crizotinib induces puma-dependent apoptosis in colon ...€¦ · crizotinib induces puma-dependent...

Cancer Therapeutics Insights

Crizotinib Induces PUMA-Dependent Apoptosis in ColonCancer Cells

Xingnan Zheng1, Kan He1, Lin Zhang2, and Jian Yu1

AbstractOncogenic alterations in MET or anaplastic lymphoma kinase (ALK) have been identified in a variety of

human cancers. Crizotinib (PF02341066) is a dual MET and ALK inhibitor and approved for the treatment

of a subset of non–small cell lung carcinoma and in clinical development for other malignancies. Crizotinib

can induce apoptosis in cancer cells, whereas the underlying mechanisms are not well understood. In this

study, we found that crizotinib induces apoptosis in colon cancer cells through the BH3-only protein

PUMA. In cells with wild-type p53, crizotinib induces rapid induction of PUMA and Bim accompanied by

p53 stabilization and DNA damage response. The induction of PUMA and Bim is mediated largely by p53,

and deficiency in PUMA or p53, but not Bim, blocks crizotinib-induced apoptosis. Interestingly, MET

knockdown led to selective induction of PUMA, but not Bim or p53. Crizotinib also induced PUMA-

dependent apoptosis in p53-deficient colon cancer cells and synergized with gefitinib or sorafenib to

induce marked apoptosis via PUMA in colon cancer cells. Furthermore, PUMA deficiency suppressed

apoptosis and therapeutic responses to crizotinib in xenograft models. These results establish a critical role

of PUMA in mediating apoptotic responses of colon cancer cells to crizotinib and suggest that mechanisms

of oncogenic addiction to MET/ALK-mediated survival may be cell type-specific. These findings have

important implications for future clinical development of crizotinib. Mol Cancer Ther; 12(5); 1–10. �2013

AACR.

IntroductionReceptor tyrosine kinases (RTK) are cell surface recep-

tors that act upon a variety of ligands including growthfactors, cytokines, and hormones. RTKs are vital regula-tors of normal cell physiology and play critical roles in thedevelopment and progression of human cancer (1, 2).MET is an extensively studied RTK and the receptor forhepatocyte growth factor (HGF; ref. 3). The activation ofMET byHGF ligation initiates various signaling cascades,such as the PI3K/AKT and RAS/RAF/MAPK pathways,to induce cell survival, proliferation,migration, and tissueregeneration (3). Aberrant activation of MET can resultfrom gene amplification, transcriptional upregulation,missense mutations, or ligand autocrine loops, and isimplicated in the pathogenesis of many human cancers

(2, 3). In colon cancer, MET overexpression and geneamplification are associated with advanced diseases andpoor prognosis (4, 5).

Recent efforts in cancer genomics continue to identifyaberrantly activated oncogenic kinases and facilitatethe development of targeted agents. This approach isexpected to ultimately deliver safer and more effectivecancer therapeutics (6). MET-targeting agents currently inclinical use include the monoclonal antibody MetAb(Roche; ref. 7) and small-molecule tyrosine kinaseinhibitors (TKI), such asARQ197 (ArQule/Daiichi Sankyo;ref. 8), INCB28060 (Incyte; ref. 9), and crizotinib(PF02341066, Pfizer; ref. 10). Crizotinib was initiallydesigned as a selective ATP-competitive MET inhibitorand later found to inhibit several related kinases, includinganaplastic lymphoma kinase (ALK; ref. 10), C-ros onco-gene1, and receptor tyrosine kinase (ROS1; ref. 11). Crizo-tinib has received the approval of the U.S. Food and DrugAdministration for the treatment of ALK-rearranged non–small cell lung carcinoma (NSCLC), and is being evaluatedin patients with other malignancies. Crizotinib has gar-nered much attention as it inhibits MET- and ALK-depen-dent tumor cell growth, migration, and invasion via bothHGF-dependent and -independent mechanisms (12). Cri-zotinib also induces apoptosis in cancer cells; however, theunderlyingmechanisms are not well understood. Current-ly, there is no reliable biomarker for crizotinib responseother than ALK or ROS1 rearrangement (11, 13, 14).

Authors' Affiliations: 1Department of Pathology, University of PittsburghCancer Institute; and 2Department of Pharmacology andChemical Biology,University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

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

X. Zheng and K. He contributed equally to this work.

Corresponding Author: Jian Yu, Hillman Cancer Center Research Pavil-ion, Suite 2.26h, 5117 Centre Ave, Pittsburgh, PA 15213. Phone: 412-623-7786; Fax: 412-623-7778; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-1146

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

www.aacrjournals.org OF1

on June 1, 2020. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst February 20, 2013; DOI: 10.1158/1535-7163.MCT-12-1146

http://mct.aacrjournals.org/

Apoptosis plays an important role in the antitumoractivities of conventional chemotherapeutic agents andtargeted therapies (1, 15, 16). The Bcl-2 family proteinsare the central regulators of mitochondria-mediatedapoptosis. The BH3-only family members are firstengaged in response to distinct as well as overlappingsignals. Several of them, such as Bim and PUMA, arepotent inducers of apoptosis by activating Bax/Bakfollowing the neutralization of antiapoptotic Bcl-2 fam-ily members (15, 17). We and others have shown thatPUMA functions as a critical initiator of apoptosis inboth p53-dependent and -independent manners in awide variety of cell types (18). PUMA transcription isdirectly activated by p53 in response to DNA damage(18), and lack of its induction renders p53-deficientcancer cells refractory to chemotherapeutic drugs andradiation. PUMA induction by nongenotoxic stimuli isgenerally p53-independent, and mediated by transcrip-tion factors such as p73 (19, 20), forkhead box O3a(FoxO3a; ref. 21, 22), and NF-kB (23, 24). Upon induc-tion, PUMA potently induces apoptosis by antagonizingantiapoptotic Bcl-2 family members and/or directlyactivating the proapoptotic members Bax and Bak,leading to mitochondrial dysfunction and caspase acti-vation cascade (18).

In this study, we investigated the underlying mechan-isms of crizotinib-induced apoptosis in colon cancer cells,and found that both p53-dependent and -independentinduction of PUMA contributes to crizotinib-inducedapoptosis. These results provide novel mechanisticinsight into the therapeutic responses of crizotinib, arationale for manipulating PUMA and BH3-only proteinsto improve the efficacy of targeted therapies, as well astherapy-induced changes in their expression as potentialbiomarkers.

Materials and MethodsCell culture and drug treatment

Human colorectal cancer cell lines, including HCT116,RKO, LoVo, DLD1, and HT29 were obtained from Amer-ican Type Culture Collection. The isogenic cell lines,including PUMA-KO (25), p53-KO (26), and p53-bindingsite knockout (BS-KO; ref. 27) HCT116 cells, PUMA-KODLD1 cells (27), and p53-KO RKO cells (28) have beendescribed. More details are found in the SupplementaryData for drug treatments. We examine loss of expressionof targeted protein by Western blotting and conductmycoplasma testing by PCR during culture routinely; noaddition authentication was done by the authors.

Western blottingWestern blotting was carried out as previously

described (29). More details on antibodies are found inthe Supplementary Data.

Real-time reverse transcription PCRTotal RNA was isolated from untreated or drug-trea-

ted cells using the Mini–RNA Isolation II Kit (cat.

#R1055, Zymo Research) according to the manufac-turer’s protocol. Total RNA (2 mg) was used to generatecomplementary DNA using SuperScript III reverse tran-scriptase (Invitrogen). The following primers were usedfor PUMA: Forward: 50-CGACCTCAACGCACAGTAC-GA-30, Reverse: 50-AGGCACCTAATTGGGCTCCAT-30,b-Actin: Forward: 50-GACCTCACAGACTACCTCAT-30, Reverse: 50-AGACAGCACTGTGTTGGCTA-30.

Transfection and siRNAThe gene-specific siRNA, including MET siRNA (30)

and PUMA siRNA (31) were synthesized by Dharmacon(Lafayette) and transfected into cells with Lipofectamine2000 (Invitrogen) according to themanufacturer’s instruc-tions. After 24 hours of transfection, cells were treatedwith PF02341066 for further analysis. More details arefound in the Supplementary Data.

Analysis of apoptosis, growth, and mitochondria-associated events

Apoptosis was analyzed by counting cells with con-densed chromatin andmicronucleation following nuclearstaining with Hoechst 33258 (Invitrogen; ref. 27). Themethods of colony formation, changes in the mitochon-drial membrane potential, and cytochrome c release havebeen previously described (25, 32).More details are foundin the Supplementary Data.

Reporter assaysPUMA reporters with or without p53-bindings sites

have been described previously (20). Reporter assayswere carried out in 12-well plates as described (33).Normalized relative luciferase units were plotted. Allreporter experiments were carried out in triplicate andrepeated 3 times.

Xenograft studiesAll animal experiments were approved by the Univer-

sity of Pittsburgh Institutional Animal Care and UseCommittee. Female 5- to 6-week-old Nu/Nu mice(Charles River)were housed in a sterile environmentwithmicroisolator cages and allowed access towater and chowad libitum. Mice were injected subcutaneously in bothflanks with 4 million wild-type (WT) or PUMA-KOHCT116 cells. Following tumor growth for 7 days, micewere treated daily by oral gavage for 9 consecutive dayswith 35mg/kg PF02341066 in 10% ethanol or 10% ethanolwithout PF02341066 (control buffer), the total volumebeing approximately 100 mL/mouse. Detailed methodson tumormeasurements, harvests, and histologic analysisare found in the Supplementary Data as previouslydescribed (34–36).

Statistical analysisStatistical analyses were carried out in Microsoft Excel.

P values were calculated by the Student t test, and wereconsidered significant if P < 0.05. The means � 1 SD aredisplayed in the figures.

Zheng et al.

Mol Cancer Ther; 12(5) May 2013 Molecular Cancer TherapeuticsOF2




ResultsPUMA is induced by crizotinib in colon cancer cellsWe first investigated the effects of crizotinib

(PF02341066, PF) on MET signaling and PUMA expres-sion in HCT116 colon cancer cells. Crizotinib treatmentled to rapid dephosphorylation of MET andAKTwithoutaffecting their total levels. However, the levels of phos-phorylated extracellular signal-regulated kinase (ERK)only decreased transiently, and recovered within 6 hours(Fig. 1A). PUMAprotein andmRNAwere inducedwithin6 hours of treatment, suggesting transcriptional regula-tion (Fig. 1A and B). Interestingly, the expression of Bcl-2family members, such as Bim and Mcl-1, also increased,whereas that of Bad, Bid, Bcl-2, and Bcl-xL remainedunchanged by 48 hours (Supplementary Fig. S1A). PUMAinduction was dose-dependent and stimulated by as littleas 1 mmol/L crizotinib (Fig. 1C). To determine whetherMET regulates PUMA expression directly, we depletedMET by siRNA.MET knockdown led to increased PUMAmRNA and protein (Fig. 1D), but not that of Bim (Sup-plementary Fig. S1B). Taken together, these data suggestthat MET inhibition selectively induces PUMA, whereas

crizotinib has a broader effect on the levels of PUMA andother BH3-only proteins.

PUMA mediates crizotinib-induced apoptosisNext, we determined the role of PUMA in crizotinib-

induced apoptosis using isogenic PUMA-KO HCT116cells and siRNA. Crizotinib treatment induced approxi-mately 15% to 65% apoptosis from 24 to 48 hours in WTHCT116 cells, which was associatedwith the activation ofcaspase-3 and caspase-9, mitochondrial membrane depo-larization, and cytochrome c release (Fig. 2A and B). Incontrast, apoptosis in PUMA-KO cells was suppressed bymore than 70% with little or no activation of caspases,mitochondrialmembranedepolarization, or cytochrome crelease at 48 hours (Fig. 2A–C). Annexin V/propidiumiodide staining confirmed the apoptotic resistance ofPUMA-KO cells (Supplementary Fig. S2A). Consistentwith blocked apoptosis, PUMA-KO cells showed muchimproved clonogenic survival (Fig. 2D). We have shownpreviously that PUMA induces Bax-dependent apoptosis(25, 29). As expected, BAX-KO HCT116 cells were alsoresistant to crizotinib-induced apoptosis (Fig. 2E and

Figure 1. PUMAwas induced by crizotinib in colon cancer cells. A, top, chemical structure of crizotinib (PF02342066). Bottom, HCT116 cells were treatedwith12 mmol/L crizotinib or PF02341066 (PF) for indicated time. The expression levels of PUMA, p-Met (T1234/1235), total Met, p-AKT (S473), total AKT, p-ERK(T202/Y204), and total ERK were analyzed by Western blotting. B, HCT116 cells were treated with 12 mmol/L PF02341066 and totalRNA was extracted at the indicated time points. PUMA mRNA expression was analyzed by semiquantitive reverse transcription PCR (RT-PCR). b-Actinwas used as a control. C, PUMA protein levels were analyzed by Western blotting in HCT116 cells treated with increasing doses of PF02341066 for24 hours. D, HCT116 cells were transfected with either a control scrambled siRNA or a Met siRNA for 24 hours. Met and PUMA expression was analyzedby Western blotting (left) and real-time PCR (right). �NS, nonspecific band for Met. �, P < 0.01, MET siRNA þ versus �. b-Actin was used as aloading control for Western blot analyses.

PUMA and Crizotinib-Induced Apoptosis

www.aacrjournals.org Mol Cancer Ther; 12(5) May 2013 OF3




Supplementary Fig. S2B). Transient PUMA knockdownwith siRNA also led to the reduced apoptosis in HCT116and LoVo cells following crizotinib treatment (Supple-mentary Fig. S2C and S2D). Despite Bim induction, Bimknockdown by siRNA did not render HCT116 cells resis-tant to crizotinib (Supplementary Fig. S1B and S2E).Collectively, these results show an essential role of PUMAand the mitochondrial pathway in crizotinib-inducedapoptosis in colon cancer cells.

p53-Dependent induction of PUMA by crizotinibBoth HCT116 and LoVo cells contain WT p53 gene,

and p53 mediates PUMA induction following DNA

damage (18). Earlier work suggested that other small-molecule MET inhibitors can cause DNA double-strandbreaks (37, 38). We found that crizotinib treatmentresulted in increased phosphorylation of p53 andH2AX, as well as p53 stabilization in HCT116, LoVo,and RKO cells, all with WT p53 (Fig. 3A and Supple-mentary Fig. S3A). Induction of PUMA, and BIM to alesser extent but not MCL-1, was observed in HCT 116cells and reduced in p53-KO HCT116 cells (Fig. 3B).Furthermore, p53 deficiency attenuated crizotinib-induced apoptosis and caspase activation in HCT116cells (Fig. 3C and Supplementary Fig. S3B), as well as inRKO cells (Supplementary Fig. S3C and S3D). In time

Figure 2. PUMA is required for the apoptotic activity of crizotinib. A, left, WT and PUMA-KO HCT116 cells were treated with 12 mmol/L PF02340166and harvested at the indicated time points. Apoptosis was analyzed by a nuclear fragmentation assay.�, P < 0.01; ��, P < 0.001, WT versus PUMA-KO.Right, activation of caspase-3 and -9 was analyzed by Western blotting in WT and PUMA-KO HCT116 cells treated with 12 mmol/L PF02341066 for48 hours. b-Actin was used as a loading control. B, WT and PUMA-KO HCT116 cells treated with 12 mmol/L PF02341066 for 24 hours were stained withMitoTracker Red CMXRos, and mitochondrial membrane potential was measured by flow cytometry. C, the cytoplasm and mitochondria werefractionated fromWT andPUMA-KOHCT116 cells treatedwith 12mmol/L PF02341066 for 36 hours. The distribution of cytochrome c (Cyt c) was analyzed byWestern blotting. Tubulin and cytochrome oxidase subunit IV (COX IV) were probed as cytoplamic and mitochondrial fraction controls. D, WT andPUMA-KOHCT116 cells treatedwith 12 mmol/L PF02340166 for 30 hours were subjected to colony formation assays as described inMaterials andMethods.Colony numbers were scored 14 days later after plating. Representative pictures of colonies (left) and quantification of colony numbers (right) areshown. E, HCT116 cells and BAX-KO HCT116 cells were treated with 12 mmol/L PF02341066 for 48 hours. Apoptosis was determined by a nuclearfragmentation assay. ��, P < 0.001, WT versus PUMA-KO.

Zheng et al.





course experiments, PUMA and Bim were induced inHCT116 p53-KO or RKO p53-KO cells after 24 hours,though at much lower levels compared with WT cells(Fig. 3D and Supplementary Fig. S3D). To further inves-tigate p53-dependent activation of PUMA, we used aseries of PUMA promoter luciferase reporters (within �2 kb; ref. 20), and found that the reporters containing the2 p53-binding sites, such as fragments "A", "abc," and"E" had high activities after crizotinib treatment (Fig.3E). Notably, the most proximal fragment "de" (� 200bp), lacking 2 p53-binding sites, still had a moderateactivity (Fig. 3E). The higher activity of the shorterfragment "abc" may be due to the removal of GC-richsequences and binding sites for transcriptional repres-sors (18). Together, these data suggest that PUMAinduction by crizotinib is mediated by both p53-depen-dent and -independent mechanisms, and p53 is themajor transcriptional activator of PUMA in WT p53colon cancer cells.

p53-Independent induction of PUMA by crizotinibTo further probe p53-independent induction of

PUMA, we treated HCT116 cells harboring a deletionof 2 p53-binding sites in the PUMA promoter (BS-KO;ref. 27) with crizotinib, and found much reduced PUMAinduction compared with WT HCT116 cells (Fig. 4A).Moreover, PUMA, but not Bim, was induced by crizo-tinib in 2 p53-mutant colon cancer cell lines DLD1 andHT29 in the absence of p53 phosphorylation or stabili-zation (Fig. 4B). Other transcription factors, such as NF-kB subunit p65 and FoxO3a can bind to respective siteslocated promixal to p53-binding sites in the PUMApromoter. Despite phosphorylation changes associatedwith activation of p65 and FoxO3a, p65 or FoxO3aknockdown did not affect PUMA induction followingcrizotinib treatment (Supplementary Fig. S4). Crizotinibalso induced apoptosis in both DLD1 and HT29 cells,which was suppressed by PUMA gene ablation orsiRNA (Fig. 4C and 4D). DLD1 cells showed lower

Figure 3. PUMA induction bycrizotinib is largely dependent onp53. A, HCT116 cells were treatedwith 12 mmol/L PF02340166 forindicated time. The expression levelsof p-H2AX (S139), p-p53 (S15), totalp53, and PUMA were analyzed byWestern blotting. B, PUMA mRNAlevels in WT and p53-KO HCT116cells treated with 12 mmol/LPF02341066 at indicated times weredetermined by real-time RT-PCR.b-Actin was used as the normalizedcontrol.��,P < 0.001;WT versus p53-KO.C,WTandp53-KOHCT116 cellswere treated with 12 mmol/LPF02340166 and harvested atindicated time points. Apoptosis wasanalyzed by a nuclear fragmentationassay. ��, P < 0.001; WT versus p53-KO. D, PUMA and Bim protein levelsin WT and p53-KO HCT116 cellstreated with 12 mmol/L PF02341066at the indicated times were analyzedbyWestern blotting. E, HCT116 cellswere transfected with the indicatedreporters for 24 hours and subjectedto 8 mmol/L PF02341066 treatmentfor 24 hours. The ratios of normalizedrelative luciferase activities (to theempty vector pBV-Luc as 1) wereplotted. b-Actin was used as aloading control for Western blotanalyses.






PUMA induction and were more resistant to crizotinib-induced apoptosis, compared with HT29 cells (Fig. 4B–D). These results indicate that PUMA plays a criticalrole in the apoptotic responses to crizotinib in both p53WT and mutant colon cancer cells.

Crizotinib synergizes with gefitinib or sorafenib toinduce apoptosis via PUMA

Cooperative signaling of MET and the EGF receptor(EGFR) can contribute to EGFR-TKI resistance (39).HCT116 cells are highly resistant to gefitinib-inducedapoptosis or PUMA expression (Fig. 5A and B). Wetherefore hypothesized that the combination of gefitiniband crizotinib may enhance apoptosis. Indeed, this com-bination induced amuch stronger induction of apoptosis,caspase activation, and PUMA and Bim in HCT116 cells,comparedwith either agent alone (Fig. 5A–C). PUMA-KOcells were highly resistant to apoptosis and long-termgrowth suppression induced by this combination (Fig.5A, B, and D). Similarly, crizotinib and erlotinib combi-

nation resulted in a strong PUMA-dependent synergy incell killing (data not shown).

Crizotinib treatment decreased phosphorylation ofboth AKT and ERK in HCT116 cells. However, ERKdephosphorylation was only transient and restored after6 hours, long before apoptosis was detected (Fig. 1A). Wereasoned that a more durable inhibition of ERK phos-phorylation may potentiate crizotinib in cell killing. Thecombination of crizotinib and sorafenib, a Raf inhibitor,markedly induced apoptosis, expression of PUMA andBim, and long-term growth suppression, compared withthe single agent (Supplementary Fig. S5). Apoptosis andlong-termgrowth suppressionwere attenuated inPUMA-KOcells (Supplementary Fig. S5A, S5B, and S5D). Of note,AKT phosphorylation was completely inhibited by all 3agents, but not ERK phosphorylation, and reduction ofeither p-AKT or p-ERK is not sufficient for the enhancedinduction of PUMA and Bim, or apoptosis in the combi-nation treatments (Fig. 5C and Supplementary S5C). Inaddition, Mcl-1 levels did not change or decreased

Figure 4. Crizotinib induces PUMA- and p53-independent apoptosis in colon cancer cells. A, PUMAprotein levels inWT andBS-KOHCT116 cells treatedwith12 mmol/L PF02341066 at the indicated times were analyzed by Western blotting. BS-KO HCT116 cells harbor the deletion of 2 p53-binding sites inthe PUMA promoter. B, p53-mutant colon cancer cell lines DLD1 and HT29 were treated with 12 mmol/L PF02341066 for 24 hours. The expressionlevels of PUMA, Bim, p-p53, and total p53 were analyzed by Western blotting. C, top, PUMA expression was analyzed by Western blotting in WTand PUMA-KO DLD1 cells. Bottom, apoptosis was analyzed by nuclear fragmentation in WT and PUMA-KO DLD1 cells treated with the indicated doses ofPF02341066 for 48 hours. ��, P < 0.001; WT versus p53-KO. D, HT29 cells were transfected with either a scrambled siRNA or PUMA siRNA for 24 hours andthen treated with 12 mmol/L PF02341066 for 48 hours. Left, Western blotting confirmed PUMA depletion by siRNA in HT29 cells. Right, apoptosis wasdetermined by a nuclear fragmentation assay. ��, P < 0.001; si-PUMA versus Scrambled. b-Actin was used as a loading control for Western blot analyses.

Zheng et al.





following crizotinib combination with gefitinib or sorafi-nib (Fig. 5C and Supplementary Fig. S5C). These resultssuggest that the combinations of crizotinib with addition-al TKIs are required to effectively target nonoverlappingsurvival pathways in cancer cells to induce apoptosis.

PUMA contributes to the antitumor activity ofcrizotinib in a mouse xenograft modelTo determine whether PUMA-mediated apoptosis

plays a critical role in the antitumor activity of crizotinibin vivo, we establishedWT and PUMA-KOHCT116 xeno-graft tumors in the flanks of BALB/c (nu/nu) nude mice.Tumors of different genotypes were established on theopposite flanks of the same mice to minimize intermousevariations in tumor uptake or drug delivery. Crizotinibwas administered orally to tumor-bearing mice daily for10 days, and tumor volumesweremonitored every 2 daysfor 3weeks. Comparedwith the buffer, crizotinib reducedgrowth of WT tumors by 81%, and PUMA-KO tumors by41% (Fig. 6A and B). Increased phospho-p53, PUMA, and

Bim expression was evident in WT tumors at day 5 oftreatment (Fig. 6C). Terminal deoxynucleotidyl transfer-ase–mediated dUTP nick end labeling (TUNEL) andactive caspase-3 staining revealed marked apoptosis inWT tumor tissues from crizotinib-treated mice, whichdecreased by more than 60% in the PUMA-KO tumors(Fig. 6D and E). These results show that the antitumoractivity of crizotinib in vivo is also dependent on the p53/PUMA axis.

DiscussionAberrantly activated oncogenic kinases are promising

drug targets for small molecules; however, biomarkersand resistance mechanisms of most clinically usefulkinase inhibitors remain largely unknown. Crizotinib hasbeen approved in ALK-rearranged NSCLC (3, 13), andclinical interest is expanding to other solid tumors withgenetic alterations in c-MET, ALK, and ROS-1 (14, 40).The antitumor activities of crizotinib include the induc-tion of cell-cycle arrest, apoptosis, and inhibition of cell

Figure 5. Crizotinib synergizes withgefitinib to induce PUMA-dependentapoptosis in colon cancer cells. A,WT and PUMA -KO HCT116 cellswere treated with 6 mmol/L PF, 20mmol/L gefitinib, or their combinationfor 48 hours. Apoptosis wasdetermined by a nuclearfragmentation assay. ��, P < 0.001;combination versus single agent inWT cells and KO versus WT incombination. Right, chemicalstructure of gefitinib. B, caspase-3activation was analyzed by Westernblotting in WT and PUMA-KOHCT116 cells treated as in A.C, HCT116 cells were treated with6 mmol/L PF02341066, 20 mmol/Lgefitinib, or their combination for24 hours. The expression levels ofp-AKT, total AKT, p-ERK, total ERK,PUMA, and Bim were analyzed byWestern blotting. D, WT and PUMA-KO HCT116 cells were treated with 6mmol/L PF02341066, 20 mmol/Lgefitinib, or their combination for 30hours and were then subjected to acolony formation assay as describedin Materials and Methods. Colonynumbers were scored 14 days afterplating. Representative pictures ofcolonies (top) and relativecolonogenic survival (bottom) of WTand PUMA-KO HCT116 cellscompared with untreated cells areshown. ��, P < 0.001; combinationversus single agent in WT cells andKO versus WT in combination.b-Actinwasusedas a loading controlfor Western blot analyses.






proliferation and invasion (3, 13). Our results indicatedthat activation of mitochondrial pathway and PUMAplays a key role in crizotinib-induced cancer cell deathin vitro and in vivo. In addition, the combinations ofcrizotinib with EGFR or Raf inhibitors resulted in thepotent induction of apoptosis in colon cancer cells viaPUMA.

Crizotinib-induced apoptosis can be attributed to Bimin lung cancer cells with MET amplification but not withMET mutations (41, 42). In colon cancer cells, Bim induc-tion, though not required, likely potentiates PUMA-dependent apoptosis by antagonizing antiapoptotic Bcl-2 family proteins including Mcl-1, whose levels remainhigh after treatment. It is likely that multiple BH3-onlyproteins are used in the apoptotic response to crizotinib,whereas a distinct member may serve as cell or tissue-specific initiator. The coregulation of Bim and PUMA bycrizotinib is interesting and somewhat unexpected, andrequires further investigation. Bim and PUMA are locatedon different chromosomes and their basal expressionlevels are quite different. It is possible that higher orderchromatin changesmaybe involved in addition to loadingof stress-induced transcription factors, such as p53 ontotheir promoters. The mechanisms of MET inhibitor-induced DNA damage response (37, 38), and p53-inde-pendent induction of PUMA and Bim remain unclear.

Despite a plethora of oncogenic activities ascribed toMET and its related kinases (3, 13), our data suggest that

crizotinib effectively inhibits MET signaling, and inducesPUMA-dependent apoptosis in colon cancer cells. Sur-prisingly, MET siRNA did not induce obvious apoptosisor p53 stabilization in HCT116 cells but only a modestPUMA induction. One possible explanation is that crizo-tinib treatment is more effective in blocking MET signal-ing thanMET siRNA. Another more likely explanation isthat crizotinib inhibits other RTKs and activates p53-dependent DNA damage responses in some cells. Inaddition, PUMA induction may further engage cyto-plasmic function of p53 to trigger apoptosis (43, 44). Theseissues might be relevant as the clinically efficacious con-centration of crizotinib used in vitro for cell killing (1–10mmol/L) are much higher than those required to selec-tively inhibit MET or ALK.

Crizotinib and other TKI combinations enhance apo-ptosis and the induction of PUMA and Bim. Interestingly,the inhibition of AKT phosphorylation is not sufficient forcell killing or strong PUMA induction. Our data areconsistent with the emerging concept that cross-talkbetween RTKs is a major mechanism for cancer progres-sion and therapeutic resistance, and successful therapywill likely require targeting multiple survival pathways(1, 6, 45). However, several challenges are facing theclinical applications of kinase inhibitors: (i) genetic altera-tions in EGFR, MET, or ALK, and possibly ROS1 areinfrequent, (ii) not all tumors with same alterationsrespond, (iii) tumor heterogeneity plus preexisting or de

Figure 6. PUMA mediates theantitumor effects of crizotinib in axenograftmodel. A, nudemicewithestablished WT or PUMA-KOHCT116 xenografts were treatedwith 35 mg/kg PF02341066 orbuffer for 10 consecutive days.Tumor volume at the indicatedtime-points was calculatedand plotted. Arrows indicatePF02341066 administration.Statistical significance isindicated for the comparisonof PF02341066-treated WTand PUMA-KO tumors. B,representative tumors at the end ofthe experiment in A. C, WT andPUMA-KO HCT116 xenografttumorswere harvested from 2miceas in A after 5 treatments. p-p53,PUMA, and Bim expression inrepresentative WT HCT116 tumorswas analyzed by Western blotting.b-Actin was used as a loadingcontrol. Representative data areshown. D, TUNEL and activecaspase-3 staining of tumorsections as harvested in C. E,quantitation of TUNEL or activecaspase-3 positive cells in D.Results of D and E were expressedas means � SD (N ¼ 3) tumors ofeach genotype.

Zheng et al.





novomutations in targets can lead to rapiddevelopment ofresistance. Therefore, it is important to identify effectivecombination therapies that target several survivalmechanisms in cancer cells to prevent the developmentof resistance. Our data suggest that the combination ofcrizotinibwith other TKIs effectively blockMET signalingand alternative survival pathways in colon cancer cells.The cell lines used in this study are not expected to containALK or ROS1 rearrangements, whereas the mechanismsdescribed warrant further investigation in this context inrelevant cancers.In conclusion, our study provides a novel antitumor

mechanism of crizotinib via PUMA-mediated apoptosisthrough both p53-dependent and -independent means.These findings are in line with other recent findings inwhich PUMA or Bim mediates the apoptotic response tovarious kinase inhibitors. Therefore, induction of PUMAand Bim may be a predictor for a favorable response tocrizotinib, and possibly other targeted agents or theircombinations. This concept is different fromusing geneticalterations or steady-state mRNA or protein levels in thetumors and measures a dynamic response that may wellbe cell-type or patient-specific (36). Because induction ofPUMA and apoptosis is significantly more robust in p53WT cells, it would be useful to determine whether crizo-tinib shows selectivity against p53 WT cancers.

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

Authors' ContributionsConception and design: L. Zhang, J. YuDevelopment of methodology: X. Zheng, K. He, J. YuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Zhang, J. YuAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): X. Zheng, K. He, L. Zhang, J. YuWriting, review, and/or revision of themanuscript:X. Zheng, K.He, J. YuAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): X. Zheng, K. He, L. Zhang, J. YuStudy supervision: J. Yu

AcknowledgmentsThe authors thank Bert Vogelstein (Howard Hughes Medical Institute,

JohnsHopkinsUniversity) for p53-KOHCT116 and p53-KORKOcells, andother members of Yu and Zhang laboratories for helpful discussions.

Grant SupportThis work is supported by NIH grant CA129829, American Cancer

Society grant RGS-10-124-01-CCE, FAMRI (J. Yu), and by NIH grantsCA106348, CA121105, and American Cancer Society grant RSG-07-156-01-CNE (L. Zhang). This project used the UPCI shared facilities that weresupported in part by award P30CA047904.

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 November 28, 2012; revised January 24, 2013; acceptedFebruary 11, 2013; published OnlineFirst February 20, 2013.

References1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation.

Cell 2011;144:646–74.2. Gschwind A, Fischer OM, Ullrich A. The discovery of receptor

tyrosine kinases: targets for cancer therapy. Nat Rev Cancer2004;4:361–70.

3. Comoglio PM, Giordano S, Trusolino L. Drug development of METinhibitors: targeting oncogene addiction and expedience. Nat RevDrug Discov 2008;7:504–16.

4. Di Renzo MF, Olivero M, Giacomini A, Porte H, Chastre E, Mirossay L,et al. Overexpression and amplification of the met/HGF receptor geneduring the progression of colorectal cancer. Clin Cancer Res1995;1:147–54.

5. Takeuchi H, Bilchik A, Saha S, Turner R, Wiese D, Tanaka M, et al. c-MET expression level in primary colon cancer. Clin Cancer Res2003;9:1480–8.

6. Hait WN, Hambley TW. Targeted cancer therapeutics. Cancer Res2009;69:1263–7.

7. Jin H, Yang R, Zheng Z, Romero M, Ross J, Bou-Reslan H, et al.MetMAb, the one-armed 5D5 anti-c-Met antibody, inhibits orthotopicpancreatic tumor growth and improves survival. Cancer Res2008;68:4360–8.

8. Munshi N, Jeay Sb, Li Y, Chen C-R, France DS, Ashwell MA, et al. ARQ197, a novel and selective inhibitor of the human c-Met receptortyrosine kinase with antitumor activity. Mol Cancer Ther 2010;9:1544–53.

9. Liu X, Wang Q, Yang G, Marando C, Koblish HK, Hall LM, et al. A novelkinase inhibitor, INCB28060, blocks c-MET-dependent signaling, neo-plastic activities, and cross-talk with EGFR and HER-3. Clin CancerRes 2011;17:7127–38.

10. Zou HY, Li Q, Lee JH, Arango ME, McDonnell SR, Yamazaki S,et al. An orally available small-molecule inhibitor of c-Met, PF-2341066, exhibits cytoreductive antitumor efficacy through anti-proliferative and antiangiogenic mechanisms. Cancer Res 2007;67:4408–17.

11. Bergethon K, Shaw AT, Ignatius Ou S-H, Katayama R, Lovly CM,McDonald NT, et al. ROS1 rearrangements define a unique molecularclass of lung cancers. J Clin Oncol 2012;30:863–70.

12. Christensen JG, Zou HY, Arango ME, Li Q, Lee JH, McDonnell SR,et al. Cytoreductive antitumor activity of PF-2341066, a novelinhibitor of anaplastic lymphoma kinase and c-Met, in experimentalmodels of anaplastic large-cell lymphoma. Mol Cancer Ther 2007;6:3314–22.

13. Ou SH. Crizotinib: a novel and first-in-class multitargeted tyrosinekinase inhibitor for the treatment of anaplastic lymphoma kinaserearranged non-small cell lung cancer and beyond. Drug Des DevelTher 2011;5:471–85.

14. Stumpfova M, J€anne PA. Zeroing in on ROS1 rearrangements in non-small cell lung cancer. Clin Cancer Res 2012;18:4222–4.

15. Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer developmentand therapy. Oncogene 2007;26:1324–37.

16. Yu J, Zhang L. Apoptosis in human cancer cells. Curr Opin Oncol2004;16:19–24.

17. Leibowitz B, Yu J. Mitochondrial signaling in cell death via the Bcl-2family. Cancer Biol Ther 2010;9:417–22.

18. Yu J, Zhang L. PUMA, a potent killer with or without p53. Oncogene2008;27:S71–S83.

19. Melino G, Bernassola F, Ranalli M, Yee K, ZongWX, Corazzari M, et al.p73 Induces apoptosis via PUMA transactivation and Bax mitochon-drial translocation. J Biol Chem 2004;279:8076–83.

20. Ming L, Sakaida T, Yue W, Jha A, Zhang L, Yu J. Sp1 and p73activate PUMA following serum starvation. Carcinogenesis 2008;29:1878–84.

21. YouH, PellegriniM, TsuchiharaK, Yamamoto K,HackerG, ErlacherM,et al. FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal. J Exp Med 2006;203:1657–63.

22. DudgeonC,WangP,SunX,PengR,SunQ,YuJ, et al. PUMA inductionby FoxO3a mediates the anticancer activities of the broad-rangekinase inhibitor UCN-01. Mol Cancer Ther 2010;9:2893–902.






23. WangP, QiuW, DudgeonC, Liu H, HuangC, Zambetti GP, et al. PUMAis directly activated by NF-[kappa]B and contributes to TNF-[alpha]-induced apoptosis. Cell Death Differ 2009;16:1192–202.

24. Dudgeon C, Peng R, Wang P, Sebastiani A, Yu J, Zhang L. Inhibitingoncogenic signaling by sorafenib activates PUMA via GSK3b and NF-kB to suppress tumor cell growth. Oncogene 2012;31:4848–58.

25. Yu J, Wang Z, Kinzler KW, Vogelstein B, Zhang L. PUMAmediates theapoptotic response to p53 in colorectal cancer cells. Proc Natl AcadSci U S A 2003;100:1931–6.

26. Bunz F, Hwang PM, Torrance C,Waldman T, Zhang Y, Dillehay L, et al.Disruption of p53 in human cancer cells alters the responses totherapeutic agents. J Clin Invest 1999;104:263–9.

27. Wang P, Yu J, Zhang L. The nuclear function of p53 is required forPUMA-mediated apoptosis induced by DNA damage. Proc Natl AcadSci U S A 2007;104:4054–9.

28. Bunz F. Human cell knockouts. Curr Opin Oncol 2002;14:73–8.29. Ming L, Wang P, Bank A, Yu J, Zhang L. PUMA dissociates Bax and

Bcl-XL to induce apoptosis in colon cancer cells. J Biol Chem 2006;281:16034–42.

30. Fornari F, Milazzo M, Chieco P, Negrini M, Calin GA, Grazi GL, et al.MiR-199a-3p regulates mTOR and c-Met to influence the doxorubicinsensitivity of human hepatocarcinoma cells. Cancer Res 2010;70:5184–93.

31. Han J, Goldstein LA, Gastman BR, Rabinowich H. Interrelated roles forMcl-1 and BIM in regulation of TRAIL-mediated mitochondrial apo-ptosis. J Biol Chem 2006;281:10153–63.

32. Yu J,Wang P, Ming L,WoodMA, Zhang L. SMAC/Diablo mediates theproapoptotic function of PUMA by regulating PUMA-induced mito-chondrial events. Oncogene 2007;26:4189–98.

33. Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B. PUMA inducesthe rapid apoptosis of colorectal cancer cells. Mol Cell 2001;7:673–82.

34. QiuW, Carson-Walter EB, Liu H, Epperly M, Greenberger JS, ZambettiGP, et al. PUMA regulates intestinal progenitor cell radiosensitivity andgastrointestinal syndrome. Cell Stem Cell 2008;2:576–83.

35. Leibowitz BJ, Qiu W, Liu H, Cheng T, Zhang L, Yu J. Uncoupling p53functions in radiation-induced intestinal damage via PUMA and p21.Mol Cancer Res 2011;9:616–25.

36. Sun J, SunQ,BrownMF,DudgeonC,Chandler J, Xu X, et al. Themulti-targeted kinase inhibitor sunitinib induces apoptosis in colon cancercells via PUMA. PLoS ONE 2012;7:e43158.

37. Medova M, Aebersold DM, Blank-Liss W, Streit B, Medo M, Aebi S,et al. MET inhibition results in DNA breaks and synergistically sensi-tizes tumor cells to DNA-damaging agents potentially by breaching adamage-induced checkpoint arrest. Genes Cancer 2010;1:1053–62.

38. Ganapathipillai SS,MedovaM, Aebersold DM,Manley PW, Berthou S,Streit B, et al. Coupling of mutated Met variants to DNA repair via Abland Rad51. Cancer Res 2008;68:5769–77.

39. Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO,et al. MET amplification leads to gefitinib resistance in lung cancer byactivating ERBB3 signaling. Science 2007;316:1039–43.

40. Yauch RL, Settleman J. Recent advances in pathway-targeted cancerdrug therapies emerging from cancer genome analysis. Curr OpinGenet Dev 2012;22:45–9.

41. OkamotoW, Okamoto I, Arao T, Kuwata K, Hatashita E, Yamaguchi H,et al. Antitumor action of the MET tyrosine kinase inhibitor crizotinib(PF-02341066) in gastric cancer positive for MET amplification. MolCancer Ther 2012;11:1557–64.

42. Tanizaki J,Okamoto I, OkamotoK, TakezawaK, KuwataK, YamaguchiH, et al. MET tyrosine kinase inhibitor crizotinib (PF-02341066) showsdifferential antitumor effects in non-small cell lung cancer according toMET alterations. J Thorac Oncol 2011;6:1624–31.

43. Chipuk JE, Bouchier-Hayes L, Kuwana T, Newmeyer DD, Green DR.PUMA couples the nuclear and cytoplasmic proapoptotic function ofp53. Science 2005;309:1732–5.

44. Chipuk JE, Green DR. How do BCL-2 proteins induce mitochondrialouter membrane permeabilization? Trends Cell Biol 2008;18:157–64.

45. Dent P, Curiel DT, Fisher PB, Grant S. Synergistic combinations ofsignaling pathway inhibitors: mechanisms for improved cancertherapy. Drug Resist Updat 2009;12:65–73.

Zheng et al.





Published OnlineFirst February 20, 2013.Mol Cancer Ther Xingnan Zheng, Kan He, Lin Zhang, et al. Cancer CellsCrizotinib Induces PUMA-Dependent Apoptosis in Colon

Updated version

10.1158/1535-7163.MCT-12-1146doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2013/02/26/1535-7163.MCT-12-1146.DC1

Access the most recent supplemental material at:

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

Subscriptions

Reprints and

[email protected] at

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

Permissions

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

.http://mct.aacrjournals.org/content/early/2013/05/01/1535-7163.MCT-12-1146To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-12-1146

http://mct.aacrjournals.org/content/suppl/2013/02/26/1535-7163.MCT-12-1146.DC1

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/early/2013/05/01/1535-7163.MCT-12-1146


crizotinib induces puma-dependent apoptosis in colon ...€¦ · crizotinib induces puma-dependent...

Documents