CT-707, a Novel FAK Inhibitor, Synergizes with Cabozantinib to … · Small Molecule Therapeutics CT-707, a Novel FAK Inhibitor, Synergizes with Cabozantinib to Suppress Hepatocellular

Small Molecule Therapeutics

CT-707, a Novel FAK Inhibitor, Synergizes withCabozantinib to Suppress HepatocellularCarcinoma by Blocking Cabozantinib-InducedFAK ActivationDan-Dan Wang1, Ying Chen1, Zi-Bo Chen1, Fang-Jie Yan1, Xiao-Yang Dai1,Mei-Dan Ying1, Ji Cao1, Jian Ma1, Pei-Hua Luo1, Yong-Xin Han2, Yong Peng3,Ying-Hui Sun3, Hui Zhang3, Qiao-Jun He1, Bo Yang1, and Hong Zhu1

Abstract

Hepatocellular carcinoma is among the leading causes ofcancer-related deaths worldwide, and the development of newtreatment regimens is urgently needed to improve therapeuticapproach. In our study, we found that the combination of a Metinhibitor, cabozantinib, and a novel FAK inhibitor, CT-707,exerted synergistic antitumor effects against hepatocellular carci-noma in vitro and in vivo. Interestingly, further studies showed thattherapeutic concentrations of cabozantinib increased the phos-phorylation of FAK, whichmight attenuate the antitumor activityof cabozantinib. The simultaneous exposure to CT-707 effectivelyinhibited the activation of FAK that was induced by cabozantinib,which contributes to the synergistic effect of the combination.Furthermore, cabozantinib increased the mRNA and protein

levels of integrin a5, which is a canonical upstream of FAK, andthe introduction of cilengitide to block integrin function couldabrogate FAK activation by cabozantinib, indicating that cabo-zantinib upregulated the phosphorylation of FAK in an integrin-dependent manner. Similar synergy was also observed on PHA-665752, another selective MET inhibitor, indicating that thisobservation might be a common characteristic of MET-targetingstrategies. Our findings not only favor the development of thenovel FAK inhibitor CT-707 as a therapeutic agent against hepa-tocellular carcinomabut also provide a new strategy of combiningMET and FAK inhibitors to potentiate the anticancer activities ofthese two types of agents for treating hepatocellular carcinomapatients. Mol Cancer Ther; 15(12); 2916–25. �2016 AACR.

IntroductionAs one of themost common cancers, hepatocellular carcinoma

is the third-leading cause of cancer-related deaths (1). Foradvanced hepatocellular carcinoma, the only first-line drug issorafenib, which unfortunately has a limited effect on hepatocel-lular carcinoma. Therefore, a novel and effective clinical treatmentstrategy for hepatocellular carcinoma is urgently needed. Cabo-zantinib (XL184), approved by the FDA to treatmedullary thyroidcancer in 2012, is amulti-target tyrosine kinase inhibitor targetingVEGFR-2, MET, RET, and other receptor tyrosine kinases (2). AstheVEGFR-2 andMET signalingpathways play a significant role inhepatocellular carcinoma pathogenesis (3), cabozantinib has

been assessed as a novel therapy for hepatocellular carcinomain a phase II clinical trial (NCT00940225). More recently, a phaseIII clinical trial, comparing the efficacy of cabozantinib versusplacebo for hepatocellular carcinoma, is recruiting hepatocellularcarcinoma patients (NCT01908426). However, the preliminarystudy suggests that the use of cabozantinib as a single agent inhepatocellular carcinoma has limited effect, and its long-termbenefit remains uncertain. Rational combinations of drugs withdifferent mechanisms of action that exert synergistic effects orovercome resistance mechanisms may improve the efficacy oftherapy.

AlthoughMET inhibitors, such as cabozantinib, exert early andpowerful antitumor activity, a recurrent problem that has limitedthe clinical practice of these inhibitors is the development ofacquired resistance, which is a common challenge in other tar-geted therapies. Various studies have been conducted to predictthe mechanisms that might cause resistance to MET-targetingtherapies and to find strategies for overcoming the resistance. Ithas been reported that a pointmutation inMET kinase (4) and/oramplification of theMET gene (5) contribute to the emergence ofresistance toward theMET inhibitor. In addition, the activation ofseveral protein kinase signal pathways is involved in acquiredresistance. In MET-addicted gastric cancer cells, the HER2 kinaseactivation can weaken the growth-inhibitory effects of PHA-665752, a selectiveMET inhibitor, and contribute to the resistanceof MET-targeting therapy (6). Similarly, in non–small cell lungcancer (NSCLC), the activation of alternative signaling pathways,

1Zhejiang Province Key Laboratory of Anti-Cancer Drug Research,College of Pharmaceutical Sciences, Zhejiang University, Hangzhou,China. 2Department of Medicinal Chemistry, Centaurus BioPharmaCo., Ltd, Beijing, China. 3Department of Discovery Biology, CentaurusBioPharma Co., Ltd, Beijing, China.

D.-D. Wang and Y. Chen contributed equally to this article.

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

CorrespondingAuthor:HongZhu, ZhejiangUniversity, 866YuHangTangRoad,Hangzhou, Zhejiang 310058, China. Phone: 8657-1882-08401; Fax: 8657-1882-08400; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-16-0282

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 15(12) December 20162916

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such as mTOR and Wnt pathways (7), is a possible molecularmechanism of resistance toMET inhibitors. These results raise thepossibility that combining tyrosine kinase inhibitors and METinhibitors could improve the antitumor effect of MET-targetingtherapies. Actually, it has been demonstrated that the combina-tion of MET and RAF inhibitors suppresses tumor cell prolifera-tion, even for MET-resistant clones in human gastric carcinomacells (8).

Focal adhesion kinase (FAK) is overexpressed and activated in apanel of tumors, and it promotes invasion, metastasis, prolifer-ation, growth, and survival in tumor cells (9). Therefore, FAK hasbeen considered a promising anticancer target, and as a result,enormous efforts have been devoted to developing novel agentsthat target FAK. CT-707 (Fig. 1B), a novel multikinase inhibitortargeting FAK, ALK, and Pyk2, was approved by the China FoodandDrugAdministration for a phase I clinical trial to treatNSCLC.A preclinical study showed that CT-707 exerted significant inhib-itory effects on FAK, and an IC50 value of 1.6 nmol/L was revealedby the in vitro kinase activity. In addition, CT-707displayed potentactivities in arresting cancer cell growth, promoting cell detach-ment, and decreasing wound healing, which were closely relatedto the biological functions of FAK in cancer cells. A preclinicalstudy showed that CT-707 also exhibits potent anticancer activ-ities in vivo, as it inhibited tumor growth and metastasis in T47D,Karpas299, and 4T1 xenograft models (data not shown). Never-theless, the antitumor activities of CT-707 combined with theother anticancer agents, particularly the other kinase-targetinginhibitor(s), have not been evaluated.

The current study revealed that CT-707 exhibits a synergisticantitumor effect on hepatocellular carcinoma when combinedwith cabozantinib, in vitro and in vivo. In an attempt to explore themolecular mechanisms underlying the combination, we foundthat therapeutic concentrations of cabozantinib increase thephosphorylation of FAK through integrin-dependent pathways,whichmight compromise the antitumor effect of cabozantinib inthe context of the significant role of FAK in tumor growth andsurvival. CT-707, as a FAK inhibitor, can remarkably decrease theactivation of FAK that is induced by cabozantinib exposure, whichmight attribute to, at least partially, the synergistic antitumoreffect. Further study showed that another MET inhibitor, PHA-665752, imposed a similar effect by activating p-FAK and syner-gizing with CT-707 on hepatocellular carcinoma, indicating thatthe induced phosphorylation of FAK might be a common char-acteristic of MET inhibitors. This also provides evidence forovercoming the limitations of MET-targeting therapies through

combining themwith FAK inhibitors. Therefore, we hypothesizedthat FAK activation attenuated the efficacy of cabozantinib onhepatocellular carcinoma and the combination of cabozantiniband FAK inhibitors would be a more promising therapeuticregimen as well as provide basic theories for clinical hepatocel-lular carcinoma treatment. Our study provided evidence for thesynergistic anticancer activities of the combination of inhibitorstargeting MET and FAK, and our findings might broaden thehorizon for clinical applications of those agents, especially whencombined with other small-molecule inhibitors.

Materials and MethodsMaterials

Cabozantinib was attained from Melone Pharmaceutical, CT-707 was kindly provided by Centaurus Biopharma, and PHA-665752 and cilengitide were purchased from Selleck Chemicals.The primary antibodies against cleaved PARP, cleaved caspase-8,cleaved caspase-3, p-P70-S6K (Thr 389), FAK, and integrin a5were purchased from Cell Signaling Technology. The primaryantibody against p-FAK (pY397) was purchased from Invitro-gen. The primary antibodies against p-AKT1/2/3 (Thr 308),AKT1/2/3, P70-S6K, p-4E-BP-1 (Ser 65), 4E-BP-1, b-actin, andhorseradish peroxidase (HRP)–labeled secondary anti-rabbit,anti-goat, and anti-mouse antibodies were purchased fromSanta Cruz Biotechnology.

Cell lines and cultureThe humanhepatocellular carcinoma cell linesHepG2 andBel-

7402 were purchased from Shanghai Institutes for BiologicalSciences (Chinese Academy of Sciences, Shanghai, China).HepG2 was normally cultured in DMEM medium, and Bel-7402 was normally cultured in RPMI1640 medium, both sup-plemented with 10% FBS in a 5% CO2 humidified incubator at37�C. All the cell lines were authenticated using DNA fingerprint-ing (variable number of tandem repeats), confirming that nocross-contamination occurred during this study.

Cell survival assayCell survival assay was assessed by sulforhodamine B (SRB)

colorimetric assay and theCCK8assay. The SRB colorimetric assaywas operated as described previously (10). For the CCK8 assay,HepG2 cells were seeded into 96-well plates and normally cul-tured overnight, following the treatment of serial concentrationsof agents for 48hours. CCK8 (20mL) solutionwas added into each

Figure 1.

Chemical structures of cabozantinib (A) and CT-707 (B).

Synergistic Antitumor Activity of XL184 with CT-707

www.aacrjournals.org Mol Cancer Ther; 15(12) December 2016 2917




well in a dark environment and then cells were cultured in a 5%CO2 humidified incubator at 37�C. The absorbance at 450 nmwas measured using a Multiskan Spectrum (Thermo ElectronCorporation) until the absorbance values remained unchanged.Assays were performed in triplicate in three independentexperiments.

Clonogenic assayCells were seeded into 6-well plates and treated with cabozan-

tinib (5 mmol/L), CT-707 (3 mmol/L), or both for 24 hours, afterwhich cells in each group were sparsely plated (approximately1,000 cells per 60-mmdish). The compound-containingmediumwas replaced every 2 to 3 days. After a normal culture for 20 days,0.1% crystal violet solution was used to stain the colonies, anddishes in each group were photographed. Then, 10% acetic acidwas used to extract the stained cells, and the absorbance at 450nmwas measured using a Multiskan Spectrum (Thermo ElectronCorporation).

Flow cytometryPropidium iodide (PI) staining and Annexin V-PI (AV-PI)

staining were used to detect the apoptosis of cells by flowcytometry on a FACSCalibur cytometer (Becton Dickinson) asdescribed previously (11).

Cell lysates and Western blot analysisCells were lysed with the loading buffer. Proteins were frac-

tionated on 10% SDS-PAGE and transferred to polyvinylidenefluoride membrane (Millipore Corporation). As indicated in theexperiment, proteins were probed with primary antibodies. HRP-labeled secondary antibodies ECL chemiluminescence detectionsystem was used to detect the relative expression of proteins.

Quantitative RT-PCRThe total RNA was prepared and detected as we previously

reported (12). The following primer was used: integrin a5, 50-ACCATCCAGTTTGACTTCCA-30 (forward) and50-TCGCTTACTGG-GAATAGCAC-30 (reverse).

Animal xenograft model and in vivo activity measurementThe human hepatocellular carcinoma cell line HepG2 xeno-

graft models were established by implanting tumor tissue sub-cutaneously inoculated into nudemice.When the tumors reachedamean size of 90mm3, themice were randomly divided into fourgroups and treated as indicated in the experiment. Tumorvolumes were measured three times a week and calculated as(length�width2)/2. The individual relative tumor volume (RTV)and T/C (the ratio of the RTV of the treated group/the RTV of thecontrol group) were calculated as previously reported. The indi-vidual tumor weight was measured after 14-day administration.The Animal Research Committee at Zhejiang University (Zhe-jiang, China) approved all animal studies, and animal care wasprovided in accordance with institutional guidelines.

Statistical analysesAll in vitro data were expressed as the mean values � SD, and

in vivo data were expressed as the mean � SEM. Data wereanalyzed using SPSS 20.0 for Windows. ANOVA followed byLSD and Dunnett test were used to make comparisons. P < 0.05was considered statistically significant.

ResultsCytotoxicity of the combination of cabozantinib and CT-707 inhuman hepatocellular carcinoma cell lines

In our primary study, we assessed the antitumor effect of thecombination of cabozantinib (Fig. 1A) and CT-707 in humanhepatocellular carcinoma cell lines, including HepG2 and Bel-7402. Cells were treated with serial concentrations of cabozanti-nib, CT-707, or both for 72hours, andSRB staining assaywas usedto detect the survival fractions of each treatment group. Thesurvival curves of cabozantinib, CT-707, and the combinationare shown in Fig. 2A. When cells were exposed to cabozantinib (5mmol/L), CT-707 (3 mmol/L), or their combination, the survivalrates were 57.3%, 39.3%, and 11.2%, respectively, in HepG2;those in Bel-7402 were 57.8%, 61.6%, and 34.2%, respectively,which suggested that the combination therapy of cabozantiniband CT-707 significantly reduced the survival fraction, comparedwith each agent alone. To specifically evaluate the synergisticantitumor effect, we calculated the combination index (CI) valuesat different ratio concentrations of cabozantinib and CT-707using CalcuSyn (13, 14). The CI values were used to assess drugsynergism, and a CI that is less than 0.90 indicates synergism. Asshown in Table 1, in both human hepatocellular carcinoma celllines, CI values were less than 0.80, indicating that cabozantiniband CT-707 functioned in a synergistic manner to reduce hepa-tocellular carcinoma cell survival.

To explore whether the combination of cabozantinib andCT-707 could induce a long-term, synergistic antitumor effect,we performed a clonogenic assay. HepG2 cells were respectivelytreated with monotherapy or cotreatment for 24 hours; then, thecells in each group were normally cultured for clone formation atthe same count. Representative pictures after 20 days in culture areshown in Fig. 2B; combination treatment resulted in significantinhibition to the proliferation of HepG2 cell clones, while themonotreatment induced moderate inhibition. Simultaneously,we calculated the rates of colony formation compared with thecontrol group. Consistent with the picture, the colony formationrates of cabozantinib, CT-707, and their combination were82.9%, 63.7%, and 24.4%, respectively, indicating that the com-bination treatment had a long-term, synergistic effect on thesurvival and proliferation of hepatocellular carcinoma cells.

The combination of cabozantinib and CT-707 results inenhanced caspase-dependent apoptosis in humanhepatocellular carcinoma cell lines

To further confirm the synergistic antitumor effect, we moni-tored the apoptosis of HepG2 and Bel-7402 cells after 48-hourexposure to cabozantinib (5 mmol/L), CT-707 (3 mmol/L), orboth. The results detected by sub-G1 analysis usingflow cytometryare shown in Fig. 3A; the apoptosis rates of control, cabozantinib,CT-707, and combination groups in HepG2 were 5.0%, 10.5%,18.4%, and41.1%, respectively, and those in Bel-7402were 4.4%,16.3%, 8.7%, and36.4%, respectively. Consistentwith the sub-G1

analysis, the results from AV-PI staining (Fig. 3B) using flowcytometry in Bel-7402 had a similar trend as the apoptosis rates(10.7% for control groups, 26.6% for cabozantinib treatment,11.2% for CT-707 treatment, and 50.6% for combination treat-ment). The results suggested that the combination of two agentsresulted in an enhanced apoptosis in hepatocellular carcinomacells compared with monotreatment. To visualize the apoptosisthrough nucleus morphology, DAPI staining for HepG2 cells was

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used, and typical pictures for this are shown in Fig. 3C. In thecombination group, extensive karyopyknosis and cellular frag-mentation were observed (as indicated by the white arrows),while other treatments failed to result in a nucleus morphologyalteration, suggesting that there was a higher rate of apoptosis inthe combination group.

Because the caspase-dependent pathway is an importantpathway involved in cell apoptosis, we monitored the activa-tion of the caspase cascade by Western blot analysis (Fig. 3D).The 48-hour combination treatment markedly increased theactivation of caspase-8, which was followed by the cleavage ofcaspase-3 and PARP, denoting apoptosis in the combinationgroups (15).

Because the tyrosine kinase FAK and MET share commondownstream signaling pathways, such as the AKT/P70-S6K/4E-BP-1 pathway (16, 17), we were inspired to monitor whether thecombination interfered with these pathways after 24 hours of

exposure. As shown in Fig. 3E, p-AKT, p-P70-S6K, and p-4E-BP-1were remarkably prohibited in the combination group in bothhuman hepatocellular carcinoma cell lines, whereas eithermono-treatment alone induced little inhibition. Considering the signif-icant role of the AKT signaling pathway in cell proliferation,survival, and apoptosis, this result provided further evidence forthe enhanced apoptosis induced by combination exposure.

The FAK phosphorylation induced by MET inhibitors might beinvolved in the synergistic antitumor effect of CT-707 and METinhibitors

To explore the underlying mechanisms, we assessed theFAK phosphorylation levels in HepG2 and Bel-7402 cells after24-hour exposure to cabozantinib, CT-707, or both by Westernblot analysis (Fig. 4A), and importantly, we found that cabo-zantinib could activate FAK phosphorylation. Given that FAKis functionally involved in tumor proliferation, growth, and

Table 1. CI values were calculated at different ratio concentrations of cabozantinib and CT-707 in HepG2 and Bel-7402 cells by CalcuSyn

HepG2 Bel-7402Cabozantinib(mmol/L)

CT-707(mmol/L) CI

Cabozantinib(mmol/L)

CT-707(mmol/L) CI

1.0 1.0 0.67 � 0.08 1.0 0.2 0.72 � 0.112.0 1.5 0.55 � 0.09 2.0 0.4 0.63 � 0.143.0 2.0 0.55 � 0.03 3.0 0.8 0.46 � 0.074.0 2.5 0.56 � 0.04 4.0 1.5 0.51 � 0.045.0 3.0 0.54 � 0.04 5.0 3.0 0.59 � 0.02

Figure 2.

Cytotoxicity of the combination ofcabozantinib and CT-707 in HepG2 and Bel-7402 cell lines. A, HepG2 and Bel-7402 cellswere treated with serial concentrations ofcabozantinib, CT-707, or both for 72 hours,and cell survival was detected using SRBstaining assay.B,HepG2 cells were exposedto cabozantinib (5 mmol/L), CT-707 (3mmol/L), or both for 24 hours, and eachgroupwas cultured at the same count for 20days. The colonies were stained with 0.1%crystal violet solution and photographed.Left, representative picture; right, colonyformation rates. �� , P < 0.001, asevaluated using Student t-test.






survival, cabozantinib-induced FAK activation might limit theantitumor activity of cabozantinib. The combination with FAKinhibitors might overcome this problem. As expected, anotheragent, CT-707, which is an inhibitor of FAK, could markedlydecrease FAK phosphorylation induced by cabozantinib, whichmight partially account for the synergetic effect (Fig. 4A).Further findings showed that when HepG2 cells were treatedwith 5 mmol/L cabozantinib, FAK phosphorylation was inc-reased within 6 hours and peaked at 24 hours (Fig. 4B).Cabozantinib-induced activation of FAK was conducted in adose-dependent manner within concentrations between 3 to 6mmol/L when treated for 24 hours (Fig. 4C).

To determine whether FAK activation is a common character-istic of MET inhibitors, we introduced another MET inhibitor,PHA-665752 (18). As shown in Fig. 4D, FAK phosphorylationwas robustly increased when HepG2 cells were treated with PHA-665752 at concentrations above 4 mmol/L for 24 hours. Notably,cabozantinib and PHA-665752 failed to affect FAK expression.Interestingly, the combination of PHA-665752 and CT-707 had asynergistic antitumor effect on both the HepG2 and Bel-7402 celllines, as the combination treatment achieved almost 100% cellkilling (Fig. 4E), which opened a new window for hepatocellularcarcinoma therapy via the combination of kinase inhibitorstargeting MET and FAK.

Figure 3.

The combination of cabozantinib and CT-707 induced enhanced apoptosis and synergistic inhibition on activating the AKT signaling pathway. A–C, HepG2and Bel-7402 cells were treated with cabozantinib (5 mmol/L), CT-707 (3 mmol/L), or both for 48 hours, and apoptosis was detected by sub-G1 analysis for both cells(A), AV/PI staining for Bel-7402 (the units of the y-axis and x-axis are fluorescence intensity. y-axis: PI staining; x-axis: Annexin V, (B), and DAPI staining for HepG2(C). D, The protein expression levels of cleaved PARP (c-PARP), cleaved caspase-8 (c-C-8), and cleaved caspase-3 (c-C-3) in both cell lines were determinedusingWestern blot analysis. E, The activation of p-AKT, p-P70-S6K, and p-4E-BP-1 was detected usingWestern blot analysis for HepG2 and Bel-7402 after 24-hourexposure to cabozantinib (5 mmol/L), CT-707 (3 mmol/L), or both. � , P < 0.05; �� , P < 0.01, as evaluated using Student t-test.

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Cabozantinib-induced FAK activation was dependent on theintegrin pathway

Then, we conducted further experiments to determine howFAK phosphorylation was activated by cabozantinib. Consid-ering that integrin is a classical upstream signaling molecule ofFAK (19), we assessed the effect of cabozantinib on integrin a5(ITGA5), one of the most important integrin subunits. Asshown in Fig. 5A and B, therapeutic concentrations of cabo-zantinib increased the protein level of integrin a5 in a time-dependent, dose-dependent manner. Furthermore, similarresults were obtained from quantitative RT-PCR analysis, as

the mRNA levels of integrin a5 were significantly upregulatedby cabozantinib and even increased by 3.2-fold in 24 hours of3 mmol/L treatment (Fig. 5C).

To determine whether FAK activation in cabozantinib-exposedcells was from the elevated mRNA and protein levels of integrina5, a selective integrin inhibitor, cilengitide (20), was furtherintroduced. HepG2 cells were either treated with cabozantinib(5.0 mmol/L, 24 hours), cilengitide (1.0 mmol/L, 1 hour), or thecombination, respectively; then, the samples were subjected toWestern blot analysis. As shown in Fig. 5D, the FAK phosphor-ylation level was increased with cabozantinib monotreatment,

Figure 4.

The effect of cabozantinib and PHA-665752 on activating FAK. A, HepG2 and Bel-7402 cells were treated with cabozantinib (5 mmol/L), CT-707 (3 mmol/L), orboth for 24 hours, and the expression levels of p-FAK and FAKwere detected usingWestern blot analysis. B,HepG2 cells were treatedwith cabozantinib (5mmol/L)for 6, 12, and 24 hours. Western blot analysis was used to assess the expression of p-FAK and FAK. C, HepG2 cells were exposed to serial concentrations ofcabozantinib (3, 4, 5, and 6 mmol/L) for 24 hours, and the expression levels of p-FAK and FAK were monitored usingWestern blot analysis. D, HepG2 and Bel-7402cells were treated with cabozantinib (5 mmol/L) or serial concentrations of PHA-665752 (3, 4, 5, and 6 mmol/L), and Western blot analysis was used toassess the expression of p-FAK and FAK. E, HepG2 and Bel-7402 cells were treated with serial concentrations of PHA-665752, CT-707, or both for 72 hoursand cell survival was assessed by SRB analysis.






and this increment in p-FAK could be completely reversed to thebasal level when cabozantinib was combined with cilengitide,while the total FAK protein levels remained unchanged. Collec-tively, these results indicated that cabozantinib exposure activatedFAK through the upregulation of the integrin a5 mRNA andprotein levels.

Because the activated FAK was endowed with the ability topromote malignancy and compromise the activities of a varietyof anticancer agents, we next questioned whether blocking theintegrin pathway with cilengitide could attenuate the activationand enhance the anticancer activity of cabozantinib. Serialconcentrations of cabozantinib, cilengitide, or a combinationwere given to HepG2 cells for 48 hours, and cell survival wasdetected using the CCK8 assay. As shown in Fig. 5E, the survivalfraction was significantly reduced in the combination groupcompared with the mono-treated groups (the survival fractionswere 44.4%, 58.2%, and 16.0% for 6.0 mmol/L cabozantinib,1.2 mmol/L cilengitide, and the combination group, respective-ly). The CI values were further calculated to assess the syner-gistic effects. As shown in Table 2, the CI values of all the testedconcentrations of cabozantinib plus cilengitide groups wereless than 0.8, which not only indicated the synergistic antican-cer activities of combining cabozantinib with cilengitide butalso implicated that the critical impairment to the anticancer

activity of cabozantinib was imposed by the activated integrinpathway.

The synergistic antitumor effect of XL184 and CT-707 inHepG2xenografts

To further assess the synergistic antitumor effect of two agents,we detected the in vivo efficacy in HepG2 xenograft nude micemodels (n¼ 5/group). As shown in Fig. 6A, the use of cabozanti-nib or CT-707 alone caused amoderate decrease in the RTV, as theT/C values and the ratio of the RTV of the treated group to the RTVof the control group were 0.5 and 0.6, respectively, at day 14 aftertreatment (Table 3). Compared with the mono-treated groups,the combination group induced a significant reduction in tumorgrowth and had a 0.2 T/C value at day 14 after treatment. Thisfinding indicated that the combination of cabozantinib and CT-707 has synergic anticancer activity in vivo. In addition, wemonitored the tumor weights of each group at the end of thetreatment and calculated the inhibition rates of each treatmentgroup compared with the control group (Table 3; Fig. 6B). Similarresults were obtained from the tumor weight analysis, and theinhibition rate was significantly increased in the combinationgroup compared with the mono-treated groups. The inhibitionrate of combination group reached 77.4%, whereas the mono-treatment of cabozantinib or CT-707 alone caused 30.7% and19.4% inhibition in the tumor weight, respectively.

To determinewhether the synergistic antitumor effect was fromincreasing tumor apoptosis in vivo, we detected the c-PARPexpression in the typical tumors of each treatment group byWestern blot analysis. As shown in Fig. 6C, the combinationtreatment significantly increased the c-PARP protein level com-pared with the mono-treatment and control groups, indicatingthat the combination of cabozantinib and CT-707 resulted inenhanced tumor apoptosis in vivo.

Figure 5.

The mechanisms of cabozantinib-induced FAKactivation. A, HepG2 cells were treated withcabozantinib (5mmol/L) for 6, 12 and 24 hours, andWestern blot analysis was performed to monitorthe expression of integrin a5 (ITGA5). B, HepG2cells were exposed to cabozantinib (3, 4, 5 and6 mmol/L) for 24 hours. Western blot analysis wasused to assess the expression of integrin a5(ITGA5). C, HepG2 cells were treated withcabozantinib at concentrations of 3 or 6 mmol/L,and RT-PCR analysis was used to detect themRNA levels of integrin a5 (ITGA5). Con, control.D, HepG2 cells were exposed to cabozantinib(5.0 mmol/L, 24 hours), cilengitide (1.0 mmol/L,1 hour), or their combination, and Western blotanalysis was used to assess the p-FAK and FAKexpression levels. E, HepG2 cells were exposedto serial concentrations of cabozantinib,cilengitide, or both for 48 hours, and cellsurvival was detected by the CCK8 assay.� , P < 0.05, as evaluated using Student t-test.

Table 2. CI values were calculated at different ratio concentrations ofcabozantinib and cilengitide in HepG2 cells by CalcuSyn

Cabozantinib (mmol/L) Cilengitide (mmol/L) CI

2.0 0.4 0.80 � 0.373.0 0.6 0.65 � 0.114.0 0.8 0.52 � 0.195.0 1.0 0.37 � 0.166.0 1.2 0.33 � 0.16

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DiscussionMET, a receptor tyrosine kinase, is a promising therapeutic

target because MET mutations and amplifications are widelyobserved in various cancers, and they promote cancer motility,growth, and invasion by activating downstream proliferation andantiapoptotic signaling pathways (21, 22). Accordingly, severalsmall-molecule inhibitors targetingMET, including cabozantinib,tivantinib, and crizotinib, are undergoing clinical trials or pre-clinical studies (23). Some of these MET inhibitors have beenevaluated in patients and have demonstrated clinical efficacy, butthe responses are variable, incomplete, and sometimes transientbecause of resistance (24). As such, increasing studies are focusedon developing combination therapeutic strategies that combineMET inhibitors with other antitumor agents to overcome thecurrent restrictions. Among these studies, a phase III clinical trialcompared the efficacy of tivantinib alone or in combination withthe EGFR inhibitor, erlotinib, in NSCLC patients, and the com-bination treatment resulted in a 6.4-week increase in the progres-sion-free survival (PFS; as the PFS of tivantinib alone and com-bination was 9.7 and 16.1 weeks, respectively; ref. 25). In anotherstudy, cabozantinib was found to activate hypoxia-induciblepathways, which may dampen its effects; consequently, the com-bination of cabozantinib plus 2ME2, a HIF-1 inhibitor, displayeda preclinical synergistic antitumor effect on hepatocellular carci-noma (26).

In the current study, remarkable synergy was obtainedthrough combining cabozantinib with a novel FAK inhibitor,

CT-707. The combination of two agents exerted synergisticactivities through enhanced apoptotic cell death, as indicatedby the observed increase in the sub-G1 population, karyopy-knosis and cellular fragmentation, as well as augmented PARPcleavage in human hepatocellular carcinoma cell lines, but ledto minimal synergistic effects in human hepatocyte HL-7702(Supplementary Fig. S1). Importantly, superior activity of thiscombination was also observed using the HepG2 xenograftmodel. The combined administration of cabozantinib and CT-707 not only resulted in a much smaller tumor size than formonotreatment groups but also markedly decreased the tumorweight, as the inhibition rate of combination treatment on thetumor weight reached 77.4% and was 2- to 3-fold higher thanfor cabozantinib or CT-707 monotreatment (30.7% and19.4%, respectively).

Previous studies have investigated the connection betweenthe FAK and MET signaling pathways. It is reported thatphosphorylation of FAK is required for cell–matrix adhesion-dependent activation of MET during transformation of breastepithelial cells (27), and MET, in turn, can induce the activationof FAK in mouse liver and hepatocellular carcinoma cell line(28), indicating that FAK and MET have cross-talk in tumorcells. In accordance with these evidences, our current studyrevealed that the simultaneous targeting of these two pathwaysmight provide extended inhibitory effects that counteract resis-tance toward MET-targeting inhibitors. Interestingly, a recentsynthetic lethal screening (29) identified two pathways as keyregulators for the antiproliferative effects of MET-targetingdrugs. One of these was the integrin signaling pathway thatacts as a survival input to promote resistance to MET inhibition.As such, the blockade of integrin function impeded the resis-tance of cancer cells against MET therapeutic antibodies, result-ing in extensive improvement in the antitumor activity. Con-sistent with these previous studies, we found that the exposureto MET inhibitors could increase the mRNA and protein levelsof the integrin. Consequently, the introduction of the integrin

Figure 6.

The synergistic antitumor effect ofcabozantinib and CT-707 in HepG2xenografts. A, Nude mice transplantedwith HepG2 xenografts were randomlydivided into four groups (n ¼ 5/group)and treated with vehicle, cabozantinib(20 mg/kg once daily for first 3 days;intragastrically (i.g.) 10 mg/kg once aday for 4th day; no administration from5th to 10th days; i.g. 10mg/kg once a dayfrom the 10th to 14th days), CT-707 (i.g.50mg/kg twice a day forfirst 3 days, 7th,8th, 11th, 12th, and 14th days; once a dayfor 4th, 6th, 9th, 10th, and 13th days; noadministration for the 5th day), or thecombination (comb) of cabozantiniband CT-707. The RTV is expressed as themean � SEM. B, Dots, tumor weights ofeach tumor in four groups; lines, meanvalues of each group. C, The expressionlevels of c-PARP in each tumor groupwere detected using Western blotanalysis. �� , P < 0.01; �� , P < 0.001, asevaluated using Student t-test.

Table 3. RTV values, T/C values, tumorweight values, and the inhibition rates ofthe tumor weight in HepG2-xenografted models

RTV T/C Tumor weight Inhibition (%)

Control 16.7 � 1.3 — 0.6 � 0.1 —

CT-707 10.2 � 1.9 0.6 0.5 � 0.1 19.4Cabozantinib 9.0 � 0.8 0.5 0.4 � 0.1 30.7Combo 3.5 � 0.8 0.2 0.1 � 0.0 77.4

Abbreviation: Combo, combination.






inhibitors significantly extended the anticancer capabilities ofcabozantinib. It is noteworthy that, in addition to integrin, thedownstream kinase was also activated as we found that METinhibition, caused by either cabozantinib or PHA-665752,induced FAK activation, the downstream signaling moleculeof integrin, which provided a novel combination strategy forimproving the efficacy of MET inhibitors. In line with thesefindings, our current study results suggested that the combina-tion of agents interfering with these two pathways might have asynergistic effect.

CT-707 is a novel anticancer drug thatwas recently approved bytheChina Food andDrugAdministration for aphase I clinical trialin NSCLC because it targets the EML-ALK fusion protein. Apreclinical study revealed that in addition to NSCLC, CT-707exhibits potent anticancer activities against a variety of cancermodels, including breast cancer, T-cell lymphoma, etc. BecauseCT-707 is a multikinase inhibitor, we are interested in pursuingthe possibility of applying CT-707 to other cancer models. In thecurrent study, CT-707 displayed superior anticancer activities onhepatocellular carcinoma models, as it not only synergized withcabozantinib to suppress hepatocellular carcinoma but alsoexerted an efficient effect in vitro and in vivo when administeredalone. Therefore, our study provides preclinical evidence for thefurther clinical use of CT-707 in cancer treatment, particularly inhepatocellular carcinoma, alone or in combination with METinhibitors.

In summary, our findings demonstrated that the combinationof cabozantinib and CT-707 exerted synergistic antitumor activityin vitro and in vivo in hepatocellular carcinoma. Further studiesshowed that cabozantinib triggered the activation of FAK throughinducing integrin expression, which might dampen its antitumoractivity in hepatocellular carcinoma. In this context, the suppres-sion of cabozantinib-activated FAK by CT-707 significantlyenhanced the anticancer activities and displayed a synergisticeffect when combined with cabozantinib. Our study provides apromising therapeutic strategy for hepatocellular carcinoma and

broadens the horizon for the clinical applications of combiningMET and FAK inhibitors.

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

Authors' ContributionsConception and design: D.-D. Wang, Y. Chen, Y.-X Han, Y. Peng, Y.-H. Sun,Q.-J. He, B. Yang, H. ZhuDevelopment of methodology: D.-D. Wang, X.-Y. DaiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D.-D. Wang, Z.-B. Chen, F.-J. Yan, X.-Y. Dai, H. ZhuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D.-D. Wang, Y. Chen, Z.-B. Chen, F.-J. Yan,M.-D. Ying, J. Cao, P.-H. Luo, Y. Peng, H. ZhuWriting, review, and/or revision of the manuscript: D.-D. Wang, Y. Chen,M.-D. Ying, J. Cao, H. Zhang, Q.-J. He, B. Yang, H. ZhuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D.-D. Wang, J. Cao, J. Ma, Q.-J. He, B. Yang,H. ZhuStudy supervision: Y. Chen, Q.-J. He, B. Yang, H. Zhu

AcknowledgmentsWe highly appreciate Hong-Hai Wu from the College of Pharmaceutical

Sciences, Zhejiang University, and Di-Feng Zhu and Ren-Hua Gai from theCenter for Drug Safety Evaluation and Research, Zhejiang University, for theirassistance with animal experiments.

Grant SupportThis work was supported by the National Natural Science of China

(81473228; to H. Zhu and 91529304; to B. Yang), Fundamental ResearchFunds for the Central Universities (to B. Yang), International Science & Tech-nology Cooperation Program of China (2014DFE30050; to B. Yang), andProject of Education Department of Zhejiang Province (Y201329974; to J. Ma).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 12, 2016; revised August 10, 2016; accepted August 24, 2016;published OnlineFirst September 16, 2016.

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2016;15:2916-2925. Published OnlineFirst September 16, 2016.Mol Cancer Ther Dan-Dan Wang, Ying Chen, Zi-Bo Chen, et al. Cabozantinib-Induced FAK ActivationSuppress Hepatocellular Carcinoma by Blocking CT-707, a Novel FAK Inhibitor, Synergizes with Cabozantinib to

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