Molecular Cancer Therapeutics - Potential Advantages of ......Preventing Cancer Cell Migration and...

Chemical Therapeutics

Potential Advantages of CUDC-101, a Multitargeted HDAC,EGFR, and HER2 Inhibitor, in Treating Drug Resistance andPreventing Cancer Cell Migration and Invasion

JingWang1, NatalieW. Pursell1, Maria Elena S. Samson1, Ruzanna Atoyan1, AnnaW.Ma1, Abdelkader Selmi2,Wanlu Xu1, Xiong Cai1, Maurizio Voi1, Pierre Savagner2, and Cheng-Jung Lai1

AbstractCUDC-101 is a novel, small-molecule, anticancer agent targeting histone deacetylase (HDAC), EGF

receptor (EGFR), and HER2. It is currently in phase I clinical development in patients with solid tumors.

Previously, we reported that CUDC-101 has potent antiproliferative and proapoptotic activity in cultured

tumor cells and in vivo xenograft models. We now show that cancer cells that have acquired resistance to

single-target EGFR inhibitors through upregulation of AXL or loss of E-cadherin remain sensitive to

CUDC-101, which inhibits MET- and AXL-mediated signaling, restores E-cadherin expression, and

reduces cell migration. CUDC-101 also efficiently inhibited the proliferation of MET-overexpressing

non–small cell lung cancer and gastric cancer cell lines and inhibited the migration and invasion of

invasive tumor cells. Taken together, these results suggest that coupling HDAC and HER2 inhibitory

activities to an EGFR inhibitor may potentially be effective in overcoming drug resistance and preventing

cancer cell migration. Mol Cancer Ther; 12(6); 925–36. �2013 AACR.

IntroductionDuring the past several years, 4 EGF receptor (EGFR)

inhibitors, gefitinib, erlotinib, cetuximab, and panitumu-mab, have been approved and have become standard-of-care for use in patients with cancer. However, the activityreported in unselected patients has been quite limited.In addition, those patients who achieve clinical benefittypically develop drug resistance (1–3). The extent ofintrinsic and acquired resistance to EGFR inhibitors thusleaves ample room for further anticancer drug develop-ment. Several potential mechanisms of resistance toEGFR inhibitors have been identified in the last decade,including secondary EGFRmutations (4–6), expression ofa constitutively active form of EGFR (EGFRvIII; ref.7),hepatocyte growth factor (HGF)-MET pathway activation(8, 9), HER2 and HER3 reactivation and overexpression-induced oncogenic shift (10), subcellular EGFR reloca-lization (11), ubiquitination-induced EGFR activity (12),increased VEGF production (13), overexpression of type 1insulin-like growth factor receptor (IGF-1R; ref. 14), and

most recently overexpression of AXL (15). Notably, EGFRinhibitor-resistant tumor cell lines generated in vitrohave shown cross-resistance to other receptor tyrosinekinase inhibitors (16), suggesting that combination ther-apy or inhibitors targeting multiple oncogenic pathwaysmay be advantageous in overcoming resistance to EGFRinhibitors.

Previously, we reported the synthesis and characteri-zation of a novel, multifunctional small molecule, CUDC-101, which simultaneously inhibits histone deacetylase(HDAC), EGFR, and HER2 (17, 18). The multifunctionalactivity of CUDC-101 offers potential advantages oversingle-target molecules. Compared with other EGFR andHER2 inhibitors, CUDC-101 is additionally able to reducethe levels of total and phosphorylated MET through itsHDAC inhibitory activity in the non–small cell lungcarcinoma (NSCLC) cell line H1993 (18). Results fromboth preclinical studies in tumor cell lines and clinicaltrials have identified MET pathway activation as animportant mechanism of resistance to EGFR inhibitorswith MET amplification being responsible for 5% to 20%of cases of resistance in NSCLC (8, 19, 20). Activation ofMET and its ligand HGF are involved in tumor cellproliferation, survival, and invasion (21). Thus, the abilityof CUDC-101 to control MET levels may prove to bebeneficial in controlling tumor cell proliferation, survival,and invasion.

MET-dependent tumor cell migration and invasionrequires phosphoinositide 3-kinase (PI3K) pathway activ-ity and is suppressed by the cell–cell adhesion receptorE-cadherin, which is also an important determinant oftumor progression (22, 23). Loss of E-cadherin is sufficient

Authors' Affiliations: 1Curis, Inc. Lexington, Massachusetts; and 2InstitutdeRecherche enCanc�erologie deMontpellier (IRCM), InstitutNational de laSant�e et de la RechercheMedicale (INSERM), U896, Universit�eMontpellierI, Montpellier, France

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

Corresponding Author: C.-J. Lai, Curis, Inc. 4 Maguire Rd, Lexington,MA 02421. Phone: 617-503-6675; Fax: 617-503-6501; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-12-1045

�2013 American Association for Cancer Research.

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on September 15, 2020. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst March 27, 2013; DOI: 10.1158/1535-7163.MCT-12-1045

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to induce cell migration and invasion (23–25), which mayalso contribute to EGFR inhibitor resistance. Erlotinib-sensitive cell lines express high levels of E-cadherin andshow limited invasion and migration capabilities, where-as many drug-resistant lines express low levels of E-cadherin and are highly invasive (26–28). These findingssuggest that the combination of EGFR inhibitors withagents that inhibit migration and invasion may help toovercome drug resistance. HDACs suppress E-cadherinexpression when recruited by transcription factors, suchas ZEB1 and Snail, to the E-cadherin promoter (29, 30).Accordingly, HDAC inhibitors increase E-cadherin levelsand inhibit migration and invasion of prostate cancer andNSCLC cell lines (31, 32). The HER2 antibody trastuzu-mab is also able to increase E-cadherin levels in HER2-overexpressing breast cancer cell lines (33). These resultssuggest that the addition of HDAC and HER2 inhibitoryactivities to EGFR inhibitors may provide therapeuticbenefits in cases of EGFR resistance.

CUDC-101 targets HDACs, EGFR, and HER2 withnanomolar potency. In the present study, we testedwhether the HDAC and HER2 inhibitory activities ofCUDC-101 could complement its EGFR inhibitory activityby suppressing drug resistance and related tumor cellmotility changes in EGFR inhibitor-resistant tumor cells.We also compared its antiproliferative and antimetastaticeffects with other clinically available EGFR and HDACinhibitors by evaluating changes in migration, invasion,and epithelial–mesenchymal transition (EMT) markers.Our results indicate that CUDC-101 is able to efficientlycontrol the proliferation and migration of erlotinib-resis-tant NSCLC HCC827 cells and reduce tumor cell prolif-eration, migration, and invasion at least partially throughdownregulating MET pathway activity and E-cadherininduction. Taken together, these results suggest thatCUDC-101 may potentially be effective in overcomingdrug resistance and preventing cancer cell migration.

Materials and MethodsCells and reagents

CUDC-101, vorinostat, erlotinib, gefitinib, and lapatinibwere synthesized as described (17). The PCR primer pairsused for detection of E-cadherin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained fromApplied Biosystems and Life Technologies (E-cadherin:Applied Biosystems Catalog # 4453320; GAPDH: LifeTechnologies Catalog # 4326317E). The real-time PCR(RT-PCR) primers used in amplification of the EGFRT790M mutation were also custom-synthesized atApplied Biosystems as previously described (34). Cancercell lines were obtained from the American Type CultureCollection within 6 months of this study and were main-tained according to the supplier’s instructions. Noauthentication was done by the authors. Drug-resistantHCC827 clones were generated by continuous exposureof early-passage cells (p3) to 1 mmol/L of drug for 2months. Single clones were selected and characterized.Cells were treatedwith 50 ng/mLHGF (R&D Systems) or

100 ng/mL EGF (R&D Systems) in serum-free culturemedium unless otherwise indicated.

Cell growth inhibition assayCells were plated at 5,000 cells per well in complete

culturing medium in 96-well tissue culture plates for 24hours and then incubatedwith indicated compounds for 3days at concentrations of up to 20 mmol/L in mediumcontaining 0.5% FBS. After 72 hours, cell viability wasmeasured using the Promega Cell Titer-Glo LuminescentCell Viability Assay. Growth inhibitory IC50s were calcu-lated using GraphPad Prism 5 software.

Immunoblotting, immunocytochemistry, andimmunohistochemistry

Immunoblotting, immunocytochemistry, and immuno-histochemistry were conducted using standard proce-dures. Blocking solutions for immunoblotting werepurchased from Li-Cor Bioscience and used with theindicated primary antibodies (Cell Signaling Technology)and IRdye 680 and 800CW secondary antibodies (CellSignaling Technology). Bovine serum albumin (BSA)/horse serum-basedblocking solutions containing the indi-cated primary antibodies, and Alexa 488 and Alexa546 secondary antibodies (Cell Signaling Technology)were used for immunocytochemistry and immunohisto-chemistry. 40, 6-diamidino-2-phenylindole (DAPI) wasused for nuclear staining.

Wound healing cell migration assayThe wound healing cell migration assays were con-

ducted as described previously (35–38). Briefly, MDA-MB-231, HCC827, or erlotinib-resistant cells were platedat 100,000 cells per well in complete medium in 24-wellplates and allowed to form a confluent monolayer. Awound was introduced by running a P200 pipette tipevenly across themonolayer.Migrationwas then inducedwith serum-free medium containing either 50 ng/mLHGF or 100 ng/mL EGF. Calcein AM (BD Biosciences)was used to label viable cells. Fluorescencewasmeasuredwith an excitation wavelength of 549 nm and an emissionwavelength of 565 nm from the bottom of plates with aBiotech Synergy2 Plate Reader.

Tumor cell migration and Matrigel invasion assaysMDA-MB-231 andHT-1080Boyden chambermigration

and Matrigel invasion assays were conducted with amodified commercially available kit (BD Biosciences;ref. 39). Briefly, MDA-MB-231 cells or HT-1080 cells inlogarithmic growth phase were trypsinized and sus-pended in serum-free medium (containing 0.04% BSA)at densities of 500,000 cells/mL or 250,000 cells/mL. FBS(10% or 5%) was added to the bottom chamber with orwithout test compounds to serve as a chemoattractant.After 21 hours of incubation in a 37�C incubator, cells thathad migrated or invaded to the lower wells were incu-bated with Calcein AM (BD Biosciences) to fluorescentlylabel viable cells. Fluorescence was measured with an

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Mol Cancer Ther; 12(6) June 2013 Molecular Cancer Therapeutics926




excitation wavelength of 549 nm and an emission wave-length of 565 nm from the bottom of the chamber.

EMT AssayHaCaT and NBT-II cells were plated at 50,000 cells per

well in low-serum (1% FBS) medium in 24-well platesfor 24 to 48 hours. The cells were treated for 2 days withor without CUDC-101, erlotinib, or vorinostat. Growthfactors [EGF or hepatocyte growth factor (HGF)] wereadded at a concentration of 20 ng/mL after one hour ofdrug treatment. After 48 hours, cells were fixed andtreated for immunofluorescence using anti-desmopla-kins (DP1 2 2.15; ARP) and anti-cytokeratins (18-0059;Novex) antibodies to visualize functional intercellularjunctions. For EMT quantification, cells were sorted into3 phenotype-based groups based on immunofluores-cence results: "full EMT" (isolated cells), "partial EMT"(intercellular junctions involving 20%–80% of a cell’sintercellular contact area), and "no EMT" (intercellularjunctions involving more than 80% of a cell’s intercel-lular contact area).

ResultsGeneration of drug-resistant NSCLC HCC827 cellsTo explore the antidrug resistance activity of CUDC-

101, we first attempted to generate CUDC-101–resistantcells fromEGFR inhibitor-sensitiveNSCLCHCC827 cells,which harbor EGFR gene amplification and Exon 19 (E19)deletion (40). As shown in Supplementary Table ST1 andSupplementary Fig. S1, the growth inhibitory IC50 ofCUDC-101 (0.09 mmol/L) is very similar to the growthinhibitory IC50s of erlotinib and gefitinib (0.03 mmol/L) inthe parental HCC827 cells. When HCC827 cells weretreated with 1 mmol/L of these compounds, less than onepercent of cells survived at the end of a 72-hour incuba-tion. However, resistant HCC827 clones arose followingtreatment with 1 mmol/L erlotinib or gefitinib for anadditional week, but not with 1 mmol/L of CUDC-101(Fig. 1B), suggesting that a subset of cells intrinsicallyresistant to EGFR inhibitors continually survive and pro-liferate despite being in the presence of erlotinib andgefitinib, but do not in the presence of CUDC-101.

Erlotinib-resistant cells are sensitive to CUDC-101treatmentTo explore the activity of CUDC-101 against EGFR

inhibitor-resistant cells, we selected 6 erlotinib-toler-ant/resistant (TR) single clones after 2 months of drugexposure and compared their sensitivity with CUDC-101 and EGFR inhibitors. All HCC827-TR clones wereno longer sensitive to erlotinib and gefitinib with a 500-fold or greater increase in growth inhibitory IC50 rela-tive to the parental cell line (Supplementary Table ST1).In contrast, their sensitivity to CUDC-101 did notchange significantly. The fact that all HCC827-TR clonesremain sensitive to CUDC-101 indicates that CUDC-101may be able to circumvent common EGFR-inhibitorresistance mechanisms.

Potentialmechanisms of drug resistance in erlotinib-TR clones

To identify the mechanisms of erlotinib resistanceactive in the HCC827 erlotinib-TR clones, we evaluateda panel of signal transduction markers related to drugresistance. As shown in Fig. 1C, all 6 resistant clonesexpress significantly higher levels of AXL, a tyrosinekinase receptor involved in promoting tumor cell growth,migration, invasion,metastasis, and angiogenesis (41, 42).Clones TR13, TR14, TR19, and TR20 also showed reducedE-cadherin and increased N-cadherin levels (Fig. 1C).Clones TR14, TR20, and TR21 had reduced EGFR proteinexpression, suggesting an intrinsic heterogeneity in theparental cell line or a loss of dependence on EGFR (Fig.1C). Very low levels of the T790M EGFR mutation weredetected in clones TR13, TR19, and TR24 compared withH1975 NSCLC cells (Fig. 1D), in which the T790Mmutantrepresents 69% of EGFR transcripts (16). This observationsuggests that these clones acquired a few copies of theT790M-mutant EGFR gene while still expressing a largeamount of erlotinib-sensitive E19-deleted EGFR protein.Regardless of their EGFR expression levels, all TR clonesshow similar levels of AKT activation (Fig. 1C). However,this AKT activity is not due toMET amplification or IGF-1Roverexpression as reported elsewhere, as no significantincrease in MET and IGF-1R protein levels were detectedin the TR clones (Fig. 1C). In fact, MET pathway signalingwasdecreased in several of theTR clones as showedby thedecreased levels of phosphorylated MET in clones TR13,TR14, TR19, and TR24 (Fig. 1C).

CUDC-101 treatment reverses drug resistance-relatedmolecular changes in erlotinib-resistant cells

To further understand the mechanisms by whichCUDC-101 circumvents EGFR inhibitor resistance, weselected a few resistant clones and characterized theirresponse to CUDC-101. As shown in Fig. 2A and Supple-mentary Fig. S2, both erlotinib and CUDC-101 inhibitedactivation of EGFR, MET, and AKT in the parental cells,whereas only CUDC-101 was able to prevent MET andAKT activation in the erlotinib-resistant cells. AXL levelswere also significantly reduced in the HCC827 erlotinib-TR cells after treatment with CUDC-101 (Fig. 2A). Fur-thermore, in one of the clone expressing low levels ofE-cadherin (TR19), CUDC-101 was able to restore andfurther increase E-cadherin mRNA and protein levelsand decrease expression of the E-cadherin suppressorZEB1 in a dose-dependent manner (Fig. 2B and C). Theseresults indicate that CUDC-101 can counteract potentialmechanisms of drug resistance in erlotinib-resistantNSCLC cells.

Because AXL, E-cadherin, and N-cadherin are knownto play important roles in drug resistance and metas-tasis (15, 43–45), we further investigated the effects ofCUDC-101 on cell migration. To this end, we showedthat CUDC-101 reduced HGF-induced migration ofTR19 cells in the in vitrowound-healing migration assaymore effectively than erlotinib (Fig. 2D). These data

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suggest that CUDC-101 may potentially overcome erlo-tinib resistance by controlling both cancer cell prolifer-ation and migration.

Cancer cells harboring MET amplification aresensitive to CUDC-101

Amplification of the MET proto-oncogene has beenidentified as amechanismof resistance to EGFR inhibitors

in both in vitro studies and patient samples (8). Theresulting MET overexpression leads to HER3 activationindependent of EGFR, allowing continued activation ofdownstream signaling in the presence of EGFR inhibitors(8). This improper activation of MET has been shown tobestow proliferative, survival, and invasive/metastaticproperties on cancer cells (46). To investigate the effectof CUDC-101 in the presence of MET amplification, we

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Figure 1. CUDC-101 circumvent drug resistance in HCC827 NSCLC cells. A, molecular structures of CUDC-101, erlotinib, gefitinib, lapatinib, and vorinostat.B, drug-resistant colony formation of parental HCC827 cells after exposure to 1 mmol/L erlotinib, gefitinib, or CUDC-101 for one week. C, immunoblotting ofindicated markers in parental and erlotinib-TR HCC827 cells. D, TaqMan RT-PCR quantification of T790M-mutant EGFR mRNA levels in parental anderlotinib-resistant HCC827 cells. H1975 NSCLC cells were used as a positive control. MW, molecular weight.

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surveyed a panel of tumor cell lines for MET expressionlevels and showed that NSCLC cell lines (H1993, NCI-H441, and EBC-1) and gastric tumor cell lines (Hs746T,SNU-5, and MKN45) expressing high levels of MET aresensitive to CUDC-101 treatment at submicromolar IC50s(Fig. 3A). In contrast, the HDAC inhibitor vorinostat andthe EGFR inhibitor erlotinib showed minimal or nogrowth inhibitory effect in most of these cell lines (Fig.3A). Treatment with CUDC-101 significantly reduced thelevels of phosphorylated and total MET protein anddecreased the levels of phospho-AKT (Fig. 3B), whichmediates MET-induced cell migration and invasion (21).Vorinostat slightly decreased the levels of these proteins,

albeit to a lesser extent than CUDC-101, whereas erlotinibhad no effect. These results indicate that tumor cellsexpressing high levels of MET are resistant to treatmentwith the EGFR inhibitor erlotinib but remain sensitive toCUDC-101 treatment.

CUDC-101 reduces tumor cell migration andinvasion

To explore the antimetastatic activity of CUDC-101, wenext investigated the effects of CUDC-101 on highlyinvasive tumor cell lines. An in vitro wound-healingmigration assay of MDA-MB-231 human breast carcino-ma cells showed that HGF- and EGF-induced migration

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Figure 2. CUDC-101 inhibits MET signaling, reduces AXL levels, attenuates cell migration, and restores E-cadherin expression in erlotinib-resistant HCC827cells. A, immunoblotting of parental and erlotinib-TR (TR13 and TR21) HCC827 cells treated with 1 mmol/L of CUDC-101 or erlotinib in the presenceof 50 ng/mL HGF for 24 hours. B, immunoblotting of E-cadherin and ZEB1 in TR19 cells treated with CUDC-101 at the indicated concentrations in thepresence of 50 ng/mL HGF. C, TaqMan RT-PCR quantification of TR19 cells treated with CUDC-101 at the indicated concentrations in the presence of50 ng/mL HGF. Statistical significance was determined by a standard t test (��,P < 0.01; �, P < 0.05). D, HGF-induced (50 ng/mL) cell migration of erlotinib-TRclone TR19 treated with 1 mmol/L CUDC-101 or erlotinib in the wound-healing migration assay.






was significantly reduced by 1 mmol/L CUDC-101,whereas erlotinib and vorinostat were less effective evenat higher concentrations (Fig. 4AandB).Using theBoydenchambermigration assay andMatrigel invasion assay, wefurther showed the effect of CUDC-101 on tumor cellmigration and invasion inMDA-MB-231 cells andHT1080human fibrosarcoma cells upon serum induction (Fig. 4CandD). The inhibition ofmigration and invasionwasnot asecondary effect of cell death or growth inhibition, as wedetected no significant decrease in cell viability under allconditions tested (Supplementary Fig. S3).

CUDC-101 modulates migration and invasionmarkers

We next characterized the molecular mechanism ofCUDC-101–mediated inhibition of tumor cell migrationand invasion inMDA-MB-231 cells.As shown in Fig. 5A, 2hours of CUDC-101 treatment decreased EGF-inducedEGFR and MET phosphorylation and decreased HGF-inducedEGFRphosphorylation. In addition to 24 hours ofCUDC-101 treatment significantlydecreasedMETproteinlevels, likely through its HDAC inhibitory activity as wereported previously (18). At the same time, p21 proteinlevels were also significantly increased, suggesting thatCUDC-101 induces cell-cycle arrest in these cells (Fig. 5A).

MDA-MB-231 cells possess a mesenchymal phenotypeand characteristically express low levels of E-cadherinand high levels of mesenchymal markers such as vimen-tin. Our results show that 24-hour treatment with bothEGF and HGF decreases E-cadherin levels whereas 24-hour treatment with CUDC-101 restores and furtherincreases E-cadherin levels in these cells (Fig. 5A). ThisCUDC-101–induced E-cadherin accumulation was alsoconfirmed by immunofluorescence staining (Fig. 5B, left).

Simultaneously, CUDC-101 also induced morphologicchanges as shown by staining for vimentin (Fig. 5B, right),a type of major intermediate filament protein that is amarker for mesenchymal cells. It has been reported thatvimentin fibers exhibit a curved filamentous structurewhen cells are not migrating and become straight andarranged along the long axis of migrating cells (47). Ourresults show that upon HGF stimulation, MDA-MB-231cells become spindle-like and the vimentin fibers straight-en and reorganize along the long axis. When treated withCUDC-101 for 24 hours, the vimentin filaments becomecurvy again, indicative of impaired migration capacity.

Consistent with previous reports (31–33), we detected aslight increase of E-cadherin levels in response to theHDAC inhibitor vorinostat, the HER2 inhibitor lapatinib,and their combination, but not in response to erlotinib(Fig. 5C). However, CUDC-101 treatment increased E-cadherin levels with significantly greater potency thanvorinostat, lapatinib, or their combination (Fig. 5C). Treat-ment with CUDC-101 also slightly decreased vimentinlevels over time (Fig. 5A andC). E-cadherin inductionwasonly observed after 24hours of treatmentwithCUDC-101,suggesting that protein synthesis may be required. RT-PCR also showed that CUDC-101 induced E-cadherinaccumulation at the transcriptional level (Fig. 5D). Inagreement with these findings, CUDC-101 treatmentdecreased the level of ZEB1 (Fig. 5A), a zinc-finger tran-scriptional repressor of E-cadherin (48). We observed asimilar decrease in ZEB1 levels following CUDC-101treatment in our erlotinib-TR HCC827 clones (Fig. 2B).Taken together, these results show the ability of CUDC-101 to block cell migration at the molecular-level andsuggest that CUDC-101–induced inhibition of tumorcell migration may be due to its suppression of ZEB1

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Figure 3. CUDC-101 inhibitsgrowth of MET-overexpressingtumor cell lines through inhibitionof MET signaling. A, growthinhibitory IC50s of erlotinib,vorinostat, and CUDC-101 inMET-overexpressing NSCLC celllines and gastric cancer cell lines.B, immunoblotting of indicatedmarkers in H1993, NCI-H441, andHs746T cells treated with 1 mmol/Lerlotinib, vorinostat, or CUDC-101for 16 hours.

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expression and the subsequent increase of E-cadherinexpression through transcriptional regulation.

CUDC-101 inhibits EGF-induced EMTBecause CUDC-101 induces expression of the epithelial

marker E-cadherin and reduces expression of the mesen-chymal marker vimentin, we next investigated whetherCUDC-101 is able to inhibit EMT in a functional assayusing normal human keratinocytes (HaCaT). In theabsence of growth factor treatment, HaCaT cells have acuboidal, cobblestone morphology with tight cell–cellcontacts characteristic of normal epithelial cells. Treat-ment with EGF leads to the loss of cell–cell contacts andthe emergence of a spindle-shaped, fibroblast-like mes-enchymal morphology. Treatment with CUDC-101 inhi-bits these EGF-induced morphologic changes andrestores the epithelial phenotype, whereas vorinostatonly partially inhibits EMT (Fig. 6A). To quantifythe EMT phenotype, the percentage of EGF-inducedpartial and full cell–cell dissociation was scored andquantified. CUDC-101 completely inhibits full dissoci-ation and decreases partial dissociation, whereas erlo-tinib and vorinostat are comparatively less effective(Fig. 6B). This result suggested that CUDC-101 mightbe able to reduce EMT.Taken together, our results show that CUDC-101 effi-

ciently inhibits the growth of EGFR inhibitor-resistantcells and MET-amplified cells and reduces tumor migra-tion and invasion, suggesting that it may be a moreeffective clinical agent than single-agent inhibitors.

DiscussionTargeted cancer therapies are promising treatment

strategies, but only a subpopulation of patients respondsto treatment and many eventually develop drug resis-tance. In this study, we showed the antidrug resistanceand antimetastatic activities of CUDC-101, a multitargetHDAC, EGFR, and HER2 inhibitor. Our results suggestthat EGFR, HER2, and HDAC inhibition synergizes toovercome EGFR inhibitor resistance. We also providedsupporting evidence for AXL overexpression and loss ofE-cadherin expression as erlotinib resistancemechanisms(15, 26–28), underscoring the connection between drugresistance and metastatic processes.In this study, AXL was overexpressed in all 6 erlotinib-

TRHCC827 clones, highlighting the importance ofAXL inresistance to EGFR inhibitors. AXL is a tyrosine kinasereceptor expressed in various types of cancer and isinvolved in promoting tumor cell growth, migration,invasion, metastasis, and angiogenesis (41, 42). Overex-pression of AXL has been reported as a mechanism ofacquired lapatinib resistance in breast cancer cells (49),acquired imatinib resistance in gastrointestinal stromaltumors (50), and most recently erlotinib resistance inNSCLC cells and patient specimens (15). In addition, ananti-AXL monoclonal antibody YW327.6S2 has beenreported to enhance the effect of erlotinib in NSCLCmodels (51). PI3K is a potential downstream mediator of

AXL signaling (52), suggesting that AXL overexpressionmight be responsible for the observed activation of thePI3K pathway in our HCC827 erlotinib-TR clones.

A recent study found that AXL expression wasincreased (2-fold or more) in 20% of samples of patientswith NSCLC following the development of resistance tothe EGFR inhibitors erlotinib or gefitinib (15), indicatingthe importance of controlling AXL overexpression intreating EGFR inhibitor–resistant NSCLC. Although themechanism of AXL overexpression is unknown, recentstudies examining AXL overexpression-mediated EGFRinhibitor resistance in NSCLC cells found that AXL wasnot amplified or mutated (15), suggesting that AXL maybe upregulated at the transcriptional level, perhapsby epigenetic mechanisms. Treatment of our HCC827erlotinib-TR clones with CUDC-101 resulted indecreased levels of AXL, raising the possibility thatCUDC-101–mediated HDAC inhibition may suppressAXL expression.

Several of the erlotinib-TR clones identified in this studyalso had reduced levels of the EGFR protein. The obser-vation of decreased EGFR protein levels has not, to ourknowledge, been reported previously as a mechanism ofEGFR inhibitor resistance and is a subject for futurestudies. However, regardless of the baseline expressionlevel in erlotinib-resistant cells, CUDC-101 was able tofurther reduce levels of total and phosphorylated EGFRand MET and downstream phosphorylated AKT in theerlotinib-TR clones where erlotinib was ineffective. Aswith AXL levels, the observed effect of CUDC-101 on thetotal protein levels of EGFR and c-MET is likely due to itsHDAC inhibitory activity as we have shown previously(18).

Some erlotinib-resistant clones generated in this studyalso lost E-cadherin expression while gaining N-cadherinexpression, indicative of increased tumor cellmotility.Weshowed here that CUDC-101 could efficiently restore E-cadherin levels and control the migration of these erloti-nib-resistant cells. We further showed that CUDC-101treatment significantly decreases the motility of otherinvasive tumor cell lines while simultaneously increasinglevels of epithelial marker and decreasing mesenchymalmarker levels, indicating that CUDC-101 may potentiallyhave antimetastatic activity.

When compared with single-target EGFR, HER2, andHDAC inhibitors, CUDC-101 more potently induced E-cadherin expression at both the protein andmRNA levels.It has been reported that bothHDACandHER2 inhibitioncan induce E-cadherin expression (31–33). Our data areconsistent with these reports and suggest that combinedHDAC and HER2 inhibitory activity may underlie theincreased potency of CUDC-101 compared with single-target inhibitors. Although there is no knownmechanisticlink between EGFR and regulation of E-cadherin or othermigration markers, E-cadherin expression has been pro-posed to be an important predictive marker for EGFRinhibitor sensitivity (26, 27). The ability of CUDC-101 toefficiently restore E-cadherin levels suggests that tumors






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that are intrinsically resistant to or have acquired resis-tance to EGFR inhibitors may still be able to respond toCUDC-101 treatment. Mechanistically, CUDC-101 treat-ment decreased the level of ZEB1, a zinc-finger transcrip-tional repressor of E-cadherin (48), resulting in thesubsequent increase of E-cadherin expression throughtranscriptional regulation.

MET overexpression was not observed in the erlotinib-TR HCC827 NSCLC cells generated in this study, likelydue to differences in methods of generating resistantclones. To mimic clinical conditions, we chose to chron-ically expose the cells to 1 mmol/L of inhibitors, instead ofgradually increasing the concentration over a longerperiod of time as has been done previously (8). However,

Figure 5. CUDC-101 induceschanges in migration and invasionmarkers. A, immunoblotting ofserum-starved MDA-MB-231 cellstreated with EGF or HGF andCUDC-101 at the indicatedconcentrations for 2 or 24 hours.B, immunofluorescence staining ofE-cadherin (left) and vimentin (right)in serum-starvedMDA-MB-231cellstreated with CUDC-101 at theindicated concentrations in thepresence ofHGF. C, immunoblottingof E-cadherin and vimentin in MDA-MB-231 cells treated with CUDC-101, erlotinib, lapatinib, vorinostat,or the combination of lapatinib andvorinostat (LþV) at the indicatedconcentrations. D, gelelectrophoresis of traditionalRT-PCR for E-cadherin (E-cad; top)and TaqMan RT-PCR analysis ofE-cadherin mRNA levels normalizedto GAPDH mRNA levels (bottom) inMDA-MB-231 cells treated withCUDC-101, erlotinib, or vorinostatat the indicated concentrations.Statistical significance wasdetermined by a standard t test(��, P < 0.001).


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Figure 4. CUDC-101 reduces the migration and invasion of MDA-MB-231 and HT1080 cells. A, CUDC-101 reduces HGF- and EGF-induced MDA-MB-231(MM-231) cell migration in an in vitro wound healing assay. CUDC-101, erlotinib (Erl), vorinostat (Vor), HGF, and EGF were added to the cells at theindicated concentrations immediately after wounding. Live cells were visualized with Calcein AM at the indicated time points. B, quantification ofwound width. C, CUDC-101 reduces serum-induced migration and invasion of MDA-MB-231 and HT-1080 cells in the Boyden chamber migration assayand Matrigel invasion assay. Live cells migrating or invading to the other side of the chamber were visualized with Calcein AM. D, quantification ofCalcein AM intensity in both the Boyden chamber migration assay and Matrigel invasion assay. Statistical significance was determined by a standard t test(�, P < 0.05; ��, P < 0.01; ��, P < 0.001).






we also showed that CUDC-101 could block proliferationand MET signaling in MET-amplified NSCLC cells andgastric cancer cells, indicating that CUDC-101 might alsobe able to efficiently control EGFR inhibitor resistancecaused by MET amplification.

Initial intrinsic resistance or subsequent acquired resis-tance to EGFR inhibitors suggests that targeting a singlenode in a tumor signal transduction pathway may noteffectively control tumor growth. In this study, drug-resistant cells arose from EGFR inhibitor-sensitiveHCC827 cells in response to prolonged exposure to sin-gle-target EGFR inhibitors but not CUDC-101, showingthe potential advantages of simultaneously targetingmul-tiple nodes of signal transduction in cancer therapy. Inaddition, our findings indicate that erlotinib-TR HCC827NSCLC cells remain sensitive to CUDC-101 treatmentdespite activation of different resistance mechanisms,showing the broad use of CUDC-101 in controllingEGFR-resistant cancer cells.

In summary, we have shown that the multitargetHDAC, EGFR, and HER2 inhibitor CUDC-101 could con-trol the proliferation and migration of EGFR inhibitor-resistant NSCLC cells, and reduce tumor cell migrationand invasion. Thus, as a multitarget single agent, CUDC-

101 has the potential to be able to overcome drug resis-tance, which will be explored in future clinical studies.

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

Authors' ContributionsConception and design: J. Wang, N.W. Pursell, A. Selmi, X. Cai, M. Voi,C.-J. LaiDevelopment of methodology: J. Wang, N.W. Pursell, M.E.S. Samson,R. Atoyan, A. Selmi, P. SavagnerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.Wang,N.W. Pursell,M.E.S. Samson, R.Atoyan,A.W. Ma, A. Selmi, W. Xu, P. SavagnerAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): J. Wang, N.W. Pursell, A. Selmi, P.Savagner, C.-J. LaiWriting, review, and/or revision of the manuscript: J. Wang, N.W.Pursell, P. SavagnerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J. Wang, N.W. Pursell, M.E.S.Samson, R. AtoyanStudy supervision: J. Wang, C.-J. Lai

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 October 26, 2012; revised March 15, 2013; accepted March 15,2013; published OnlineFirst March 27, 2013.

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Figure 6. CUDC-101 decreasesEMT in HaCaT cells. A,immunofluorescence stainingof HaCaT normal humankeratinocytes treated with20 ng/mL EGF and 0.2 mmol/LCUDC-101, vorinostat (SAHA),or erlotinib. Desmoplakins (green)were used to locate intercellularjunctions, cytokeratins (red) wereused to estimate cytoplasmicexpansion, and DAPI (blue) wasused as a nuclear marker. B,quantification of partial andfull EMT. On the basis ofimmunofluorescence staining,cells were classified as isolated,partial EMT, or total EMT, asdefined in Materials and Methods.

Wang et al.





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2013;12:925-936. Published OnlineFirst March 27, 2013.Mol Cancer Ther Jing Wang, Natalie W. Pursell, Maria Elena S. Samson, et al. Cancer Cell Migration and Invasionand HER2 Inhibitor, in Treating Drug Resistance and Preventing Potential Advantages of CUDC-101, a Multitargeted HDAC, EGFR,

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