Cabozantinib Suppresses Tumor Growth and Metastasis in ...clincancerres.aacrjournals.org/content/clincanres/20/11/2959.full.pdf · Cabozantinib Suppresses Tumor Growth and Metastasis

Cancer Therapy: Preclinical

Cabozantinib Suppresses Tumor Growth and Metastasisin Hepatocellular Carcinoma by a Dual Blockade of VEGFR2and MET

Qingfeng Xiang1, Weiqiang Chen4, Meng Ren2, Jingnan Wang3, Hongwu Zhang3, David Y.B. Deng3,Lei Zhang1, Changzhen Shang1, and Yajin Chen1

AbstractPurpose: MET signaling has been suggested a potential role in hepatocellular carcinoma (HCC) and

associated with prometastasis during antiangiogenesis therapy. We investigated the potential association

between MET expression and therapeutic response to sorafenib in patients with HCC. Antitumor effects of

cabozantinib, a dual inhibitor of MET and VEGFR2, were examined in cultured HCC cells as well as in vivo

models.

Experimental Design: Total MET and phosphorylatedMET (p-MET) weremeasured in 29 resected HCC

specimens, and correlatedwith response to sorafenib as postoperative adjuvant therapy. In the second set of

experiments using cultured HCC cells, andmouse xenograft andmetastatic models, effects of cabozantinib

were examined.

Results: High level of p-MET in resected HCC specimens was associated with resistance to adjuvant

sorafenib therapy. In cultured HCC cells that expressed p-MET, cabozantinib inhibited the activity of MET

and its downstream effectors, leading to G1-phase arrest. Cabozantinib inhibited tumor growth in p-MET–

positive and p-MET–negative HCC by decreasing angiogenesis, inhibiting proliferation, and promoting

apoptosis, but it exhibited more profound efficacy in p-MET–positive HCC xenografts. Cabozantinib

blocked the hepatocyte growth factor (HGF)–stimulated MET pathway and inhibited the migration and

invasion of the HCC cells. Notably, cabozantinib reduced the number of metastatic lesions in the lung and

liver in the experimental metastatic mouse model.

Conclusions: Patients with HCC with high level of p-MET are associated with resistance to adjuvant

sorafenib treatment. The dual blockade of VEGFR2 and MET by cabozantinib has significant antitumor

activities in HCC, and the activation of MET in HCC may be a promising efficacy-predicting biomarker.

Clin Cancer Res; 20(11); 2959–70. �2014 AACR.

IntroductionHepatocellular carcinoma (HCC) is the fifth most

common cancer and the third most common cause ofcancer-related death worldwide (1). Despite improve-ments in diagnostic and therapeutic strategies, the prog-nosis of HCC still remains poor (2–4). Inhibiting angio-

genesis has been used as a strategy in the treatment ofHCC (5, 6). For example, sorafenib, a VEGF receptor(VEGFR) inhibitor with activity against platelet-derivedgrowth factor receptor (PDGFR), c-Kit receptor, RAF andp38 signal transduction pathways, has become a standardtreatment in patients with advanced HCC (7). Eventhough sorafenib improves the median survival inadvanced HCC, the median overall survival remains lessthan 1 year partly due to that many patients eventuallybecome resistant to this drug (8, 9). In addition, sorafe-nib, like other VEGFR inhibitors such as sunitinib andcediranib, possesses the possibility to increase the inva-siveness and/or metastatic potential of tumors (10–12).Thus, developing inhibitors that simultaneously inhibitVEGF and other pathways involved in tumor invasionand metastasis may confer broad and potent antitumorefficacy.

MET, a transmembrane tyrosine kinase receptor for hepa-tocyte growth factor (HGF), has been observed to play animportant role in the development of human cancersand drug resistance in cancer cells (13–15). Moreover,

Authors' Affiliations: Departments of 1Hepatopancreatobiliary Surgery;2Endocrinology, Sun Yat-sen Memorial Hospital; 3Research Center ofTranslationalMedicine, the First AffiliatedHospital, SunYat-senUniversity,Guangzhou; and 4Department of General Surgery, Zhongshan Boai Hos-pital, Southern Medical University, Zhongshan, China

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

Corresponding Authors: Yajin Chen or Changzhen Shang, Departmentof Hepatopancreatobiliary Surgery, Sun Yat-sen Memorial Hospital,Sun Yat-sen University, 107 Yanjiang West Road, Guangzhou 510120,China. Phone: 86-20-3407-1169; Fax: 86-20-3407-1091; E-mail:[email protected] or [email protected]

doi: 10.1158/1078-0432.CCR-13-2620

�2014 American Association for Cancer Research.

ClinicalCancer

Research

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Published OnlineFirst April 3, 2014; DOI: 10.1158/1078-0432.CCR-13-2620





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overexpression of MET has been reported in various typesof human cancers, including HCC (13, 14). BlockingMET expression by gene therapy reduces cell proliferation,colony formation, and migration in vitro and suppressestumor growth, angiogenesis, and metastasis in vivo in mul-tiple HCC cell lines (16–18). Interestingly, emerging evi-dence demonstrated that tumor vascular pruning causedby inhibition of the VEGF pathway led to the inductionof hypoxia and subsequently triggered MET expression,which enhances tumor invasion and metastasis (10,11, 19). Therefore, blockade of both MET and VEGFRpathways may achieve better treatment outcome in HCC.

Tivantinibwas initially reported as aMET-selective inhib-itor anddemonstrated anearly doubling of progression-freesurvival and overall survival in patients withHCCwith highexpression of MET (20). However, subsequent studies con-firmed that tivantinib is not a MET inhibitor but an anti-mitotic agent that kills tumor cells independently of MET(21, 22). Cabozantinib is an oral small-molecule tyrosinekinase inhibitor that blocks phosphorylation of MET andVEGFR2 and also has activity against AXL, RET, and KIT(23). Results from phase II and III clinical trials demon-strated that cabozantinib exhibited encouraging clinicalactivity in multiple human cancers including castration-resistant prostate cancer, medullary thyroid cancer, andHCCwithmanageable side effects (24–26). Although cabo-zantinib has clinical benefit, the antitumor mechanism ofcabozantinib in HCC has not been fully elucidated. Fur-thermore, antitumor agents that administered to patientsbefore knowing their mechanism of action may be mis-leading in the development of predictive biomarkers. In thepresent study, we investigated whether cabozantinib couldinhibit tumor growth and metastasis, and explored themolecularmechanismof antitumor activity of cabozantinibin HCC.

Materials and MethodsPatients and specimens

Archival HCC specimens were obtained from 29 patientswho accepted potentially curative treatment of hepaticresection at Sun Yat-sen Memorial Hospital of Sun Yat-senUniversity (Guangzhou, China) between January 2008 andDecember 2012. These patients were pathologically diag-nosed as HCCwith microvascular invasion. Becausemicro-vascular invasion is one of the most powerful factors asso-ciated with the recurrence of HCC after resection (27),patients consented to take sorafenib as the adjuvant ther-apy. The criteria for following-up, definition of sorafenibresistance and high expression ofMETwere described in theSupplementary Materials and Methods. The characteristicsof all patients were summarized in Supplementary Table S1.

Cell lines and culture conditionsSK-HEP1 and HepG2 cells were obtained from the Amer-

ican Type Culture Collection. MHCC97L and MHCC97Hcellswere supplied by theCell Bankof theChineseAcademyof Sciences (Shanghai, China). These cell lines were main-tained in Dulbecco’s Modified Eagle Medium (DMEM;GIBCO BRL) supplemented with 10% FBS (GIBCO BRL),100 U/mL penicillin, and 100 U/mL streptomycin.HUVECs (human umbilical vein endothelial cells) werepurchased from ScienCell Research Laboratories and weremaintained in EBM-2 medium (Cambrex Bio Science Inc.)according to themanufacturer’s instructions. The four HCCcell lines were authenticated using short tandem repeatDNA testing by Beijing Microread Gene Tech., Co., Ltd. in2013. HUVECs were not authenticated by the authors. Allcell cultures were maintained at 37�C in a CO2 incubatorwith a controlledhumidified atmosphere composedof 95%air and 5% CO2.

Reagents and antibodiesReagents and antibodies used in this studywere described

in the Supplementary Materials and Methods.

Cell viability, colony formation, cell cycle, andapoptosis analyses

Cell viability, colony formation, cell cycle, and apoptosisanalyses were performed as described in the SupplementaryMaterials and Methods.

Western blot analysisCells or isolated independent tissues (lungs and tumors)

from vehicle control- and cabozantinib-treated mice werelysed with RIPA Lysis Buffer (Santa Cruz Biotechnology)containing protease inhibitors (Complete; Roche) andphosphatase inhibitors (PhosStop; Roche). The proteinconcentration was determined using a bicinchoninic acidassay (Beyotime Biotechnology) and equalized before load-ing. A total of 25 to 50 mg of protein were separated by SDS–PAGE and transferred onto polyvinylidene difluoridemem-branes. Membranes were blocked and blotted with therelevant antibodies. Horseradish peroxidase–conjugatedsecondary antibodies were detected with an enhanced

Translational RelevancePatients with hepatocellular carcinoma (HCC) with

an active hepatocyte growth factor (HGF)/MET signalingpathway have a significantly worse prognosis. Moreover,MET activation triggered by antiangiogenic therapies,such as sunitinib and sorafenib, can contribute tometas-tasis. In this study, we first verified an association ofphosphorylated MET (p-MET) with resistance to sorafe-nib as postoperative adjuvant therapy in a set of humanHCC samples. In the next set of experiments, we exam-ined antitumor effects of cabozantinib, a dual inhibitorof MET and VEGFR2, using cultured HCC cells, mousexenograft andmetastatic models. Both the in vitro and invivo results showed that cabozantinib could inhibit HCCcells growth and metastasis. On the basis of an under-standing of the mechanism of the drug and its effective-ness inmultipleHCC cells and in vivomodels, we believethat cabozantinib should be a promising strategy for thetreatment of HCC in the future clinical practice.

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chemiluminescence reagent (Millipore Corp.). GAPDH(glyceraldehyde-3-phosphate dehydrogenase) was used asa loading control. All antibody dilutions were 1:1,000except for the GAPDH antibody, which was used at adilution of 1:5,000.

In vitro migration and invasion assaysWound-healing and Transwell assays were used to exam-

ine migration of HCC cells. The invasiveness of cells wasdetermined as described in the Supplementary Materialsand Methods.

Animal experimentsAnimal care. Female BALB/c athymic nude mice, 5- to

6-week-old (Experimental Animal Center of Sun Yat-senUniversity, China), were used for in vivo studies. All animalswere fed a standard diet ad libitum and housed in a tem-perature-controlled animal facilitywith a12/12hours light/dark cycle. All procedures were performed according to theNIH Guide for Care and Use of Laboratory Animals andwere approved by the Bioethics Committee of Sun Yat-senUniversity.

Tumor implantation and growthMHCC97H and HepG2 xenograft models were estab-

lished by subcutaneous injection of tumor cells (5 �107/mL) in PBS with Matrigel at a 1:1 ratio. The cellsuspension was injected in a total volume of 0.2 mL intothe right flank of the mice and was allowed to grow for 2weeks to reach a tumor size of approximately 80 to 200mm3. The mice were then randomized into three groups(n ¼ 8/group): vehicle control (ddH2O, orally), cabozan-tinib (10 mg/kg/d, orally), or cabozantinib (30 mg/kg/d,orally) for 14 consecutive days. Tumor dimensions andbody weights were measured every 2 days starting withthe first day of treatment. Tumor volume (mm3) was

calculated by the following formula: ðl� w2Þ=2, where land w refer to the larger and smaller dimensions collectedat each measurement. The mice were sacrificed after14 days of treatment. Solid tumors were excised, weighed,and either processed for paraffin embedding or snapfrozen and stored at �80�C.

Inhibition of expression of VEGFR2, MET, and itsdownstream pathway in vivoFor this experiment, treatment was not initiated until

tumors reached 300 to 400 mm3 in size, and cabozanti-nib (30 mg/kg) or vehicle was administered once dailyfor only 3 days. The mice (n ¼ 3/group) were sacrificed3 hours after the last treatment, and lungs and solidtumors were homogenized in lysis buffer for Westernblot analysis.

Experimental metastasisSK-HEP1 cells (1 � 106 cells) in 300 mL PBS were

injected directly into the tail veins of 5- to 6-week-oldfemale nude mice (28). This injection was immediatelyfollowed by randomization (n ¼ 6/group) and oral treat-

ment with cabozantinib (30mg/kg), sorafenib (30mg/kg),or ddH2O. Mice were sacrificed after daily treatment for 28days, and their livers and lungs were weighed and sampledfor tissue sectioning. To examine the metastases, 100sequential sections (5 mm) were cut from the lungs andlivers of each mouse, and every 10th section was stainedwith hematoxylin and eosin (H&E). Expression of phos-phorylated MET (p-MET) in each group was determined byimmunohistochemistry (IHC).

Immunohistochemical analysisFrozen, 5-mm thick sections of tumor samples were pre-

pared to determine vessel density with an anti-CD31(1:100) antibody. To evaluate proliferation and apoptosis,5-mm paraffin-embedded sections were stained with anti–Ki-67 (1:50) and anti-cleaved PARP (1:50) antibodies,respectively. After blocking endogenous peroxidase activity,the sections were incubated overnight with the primaryantibodies at 4�C. Detection was completed with thePolink-2 Plus IHC Detection System (Beijing ZhongshanBiotechnology Co.) according to the manufacturer’sinstructions. Sections were visualized by adding diamino-benzidine (DAB kit; Beijing ZhongShan BiotechnologyCo.). Negative controls were obtained by omitting theprimary antibody. Staining was evaluated by two indepen-dent observers. To quantify the mean vessel density (MVD)in sections stained for CD31, 10 random fields per tumorsample at �200 magnification were captured and quanti-fied as CD31-positive area/total area by Image-Pro Plussoftware (Media Cybernetics, Inc.). For Ki-67 and cleavedPARP, only nuclear immunoreactivity was considered pos-itive. The proliferation index and apoptosis index corre-sponded to the number of labeled Ki-67 or cleaved PARPcells among at least 500 cells per region and are expressed aspercentages.

Statistical analysisStatistical analyses were performed with mean � SD

values using the Student t test and two-way ANOVA withthe Bonferroni correction. Statistical significance was con-cluded at P < 0.05.

ResultsThe expression of p-MET in resected HCC specimens isassociated with resistance to adjuvant sorafenibtherapy

The expression of MET in HCC specimens was detectedby immunostaining. We observed that MET is overex-pressed in most of tumor tissues (data not shown),whereas p-MET is highly expressed in approximately27.6% patients with HCC. Notably, we found that pos-itive p-MET staining was associated with the therapeuticresponse to sorafenib. Specifically, seven of 12 (58.3%)cases with sorafenib resistance have high p-MET expres-sion, whereas only one of 17 (5.88%) sorafenib-sensitivecases were observed to have positive staining for p-MET(Supplementary Fig. S1).

Antitumor Activity of Cabozantinib in HCC

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Antiproliferative effect of cabozantinib on HCC cellsin vitro

The effect of cabozantinib on proliferation in each HCCcell line is shown in Fig. 1. Cabozantinib inhibited cellgrowth in a concentration-dependentmanner inMHCC97Land MHCC97H cells, with IC50 values of 13.47 and 9.466nmol/L, respectively. SK-HEP1 andHepG2 cells weremuchless sensitive to cabozantinib (IC50 ¼ 4,306 and 5,040nmol/L, respectively). Similar results were obtained fromthe colony formation assay (Supplementary Fig. S2). Toanalyze the mechanisms by which cabozantinib inhibitedcell proliferation, flow cytometric analysis was conductedto analyze the cell cycle and apoptosis of cells aftertreatment with various concentrations of cabozantinib.As shown in Fig. 2A, in both MHCC97L and MHCC97Hcells, cabozantinib markedly increased the percentage ofcells in the G1-phase, whereas decreasing the percentageof cells in the S-phase. Cyclin D1 is a critical regulator ofthe G1–S transition (29). Upregulation of cyclin Dl resultsin rapid growth of a subset of HCC (30). Western blotanalysis indicated that cyclin D1 expression in MHCC97Land MHCC97H cells was reduced after treatment withcabozantinib for 24 hours (Supplementary Fig. S3A).Notably, after treatment with cabozantinib, SK-HEP1 andHepG2 cells showed a decrease in the G1-phase and anincrease in the G2-phase (Fig. 2B). Cabozantinib inducedapoptosis in SK-HEP1 and HepG2 cells, but not inMHCC97L and MHCC97H cells (Fig. 2C and D andSupplementary Fig. S3B). The data presented above col-lectively suggest that different mechanisms seem to be

involved in the antiproliferative effect of cabozantinib onMHCC97L, MHCC97H, SK-HEP1, and HepG2 cells.

Cabozantinib inhibits MET and VEGFR2phosphorylation and their downstream effectorsin vitro

Western blot analysis demonstrated that cabozantinib-sensitive MHCC97L and MHCC97H cells displayed a dra-matic elevation in MET phosphorylation, compared withSK-HEP1 and HepG2 cells. No VEGFR2 expression wasdetected in cultured HCC cells (Fig. 3A), which indicatesthat the inhibitory activity of cabozantinib on VEGFR2 isnot involved in its antiproliferative effects on these HCCcells in vitro.

Next, we investigated the effect of cabozantinib treatmenton MET-dependent signaling pathways. Marked suppres-sion of p-MET was observed in MHCC97L and MHCC97Hcells tested after 4 hours incubation with cabozantinib atconcentrations as low as 10 to 100 nmol/L. Moreover,treatment with these doses also effectively abrogated thephosphorylation of downstream effectors, such as STAT3,AKT, and ERK1/2 (Fig. 3B). Thus, constitutive activation ofthese proliferative and survival effectors in MHCC97L andMHCC97H cells seems to depend specifically on METsignaling. In contrast, in SK-HEP1 and HepG2 cells, inwhich MET is not constitutively phosphorylated, cabo-zantinib at a dose of 100 nmol/L had no demonstrableeffect on the phosphorylation of STAT3, AKT, or ERK1/2,indicating that these proliferative and survival effectorsare likely activated through alternative growth factor

Figure1. Cabozantinib inhibitsHCCcell proliferation. MHCC97L,MHCC97H, SK-HEP1, and HepG2cells were treated with differentconcentration of cabozantinib for72 hours in DMEM containing 10%FBS. Eachpoint, themean�SD forthree independent experiments.IC50 was calculated by nonlinearregression analysis usingGraphPad Prism software.

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Figure 2. Effects of cabozantinib on cell-cycle progressionandapoptosis inHCCcells. A andB, effect of cabozantinib on the cell cycle.MHCC97L,MHCC97H,SK-HEP1, and HepG2 cells were treated with either 0.1% DMSO (dimethyl sulfoxide) or cabozantinib for 24 hours. After treatment, cells were harvested,fixed, and stained with propidium iodide (PI) for flow cytometric analysis. Data were analyzed using ModFit and were reported as the mean � SD.C andD, apoptosis of HCCcells detected by the Annexin V–FITC/propidium iodide–binding assay. Cells were treatedwith either 0.1%DMSOor cabozantinibfor 48 hours at the indicated concentrations, and then stained with Annexin V–FITC and propidium iodide. The rate of apoptosis was determined usinga flow cytometer, and data were analyzed using Kaluza software and were reported as the mean � SD. The results are representative of three independentexperiments. �, P < 0.05 and ��, P < 0.01, versus control.






receptors. An increase in the concentration of cabozanti-nib, up to 5,000 and 10,000 nmol/L, had a significantimpact on the phosphorylation of STAT3, AKT, andERK1/2 in both SK-HEP1 and HepG2 cells (Fig. 3B). Thiseffect may be because cabozantinib has a nonspecificinhibitory effect on these effectors or exerted through theinhibition of other cancer-specific cabozantinib targets,such as AXL, RET, and KIT.

In a cytokine-stimulated tyrosine kinase activity assay, wefound that cabozantinib treatment resulted in the markedinhibition of cytokine-stimulated phosphorylation of METand VEGFR2 and their resultant downstream effectors inHUVECs (Fig. 3C). In a concentration-dependent manner,cabozantinib eliminated HGF-induced MET phosphoryla-tion and its downstream effectors STAT3, Akt, and Erk-1/2in both SK-HEP1 and HepG2 cells (Fig. 3D).

Cabozantinib inhibits HGF-induced cell motility andinvasion

Because MET is not necessary for proliferation of SK-HEP1 and HepG2 cells, we used these two cell lines to test

whether cabozantinib has an effect on motility and inva-sion. We observed that HGF enhanced the migration andinvasion of SK-HEP1 and HepG2 cells as evaluated by awound-healing and Transwell assays. Moreover, at a con-centration that has minimal impact on growth, cabozanti-nib inhibited HGF-inducedmigration and invasion in bothSK-HEP1 andHepG2 cells (Fig. 4). These findings reflect thepotential antimetastatic effect of cabozantinib inHCC cells.

In vivo efficacy andmechanism of cabozantinib againstMHCC97H and HepG2 xenografts

To examined that cabozantinib inhibits VEGFR2 andMET signaling activity in vivo, establishedMHCC97H xeno-grafts (n¼ 3/group) were treated daily with an oral dose ofvehicle or cabozantinib at 30mg/kg for 3 days. As shown inSupplementary Fig. S4, administration of cabozantinibresulted in significant inhibition of VEGFR2 and METphosphorylation in mice lungs and tumors, respectively,comparedwith the vehicle-treated control group.Moreover,the inhibition of downstreamMET effectors, such as STAT3,AKT, and ERK1/2, was also detected in MHCC97H tumors.

Figure 3. The expression profile ofreceptor tyrosine kinases (RTK)and the effect of cabozantinib onRTKs signaling in HUVECs andHCC cells. A,Western blot analysiswas performed to detect theexpression profile of VEGFR2 andMET in HUVECs and HCC cells.B, Western blot analysis wasconducted to measure effect ofcabozantinib on phosphorylationof MET and downstream effectorsin MHCC97L, MHCC97H, SK-HEP1, and HepG2 cells. Cells weretreated with either 0.1% DMSO orthe indicated concentrations ofcompounds in DMEM containing10%FBS for 4 hours before proteinextraction. C, HUVECswere grownwith either 0.1% DMSO or theindicated doses of cabozantinib for4 hours, followed by the addition of50 ng/mL VEGF or HGF 10minutesbefore cell lysis for Western blotanalysis. D, cells were starved inmedium containing 1% FBS for12 hours before adding either 0.1%DMSO or cabozantinib. Afterincubation for 4 hours andstimulation with 50 ng/mL HGF for10 minutes, cells were lysed forWestern blot analysis.Independent experiments wereperformed at least three times, andthe results from a representativeexperiment are shown.

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The results in Supplementary Table S2 and Fig. 5A dem-onstrate that cabozantinib at concentrations of both 10 and30mg/kg displayed a good anticancer effect on MHCC97Hxenografts; their tumor growth inhibition (TGI) rates were53.4% and 84.6%, respectively. Treatment of HepG2 xeno-grafts with an identical treatment scheme led to 27.5% and59.1% TGI, compared with the vehicle-treated controlgroup (Supplementary Table S2; Fig. 5B). These differencesin efficacy imply that the overexpression of p-MET mayidentify a sensitivity index for cabozantinib treatment inHCC.We next evaluated the antiangiogenic, antiproliferative,

and proapoptotic effects of cabozantinib in treated tumorxenografts. Immunohistochemical analyses revealed thatcabozantinib decreased the MVD in MHCC97H xeno-grafts by 49.9% and 90% at doses of 10 and 30 mg/kg,respectively, compared with the vehicle-treated control

(Fig. 6). MVD in HepG2 xenografts was reduced by 58.3%and 87.1% at doses of 10 and 30 mg/kg, respectively,compared with the vehicle-treated control. In addition,MHCC97H and HepG2 xenografts treated with cabozan-tinib revealed a significant reduction in proliferation(the percentage of Ki-67–positive cells) and an increasein apoptosis (the percentage of cleaved PARP-positivecells).

Cabozantinib prevents metastasis of SK-HEP1cells to the lung and liver

To determine whether cabozantinib treatment couldreduce metastasis, SK-HEP1 cells were directly injected intothe tail vein of female nude mice. After injection, micereceived sorafenib or cabozantinib treatment for 4 weeks.We found that the formation of metastases in the lung and

Figure 4. Cabozantinib depressesHGF-induced cell migration andinvasion of SK-HEP1 and HepG2cells. A, cabozantinib inhibits HGF(50 ng/mL)-stimulated cellmigration in the wound-healingassay. Shown here arerepresentative images of threeindependent experiments. BandC,cabozantinib inhibits HGF (50 ng/mL)-stimulated SK-HEP1 andHepG2 cells migration andinvasion in a Transwell assay. Therepresentative photographs ofmigration and invasion cells wereshown as B and C, respectively.Bar graphs, the average number ofstained cells was calculated fromthree independent experiments,with 10 fields counted perexperiment, and are reported asthe mean � SD. ��, P < 0.01; scalebars, 50 mm.






liver was reduced by 53.7% and 52.9% in the cabozantinib-treated group, respectively, compared with the vehicle-trea-ted group (Fig. 7). Interestingly, lungs and livers frommicein the sorafenib-treated group displayed an apparentincrease in metastatic foci. Additional evidence of the inhi-bition of metastasis in the cabozantinib-treated group wasconfirmed by the significant difference in whole-lung wetweights among control, sorafenib, and cabozantinib treat-ment groups (Fig. 7A). Comparison of p-MET, as assessedby IHC, in mice treated with ddH2O or sorafenib corrob-orated the known positive relationship between VEGFsignaling inhibition and MET activation (31–33). Aftersorafenib treatment for 28 days, staining for p-MET wasstrong and widespread in lung tissue and metastatic foci inthe liver. Staining for p-MET was weak or absent in thecabozantinib-treated group (Supplementary Fig. S5). Cabo-zantinib treatment was well tolerated, as determined bystable body weights throughout the 28 days treatmentperiod.

DiscussionTargeting angiogenesis has become an established ther-

apeutic approach to fighting solid tumor growth in patientswith cancer, and the systemic therapy with sorafenib repre-sents amilestone in advancedHCC.However, the benefit ofsorafenib in clinical therapy ismarginal and transient (8, 9).The MET pathway has been found involved in gefitinibresistance in lung cancer (15). In line with this, we revealedthat high level of activated MET in HCC is associated withresistance to adjuvant sorafenib treatment. Moreover, wedemonstrated that cabozantinib, a dual inhibitor of METandVEGFR2, could inhibit the growth,migration, invasion,and metastasis of HCC both in vitro and in vivo.

In tumor cell growth inhibition assays, cabozantinibcould inhibit the growth of p-MET expressing cells(MHCC97L and MHCC97H) at low concentrations, butrequired much higher concentration in p-MET–negativecells (SK-HEP1 and HepG2). Flow cytometry analysisrevealed that cabozantinib suppressed the proliferation of

MHCC97L and MHCC97H cells by causing G1-phase cell-cycle arrest without inducing apoptosis. The expression ofcyclinD1,which is a critical regulator of theG1–S transition,was markedly blocked by cabozantinib. These results aresupported by the study of Zhang and colleagues, whoshowed that MET knockout induces significant G1 arrestand a decrease of cyclin D1 in MHCC97L and MCC97Hcells (16). Cabozantinib displayed a significant concentra-tion-related antiproliferative effect on MHCC97L andMHCC97H cells at approximately 10 nmol/L, which wasaccompanied by a reduction of phosphorylation of METand its downstream effectors STAT3, Akt, and Erk1/2. Thesefindings highlight that cabozantinib suppressed the prolif-eration ofMHCC97L andMHCC97H cells by impeding theMET pathway.

Consistent with the in vitro study, cabozantinib showedan anti-MET and -VEGFR2 pathway activity in theMHCC97H xenografts model. The treatment of MHCC97Hxenografts with cabozantinib resulted in a more pro-nounced TGI compared with its efficacy on HepG2 xeno-grafts, suggesting that p-MET amplificationmay be amolec-ular marker of susceptibility to cabozantinib treatment inHCC cells. Both MHCC97H and HepG2 xenografts treatedwith cabozantinib showed reduced microvessel density,suppressed proliferation, and increased apoptosis. On thebasis of our findings,we propose that the antitumor effect ofcabozantinib on p-MET–positive MHCC97H xenograftsseems to be mediated by inhibiting tumor angiogenesis(anti-VEGFR2 effect) and by directly inhibiting tumorcell proliferation (anti-MET effect). For p-MET–negativeHepG2 xenografts, impeding stromal angiogenesis throughVEGFR2 inhibition may contribute to the dominant abro-gation of tumor growth. Because patients with HCC mayhave MET-negative disease (34), we propose that METactivation may be a useful biomarker in cabozantinib clin-ical trials in identifying patientswithHCCwithpotential forthe greatest benefit, by predicting durable tumor shrinkageand increased tumor response rate. However, the degree towhich inhibition of the MET signaling pathway contributesto antiangiogenesis and inhibition of other cabozantinib

Figure 5. Cabozantinib abrogatestumor growth in the xenograftmouse model. A and B, MHCC97Hand HepG2 cells weresubcutaneously implanted in nudemice as described in Materials andMethods. Mice bearing tumorsxenografts were treated daily withvehicle or either 10 or 30 mg/kg ofcabozantinib for 14 days. Theimage shows the tumor size ofMHCC97H and HepG2 xenograftsat the end of the experiment. Data,themean�SDof the tumor volumeand tumor weight for each group ofeight experimental animals.�, P < 0.05; ��, P < 0.01; the treatedgroup versus the control group.

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targets contributes to tumor growth inhibition in vivoremains to be clarified.It has been reported that cabozantinib suppressed cell

migration and invasion in various types of tumor cells(23, 35). Consistently, our results showed that cabozan-tinib inhibited HGF-stimulated migration and invasionin HCC cell lines. Notably, treatment of SK-HEP1 andHepG2 cells with low concentration of cabozantinib hadminimal impact on growth but strongly reduced migra-tion and invasion potential, suggesting a role of cabo-zantinib on cell motility and invasion without affectingproliferation.Recent studies have demonstrated that antiangiogen-

esis agents could increase local invasion and distantmetastasis during or after treatment (11, 12, 36, 37). For

example, VEGFR inhibitors, such as sorafenib and suni-tinib, can result in upregulation of MET, leading topromotion of metastasis (19, 32, 38). In support of this,we observed that sorafenib treatment promoted metasta-sis in the lung and liver, and accompany with activationof MET in experimental metastasis models with SK-HEP1cells. This finding argues that simultaneously targetingMET and VEGFR2 may circumvent the "metastatic escapepathways." Indeed, mice treated with cabozantinib hadfewer metastastic foci in lung and liver tissues comparedwith control- and sorafenib-treated groups, suggestingthat cabozantinib could reduce tumor metastasis mainlythrough inactivation of MET. Interestingly, our observa-tions raise the possibility that MET could be activatedduring tumor progression and sorafenib treatment,

Figure 6. Effects of cabozantinib onangiogenesis, cell proliferation,and apoptosis of MHCC97H andHepG2 xenografts. Mice bearingtumor xenografts were treated asdescribed in Materials andMethods. A, representativepictures of blood vessels stainedwith anti-CD31; B, proliferativecells stained with Ki-67; C,apoptotic cells stained with anti-cleaved PARP antibodies intumors. The bar graph, mean� SDof quantification of CD31-positiveareas, Ki-67– and PARP-positivecells from immunohistochemicalanalysis of tumors. �, P < 0.05;��, P < 0.01; the treated groupversus the control group; scalebars, 100 mm.






leading to enhanced metastasis, thereby identifying apotential target for therapeutic intervention.

In summary, our study revealed that the high level ofp-MET in HCC tissue could be a prognosticator of resis-tance to adjuvant sorafenib therapy. The inhibition ofboth VEGFR2 and MET signaling pathways by cabozan-tinib could have considerable therapeutic effects in HCCin vitro and in vivo. Also, the presence of MET activation in

HCC may be a promising biomarker for predicting theresponse to cabozantinib treatment. Altogether, cabozan-tinib could be a useful agent for inhibiting tumor growth,angiogenesis, and metastasis in HCC with dysregulatedMET and VEGFR2 signaling pathways.

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

Figure 7. Effect of sorafenib and cabozantinib on the metastasis of SK-HEP1 in nude mice. Following the experimental design as described in Materials andMethods, nude mice were sacrificed at day 28 to evaluate lung and liver metastases. The image is a representative H&E-stained section of lung (A) and livermetastases (B), and the average number of foci is presented as themean�SD. �,P<0.05; ��,P<0.01; the treatedgroup versus the control group; scale bars inA and B �40, 500 mm; scale bars in A and B �200, 100 mm.


Xiang et al.




Authors' ContributionsConception and design: M. Ren, Y. Chen, C. ShangDevelopment of methodology: Q. Xiang, Y. ChenAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): Q. Xiang, J. WangAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): Q. Xiang, W. Chen, J. Wang, H. ZhangWriting, review, and/or revision of the manuscript: Q. Xiang, W. Chen,M. Ren, J. Wang, L. Zhang, Y. ChenAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): W. Chen, J. Wang, L. Zhang,Y. Chen, C. ShangStudy supervision: W. Chen, D.Y.B. Deng, Y. Chen

AcknowledgmentsThe authors thank the support from the Key Laboratory of Malignant

TumorGeneRegulation andTarget TherapyofGuangdongHigher Education

Institutes, Sun Yat-sen University (Guangzhou, China). The authors alsothank the expert assistance provided by Dr. Dong Yin in the revision of thisarticle.

Grant SupportThis work was supported by grants from the National Natural Science

Foundation of China (no. 81372562) and the Key Science and TechniqueResearch Project of Zhongshan (no. 20122A002).

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 September 25, 2013; revised January 31, 2014; accepted March22, 2014; published OnlineFirst April 3, 2014.

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Correction

Correction: Cabozantinib Suppresses TumorGrowth and Metastasis in HepatocellularCarcinoma by a Dual Blockade of VEGFR2and MET

In this article (Clin Cancer Res 2014;20:2959–70), which was published inthe June 1, 2014, issue of Clinical Cancer Research (1), a reader alerted us topotential image manipulation in the control �200 and sorafenib (30 mg/kg)�200 panels in Fig. 7A. AACR Publications staff members and editors reviewedthe figure and agreed that sections of the panels appeared to be identical.The authors admitted that an image of the sorafenib (30 mg/kg) �200 panelwas mistakenly included in the control �200 panel with their submission. Theauthors have provided the correct version of the control �200 panel for Fig. 7Abelow. The results and conclusions put forth in this article remain unchanged. Theauthors regret these errors.

Reference1. Xiang Q, Chen W, Ren M, Wang J, Zhang H, Deng DY, et al. Cabozantinib suppresses tumor

growth and metastasis in hepatocellular carcinoma by a dual blockade of VEGFR2 and MET.Clin Cancer Res 2014;20:2959–70.

Published online May 2, 2016.doi: 10.1158/1078-0432.CCR-16-0034�2016 American Association for Cancer Research.

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ClinicalCancerResearch

Clin Cancer Res; 22(9) May 1, 20162312

2014;20:2959-2970. Published OnlineFirst April 3, 2014.Clin Cancer Res Qingfeng Xiang, Weiqiang Chen, Meng Ren, et al. Hepatocellular Carcinoma by a Dual Blockade of VEGFR2 and METCabozantinib Suppresses Tumor Growth and Metastasis in

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