Cyclooxygenase-2 Inhibition Inhibits C-Met Kinase Activity and Wnt Activity in Colon Cancer Jurriaan B

Research Article

Cyclooxygenase-2 Inhibition Inhibits c-Met Kinase Activity and Wnt Activity in Colon Cancer Jurriaan B. Tuynman,1,2 Louis Vermeulen,1 Elles M. Boon,3 Kristel Kemper,1 Aeilko H. Zwinderman,4 Maikel P. Peppelenbosch,5 and Dirk J. Richel1

1Laboratory of Experimental Oncology, Departments of 2Surgery, 3Pathology, and 4Clinical Epidemiology and Biostatistics, Academic Medical Center, Amsterdam, the Netherlands and 5Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands

Abstract (NSAIDs; refs. 3–5). However, precise molecular mechanisms still Activity of receptor tyrosine kinases (RTK) in colorectal cancer remain to be established. (CRC) is associated with enhanced tumor growth and a poorer The main event and the most characterized mechanism of prognosis. In addition, cyclooxygenase-2 (COX-2) expression carcinogenesis in CRC is the disruption of a functional adenopolyposis coli (APC) complex due to a mutation of the contributes to tumor growth and invasion. COX-2 inhibitors APC b-catenin exhibit important anticarcinogenic potential against CRC, but gene or the gene, leading to inappropriate the molecular mechanism underlying this effect and the activation of the Wnt signaling pathway (6, 7). This results in h relation with RTK signaling remain the subject of intense nuclear accumulation of -catenin, which associates with the research effort. Therefore, the rapid effects of COX-2 inhibition T-cell factor (TCF)-4, leading to transcription of genes involved in in CRC on the complement of all cellular kinases were dedifferentiation, cell proliferation, and survival, events involved in investigated using a kinase substrate peptide array, Western carcinogenesis. In addition, overexpression or activating muta- blotting, transfection, small interfering RNA assays, and CRC tions of RTKs, such as the EGFR, the insulin-like growth factor cell lines. The resulting alterations in the kinome profile receptor (IGFR), and c-Met, a receptor for hepatocyte growth revealed that celecoxib, a selective COX-2 inhibitor, impairs factor (HGF), contribute to the survival and progression of CRC phosphorylation of substrates for the RTKs c-Met and insulin- (8–11). c-Met is frequently overexpressed in advanced stages of like growth factor receptor, resulting in decreased downstream CRC and is correlated with a poor prognosis (12). c-Met activation signaling. The decrease in c-Met activation is accompanied by HGF or by amplification results in cell dissociation, migration, with an increase in glycogen synthase kinase 3B kinase activity scattering, and invasion, important processes in cancer progres- together with a rapid increase in phosphorylation of B-catenin. sion and metastasis (9–11, 13). In agreement, a significant reduction of B-catenin-T-cell Evidence has emerged suggesting functional interaction factor–dependent transcription is observed both with cele- between COX-2 enzymatic activity, RTK signaling, and Wnt activity coxib and selective inhibition of c-Met phosphorylation by (14–17). COX-2 activity enhances the cross-talk between the small molecules. Hence, corepression of c-Met–related and B- membrane tyrosine kinases c-Met and EGFR, resulting in nuclear h catenin–related oncogenic signal transduction seems a major accumulation of -catenin; however, precise mechanisms remain effector of celecoxib in CRC, which provides a rationale to use unclear (18). Previous studies have shown that relative high c-Met inhibitors and celecoxib analogous to target c-Met and concentrations of NSAIDs are able to down-regulate the Wnt Wnt signaling in a therapeutic setting for patients with CRC. signaling cascade in colon cancer cells (19–22). Although c-Met [Cancer Res 2008;68(4):1213–20] activation has been shown to enhance Wnt signaling in non–APC- mutated, non-CRC cells, neither the relationship between c-Met activation and its downstream signal transduction and Wnt Introduction signaling nor the effect of COX-2 inhibition on c-Met signaling in Colorectal cancer (CRC) remains a leading cause of cancer- CRC has been clarified (23). related death worldwide despite recent advances in adjuvant Because COX-2 has a crucial role in the carcinogenesis and chemotherapeutic regimens. In the last decade, extensive research progression of CRC, exploration of its mechanisms can lead to has been made in the development of new therapeutic regimens for new therapeutic targets to improve survival of CRC. Celecoxib, a CRC. Targeted therapy against receptor tyrosine kinases (RTK), selective COX-2 inhibitor, has been shown to posses the highest such as epidermal growth factor receptor (EGFR) and vascular anticarcinogenic capacity of NSAIDs and coxibs. Therefore, we have endothelial growth factor receptor, has been shown to improve explored the effects of the selective COX-2 inhibitor celecoxib on survival in patients with CRC (1, 2). In addition, a large amount of signal transduction in CRC cells. To this end, a peptide array was data is available describing both clinically chemopreventive and used, which contains 1,176 different kinase substrate consensus preclinically anticancer activities of selective cyclooxygenase-2 sequences, generating a comprehensive overview of the total (COX-2) inhibitors and nonsteroidal anti-inflammatory drugs complement of cellular kinase activity and thus enabling analysis of celecoxib effects without a priori assumptions as to signaling pathways affected by the compound. The generated data have been extensively validated (24–28). Our results suggest that celecoxib Requests for reprints: Jurriaan B. Tuynman, Department of Surgery, G4, Academic represses c-Met–dependent signaling, resulting in down-regulation Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands. Phone: of oncogenic Wnt signaling in CRC. These findings both stress the 31-205669111; Fax: 31-205669111; E-mail: [email protected]. I2008 American Association for Cancer Research. importance of the tyrosine kinase receptor c-Met in carcinogenesis doi:10.1158/0008-5472.CAN-07-5172 and provide a rationale to target c-Met in therapeutic modalities. www.aacrjournals.org 1213 Cancer Res 2008; 68: (4). February 15, 2008

Materials and Methods (Bio-Rad). To study kinase activity, 50 AL of cell lysate (5 mg/mL protein) were cleared on a 22-Am filter on ice, after which 10 AL of activation mix Reagents. Aspirin and NS398 were purchased from Sigma. Celecoxib were added, containing 50% glycerol, 50 Amol/L ATP, 60 mmol/L MgCl2, (pure reagent) was kindly provided by Pharmacia. Primary antibodies 0.05% (v/v) Brij-35, 0.25 mg/mL bovine serum albumin, and 2,000 ACi/mL h 33/37 against the following targets were used: phosphospecific -catenin (Ser / [g-33P]ATP. The resulting samples were incubated on the PepChip arrays Thr410), non-phospho-h-catenin, phospho-insulin receptor (INSR; Tyr1361), 845 1234/5 (Pepscan) containing 1,176 kinase substrate peptides in duplicate for 2 h in a phospho-EGFR (Tyr ), EGFR, phospho-c-Met (Ser ), c-Met, phospho- humidified stove at 37jC. Subsequently, the arrays were washed in 2 mol/L 6270 473 505 Gab-1 (Tyr ), Gab-1, phospho-Akt (Ser ), Akt, phospho-Lck (Tyr ), NaCl, 1% Triton X-100, PBS, 0.1% Tween 20, and H O, after which slides were 527 h h 9 2 phospho-Src (Tyr ), phospho-glycogen synthase kinase 3 (GSK3 ; Ser ), dried and exposed to a phosphoimaging screen for 24 h. Experiments were h 380 GSK3 , phospho-PTEN (Ser ), and PTEN (Cell Signaling Technology). performed in duplicate and repeated independently (n = 3). The robustness h Antibodies against c-Myc (9E10), anti-c-Met (c12), and -actin (c19; Santa of this array technology was confirmed by repeating each experiment (each Cruz Biotechnology) were also used. LY294002 and small interfering RNA time with a different print run of the array) and by using different dilutions of h (siRNA) GSK3a/ were purchased from Cell Signaling Technology. A novel the same lysate and different exposure times to ensure that spots were small molecule inhibiting c-Met phosphorylation (PHA665752) was kindly analyzed in the linear range of the enzymatic kinase reaction. provided by Pfizer, Inc. Horseradish peroxidase (HRP)-conjugated second- Data acquisition and statistical analysis. Imaging of the incorporated ary antibodies were all derived from DAKO. [g-33P]ATP was performed using a phosphoimager (Fuji) at 50 Am/pixel. Cell culture. Human colon cancer cell lines DLD1 (COX-2 negative) and Data analysis was performed using Microsoft Excel and Statistical Package HT29 (COX-2 positive; American Type Culture Collection) were cultured in for the Social Sciences 11.5 (SPSS, Inc.). After image acquisition and DMEM (Sigma-Aldrich) supplemented with 10% FCS (Integro), 5 mmol/L quantification using ArrayVision software 6.0 (Molecular Dynamics), each L-glutamine, 100 units/mL penicillin, and 100 Ag/mL streptomycin spot was corrected for its individual background. To reduce variation j (Invitrogen Corp.) at 37 C in a 5% CO2 atmosphere. Cells were passaged induced by incubation and imaging conditions, the data of each array were 20 times maximally. The concentration range of celecoxib in the experiment corrected with a linear trimmed median normalization method for each was chosen to resemble the in vivo situation (5–50 Amol/L). array and each individual experiment. After excluding inappropriate Cell viability in cell culture. The cell viability was assessed by duplicates for further analysis, the resulting data matrix consisted of the mitochondrial function, measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- phosphorylation levels of 1,176 peptides induced by the HT29 lysate treated nyltetrazolium bromide (MTT) reduction activity. Briefly, cells were seeded with celecoxib relative to the control. To identify biologically relevant in a 14-well plate and stimulated with increasing concentrations of activity changes, we calculated the differences in variance relative to the celecoxib (5–50 Amol/L). After 48 h, 0.5 mg/mL MTT (Sigma-Aldrich) was regression curve (SD > 1.96). Data were considered relevant for analysis added for 30 min at 37jC. Subsequently, the medium was aspirated and the when the signal to noise ratio in both conditions was >1.8 and a z1.7 fold cells were lysed in isopropanol/0.04 mol/L HCl. change was observed in both conditions. Kinome array analysis. A kinase substrate peptide array consisting of Transfection and luciferase activity assays. The HT29 and DLD1 colon 1,176 peptides with specific phosphorylation sites was used to comprehen- carcinoma cells were transiently transfected with Renilla constructs and sively evaluate the effects of aspirin and celecoxib on the kinome, as have 5 Ag of either TOPflash or FOPflash TCF reporter constructs (Upstate been previously described (24–26). After celecoxib treatment, the cells were Biotechnology) using Lipofectamine and Plus reagent (Invitrogen). Five washed in ice-cold PBS and harvested in lysis buffer (Cell Signaling hours after transfection, complete culture medium was added and the cells Technology) supplemented with protease and phosphatase inhibitors were stimulated. Forty-eight hours after stimulation, cells were lysed in lysis (Roche). Protein concentrations were determined using Bradford analysis buffer (Luciferase Assay System, Promega), and 20 AL of each lysate were

Figure 1. Celecoxib affects cell viability in HT29 and DLD1 colon cancer cells. A, cell viability was assessed by mitochondrial function, measured by MTT reduction activity after 72 h of aspirin treatment with 50 and 500 Amol/L (1 and 10 times serum levels achievable in patients), which did not influence cellular viability incubation. NS398, a selective COX-2 inhibitor, did not decrease cellular viability, whereas incubation with celecoxib significantly reduced cellular viability both at 5 and 50 Amol/L (1 and 10 times serum levels achievable in patients). B, celecoxib concentration-dependent and FCS concentration-dependent decline of cellular viability after 72 h.

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Figure 2. Selective COX-2 inhibition alters the kinase activity profile. Each dot represents the amount of phosphorylation of a specific substrate induced by celecoxib and control (Y-axis) detected analyzed by using a peptide array and phosphoimaging and quantification software. The gray spots are significantly altered. The gray lines through the gray dots represent the variation between three different experiments.

monitored for luciferase activity using luciferase assay substrate buffer viability were dependent on the amount of FCS present in the (Promega). Light units were recorded using a luminometer. Transfection medium. Higher amounts of FCS reduced the celecoxib-induced Renilla efficiency was determined by cotransfection with the luciferase reduction in mitochondrial activity significantly (Fig. 1B). We reporter vector pRL-CMV (Promega). The resulting transfection efficiency concluded that our experimental setup was valid for investigating was used to correct for differences in transfection in each culture well in celecoxib-specific effects on CRC cell physiology. each experiment. All experiments were performed in duplicate and repeated independently thrice. Analysis of kinome alterations on aspirin and celecoxib Western blot analysis. Equal amounts of lysates of HT29 and DLD1 cells administration in HT29 cells. Because almost all cellular were loaded onto SDS-PAGE and subsequently transferred to a poly- biochemical pathways are under strict control of reversible vinylidene difluoride membrane (Millipore Corp.). The membranes were phosphorylation of rate-limiting enzymes, we assumed that the blocked with TBS supplemented with 0.05% Tween 20 (wash buffer) and 1% effects of celecoxib would be reflected in altered activity of cellular casein. Primary antibody incubation was done overnight at 4jC, diluted kinases. Therefore, we have examined the effects of celecoxib on 1:1,500 in blocking buffer. Subsequently, the membranes were washed and rapid signal transduction in cell lysates of HT29 cells after short- incubated with a HRP-conjugated secondary antibodies (DAKO) diluted in term treatment with 25 Amol/L celecoxib using a peptide array blocking buffer. The bands were visualized using Lumilight Plus substrate containing 1,176 different, spatially addressed oligopeptide kinase (Roche) and chemiluminescence detection by a Lumi-Imager (Roche). Each substrates in duplex as described (24–26). After in vitro phosphor- sample was corrected for loading by comparison with the appropriate h-actin signal. ylation of the peptide substrates by kinase activities in cell lysates 33 Small interfering RNA transfection. SignalSilence GSK3 siRNA kit (Cell and [g- P]ATP and substrate phosphorylation quantification, the Signaling Technology) was used to down-regulate GSK3a/h protein in CRC Spearman’s correlation between independently performed experi- cell lines. In brief, HT29 cells were transfected with siRNA ( final ments was always in excess of 0.85. Therefore, the data were concentration of 100 nmol/L) using transfection reagent (Cell Signaling considered as valid for analysis of the celecoxib-induced effects on Technology). After 12 h, the cells were treated with celecoxib. After 2 days, CRC cell kinase activity (n = 3). Thirty minutes of celecoxib cells were harvested and subjected to Western blot analysis. Control cells treatment resulted in significant changes in the cellular phosphor- were transfected with nonspecific siRNA and cultured under the identical ylation patterns of HT29 cells, reflecting the potent effects of conditions. celecoxib on CRC cellular signal transduction (Fig. 2). Table 1 provides the top 20 affected substrates. Strikingly, a celecoxib- Results dependent, significant decrease in phosphatidylinositol 3-kinase The effects of celecoxib on viability of CRC cell lines. Before (PI3K)-associated signal transduction, including reduced phosphor- embarking on peptide array analysis of celecoxib effects on cellular ylation of substrates for IGFR/c-Met, protein kinase C~ (PKC~), and physiology, we decided to first characterize the effects of NSAID Akt. In agreement, a concomitant increase in phosphorylation of a treatment on cell proliferation and survival in our model system substrate for GSK3h was apparent after celecoxib treatment. Taken using a MTT assay. Celecoxib potently reduced mitochondrial together, the peptide array analysis suggests that celecoxib acts on activity on 72 h of incubation, which was observed in both HT29 growth factor–related signal transduction by inhibition of IGFR- cells (COX-2 positive) and DLD1 cells (COX-2 negative), whereas induced and c-Met–induced kinase activity. the administration of aspirin or NS398, another selective COX-2 Dephosphorylation of c-Met on the Y1234 autophosphoryla- inhibitor, had little effect (Fig. 1A). This supports the well-described tion site is induced by celecoxib in HT29 CRC cells. The results phenomenon that the anticancer activity of celecoxib is more of the peptide array indicate that celecoxib inhibits IGFR and c-Met potent than other NSAIDs and is COX-2 independent (3). autophosphorylation followed by diminished activation of kinases Interestingly, it was noted that the effects of celecoxib on cellular downstream in the PI3K signaling pathway (Table 1). To confirm www.aacrjournals.org 1215 Cancer Res 2008; 68: (4). February 15, 2008

Table 1. Peptide substrates with significantly altered Table 1. Peptide substrates with significantly altered phosphorylation by lysates from CRC cells treated with phosphorylation by lysates from CRC cells treated with selective COX-2 inhibitor for 30 min selective COX-2 inhibitor for 30 min (Cont’d)

Fold PEP_NR Sequence PH_SITE Kinase Fold PEP_NR Sequence PH_SITE Kinase change change

0.02 518 VTRRTLSMD T-773 PKA 2.77 961 PRKGSPKRG S-23 Sperm-specific 0.08 151 TNHIYSNLA Y-936 3.20 184 LRRASLGAA 0.09 248 PRTPGGRR 4.70 69 GDSSYKNIH Y-697 c-Fms 0.14 848 AQAASPAKG S-386 cdc2 5.64 891 EAVTSPRFI S-31 0.22 1087 TKFASDDEH S-366 CKII 5.65 845 YTRFSLARQ S-24 PKC 0.22 64 SRGDYMTMQ Y-987 INSR 17.99 242 VTPRTPPPS T-97 ERT 0.23 680 LVMQTAAGT T-166 0.24 246 SPRTPGGRR 0.24 22 EEEAYGWMD Y-87 v-Src NOTE: The fold induction of each substrate, their corresponding 0.29 603 RRVTSATRR S-8 PKA protein kinases, and chip peptide number are provided. 0.29 791 NSYGSRRGN S-711 PKC Abbreviation: PKA, protein kinase A. 0.31 56 RDEEYGYEA Y-411 0.35 1150 PSLPTPPTR T-208 0.36 1084 FGSRSLYGL S-59 this observations, short-term (30 min) incubation experiments 0.36 122 DSLIYDDGL Y-1029 were performed in HT29 cells and phosphorylation levels were 0.36 472 VRKRTLRRL T-678 PKC evaluated by Western blot analysis using phosphospecific anti- 0.37 1086 PNVSYIASR Y-447 bodies (Fig. 3A). In agreement with the peptide array analysis, 0.37 134 SAYGSVKAY S-25 PKC 0.38 1079 SDEEH S-1 celecoxib completely eliminated c-Met Y1234/5 autophosphorylation 0.39 316 PLSRTLSVS S-7 PKC/CAMII within a 30-min time frame. As the peptide substrate used in the 0.39 1048 SGYISSLEY S-206 CKII array does not distinguish well between the c-Met receptor and 0.40 593 TADISEDEE S-362 CKII INSR, the autophosphorylation status of the INSR was investigated, 0.41 1094 TKSASFLKG S-262 Calcium-dependent providing functional validation. Only a minor decrease in INSR 0.41 146 TNEEYLDLS Y-769 autophosphorylation was observed (possibly the result of cross- 0.42 577 LRRFSLATM S-128 PKA reactivity of the INSR antibody with pY1234/5 c-Met; data not 0.42 314 PLSRTLSVRSL shown). The EGFR phosphorylation was not affected. Important 0.43 450 RRLSSLRA S-235 regulating kinases involved in other pathways, such as Src and Lck, 0.45 1113 SSEITTKDL T-12 CKII were altered in activation, as shown by their phosphorylation 0.45 269 LSGFSFKKN S-169 PKC status. Moreover, the activity of PTEN, the phosphatase regulating 0.46 613 YVTTSTRTY S-33 PKC A 0.46 536 REVSSLKNK S-1917 PKC the PI3K signal transduction, was not changed (Fig. 3 )on 0.46 474 RKRTLRRL T-678 celecoxib administration, indicating a direct and specific effect on 0.46 1058 TAYGTRRHL T-184 PKC c-Met and validating the results of the peptide array. 0.47 684 PSRRSRSRS S-76 RS Kinetic analysis of celecoxib action on impaired c-Met Y1234/5 0.47 561 DDAYSDTET S-86 CKI autophosphorylation showed that, already after 2 min after 0.47 1160 RKLKSQGTR S-8 0 celecoxib treatment, a markedly reduced autophosphorylation is 0.48 1026 AVMVSHYIH S-56 evident (Fig. 3B). In addition, a concentration-dependent decrease 0.49 491 TRAPSRTAS S-450 MFPK of phospho-c-Met by celecoxib was observed (Fig. 3C). This effect 0.49 289 RKRTRKE of celecoxib on c-Met autophosphorylation was corroborated by 0.50 366 RRRASVA 0.51 403 RRSSSRPIR S-8 PKA experiments in which we directly addressed the phosphorylation 0.52 872 STNDSLL S-411 GRK5 status of the adaptor protein Gab-1, which is activated on c-Met 0.53 852 NKQGYKARQ Y-187 activation, enabling signal transduction to multiple pathways. A B 0.53 285 RKASRKE Figure 3 and shows that Gab-1 Y307 phosphorylation was 1.95 511 IHQRSRKRL S-315 rapidly down-regulated by celecoxib with kinetics closely resem- 2.04 911 HATPSPPVD S-357 GSK3 bling the effects observed on c-Met autophosphorylation. In c-Met, 2.04 523 SRGKSSSYS S-576 PKC signal transduction phosphorylation of Gab-1 in turn produces 2.14 1073 DDINSYEAW S-114 stimulation of the PI3K pathway followed by Akt phosphorylation 2.17 944 GGIRSLNVA S-113 Isocitrate (29). In agreement with the observed celecoxib-induced blockage of 2.18 271 VESLSSSEE S-32 constitutive c-Met RTK activity and also with the reduced 2.31 822 LSGLSFKRN S-103 PKC 2.31 340 RTGRSGSV phosphorylation of an Akt-specific substrate on the peptide array 2.33 98 TEDQYSLVE Y-607 c-Src following celecoxib treatment, Akt phosphorylation was substan- 2.36 775 RRRASQLKI S-434 PKC tially reduced following celecoxib treatment when assayed on 2.46 924 STSLSPFYL S-45 Crystalline Western blot (Fig. 3A). Similarly, the increase in GSK3h activity as 2.47 968 KGGSYSQAA Y-344 shown in the peptide array experiment corresponded a decrease of 2.50 900 ETRFTDTRK T-57 CaM-III GSK3h phosphorylation of the Akt target S9 in GSK3h on celecoxib 2.68 705 TRRISQTSQ S-2809 CaM-II treatment, an event associated with decreased activity of this enzyme (30).

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Together, these results show that the findings of the peptide translocation of the protein. Interestingly, celecoxib-induced c-Met array correspond with those of conventional techniques. Selective dephosphorylation was followed by a substantial increase of the COX-2 inhibition by celecoxib efficiently down-regulates c-Met levels of phospho-h-catenin (Fig. 3C). Because phosphorylation of receptor activity, resulting in both decreased activity of the h-catenin enables breakdown of the protein and thus to a decrease extracellular signal-regulated kinase (ERK) kinases and PI3K of TCF-h-catenin–dependent transcription, the effect of celecoxib pathway, the latter evident from decreased Akt and GSK3h on the transcription activity of TCF-h-catenin was explored. phosphorylation. These results also strongly suggest that the Therefore, DLD1 and HT29 cells were transfected with a specific celecoxib-induced inhibition of c-Met–dependent signaling TCF reporter (TOP) construct and corrected results for cell death decreases the level of Wnt activity in colon cancer cells because using a constitutive Renilla firefly construct as well as by using a the levels of phospho-h-catenin are increased on celecoxib construct containing a scrambled TCF-responsive motif (FOP). administration. Following celecoxib treatment for 36 h, h-catenin/TCF–activated Wnt signaling is decreased by celecoxib. The results TOPflash activity was abrogated in both DLD1 and HT29 cells described above suggest that c-Met activation by phosphorylation (Fig. 4A). The inhibition of TCF-mediated transcription was is a relevant target for celecoxib with respect to CRC cell observed after treatment of relative low concentrations cytotoxicity but leave the mechanism by which diminished c-Met (12.5 Amol/L) of celecoxib by HT29 cells, whereas 25 Amol/L was activation may have an anticancer effect unexplored. As it is well needed in DLD1 cells. established that activity of the Wnt pathway is critical factor for This indicates a specific effect of celecoxib on the TCF- CRC cell survival and proliferation, the effects of celecoxib on the mediated transcription in both HT29 and DLD1 cell lines, which activity of this pathway were determined. The major defect in the coincides with the increase in h-catenin phosphorylation. In regulation of the Wnt pathway in APC-mutant cells is the decrease agreement, a dose-dependent, celecoxib-induced down-regulation in levels of phospho-h-catenin, causing diminished breakdown of of the established Wnt targets, Myc and cyclin D1, was observed on h-catenin by the proteosome and subsequent increased nuclear protein levels (Fig. 4B). Thus, the inhibition of c-Met signaling by

Figure 3. Western blot analysis confirms kinomic analysis and identifies Met and downstream Akt, GSK3, as a target for COX-2 inhibition. A, Western blot analysis with phosphospecific antibodies of HT29 cells incubated with celecoxib or control for 30 min confirms the results of the peptide array. B, kinetic analysis of celecoxib action on impaired Met Tyr1234/5 phosphorylation showed that, already after 2 min after celecoxib treatment, a markedly reduced phosphorylation is evident. C, the decrease of phospho-Met by celecoxib is concentration dependent. D, GSK3h mediates the celecoxib-induced phosphorylation of h-catenin (Ser37-39). HT29 cells were transfected with siRNA against GSK3h and subsequently treated with celecoxib for 30 min. It is shown that celecoxib treatment did not result in increased h-catenin phosphorylation in cells transfected with GSK3h siRNA (left three lanes) compared with cells transfected with scrambled siRNA as a control (right three lanes). When transfected with siRNA, GSK3h is still present but, after 36 h, in less quantity. Celecoxib did not induce h-catenin phosphorylation in combination with GSK3h siRNA, whereas the cells transfected with scrambled siRNA show phosphorylation of h-catenin on celecoxib treatment.

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cells transfected with scrambled siRNA. These results show that GSK3h is functionally active and necessary to induce phosphorylation of h-catenin in these cells. In addition, treatment with LiCl, an inhibitor of GSK3h, did result in similar decreased h-catenin phosphorylation (data not shown). As shown in Fig. 3D, celecoxib did not induce h-catenin phosphorylation in combination with GSK3h siRNA. This confirms that GSK3h mediates the phosphorylation of h-catenin and suggests that the effects of celecoxib on h-catenin/TCF–dependent transcription are mediated upstream of GSK3h. c-Met activation enhances Wnt signaling in colon cancer cell lines. To determine whether HGF was able to stimulate TCF transcription, by stimulating c-Met, HT29 cells were transfected with a TCF reporter vector, TOPflash, which contains a combination of TCF-binding elements. Exposure to HGF increased the transcriptional activity of TOPflash f10-fold (Fig. 5). As a positive control, cotransfection with a LEF-1 expression vector increased the activity of TOPflash f40-fold. In contrast, the reporter construct containing mutated TCF sites, FOPflash, had very low activity in HT29 cells; treatment with HGF did not significantly increase luciferase activity of this construct. To investigate the effect of endogenous HGF on TCF activity, we treated HT29 cells with PHA665752, a selective c-Met kinase inhibitor. Previous studies have shown that the PHA665752 is

Figure 4. Wnt activity is decreased in DLD1 and HT29 cells on celecoxib administration. A, TCF reporter activity following treatment of HT29 and DLD1 cells. Treatment with increasing concentrations of celecoxib for 36 h. Cells were lysed and TOPflash and FOPflash activities were recorded in a luminometer. Both figures display the relative luciferase activity of cells transfected with TOPflash or FOPflash. B, after 48 h, a dose-dependent celecoxib-induced down-regulation of the established Wnt targets, Myc and Met, was observed in protein levels as determined by Western blot analysis. Actin levels are shown as a loading control. celecoxib coincides with reduced activity of Wnt/h-catenin pathway. Celecoxib induces decreased activity of GSK3B resulting in increased phospho-B-catenin. The observed increase in the phosphorylation of h-catenin together with decreased phosphorylation of both Akt and one of its downstream targets, GSK3h, suggest that the GSK3h enzyme has remaining functional activity to the phosphorylation of h-catenin in cell lines with an APC mutation. To investigate whether GSK3h is essential for the observed increase in phosphorylation of h-catenin on COX-2 inhibition, despite the presence of the APC mutation, HT29 cells Figure 5. Met activation stimulates Wnt activity. HT29 and DLD1 cells were were transfected with siRNA against GSK3h and subsequently treated with PHA665752, a selective Met kinase inhibitor, to investigate the treated with celecoxib for 30 min. In Fig. 3D, it is shown that effect of endogenous HGF and subsequent Met activation on TCF/h-catenin h activity as displayed by the TOP/FOP ratio. All transfections were corrected celecoxib treatment did not result in increased -catenin phos- for Renilla transfection efficiency. The addition of Met inhibition in the presence phorylation in cells transfected with GSK3h siRNA compared with of HGF completely abrogated the effect of HGF on TCF transcriptional activity.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2008 American Association for Cancer Research. COX-2 Inhibition Inhibits c-Met Kinase and Wnt Activity specific for c-Met, not blocking other kinases. It is shown that the epithelial cells with a functional APC complex, HGF-dependent addition of PHA665752 in the presence of HGF completely inhibition of GSK3h activity results in diminished phosphorylation abrogated the effect of HGF on TCF transcriptional activity, of h-catenin and increased nuclear accumulation of the protein thereby confirming that the HGF-induced effect is specific and acts (36). In our study, we observed an increased GSK3h activity by activation of c-Met. following celecoxib treatment in the peptide array experiments. These results were confirmed by Western blot analysis showing that h Discussion celecoxib diminishes phosphorylation of GSK3 S9, an event associated with increased GSK3h activity (37). Thus, it is possible Although the antagonistic effect of COX-2 inhibitors on the that also in CRC cells harboring APC mutation increased GSK3h mortality from CRC is undisputed, the nature of the underlying activity mediates the increase of h-catenin phosphorylation and molecular mechanism remains unestablished. In the present study, subsequent decreased transcriptional activity of the Wnt pathway. we have aimed to clarify this issue by investigating the effects of Hence, a possible interpretation of the present study is that celecoxib, a COX-2 inhibitor with both preventive and therapeutic inhibition of c-Met autophosphorylation followed by reduced c-Met properties, on phosphoregulated signal transduction in CRC cells activation contributes to celecoxib-dependent growth reduction without a priori assumptions as to the kinase pathways affected by and cytotoxicity in CRC cells. the treatment. To this end, we used a relatively novel peptide array To translate the results shown in vitro, we have chosen celecoxib approach investigating multiple kinase activities in parallel in concentration that can be achieved in vivo as shown in serum from combination with conventional techniques to validate the findings patients (38). Furthermore, we have previously shown in a clinical and to translate kinase activity to mechanisms in CRC. The results phase 2 trial in patients with esophageal adenocarcinoma that both obtained suggested that celecoxib targets c-Met autophosphor- COX-2 and Met expression in cancer tissue, both highly expressed in ylation, resulting in impaired downstream activity of the Gab/PI3K esophageal adenocarcinoma, were down-regulated after 4 weeks of pathway. celecoxib treatment (17). A phase 2 trial in which patients with CRC The identification of c-Met autophosphorylation and subsequent have been treated with celecoxib neoadjuvantly is currently under signaling through the PI3K pathway as a target for celecoxib was investigation to show molecular effects in tumor tissue in vivo. unexpected but makes sense as viewed from the body of currently The mechanism by which celecoxib influences c-Met autophos- available literature. The expression of c-Met is an important phorylation remains unclear as the effect of celecoxib is overcome prognostic factor for CRC progression and metastasis (12, 31, 32). by high concentrations of HGF. Diminished prostaglandin E Similar results are found for other adenomatous cancer types. 2 (PGE ) production by inhibition of the COX-2 activity may be Furthermore, PI3K activity is a well-known phenomenon in CRC 2 implicated. Indeed, PGE enhances activation of c-Met, possibly via and literature is available showing that celecoxib treatment reduces 2 activation of prostanoid receptors, in turn cross-linking growth activity of this pathway (33). The results presented in the current factor receptors and subsequent downstream signal transduction study suggest that activity of this pathway is partly driven by the (18, 36). The observation that HGF is capable of inducing Wnt basal c-Met activity. We have previously shown that c-Met signaling, which can be reversed on c-Met inhibition, provides expression can be regulated by the Wnt pathway, whereas also evidence that c-Met inhibition is essential for the effects described data have been presented that activation of c-Met results in in this study regardless of PGE . enhanced activity of the Wnt pathway (12, 23, 32). Thus, a positive 2 The present study shows that corepression or c-Met and h- feedback between activation of the pathway on one hand and catenin oncogenic signal transduction is a major effector of activity of c-Met signaling on the other hand seems to exist. This celecoxib-induced effects in CRC cells. Despite the negative reports notion is reinforced by our observation that celecoxib treatment showing an increased cardiovascular risk of COX-2 inhibitors, resulted in a rapid reduction of c-Met phosphorylation and which indeed negates the use of COX-2 inhibitors for CRC subsequent downstream signaling through both ERK and Akt and prevention, celecoxib administration as (neo-) adjuvant therapy GSK3h. This decrease is followed by a diminished transcriptional or in combination with other drugs for advanced disease could be activity of the Wnt pathway as assessed by the TOP/FOP assay and of therapeutic benefit for patients with CRC. The importance of expression levels of established Wnt targets as well as by the c-Met as a target for selective therapy in patients with CRC will stimulation of h-catenin phosphorylation in celecoxib-treated cells. need further clinical investigation. Although we have shown that presence of GSK3h is essential for the celecoxib-induced phosphorylation of h-catenin, these findings are in disparity with the presence of APC mutations in these cells, Acknowledgments which are generally assumed to abolish the capacity of GSK3h to Received 9/5/2007; revised 11/28/2007; accepted 12/19/2007. interact and thus phosphorylate h-catenin (34). However, recently, The costs of publication of this article were defrayed in part by the payment of page Li et al. (35) showed that GSK3h is still capable of regulating charges. This article must therefore be hereby marked advertisement in accordance h with 18 U.S.C. Section 1734 solely to indicate this fact. phosphorylation of -catenin despite the presence of an APC We thank J. Joore (Pepscan Systems, Lelystad, the Netherlands) for the technical mutation in CRC cells, supporting our findings. In mammary support.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2008 American Association for Cancer Research. Cyclooxygenase-2 Inhibition Inhibits c-Met Kinase Activity and Wnt Activity in Colon Cancer

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