CTEN Prolongs Signaling by EGFR through Reducing Its ... · Tumor and Stem Cell Biology CTEN Prolongs Signaling by EGFR through Reducing Its Ligand-Induced Degradation Shiao-Ya Hong1,

Tumor and Stem Cell Biology

CTEN Prolongs Signaling by EGFR through Reducing ItsLigand-Induced Degradation

Shiao-Ya Hong1, Yi-Ping Shih1, Tianhong Li2,3, Kermit L. Carraway III1,3, and Su Hao Lo1,3

AbstractActivation of EGF receptor (EGFR) triggers signaling pathways regulating various cellular events that

contribute to tissue development and function. Aberrant activation of EGFR contributes to tumor progres-sion as well as therapeutic resistance in patients with cancer. C-terminal tensin-like (CTEN; TNS4) is a focaladhesion molecule that is a member of the tensin family. Its expression is upregulated by EGF and elevatedCTEN mediates EGF-induced cell migration. In the presence of CTEN, we found that EGF treatment elevatedthe level of EGFR protein but not mRNA. The extended half-life of activated EGFR sustained its signalingcascades. CTEN reduced ligand-induced EGFR degradation by binding to the E3 ubiquitin ligase c-Cbl anddecreasing the ubiquitination of EGFR. The Src homology 2 domain of CTEN is not only required for bindingto the phosphorylated tyrosine residue at codon 774 of c-Cbl, but is also essential for the tumorigenicityobserved in the presence of CTEN. Public database analyses indicated that CTEN mRNA levels are elevatedin breast, colon, lung, and pancreas cancers, but not correlated with EGFR mRNA levels in these cancers. Incontrast, immunohistochemistry analyses of lung cancer specimens showed that CTEN and EGFR proteinlevels were positively associated, in support of our finding that CTEN regulates EGFR protein levels through aposttranslational mechanism. Overall, this work defines a function for CTEN in prolonging signaling fromEGFR by reducing its ligand-induced degradation. Cancer Res; 73(16); 5266–76. �2013 AACR.

IntroductionEGF receptor (EGFR) tyrosine kinase engages a vast array of

signaling pathways to regulate tissue development andhomeostasis (1, 2). EGFR signaling is normally induced byligand binding (such as EGF), leading to receptor dimerization,autophosphorylation, activation of downstream signalingmolecules, and cellular events such as proliferation,migration,and differentiation. Given its importance, EGFR signaling istightly regulated. Ligand binding not only induces receptoractivation but also triggers suppressionmechanisms to ensureprecise control of EGFR signaling output (3). One of thenegative regulations is governed through ubiquitin-dependentEGFR degradation, which is largely mediated by the RINGfinger E3 ubiquitin ligase c-Cbl. c-Cbl binds to tyrosine-phos-phorylated EGFR through its tyrosine kinase-binding (TKB)domain, allowing recruitment of ubiquitin-conjugating enz-ymes and transfer of ubiquitin to phosphorylated EGFR.Ubiquitination of EGFR then facilitates its internalization anddegradation in lysosomes (4). Disruption of this negative

regulatory system could trigger cellular events that contributeto tumor formation.

Focal adhesions connect extracellular matrix to cytoskeletalnetworks and play critical roles in cell adhesion, migration,proliferation, and survival. They are also involved in cross-talkwith growth factor receptors, such as EGFR, to elicit a widerrange of cellular responses. C-terminal tensin-like (CTEN) isthe smallest protein in the tensin focal adhesion family (5)known to regulate cell adhesion and migration (6). Lessamount of, if any, CTEN is expressed in normal tissues includ-ing breast, lung, ovary, and colon (7). However, its expression isprofoundly increased when these tissues develop tumors. Incolon cancer, 76% of patients' tumor samples exhibit signifi-cantly elevated CTEN protein levels (8), which is associatedwith poor prognosis (9). In invasive breast cancer, CTENexpression correlates with high EGFR and HER2 levels, as wellas with metastasis to lymph nodes (6), and is associated withpoor prognosis (10).Manipulation of CTENprotein levels alterscell invasion, epithelial–mesenchymal transition (EMT), andcolony formation capacities of colon cancer cells (8, 11).Activation of EGFR by EGF leads to an expressional switchfrom tensin 3 (TNS3) to CTEN, and this upregulation of CTENwith reduced TNS3 contributes to EGF-promoted mammarycell migration (6). Mitogen-activated protein/extracellular sig-nal–regulated kinase (MEK) activity is required for EGF-induced CTEN overexpression (6). Consistently, it is reportedthat gain-of-function mutations in K-RAS and B-RAF (bothupstream molecules of MEK) are associated with CTEN over-expression (12). Altogether, these findings closely link CTEN toEGFR signaling cascades.

Authors' Affiliations: 1Department of Biochemistry and Molecular Med-icine, 2Division of Hematology & Oncology, Department of Internal Med-icine, and 3Comprehensive Cancer Center, School of Medicine, Universityof California-Davis, Sacramento, California

Corresponding Author: Su Hao Lo, Department of Biochemistry andMolecular Medicine, University of California-Davis, 4635 Second Ave.Room 3210, Sacramento, CA 95817. Phone: 916-734-3656; Fax: 916-734-5750; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-4441

�2013 American Association for Cancer Research.

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Published OnlineFirst June 17, 2013; DOI: 10.1158/0008-5472.CAN-12-4441

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In the present study, we have identified a novel role for CTENin EGFR regulation. Our data indicate that CTEN suppressesligand-induced EGFR degradation by reducing receptor ubi-quitination, which is accomplished via the ability of CTEN tointeract with c-Cbl in a phosphotyrosine-SH2–dependentmanner. Overexpression of CTEN enhances cancer cell migra-tion, invasion, as well as colony formation activities, which canbe reversed by EGFR inhibitor. In addition, the Src homology 2(SH2) domain of CTEN is essential for promoting these tumor-igenic properties. The clinical relevance of our findings issupported by the upregulation of CTEN mRNA in a variety ofcancer types and the correlation of elevated CTEN and EGFRprotein levels in lung cancer.

Materials and MethodsReagents and plasmid constructsAntibodies against EGFR, c-Cbl, ubiquitin (EMD Millipore),

ERK1/2, pY845-EGFR, pY992-EGFR, pY1045-EGFR, pY1068-EGFR, pY1173-EGFR, phospho-tyrosine (Cell Signaling Tech-nology), Flag (Sigma-Aldrich), and phospho-ERK1/2 (SantaCruz Biotechnology) were purchased from indicated commer-cial suppliers. Recombinant human EGF was purchased fromPromega Corporation. Lactacystin was purchased from Sigma-Aldrich. Cetuximab (Erbitux) was from ImClone System Incor-porated. Flag-tagged CTEN full-length, N-terminal, C-terminalplasmids were constructed by PCR amplification of the EGFP–CTEN (13). PCR products were then directionally cloned intoEcoRI and BamHI sites in the vector, pFLAG-CMV-2 (Sigma-Aldrich). Hemagglutinin (HA)-tagged c-Cbl mutants were con-structed by site-directedmutagenesis with the HA-tagged wild-type (WT) c-Cbl.

Cell culture and DNA transfectionAll cell lines were purchased from American Type Culture

Collection and authenticated by this established providerthrough cytogenetic analysis. They were cultured and usedwithin 6 months after resuscitation in appropriate media:Dulbecco's Modified Eagle Medium (DMEM) 4.5 g/L glucosefor HEK 293T, SW480, and A549 cells; RPMI-1640 for 5637cells; McCoy's 5A medium for HCT116 cells supplementedwith 10% FBS (Sigma-Aldrich), 1% penicillin/streptomycin,and 2 mmol/L L-glutamine. All cells were maintained at37�C in humid air with 5% CO2 condition. For the EGF-treatment experiments, cells were starved in serum-free mediafor 24 hours and then replaced with media containing EGF,prepared freshly from an aqueous 100 mg/mL recombinanthuman EGF solution. Transfections were carried out usingLipofectamine 2000 (Invitrogen) reagent according to the man-ufacturer's protocol.

RNA extraction and RT-PCRTotal cellular RNAs were extracted using TRIzol reagent

(Invitrogen) according to the instructions of the user manual.cDNAs were generated from 2 mg RNA using the High CapacitycDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR (RT-PCR) analyses were conducted with SYBR GreenPCR Master Mix using PCR primers on an ABI Prism HT7900Sequence Detection System (Applied Biosystems). EGFR prim-

er sequences were as follows: 50-CGGGACATAGTCAGCAGTG-30 (forward) and 50-GCTGGGCACAGATGATTTTG-30 (reverse).Relative EGFR mRNA expression was determined by the com-parative Ct method using RQ Manager software (AppliedBioscience).

CoimmunoprecipitationCell lysates of each 100-mm dish were prepared in 1 mL NP-

40 lysis buffer (50 mmol/L Tris–HCl, pH 7.4, 150 mmol/L NaCl,0.1% NP-40, and 1 mmol/L EDTA) supplemented with 1�protease and phosphatase inhibitor cocktail (Roche AppliedScience) by gentle pipetting. The cell lysates were then incu-bated on ice for 20minutes and centrifuged at 13,000 rpm for 10minutes at 4�C. The supernatants were collected and pre-cleared with protein A-agarose for 30 minutes at 4�C withagitation. The precleared aliquots were then immuneprecipi-tated with M2 Flag affinity beads or specific antibodies-con-jugated agarose for 2 to 4 hours at 4�C with agitation. Beadswere washed three times by using lysis buffer supplementedwith 300mmol/L NaCl, and pellets were boiled for 5minutes in10 mL 2� SDS sample buffer for the analysis.

GST pull-down assaysThe glutathione S-transferase (GST) fusion proteins contain-

ing the SH2 domain or phosphotyrosine binding (PTB)domains of CTEN have been previously described (14). GSTfusion proteins, or GST alone, were expressed in Escherichiacoli BL21 strain and affinity-purified using glutathione-Sephar-ose 4B beads (Amersham Pharmacia Biotech) according to themanufacturer's instructions. For in vitro pull-down assay, 750mg of EGF-treatedwhole-cell extracts in NP-40 lysis buffer fromHEK 293T cells were added to 25 mg of affinity-purified fusionproteins bound to glutathione-Sepharose beads. The boundproteins were separated by electrophoresis and examined byimmunoblotting. The peptide competition assay was con-ducted by preincubating the GST fusion proteins, which boundto the glutathione-Sepharose, with 100mg of indicated peptidesor phosphopeptides before adding the cell lysates. The peptidesand phosphopeptides were commercially synthesized (Gen-Script): c-Cbl Y700, CGEEDTEYMTPSSR (700XP), CGEEDTE-pYMTPSSR (700P); c-Cbl Y774, CENEDDGpYDVPKPP (774XP),CENEDDGpYDVPKPP (774P). The purities of all peptides weremore than 80% and their composition were verified by massspectrometry.

Migration, invasion, and colony formation assaysHCT116 cells (5 � 104 cells) were transfected with EGFP-

CTEN WT, R474A mutant, or control vector for 4 to 6 hoursbefore seeding onto the upper chamber of a Transwell (8-mmpore size; BD Biosciences). The cells in the upper chamberwere maintained in a serum-free medium and the lowerchamber was filled with culture medium supplemented with10% FBS. After 24 hours, the nonmigrating cells were removedfrom the upper surface of themembrane by scrubbing and themigrated cells on the lower surface were stained with crystalviolet (0.25% crystal violet, 3.7% formaldehyde, and 80%methanol) and counted. For Transwell invasion assay, thetransfected cells were seeded onto the upper chamber of a

CTEN Regulates EGFR Signaling

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Figure 1. CTEN attenuates EGF-induced EGFR downregulation in a transcription-independent manner. A, dose response of EGFR downregulation.Mock or Flag-CTEN–overexpressing HEK 293T cells were stimulated with indicated dosages of EGF for 1 hour. Left, representative immunoblotanalyses; right, bar graphs showing the relative protein levels of EGFR.B, kinetics of EGFRprotein levels.Mock or Flag-CTEN–overexpressingHEK293T cellswere treated with 10 ng/mL of EGF for indicated time. Left, immunoblot analyses with the relevant antibodies; right, quantification of the EGFR protein. Theintensity of each band was quantified, and expressed as a percentage using the signal at dose 0 or time 0 as a reference. C, kinetics of pEGFR levels.The mock or transfected cells were stimulated with 10 ng/mL of EGF for indicated time. tEGFR were immunoprecipitated (IP) from equal amounts of cell

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Cancer Res; 73(16) August 15, 2013 Cancer Research5268




Transwell. The experimental process is comparable with theTranswell migration assay described earlier, except that themembrane of the Transwell was precoated with 2 mg/wellserum-reduced Matrigel (BD Biosciences), and invasion wasassessed by cell counting after 48-hour incubation. For colonyformation assay, cells (5 � 103/well) were seeded in 6-wellplates and subjected to drug selection for 2 to 3 weeks, withG418 (400 mg/mL) selective medium refreshed every 3 days.The stable G418-resistant colonies were counted after stain-ing with crystal violet, washed with water, and air-dried. ForEGFR inhibition, 5 mg/mL of cetuximab was added to bothupper and lower chambers inmigration and invasion assay. Incolony formation assay, 5 mg/mL of cetuximab was added tothe selection medium to inhibit EGFR signaling.

Microarray analysisThe gene expression datasets were downloaded from the

National Center for Biotechnology Information (NCBI) GeneExpression Omnibus (GEO). We chose four different types ofcancer datasets that have balanced number of disease andcontrol samples in each dataset. Figure 5 showed the geneexpression datasets that were used in this study, includingbreast cancer (GSE15852), colon cancer (GSE10950), non–small cell lung cancer (GSE12236), and pancreatic ductaladenocarcinoma (GSE28735). Partek Genomics Suite (Parteksoftware, version 6.6 Beta) was used to analyze gene expres-sion values and to generate dot plots. Expression differencesbetween tumor and adjacent normal tissues were assessed byone-way ANOVA. Statistical analysis was conducted usingStudent t test. A P value less than 0.05 was consideredsignificant. All reported P values are two-tailed.

Immunohistochemistry and evaluationHuman lung cancer (8 small cell carcinomas, 13 adenocar-

cinomas, 18 squamous cell carcinomas, and 9 other type non–small cell carcinomas) and adjacent paired tissue microarrayslides were purchased from Pantomics Inc. The formalin-fixed,paraffin-embedded tissue slides were first incubated at 65�Cfor 30 minutes. Then, the samples were deparaffinized andplaced in a pressure cooker containing 10 mmol/L bufferedsodiumcitrate solution (pH 6.0) for antigen retrieval. The slideswere immersed in 3% hydrogen peroxide for 10 minutes toeliminate endogenous peroxidase activity. After that, the slideswere blocked before being incubated overnight at 4�Cwith 1:50diluted rabbit monoclonal anti-CTEN antibody (Spring Bio-science) or with mouse monoclonal anti-WT EGFR antibody(DAK-H1-WT from Dako). Detection was carried out on nextday with streptavidin-biotinylated peroxidase-conjugated

reagents with 3-amino-9-ethyl carbazole (AEC) as the chro-mogen (Vector Laboratories Inc.). The samples were observedusing a Zeiss Axioplan2 optical microscope imaging systemwith a real-color AxioCam high-resolution charge-coupleddevice camera (Carl ZeissMicroImaging). Slideswere evaluatedand the staining intensity was ranked on a scale ranging from 0to 3 (0, negative; 1, weak; 2, moderate; and 3, strong intensitystaining). The score of staining more than 1 was identified asupregulated in CTEN or EGFR expression. Statistical analysiswas conducted using GraphPad Prism (version 5.01, GraphPadSoftware Inc.). The statistical relationships between CTEN andEGFR of immunohistochemical (IHC) data were evaluatedthrough the two-sided Fisher exact test. P values less than0.001 were considered statistically highly significant.

ResultsCTEN expression modulates EGFR protein levels duringEGF treatment

It has previously been shown that EGF-induced CTENupregulation promotes cell migration (6). To further investi-gate the potential role of CTEN in EGFR signaling and itsregulation mechanism, recombinant CTEN was expressed inHEK293T cells, which did not express endogenousCTEN. Thencells were treated with various concentrations of EGF for 1hour. As shown in Fig. 1A, EGF-induced EGFR downregulationwas significantly reduced in the presence of CTEN. At this 1hour time point, maximal suppression of EGFR downregula-tion by CTEN was observed at 10 ng/mL EGF treatment.Consistently, the activation of downstream kinases, such asextracellular signal–regulated kinase (ERK)1/2 (measured byits phosphorylation level), wasmarkedly higher in the presenceof CTEN (Fig. 1A). Time course experiments indicated that thehalf-life of EGFR was extended from 45 to 90 minutes in thepresence of EGF when CTEN was overexpressed (Fig. 1B).Because EGFR tyrosine phosphorylation is an immediate eventupon ligand binding and also represents the activated form ofEGFR, the levels of tyrosine-phosphorylated EGFR were exam-ined. As shown in Fig. 1C, total tyrosine phosphorylation levelsof EGFR (pEGFR) were significantly enhanced in the presenceof CTEN. The increase of pEGFR seemed to be a reflection ofEGFR protein level. Once the pEGFR levels were normalized tototal protein levels (tEGFR), there were no differences betweencells with or without CTEN. By using phospho site-specificantibodies, higher phospho-Y845 (pY845), pY992, pY1045,pY1068, and pY1173 levels together with increased EGFRamounts were detected in CTEN transfectants than in mockcells (Fig. 1D). Altogether, these results showed that thepresence of CTEN significantly diminished EGF-induced EGFR

lysates with anti-EGFR antibody, followed by immunoblotting with anti-phosphotyrosine antibodies. Left, representative immunoblot analyses; right, bargraphs showing the ratios of pEGFR/tEGFR at indicated time points. The intensity of each bandwas quantified and shown as a fold change from the 5minutetime point of mock cells. D, phosphorylation levels of specific tyrosine sites on EGFR. Equal amounts of cell lysates from mock cells and CTEN-transfectants were immunoblotted with indicated antibodies. E, downregulation of EGFR in the absence of CTEN. Of note, 50 ng/mL of EGF were treated incontrol (si-ctrl) or CTEN (si-CTEN) siRNA knockdown SW480, A549, and 5637 cells for 1 hour. Band intensities of EGFR were quantified and the degrees ofEGFR downregulation (EGF treated/untreated) were shown as ratios. F, RT-PCR analyses of EGFR mRNA levels. Control or CTEN siRNA knockdownSW480, A549, and 5637 cells were untreated or treated with EGF (50 ng/mL) for 1 hour. EGFRmRNA levels were analyzed by RT-PCR and normalized to theinternal control, b-actin mRNAs. Error bars represent mean � SEM. Results for A–E are representative of 2 independent experiments each and for F isthe average of 3 independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.






downregulation, sustained activated receptor, and thereforeprolonged EGFR signaling.

The effect of CTEN on EGFR protein and mRNA levels wasalso examined in colon (SW480), lung (A549), and bladder(5637) cancer cell lines that overexpress endogenous CTEN.Both basal and inducible downregulation of EGFR proteinlevels were significantly enhanced when CTEN expression wassilenced by siRNA (Fig. 1E). Furthermore, EGFR mRNA levelswere not changed after CTEN was silenced in these cell lines(Fig. 1F), indicating that the effect is independent of transcrip-tional control.

CTEN reduces ligand-induced ubiquitination of EGFRand modulates c-Cbl–EGFR interaction

It is known that ubiquitination is a critical step for EGF-induced degradation of EGFR. To examine the effect of CTENon EGFR ubiquitination, HEK 293T cells were transfected withCTEN and then treated with or without EGF. Equal amounts ofEGFR were immunoprecipitated and their ubiquitinationlevels were measured. As shown in Fig. 2A, CTEN significantlyreduced the ubiquitination of EGFR in the presence of EGF.This suggests that CTEN protects ligand-induced EGFR deg-radation through reducing its ubiquitination. c-Cbl is theprimary E3 ubiquitin ligase that binds directly to pY1045 and

indirectly to pY1068 sites of EGFR and then ubiquitinates thereceptor upon ligand stimulation. Hence, the phosphorylationlevels of both sites were examined to test whether CTENmightcompromise c-Cbl–binding sites on EGFR. Equal amounts ofEGFR were immunoprecipitated and their pY1045 and pY1068levels were measured in a time course experiment (Fig. 2B). Inagreement with our findings in Fig. 1C, no remarkable phos-phorylation difference at these sites between control andCTEN transfectants was observed. In the same experiment,the interaction between c-Cbl and EGFRwas analyzed. Overall,increasing amounts of c-Cbl were interacted with EGFR incontrol cells, whereas c-Cbl–EGFR interaction levels werereducing in CTEN transfectants during the time course (Fig.2B). Nonetheless, significant more c-Cbl proteins were boundto EGFR in the present of CTEN before 15 minutes. After 15minutes, more c-Cbl–EGFR interaction was detected in thecontrol cells. These data strongly suggest that CTEN mayregulate the dynamic of c-Cbl–EGFR interaction.

CTEN interacts with the E3 ubiquitin ligase c-Cbl in aSH2-phosphotyrosine–dependent fashion

Because (i) EGF-induced EGFR ubiquitination level is com-promised in the presence of CTEN, (ii) CTEN modulates thec-Cbl–EGFR interaction without altering pY1045 and pY1068

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Figure 2. CTEN reduces ubiquitination but not tyrosine phosphorylation levels of EGFR induced by EGF. A, ubiquitination of endogenous EGFR afterEGF treatment. HEK 293T cells transiently transfected with control vector, Flag-CTENWT, or Flag-CTENR474Amutant were preincubatedwith 10 mmol/L oflactacystin for 1 hour. The cells were treated with or without 10 ng/mL of EGF for 15 minutes. Cell lysates were immunoprecipitated (IP) withantibodies specific for EGFR, followed by immunoblotting with anti-Ub (ubiquitin) or anti-EGFR antibodies. The intensities of the bands were compared withthose in EGF-treated control cells. Total cell lysateswere immunoblottedwith anti-Flag (for Flag-CTEN) or anti-GAPDHantibodies. B, kinetics of EGF-inducedEGFRphosphorylation andc-Cbl–EGFRbinding in thepresenceofCTEN. Themockor Flag-CTEN–transfectedHEK293Tcellswere stimulatedwith 10ng/mLof EGF for indicated time. To pull down equal amount of EGFR from mock and CTEN-overexpressing cells at each time point, adjusted amounts ofcell lysateswere immunoprecipitatedwith antibodies specific for EGFR, followedby immunoblottingwith anti-c-Cbl, pY1045-EGFR, pY1068-EGFR, and totalEGFR. Top, representative immunoblot analyses. The intensity of each band was quantified and the changes between CTEN and mock transfectionsampleswere shown as ratios (CTEN-overexpressing/mock) before normalized to EGFR levels. Bottom, analysis showing the fold change tomock sample ateach time point after normalized to corresponding EGFR. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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levels, and (iii) c-Cbl protein levels are not altered by CTENexpression (Fig. 3), we investigated whether CTEN could inter-act with c-Cbl and therefore affect EGFR ubiquitination. To

show the interaction betweenCTEN and c-Cbl, Flag-tagged full-length, N-terminus, or C-terminus of CTEN were transfectedinto HEK 293T cells (Fig. 3A). Recombinant CTEN and its

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Figure 3. TheSH2 domain of CTEN is essential for the interactionwith tyrosine-phosphorylated c-Cbl. A, the domain structures of CTEN and c-Cbl proteins. c-Cbl contains an N-terminal TKB region, a RING finger, a proline-rich region, and a C-terminal UBA (ubiquitin-associated) domain. Four tyrosinephosphorylation residues on c-Cbl are listed. CTEN possesses an SH2 domain and a PTB domain. Mutations of arginine 474 and 650 residues within CTENabolish the binding activities of the SH2 and PTB domains, respectively. CTEN N-terminus (Nter) and C-terminus (Cter) fragment mutants are illustrated. B,coimmunoprecipitation with CTEN fragments. HEK 293T cells were transfected with Flag-tagged CTEN WT, N-terminus (Nter), or C-terminus (Cter)fragments and then treatedwith orwithout 10ng/mLEGF for 15minutes.Cell lysateswere immunoprecipitated (IP)with anti-Flagantibodyand immunoblottedwith anti-c-Cbl antibody. Immunoblot analyses of c-Cbl and Flag-CTEN in total cell lysates are shown. C, same as in B except that Flag-tagged CTEN WT,SH2 mutant (R474A), and PTB mutant (R650A) were used. D, GST pull-down assay. GST or GST fusion proteins were incubated with equal amounts oflysates from EGF-treated or -untreated HEK 293T cells. Bound proteins were subjected to immunoblotting with anti–c-Cbl antibody (top right). Thebait proteinsweredetectedbyPonceau-Sstaining (bottom). E, coimmunoprecipitationofWTandvarious tyrosinemutantsofHA-tagged c-Cbl. HA-c-CblWT,Y674F, Y700F, Y731F, or Y774F were cotransfected with Flag-CTEN into HEK 293T cells. Lysates from untreated cells or cells treated for 15 minuteswith 10 ng/mLof EGFwere immunoprecipitatedwith anti-Flag antibody and immunoblottedwith anti-HA antibody. Immunoblot analyses ofHA-c-Cbl andFlag-CTEN in total cell lysate are shown. F, phosphopeptide competition assay. GST or GST–SH2 CTEN proteins were preincubated with c-Cbl phosphopeptides(700P, 774P) or nonphosphorylated peptides (700XP, 774XP) for 30 minutes. Lysates from EGF-stimulated (10 ng/mL, 15 minutes) HEK 293T cells wereadded and the incubation continued for another 90minutes. GST or GST–SH2 bound proteinswere separated by SDS-PAGE and identified by immunoblottingwith anti-c-Cbl antibody (top). The bait proteins were detected by Ponceau-S staining (bottom). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.






associated proteins were immunoprecipitated with anti-Flagantibody. As shown in Fig. 3B, c-Cbl was coimmunoprecipitatedwith Flag-tagged full-length CTEN and this interaction wasmarkedly enhanced by EGF treatment, suggesting that thec-Cbl–CTEN interaction might be phosphorylation-dependent.Interestingly, the interaction was retained when the full-lengthCTENwas replacedwith its C-terminal fragment (residues 438–715), but was almost abolished when the N-terminal region(1–438) of CTEN was used (Fig. 3B). The C-terminal region ofCTEN contains two functional domains, the SH2 and PTBdomains. We used CTEN loss-of-function mutations (R474Afor the SH2 domain and R650A for the PTB domain) to identifythe c-Cbl–binding domain. As shown in Fig. 3C, SH2 mutant(R474A) of CTEN clearly lost the ability to interact with c-Cbl,whereas PTB mutant (R650A) sustained the binding to c-Cblafter EGF induction (Fig. 3C). These results indicate that theSH2 domain of CTEN is essential for ligand-induced binding toc-Cbl. We further confirmed the interaction between the CTENSH2domain andc-Cbl using aGST fusion pull-downassay. GST,GST–SH2, and GST–PTB fusion proteins were expressed inand purified from E. coli. The fusion proteins were incubatedwith cell lysates from EGF-treated or -untreated HEK 293Tcells, and the pull-down assay was conducted as described inMaterials and Methods. Indeed, CTEN GST–SH2, but not GSTor GST–PTB, fusion proteins were able to pull down c-Cbl(Fig. 3D), suggesting that the SH2 domain of CTEN binds totyrosine phosphorylated c-Cbl.

c-Cbl is a prominent substrate of EGFR tyrosine kinaseand tyrosine 674 (AIY674SLAAR), 700 (JEY700MTPS), 731(CTY731EAMY), and 774 (DGY774DVPK) residues are fourmajorphosphorylation sites (15–17). To identify the potential phos-phorylation-dependent binding sites on c-Cbl, we individuallymutated tyrosine 674, 700, 731, and 774 to phenylalanine andexamined their CTEN-binding activities. c-Cbl Y774F mutantseemed to lose binding to CTEN after ligand stimulation (Fig.3E), indicating that tyrosine 774 of c-Cbl could be a crucialresidue for EGF-induced phosphorylaiton-dependent interac-tion with CTEN. To verify the role of Y774 residue in CTENbinding, synthetic peptides containing the Y700 or Y774 siteswith or without phosphorylation were used to compete withc-Cbl binding to GST–SH2 (Fig. 3F). Only phosphorylatedY774 peptide (774P) was able to block the interaction betweenCTEN and c-Cbl, further confirming that Y774 of c-Cbl is aspecific phosphotyrosine residue for binding to SH2 domain ofCTEN.

A functional SH2 domain of CTEN is essential forreducing EGF-induced EGFR ubiquitination/degradation and promoting CTEN-mediatedtumorigenicity

Our results showing c-Cbl–CTEN interaction suggest thatthe effect of CTEN on EGF-induced EGFR downregulationmaydepend on CTEN SH2 domain binding to phosphorylatedY774 on c-Cbl. To test this possibility, EGFR ubiquitinationin the presence of CTEN SH2mutant (R474A) was analyzed. Asshown in Fig. 2A, R474A no longer exhibits the potential toreduce the ubiquitination of activated EGFR. We furtherexamined the effect of CTEN R474A on EGFR protein and

ERK1/2 activation levels in the presence of EGF. Downregula-tion of EGFR in response to EGF was reduced in CTEN-transfected HEK 293T cells when compared with those ofexperimental groups that transfected with control vector andR474Amutant (Fig. 4A). Consistent with the diminished down-regulation of EGFR, phosphorylation of the EGFR effectorERK1/2 in response to EGF was markedly increased in thepresence of WT CTEN, but not R474A mutant (Fig. 4A). Theseresults show that the effect of CTEN on EGF-induced EGFRubiquitination, degradation, and signaling is primarily medi-ated through the CTEN SH2 domain.

Accumulating reports have indicated that CTENoverexpres-sion may be correlated to various types of cancer development(8, 10, 18, 19). Our results show that the interaction between theSH2 domain of CTEN and pY774 c-Cbl is important in atten-uating EGFR degradation and prolonging EGFR signaling,which might play important roles in tumor progression. Thus,we set up a series of experiments to examine the potential roleof CTEN SH2 domain in tumorigenic properties. Ectopicexpression of CTEN in HCT116 (low CTEN) colon cancer cellssignificantly promoted cell migration, invasion, and colonyformation activities (Fig. 4C–E). In contrast, no enhancementeffects on these activities were observed in cells transfectedwith CTEN SH2 mutant (R474A), showing the essential role ofthe SH2 domain on CTEN-mediated tumorigenicity in cancercells. To further validate the involvement of EGFR signaling inthese CTEN-mediated phenotypes, the experiments wererepeated in the presence of cetuximab, an anti-EGFR antibody.Although cetuximab had no effect on HCT116 cell migrationand invasion, it suppressed HCT116 colony formation activity(Fig. 4C–E). More importantly, the effects of CTEN overexpres-sion were reversed by cetuximab, implicating a direct linkbetween CTEN and EGFR signaling in these processes.

CTENmRNA levels areupregulated in a variety of cancersand its protein expression is correlatedwith that of EGFRin human lung samples

Several lines of evidence have indicated that CTEN mRNAexpression is upregulated in various cancers (8, 10, 18, 19). Toextend these findings, we took advantages of four microarraydatasets available from GEO and analyzed CTEN and EGFRexpression profiles using the Partek Genomics Suite software.Compared with paired normal tissues, we found that CTENmRNA expression is significantly upregulated in tumors frombreast (GSE15852; ref. 20), colon (GSE10950; ref. 21), lung(GSE12236; ref. 22), and pancreas (GSE28735; Fig. 5; ref. 23).Nonetheless, EGFR mRNA levels were downregulated withstatistical significance in breast cancer, but not in colon, lung,andpancreas datasets. These results show that CTEN transcriptwas increased, but not correlated with EGFR mRNA levels inthese cancers. Because our cell culture studies suggested thatCTENexpression altered EGFRprotein but notmRNA levels, weanalyzed the relationship between CTEN and EGFR proteinlevels by IHC staining in normal and lung cancer consecutivetissue section samples. To rule out the possibility that truncatedvariants of EGFR such as EGFR-vIII may escape from ligand-induceddownregulation,we limitedour analysis toWTEGFR inthese samples by using an anti-WT EGFR antibody (DAK-H1-

Hong et al.





WT). Figure 6A shows representative IHC staining images ofCTEN and EGFR. In a total of 96 cases, CTEN and EGFR areupregulated in 50 (52.1%) and 53 (55.2%) samples, respectively(Fig. 6B). Among them, 39 (40.6%) samples show both elevatedCTEN and EGFR IHC staining, whereas 32 (33.3%) samplesdisplay no change or downregulation on CTEN and EGFRtogether (Fig. 6B). The statistical analyses indicate a stronglypositive correlation between CTEN and EGFR protein levels inthese lung samples, supporting the cell culture finding thatCTEN upregulation increases EGFR protein level.

DiscussionPrevious studies have shown that CTEN expression is

induced by EGF treatment in a variety of normal and cancercells, including MCF10A, RWPE-1, HeLa, and SW480 cells(6, 24). Upregulated CTEN disrupts the interactions betweenintegrin receptors and actin cytoskeleton (6) and promotescancer cell migration, invasion, and colony formation(6, 8, 9, 11, 24). In this report, we have identified a novelfunction of CTEN in negatively regulating c-Cbl–mediatedEGFR degradation. By binding to activated c-Cbl, CTEN isable to decrease EGFR ubiquitination and degradation. At this

point, we do not know exactly how c-Cbl–CTEN interactionleads to the reduction of EGFR ubiquitination. It is possiblethat, by binding to c-Cbl, either CTEN sequesters c-Cbl awayfrom activated EGFR or/and reduces its E3 ligase activity. Theformer seems to be supported by our results showing that c-Cbl–EGFR interaction was markedly reduced in the presenceof CTEN after 15-minute EGF treatment. Because c-Cbl–CTENinteraction depends on c-Cbl tyrosine phosphorylation, whichis relied on its binding to activated EGFR, this may explain thedelay disruption of c-Cbl–EGFR interaction by CTEN. None-theless, CTEN could somehow transiently (�15 minutes) butrobustly enhance c-Cbl–EGFR interaction. How CTEN canaccomplish these dynamic processes remains to be investigat-ed. The function of c-Cbl in promoting EGFR degradation isknown to be suppressed in several ways (25), including (i)dephosphorylation of crucial phosphotyrosine sites on c-Cbl bySH2-containing phosphatase-1 (SHP1), (ii) targeting of c-Cblfor degradation by the E3 ligase atrophin-1–interacting pro-tein-4 (AIP4), (iii) binding of c-Cbl to Sprouty 1/2 pTyr 55 sitesthat compete with the pTyr 1045 site on EGFR, and (iv) theformation of a complex between c-Cbl and PAK-interactingexchange factor (bPIX), PAK1 and Cdc42. Our study presents a

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Figure 4. The SH2 domain of CTENplays a critical role in regulatingEGFR signaling and CTEN-mediated tumorigenic properties.A, the potential of EGFR signalingby CTEN overexpression. HEK293T cells transfected with controlvector, Flag-CTEN WT or SH2mutant (R474A) were treated with10 ng/mL of EGF for 1 hour. Equalamounts of total cell lysates wereanalyzed by immunoblotting withthe indicated antibodies. Bandintensities of EGFR and pERK1/2were quantified. The degrees ofEGFR downregulation (EGF-treated/untreated) were shown asratios. The changes of pERK1/2 inCTEN WT- or R474A-mutanttransfectants were compared withcontrol cells. B, representativeimmunoblot analyses showingequal recombinant protein levelsexpressed in cells used in C–E.C–E, cell migration (C), invasion(D), and colony formation (E) ofHCT116-EGFP vector, HCT116-EGFP-CTEN-WT, and HCT116-EGFP-CTEN-R474A transfectantswith or without EGFR inhibitortreatment. Numbers of cells orcolonies were counted andcompared with that of HCT116-EGFP vector cells. C–E arepresented as the mean and 95%confidence intervals (error bars)from 3 independent experiments.P values were calculated usingtwo-sided Student t test. �, P <0.05; ��, P < 0.01; ��, P < 0.001.GAPDH, glyceraldehyde-3-phosphate dehydrogenase.






novel mechanism in modulating EGFR protein levels andprovides additional layer of feedback control in EGFR signal-ing. In addition to EGFR, c-Cbl contributes to ubiquitinationand downregulation of receptors for platelet-derived growthfactor, VEGF, fibroblast growth factor, hepatocyte growthfactor, macrophage colony-stimulating factor 1, neutrophin,and insulin, as well as members of non–receptor tyrosinekinase Src family (26). It will be interesting to examine whetherCTEN may regulate the stabilities of these proteins throughbinding to c-Cbl.

We have shown that a functional SH2 domain is essential forCTEN's role in promoting cancer cell migration, invasion, as

well as colony formation, and that CTEN SH2 domain binds toc-Cbl and prolongs EGFR signaling. These results indicate thatthe prolonged EGFR signaling is responsible for transforma-tion properties, which is supported by the fact that EGFRinhibitor could reverse CTEN-mediated phenotypes. More-over, SH2 domains of tensin members also interact withphosphoinositide 3-kinase (PI3K), p130Cas, focal adhesionkinase, phosphoinositide-dependent kinase-1 (PDK-1), anddownstream of tyrosine kinase 2 (Dok-2; refs. 27–29). Thesebinding partners may all contribute to CTEN-enhanced cancerphenotypes. On the other hand, SH2 domains of CTEN andtensins bind to the tumor suppressor DLC1 (deleted in liver

GSE15852

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(n=43 pairs)

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Figure 5. CTEN mRNA expression is upregulated in human tumor samples. The gene expression data of 43 breast cancer (GSE15852), 24 colon cancer(GSE10950), 20 non–small cell lung cancer (GSE12236), and 45 pancreatic ductal adenocarcinoma (GSE28735) with matched normal sampleswere obtained from the NCBI GEO database. Dot plots for CTEN and EGFR mRNA expression were created from the indicated gene expressiondatasets. N, normal; T, tumor. The changes in CTEN and EGFR in four gene expression datasets were shown in boxes. Comparisons of mean values andSDs between the normal and tumor groups were analyzed using the Partek Genomics Suite software. Statistical significance of the differenceswas analyzed using paired Student t test.

Hong et al.





cancer 1) in a phosphotyrosine-independent manner, which isrequired for DLC1's tumor suppression activity (14). Thefunction of recruiting DLC1 to focal adhesion for preventingcell transformation seems contradictory to the phenotypesobserved in Fig. 4. One likely reason is that DLC1 is down-regulated or absence in many cancer cell lines includingHCT116 cells used in this study. Nonetheless, these findingsalso implicate the complexities of CTEN regulatory networks innormal cells.Upregulation and aberrant activation of EGFR are highly

associated with tumor progression as well as therapeuticresistance in human patients with cancer (30, 31). EGFR-targeted therapies are used to treat several types of cancers

including colorectal, breast, and lung cancer. Althoughshowing promising effects in some patients, many otherpatients still suffer from side effects and resistance asso-ciated with anti-EGFR agents. The reasons that underlinethe anti-EGFR resistance have not yet been clarified, butmay be related to much higher EGFR levels/activitiesand RAS/RAF mutations in some patients (32). Consideringthat CTEN (i) regulates activated EGFR levels, (ii) is adownstream of the RAS/RAF/MEK pathway, (iii) plays rolesin tumor migration/invasion, and (iv) is expressed at verylow levels in most normal tissues, targeting CTEN alongwith EGFR might be a more efficient approach for cancertherapies.

B

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Figure 6. CTEN and EGFR protein levels show a positive correlation in human lung cancer and normal tissue microarray. A, representative imagesof CTEN and EGFR immunohistochemistry: negative (0), weak (1), moderate (2), and strong (3). Scale bars, 200 mm. B, statistical analysis andrepresentative images for CTEN and EGFR correlation. Results were acquired from the IHC staining in 96 cases of human lung tissue microarray.Note: IHC analysis comparing lung cancer and cancer-adjacent tissue to normal tissues indicated that increased EGFR expression in CTEN-upregulated lung cancer was statistically highly significant (P < 0.001). The relationship between EGFR and CTEN expression was evaluated throughthe Fisher exact test.






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

Authors' ContributionsConception and design: S.-Y. Hong, T. Li, S.H. LoDevelopment of methodology: S.-Y. Hong, Y.-P. ShihAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.-Y. Hong, T. Li, K.L. Carraway IIIWriting, review, and/or revision of the manuscript: S.-Y. Hong, Y.-P. Shih,T. Li, K.L. Carraway III, S.H. LoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S.-Y. HongStudy supervision: S.H. Lo

Grant SupportY.-P. Shih is a T32 fellowship recipient (T32CA108459). T. Li is supported by

UL1 RR024146 from the National Center for Research Resources and Universityof California-Davis Comprehensive Cancer Center Developmental Award fromNational Cancer Institute (P30CA093373). This study was supported by NIHgrants CA102537 (S.H. Lo) and CA123541 (K.L. Carraway III).

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 indicate thisfact.

Received December 4, 2012; revised May 17, 2013; accepted June 3, 2013;published OnlineFirst June 17, 2013.

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2013;73:5266-5276. Published OnlineFirst June 17, 2013.Cancer Res Shiao-Ya Hong, Yi-Ping Shih, Tianhong Li, et al. Ligand-Induced DegradationCTEN Prolongs Signaling by EGFR through Reducing Its

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CTEN Prolongs Signaling by EGFR through Reducing Its ... · Tumor and Stem Cell Biology CTEN Prolongs Signaling by EGFR through Reducing Its Ligand-Induced Degradation Shiao-Ya Hong1,