE-cadherin and claudin-5 clusters. Blood 2011;118:3990–4002.

27. Goh YY, Pal M, Chong HC, Zhu P, Tan MJ, Punugu L, et al. Angiopoietin-like 4 interacts with integrins beta1 and beta5 to modulate keratinocytemigration. Am J Pathol 2010;177:2791–803.

28. Danen EH. Integrin signaling as a cancer drug target. ISRN Cell Biol2013;2013:14.

29. Siveen KS, Sikka S, Surana R, Dai X, Zhang J, Kumar AP, et al. Targeting theSTAT3 signaling pathway in cancer: role of synthetic and natural inhibitors.Biochim Biophys Acta 2014;1845:136–54.

30. Marcinkiewicz C, Weinreb PH, Calvete JJ, Kisiel DG, Mousa SA, TuszynskiGP, et al. Obtustatin: a potent selective inhibitor of alpha1beta1 integrinin vitro and angiogenesis in vivo. Cancer Res 2003;63:2020–3.

31. Knudson AG. Two genetic hits (more or less) to cancer. Nat Rev Cancer2001;1:157–62.

32. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003;3:721–32.

33. Mitra SK, Hanson DA, Schlaepfer DD. Focal adhesion kinase: incommand and control of cell motility. Nat Rev Mol Cell Biol 2005;6:56–68.

34. Liao YC, Shih YW, Chao CH, Lee XY, Chiang TA. Involvement of the ERKsignaling pathway in fisetin reduces invasion and migration in thehuman lung cancer cell line A549. J Agric Food Chem 2009;57:8933–41.

35. Behera R, Kumar V, Lohite K, Karnik S, Kundu GC. Activation of JAK2/STAT3 signaling by osteopontin promotes tumor growth in human breastcancer cells. Carcinogenesis 2010;31:192–200.

36. Wong RS. Apoptosis in cancer: from pathogenesis to treatment. J Exp ClinCancer Res 2011;30:87.

37. Herr I, Debatin KM. Cellular stress response and apoptosis in cancertherapy. Blood 2001;98:2603–14.

38. Suiqing C, Min Z, Lirong C. Overexpression of phosphorylated-STAT3 correlated with the invasion and metastasis of cutaneous squa-mous cell carcinoma. J Dermatol 2005;32:354–60.

39. Valdembri D, Serini G, Vacca A, Ribatti D, Bussolino F. In vivo activation ofJAK2/STAT-3 pathway during angiogenesis induced by GM-CSF. FASEB J2002;16:225–7.

40. Jarnicki A, Putoczki T, Ernst M. Stat3: linking inflammation to epithelialcancer - more than a "gut" feeling? Cell Div 2010;5:14.

41. Niu G, Wright KL, HuangM, Song L, Haura E, Turkson J, et al. ConstitutiveStat3 activity up-regulates VEGF expression and tumor angiogenesis.Oncogene 2002;21:2000–8.

42. Kishore R, LosordoDW. Gene therapy for restenosis: biological solution toa biological problem. J Mol Cell Cardiol 2007;42:461–8.

www.aacrjournals.org Cancer Res; 77(21) November 1, 2017 5845

ANGPTL1 Suppresses HCC Angiogenesis and Metastasis

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579

2017;77:5831-5845. Published OnlineFirst September 13, 2017.Cancer Res   Qian Yan, Lingxi Jiang, Ming Liu, et al.   Angiogenesis and Metastasis by Inhibiting JAK2/STAT3 Signaling

1 to Suppress HCCβ1α Interacts with Integrin ANGPTL1

  Updated version

  10.1158/0008-5472.CAN-17-0579doi:

Access the most recent version of this article at:

  Material

Supplementary

  http://cancerres.aacrjournals.org/content/suppl/2017/09/13/0008-5472.CAN-17-0579.DC1

Access the most recent supplemental material at:

   

   

  Cited articles

  http://cancerres.aacrjournals.org/content/77/21/5831.full#ref-list-1

This article cites 42 articles, 11 of which you can access for free at:

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.org

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/77/21/5831To request permission to re-use all or part of this article, use this link

on November 11, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0579