The Dual EGFR/HER2 Inhibitor Lapatinib Synergistically ... · 06/05/2011 · Melissa J. LaBonte 1, Peter M. Wilson , Will Fazzone1, Jared Russell2, Stan G. Louie2, Anthony El-Khoueiry

Therapeutics, Targets, and Chemical Biology

The Dual EGFR/HER2 Inhibitor Lapatinib SynergisticallyEnhances the Antitumor Activity of the Histone DeacetylaseInhibitor Panobinostat in Colorectal Cancer Models

Melissa J. LaBonte1, Peter M. Wilson1, Will Fazzone1, Jared Russell2, Stan G. Louie2,Anthony El-Khoueiry3, Heinz-Josef Lenz3, and Robert D. Ladner1

AbstractAs key molecules that drive progression and chemoresistance in gastrointestinal cancers, epidermal growth

factor receptor (EGFR) and HER2 have become efficacious drug targets in this setting. Lapatinib is an EGFR/HER2 kinase inhibitor suppressing signaling through the RAS/RAF/MEK (MAP/ERK kinase)/MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase)/AKT pathways. Histone deacetylase inhibitors(HDACi) are a novel class of agents that induce cell cycle arrest and apoptosis following the acetylation of histoneand nonhistone proteins modulating gene expression and disrupting HSP90 function inducing the degradationof EGFR-pathway client proteins. This study sought to evaluate the therapeutic potential of combining lapatinibwith the HDACi panobinostat in colorectal cancer (CRC) cell lines with varying EGFR/HER2 expression andKRAS/BRAF/PIK3CAmutations. Lapatinib and panobinostat exerted concentration-dependent antiproliferativeeffects in vitro (panobinostat range 7.2–30 nmol/L; lapatinib range 7.6–25.8 mmol/L). Combined lapatinib andpanobinostat treatment interacted synergistically to inhibit the proliferation and colony formation in all CRCcell lines tested. Combination treatment resulted in rapid induction of apoptosis that coincided with increasedDNA double-strand breaks, caspase-8 activation, and PARP cleavage. This was paralleled by decreased signalingthrough both the PI3K and MAPK pathways and increased downregulation of transcriptional targets includingNF-kB1, IRAK1, and CCND1. Panobinostat treatment induced downregulation of EGFR, HER2, and HER3 mRNAand protein through transcriptional and posttranslational mechanisms. In the LoVo KRAS mutant CRCxenograft model, the combination showed greater antitumor activity than either agent alone, with no apparentincrease in toxicity. Our results offer preclinical rationale warranting further clinical investigation combiningHDACi with EGFR and HER2-targeted therapies for CRC treatment. Cancer Res; 71(10); 1–14. �2011 AACR.

Introduction

Colorectal cancer (CRC) was the third leading cause ofcancer incidence and second deadliest malignancy in theUnited States with an estimated 143,000 new cases and51,000 deaths in 2010 (1). Despite advances in chemothera-peutic options for CRC, a high incidence of drug resistance anddisease progression remain a major stumbling block to effec-tive disease control. Currently, patientswithmetastatic diseasehave a 5-year survival of less than 10% (2–5). Many patients fail

all standard therapeutic options, while remaining candidatesfor continued therapy. There is a critical need formore effectivetreatment strategies for patients with metastatic CRC.

Members of the human epidermal receptor (HER) familyincluding the epidermal growth factor receptor (EGFR) and to alesser extent the HER2 and HER3 play key roles in driving theoncogenic pathways important for CRC growth and prolifera-tion, survival, angiogenesis, invasion, and metastasis (6). Tar-geting EGFR has shown efficacy in CRC patients with the use ofthe monoclonal antibodies cetuximab (7, 8) and panitumumab(9, 10). However, the therapeutic efficacy of these agents iscurrently limited toa subsetofCRCpatients and thepresenceofintrinsic resistance (such as KRASmutation) or the subsequentdevelopment of resistance represents a serious therapeuticchallenge (11). The identification of alternative approachesthat further disrupt EGFR-dependent tumor cell growth iscritical and may have significant clinical impact. The tyrosinekinase inhibitor (TKI) lapatinib (Tykerb) targets EGFR andHER2 and is approved for metastatic breast cancer and isunder clinical investigation in a variety of tumor types includingCRC (12, 13).

A novel class of anticancer agents that may improve theefficacy of HER-targeted agents are histone deacetylase

Authors' Affiliations: 1Department of Pathology; 2Clinical Pharmacy andPharmaceutical Sciences; and 3Division of Medical Oncology, NorrisComprehensive Cancer Center, University of Southern California, LosAngeles, California

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Robert D. Ladner, Department of Pathology,Room 5322, Norris Comprehensive Cancer Center, 1441 Eastlake Avenue,University of Southern California, Los Angeles, CA 90089. Phone: 323-865-3116; Fax: 323-865-0522; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-2430

�2011 American Association for Cancer Research.

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Published OnlineFirst April 4, 2011; DOI: 10.1158/0008-5472.CAN-10-2430

http://cancerres.aacrjournals.org/

inhibitors (HDACi). HDACi elicit their anticancer activitythrough the hyperacetylation of both histone and nonhis-tone proteins resulting in alterations in chromatin structure,transcriptional activity, gene expression changes, growtharrest, and apoptosis (14–16). HDACi can also induce theacetylation of heat shock protein 90 (HSP90), which is acritical molecular chaperone involved in maintaining cellu-lar homeostasis (17). Disruption of the HSP90 chaperonecomplex through acetylation results in the destabilization ofclient proteins critical for cancer cell survival and continuedproliferation including members of the HER family (18, 19).Importantly, variation in HSP90 dependency has beenreported in specific tumor subtypes that harbor HER-signal-ing aberrations including EGFR tyrosine kinase mutations innon–small cell lung cancer (NSCLC) and HER2 amplificationin breast cancer (19, 20). In addition to protein destabiliza-tion, HDACi are reported to induce potent transcriptionaleffects in multiple pathways that disrupt tumor progressionincluding HER signaling, dNTP biosynthesis, angiogenesis,invasion, and mitosis (21–23). These transcriptional effectsare reported to occur through inhibition of new transcriptsynthesis and destabilization and accelerated decay ofmature transcripts (22, 23).

Panobinostat (LBH589) functions as an HDACi-targetingclass I and II HDACs and has shown activity in hematologicand nonhematologic tumor models and is under extensiveclinical evaluation (24, 25). HDACi have shown synergisticantitumor efficacy with a variety of structurally diverse antic-ancer agents. Previously we reported that HDACi synergizedwith fluoropyrimidines in CRC cancer cells through down-regulation of the fluoropyrimidine target enzyme thymidylatesynthase (22) and we tested these observations in the clinicalsetting (26). Additional synergistic interactions with HDACihave been reported including combinations with the protea-some inhibitor bortezomib (27), and TRAIL (tumor necrosisfactor–related apoptosis-inducing ligand) agonist antibodies(28) and the EGFR TKI gefitinib in head and neck cancer (29).The molecular basis for these synergistic interactions isattributed to HDACi-induced changes in expression or activityof a specific drug target. Importantly, a recent study showedthat panobinostat induced downregulation of EGFR in mutantEGFR lung cancer cells and that combined treatment with theEGFR TKI erlotinib resulted in synergistic antiproliferativeeffects (19). An additional study also reported that the HDACiLAQ824 induced downregulation of HER2 in HER2-amplifiedbreast cancer cells resulting in a synergistic interaction withtrastuzumab (30).

As EGFR and HER2 are of key importance in promotingtumor cell proliferation and survival in CRC, we tested thehypothesis that combined targeting of EGFR and HER2 bylapatinib in combination with panobinostat would have syner-gistic antiproliferative effects in CRC models with differentmutational statuses.

Materials and Methods

Additional details can be found in the SupplementaryMethods.

Compounds and reagentsPanobinostat (LBH589) was provided by Novartis Pharma-

ceuticals. Vorinostat was provided by the National CancerInstitute. Entinostat (MS-275) and lapatinib were purchasedfrom LC Laboratories. CellTiter 96 Aqueous MTS was pur-chased from Promega. Epidermal growth factor (EGF) and(hydroxypropy)methylcellulose (HPMC) were purchased fromSigma-Aldrich. 17-(Allylamino)-17-demethoxygeldanamycin(17-AAG) was purchased from A.G. Scientific, Inc. Halt pro-tease and phosphatase inhibitor cocktail was purchased fromThermo Scientific.

Cell linesThe human CRC cell lines DLD-1, HCT116, HT29, LoVo, and

RKO were purchased from American Type Culture Collection.DLD-1, LoVo, and RKO were maintained in Dulbecco's mod-ified Eagle's medium (DMEM). HCT116 and HT29 were main-tained in McCoy's 5A. H630 CRC cells were a gift from EdwardChu at the Yale Cancer Center and maintained in DMEM.Media was supplemented with 10% FBS (Lonza) with peni-cillin/streptomycin, sodium pyruvate, and L-glutamine (Invi-trogen). Cells were maintained in a humidified Formaincubator (Forma) at 37�C with 5% CO2 and routinelyscreened for mycoplasma using the MycoALERT detectionkit (Lonza).

Growth inhibition assay and drug combination analysisThe CellTiter 96 AQueousMTS assay (Promega) was carried

out according to the manufacturers guidelines and as pre-viously described (22). IC50(72 hours) values were calculatedfrom sigmoidal dose–response curves utilizing Prism (Graph-pad). The combination effect was determined by the combi-nation index (CI) method (31) utilizing Calcusyn software(Biosoft). Fraction affected (FA) was calculated from thepercent growth inhibition: FA ¼ (100 – % growth inhibi-tion)/100. CI values less than 1, synergism; 1–1.2, additive;and more than 1.2, antagonism.

Colony formation assayThe colony formation assay was carried out as previously

described (32). Three concentrations of panobinostat wereselected to induce dose-dependent decreases in colonyforming capacity. Because lapatinib is considered a targetedagent, a fixed clinically meaningful dose of 3 mmol/L wasused to evaluate the combined drug effect. Following atransient 24 hours drug exposure, culture media wasreplaced with drug-free medium and cells were allowed togrow for 7 to 14 days. Colonies were then fixed, stained,counted, and drug-treated samples compared directly tountreated controls set at 100%.

Flow cytometric/sub-G1 analysisCell cycle and viability were determined by flow cytometric

analysis of DNA content after a 24-hour drug exposure aspreviously described (33). Cells were harvested and analyzedusing a Coulter EPICS ELITE flow cytometer (Beckman Coul-ter). Cell populations were quantified using Expo32 software(Beckman Coulter). Cells with DNA content less than 1 were

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considered apoptotic. Statistical analysis consisting of a2-tailed ANOVA was carried out using Prism (Graphpad).

ImmunoblottingImmunoblotting was preformed as previously described

(33). Cell lysates were prepared using radioimmunopreci-pitation assay (RIPA) buffer supplemented with Halt Pro-tease and Phosphatase Inhibitor. Protein concentrationswere quantified according to the BCA Protein Assay Kit(Pierce).

Quantitative real-time PCRRNA was isolated using TRizol according to manufacturer's

instructions (Invitrogen). cDNA was reverse transcribed using500 ng RNA using qScript cDNA Synthesis Kit according to themanufacturer's instructions and analyzed using PerfeCTaSYBR Green Supermix (Quanta Biosciences Inc.) and anApplied Biosystems 7500 PCR Detection System (AppliedBiosystems Inc.). Target genes (Supplementary Table S1) werenormalized to GAPDH (glyceraldehyde 3 phosphate dehydro-genase) and quantified using the comparative Ct method (34).Histograms and statistical analysis (2-tailed paired Student's ttest) were carried out using Prism (Graphpad).

In vivo analysisXenograft experiments were conducted in male C57Bl/6

BALB/c mice (Taconic Labs) that were 6- to 8-week old.Subcutaneous LoVo CRC xenografts were established andallowed to grow until they reached�100mm3 (day 0). Animalswere randomized to treatment groups: vehicle, panobinostat,lapatinib, and combination of panobinostat and lapatinib (n¼6, group). Panobinostat was administered at 2.5 mg/kg byintraperitoneal (i.p.) injection q.d. for 5 consecutive days aweek. Lapatinib was administered by oral gavage at 30 mg/kgBID for the duration of the study. Two perpendicular dia-meters of tumors were measured every 2 days with a digitalcaliper by the same investigator. Tumor volume (TV) was

calculated according to the following formula: TV (mm3) ¼[length (mm) � width (mm)2]/2. Animal body weight wasmeasured every 2 days as an index of toxicity.

Results

IC50(72 hours) growth inhibitory effects ofpanobinostat and lapatinib in CRC cell lines

Prior to evaluating lapatinib in combination with panobi-nostat, we first analyzed the antiproliferative activity of bothagents separately in a panel of 6 heterogenous CRC cell lines(Table 1). DLD-1, H630, HCT116, HT29, LoVo, and RKO CRCcells were exposed to increasing concentrations of lapatiniband panobinostat for 72 hours and growth inhibition wasmeasured by MTS assay. In all CRC cell lines tested, panobi-nostat showed concentration-dependent growth inhibitoryactivity with IC50(72 hours) values ranging between 4.2 and26 nmol/L (Table 1). Lapatinib exerted concentration-depen-dent growth inhibitory activity with IC50(72 hours) valuesranging from 5.5 to 25.9 mmol/L (Table 1). Consistent withour previous observations (33), the IC50(72 hours) valuesobtained for lapatinib in this short-term growth inhibitionassay are representative of cells which are relatively resistantto the growth inhibitory effects of lapatinib.

Panobinostat combined with lapatinib synergisticallyinhibits CRC cell proliferation and colony formation

We previously determined the EGFR and HER2 proteinexpression in a panel of CRC cell lines and showed that 4of 5 cell lines analyzed (DLD-1, H630, HCT116, HT-29, andLoVo) had significant EGFR expression with the exception ofthe H630 cell line which had low EGFR expression butexpressed HER2 at a significant level compared to the otherCRC cell lines (Table 1; ref. 33).

CRC cell lines were subsequently treated with increasingconcentrations of panobinostat and lapatinib alone andin combination for 72 hours and growth inhibition was

Table 1.Mutational status ofKRAS,BRAF, and PIK3CA and sensitivity of lapatinib (LAP) and panobinostat(LBH589) in 6 CRC cell lines

CRCcell line

Protein expressiona Mutation statusb IC50(72 hours)c

EGFR HER2 KRAS BRAF PI3KCA LAP (mmol/L) LBH589 (nmol/L)

DLD-1 þþ þ G13D Wt E454K 13.7 � 0.1 26.0 � 0.4H630 � þþþ Wt Wt Wt 11.3 � 0.3 5.3 � 0.1HCT116 þ þ G13D Wt H1047R 25.9 � 0.5 4.2 � 0.5HT29 þþ þ Wt V600E Wt 13.7 � 0.4 6.6 � 0.1LoVo þþþ þ G13D Wt Wt 8.9 � 0.2 9.3 � 0.1RKO þ � Wt V600E H1074R 9.2 � 0.5 10.5 � 0.2

aPreviously reported (29, 30).bMutation data compiled from Cancer Genome Project database http://www.sanger.ac.uk/genetics/CGP/CellLines/.cIC50 ¼ concentration of drug required to inhibit growth by 50% compared with vehicle treated controls and calculated in Prism 5.0(Graphpad). Values are the mean of 4 independent experiments � SEM.

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measured by MTS assay. The median-effect analysis method(31) was utilized to evaluate the combined drug effect.Simultaneous treatment with panobinostat and lapatinibresulted in synergistic increases in growth inhibition at 0.5FA and synergistic CI values of less than 1 over the majorityof concentrations tested in all CRC cell lines examined(Fig. 1). Of note, this interaction was observed in cell linesharboring activating mutations in the KRAS, BRAF, and

PIK3CA oncogenes. Temporal sequencing of lapatinib andpanobinostat was also explored in the H630 cell line andwhile concomitant treatment with both agents yielded thehighest FA, preincubating cells with either agent followedby the inverse also yielded additive to synergistic effectscompared to either agent alone (Supplementary Fig. S1). Inaddition, 2 additional HDACi were also evaluated in com-bination with lapatinib in the H630 and LoVo cell lines. In

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Figure 1. Synergistic growth inhibitory effects of panobinostat (LBH589) combined with lapatinib (LAP) in CRC cell lines. Growth inhibition was determined byMTS assay for 72 hours. Six CRC cell lines were exposed to increasing doses of LBH589 and LAP. Data points represent mean � SD percent growthinhibition (n ¼ 3 experiments) compared to controls at 100%. The combined drug effect was analyzed using the CI equation and presented with FA forcombinations. CI values less than 1 ¼ synergism; 1–1.2 ¼ additive; and more than 1.2 ¼ antagonism.

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the H630 cell line, lapatinib in combination with entinostatshowed synergistic interactions across the range of concentra-tions evaluated. In contrast, the combination with vorinostatyielded primarily additive effects and only at elevated concen-trations. In LoVo cells, lapatinib combined with entinostatyielded increases in the FA and additive to synergistic inter-actions across most concentrations tested. Lapatinib com-bined with vorinostat in the LoVo cells did not show anysignificant increase in the FA (Supplementary Fig. S2).A long-term clonogenicity assay was subsequently carried

out to assess the capacity of panobinostat and lapatinibcombinations to cause irreversible growth arrest in 4 CRCcell lines. Combined drug analysis was carried out using 3increasing concentrations of panobinostat that induced dose-dependent decreases in colony forming capacity. The con-centrations selected were well within clinically relevant doses(24). The selected panobinostat concentrations were alsocombined with 3 mmol/L lapatinib, a fixed clinically relevantdose for 24 hours followed by drug removal and outgrowth indrug-free medium (12). In all cell lines evaluated, increasingdoses of panobinostat alone resulted in a dose-dependentsuppression of colony formation (Fig. 2A). Combining 3mmol/L lapatinib with the increasing concentrations of pano-binostat resulted in significant suppression of colony forma-tion in all 4 cell lines examined (Fig. 2A and SupplementaryFig. S3).

Lapatinib enhances panobinostat-induced apoptosis inCRC cellsDLD-1, H630, HCT116, and LoVo CRC cells were treated

with 3 mmol/L lapatinib, 10 and 15 nmol/L panobinostat andcombinations for 24 hours and DNA content was analyzedby flow cytometry to measure the onset of apoptosis. Treat-ment with lapatinib did not induce any significant alterationsin the percentage of cells in sub-G1 (indicative of apoptosis)compared to control in any of the CRC cell lines (Fig. 2B).Treatment with 10 and 15 nmol/L panobinostat resulted in adose-dependent increase in the percentage of cells in sub-G1

in 3 of the 4 CRC cell lines. Interestingly the DLD-1 cellsshowed resistance to panobinostat-induced apoptosis withless than 5% of cells in sub-G1. However, the combinationsof 3 mmol/L lapatinib with both 10 and 15 nmol/L panobino-stat in the DLD-1 cells increased the number of apoptoticcells in sub-G1 to 24.1% and 30.1%. Significant increases inapoptosis were also observed in the H630, HCT116, and LoVocells with the addition of 3 mmol/L lapatinib to 15 nmol/Lpanobinostat increasing the number of cells in sub-G1 from28.6%, 24.9%, and 27.5% with panobinostat alone to 52.1%,43.4%, and 50.9%, respectively (Fig. 2B).The induction of DNA double-strand breaks and apoptosis

was assessed by Western blotting for gH2A.X (Ser139) andactivation of caspase-8 and PARP cleavage. H630 and LoVoCRC cells were treated as above for 18 and 24 hours. Lapatinibtreatment resulted in no detectable increase in gH2A.X whencompared to controls whereas panobinostat treatment aloneincreased gH2A.X as early as 18 hours in both H630 and LoVocells. Lapatinib plus panobinostat induced gH2A.X to a greaterextent in both cell lines when compared to either single agent.

Lapatinib treatment alone in H630 and LoVo cells did notinduce cleavage of caspase-8 or PARP (Fig. 2C). As expected, 15nmol/L panobinostat induced low levels of PARP cleavageconsistent with the low level of apoptosis occurring. However,combination treatment increased both caspase-8 and PARPcleavage at 18 and 24 hours in both cell lines.

Panobinostat in combination with lapatinibsynergistically inhibits the growth of LoVo xenografts innude mice

LoVo CRC xenografts were established as outlined in theMaterials and Methods. Panobinostat was administered at2.5 mg/kg by i.p. injection q.d. for 5 days each week andlapatinib was administered at 30 mg/kg BID by oral gavage forthe duration of the study. Single-agent lapatinib and pano-binostat resulted in modest tumor growth inhibition whencompared to vehicle-treated controls (Fig. 3A). However,coadministration of lapatinib and panobinostat resulted inthe greatest tumor growth inhibition compared to vehiclecontrols. At the end of the 24-day treatment period, lapatinibmonotherapy resulted in a 4.1% reduction in mean TV to1,075.3 � 163.3 mm3 compared to the vehicle control groupwith a mean TV of 1,121.7 � 288.9 mm3 (Fig. 3A). Panobino-stat monotherapy resulted in a reduction in mean TV of 23.8%to 954.6 � 275.9 mm3 when compared to the vehicle-treatedgroup. However, panobinostat plus lapatinib resulted in areduction in mean TV of 49.8% to 563.2 � 111.6 mm3 whencompared to the vehicle-treated group (P < 0.05). Lapatinibplus panobinostat also resulted in a highly significant increasein tumor delay (Td) with a ratio of observed:expected of 1.15,indicative of a synergistic increase in antitumor activity(Fig. 3B). Importantly, combination treatment did not causeany significant difference in body weight compared to mono-therapy (P ¼ 0.48; Fig. 3C).

HDAC inhibition modulates ERBB family gene andprotein expression

We subsequently investigated the effects of panobinostaton both HER-family gene and protein expression in CRCmodels. DLD-1, H630, HCT116, and LoVo cells were treatedwith increasing concentrations of panobinostat for 24 hoursand the mRNA expression of ERBB1 (EGFR) and ERBB2(HER2) was analyzed by quantitative real-time PCR (qRT-PCR). Although the DLD-1 and LoVo cells showed an initialinduction of ERBB1mRNA at the lower doses of panobinostat(10–25 nmol/L), treatment with 100 nmol/L panobinostatresulted in a significant reduction of ERBB1 mRNA in all celllines examined (Fig. 4A). ERBB2 mRNA expression was sig-nificantly downregulated in DLD-1, H630, and LoVo cells with100 nmol/L panobinostat (Fig. 4B).

The effect of increasing concentrations of panobinostat onEGFR and HER2 protein expression in DLD-1, H630, HCT116,and LoVo cells was evaluated. Panobinostat treatment for 24hours resulted in a dose-dependent decrease in both EGFRand HER2 protein in all cell lines examined (Fig. 4C). Impor-tantly, at the concentration of 15 nmol/L panobinostat usedfor the apoptotic analyses, all cell lines showed downregula-tion of EGFR and HER2 protein. DLD-1 and LoVo cells, which






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Figure 2. Panobinosat (LBH589)and lapatinib (LAP) synergisticallysuppress colony formation andinduce apoptosis in CRC cell lines.A, DLD-1, H630, HCT116, andLoVo CRC cells were treated withLBH589 and 3 mmol/L LAP for24 hours and then replaced withdrug-free media for 7 to 14 days.Data presented as the percentageof colony formation compared tocontrols, mean � SEM from n ¼ 3experiments. Statisticalsignificance was determinedby 2-way ANOVA, *, P < 0.05;**, P < 0.01; ***, P < 0.001. B,percentage of cells in sub-G1 at24 hours are represented as themean � SD from 2 independentexperiments, *, P < 0.05;**, P < 0.01; ***, P < 0.001 2-wayANOVA. C, Western blot analysisof g-H2A.X, caspase-8, and PARPfollowing treatment for 18 and24 hours. b-Tubulin controlledfor loading.

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both showed increased ERBB1 mRNA with 15 nmol/L pano-binostat, showed measurable decreases in the EGFR protein.

The combination of panobinostat and lapatinibenhances the downregulation of ERBB1 and ERBB2 geneexpressionThe effect of combined lapatinib and panobinostat treat-

ment on both EGFR and HER2 mRNA and protein expressionwas analyzed. H630 and LoVo cells were treated with 3 mmol/Llapatinib and 10 and 15 nmol/L panobinostat alone and in

combination for 18 and 24 hours. mRNA expression of ERBB1and ERBB2 was analyzed by qRT-PCR. Treatment with lapa-tinib alone had no significant effect on ERBB1 and ERBB2mRNA expression. ERBB1 and ERBB2 mRNA expression wasdownregulated approximately 2-fold following treatment with15 nmol/L panobinostat in the H630 cells. In the LoVo cells,ERBB1 mRNA was increased 2-fold and ERBB2 mRNA wasdownregulated 2-fold by 15 nmol/L panobinostat treatment.Combination treatment resulted in an increased downregula-tion of ERBB2 in both cell lines than panobinostat treatment

Figure 3. Panobinostat (LBH589)and lapatinib (LAP) synergisticallyenhance antitumor activity in LoVoCRC xenograft model. Male nu/numice with subcutaneous LoVoCRC tumors (100 mm3) weretreated with vehicle, LAP,LBH589, or their combination (n ¼6, group). LAP (30 mg/kg) wasadministered by oral galvage BIDand 2.5 mg/kg LBH589 wasadministered i.p. for 5 consecutivedays per week. A, TV isrepresented as mean � SEM foreach group. Statisticalsignificance was determined by 2-way ANOVA, *,P < 0.05; **, P < 0.0;***,P < 0.001. B, Td1¼ time in daysto reach a TV 5 times the initialvolume on day 1. Expected Td2 ¼the mean vehicle þ (mean LAP –

mean vehicle) þ (mean LBH589 –

mean vehicle). Observed Td3 ¼the combination of 30 mg/kg LAPand 2.5 mg/kg LBH589. Ratio4 ofobserved:expected Td. Ratiomore than 1 indicates synergismand less than 1 indicatesantagonism. C, mouse bodyweight represented as the percentinitial body weight at day 24compared to day 1.

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alone. In the H630 cells, ERBB1mRNA was downregulated to agreater extent than panobinostat treatment alone as a resultof combination treatment. Interestingly, the induction ofERBB1 mRNA observed with panobinostat treatment in theLoVo cells was completely abrogated with combination treat-ment (Fig. 5A).

To confirm the decrease in EGFR/HER2 signaling, wesubsequently analyzed the mRNA expression of 3 knowndownstream transcriptional targets of EGFR signaling(35–37). Lapatinib treatment alone had no significant effectson CCND1 and IRAK1 gene expression in both the LoVo andH630 cell. However, in the LoVo cells, lapatinib treatment

alone induced a 3-fold downregulation of NF-kB1 mRNAexpression. Panobinostat alone induced significant downre-gulation of CCND1, NF-kB1, and IRAK1 (Fig. 5A). As expected,combination treatment enhanced the downregulation ofCCND1, NF-kB1, and IRAK1 gene expression greater thanpanobinostat alone, consistent with the enhanced disruptionof HER signaling (Fig. 5A).

The combination of lapatinib and panobinostatsuppresses signaling downstream of EGFR and HER2

To further evaluate the enhanced effects of combinationtreatment, the protein expression of EGFR and HER2 and

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Figure 4. Panobinostat (LBH589)modulates EGFR and HER2mRNA and protein expression.DLD-1, H630, HCT116, and LoVoCRC cell lines were treated withLBH589 for 24 hours. Followingtreatment, mRNA expression wasdetermined for ERBB1 (A) andERBB2 (B). Histogram barsrepresent the mean� SD for n¼ 2analyzed in triplicate. GAPDHmRNA expression was used tonormalize. Statistical significancewas determined by Student's ttest of treated samples whencompared to control, *, P < 0.05;**, P < 0.01; ***, P < 0.001. C,Western blot analysis of the effectof LBH589 on EGFR and HER2protein expression. Densitometricanalysis was carried out usingScion Image normalized tob-tubulin to control for loading.

LaBonte et al.





activation status of downstream signaling proteins, AKT andMAPK (mitogen activated protein kinase), was subsequentlyanalyzed by Western blot. H630 and LoVo CRC cells weretreated with 3 mmol/L LAP and 15 nmol/L panobinostat aloneand in combination for 18 and 24 hours. Posttreatment, bothcell lines showed downregulation of EGFR and HER2 withcombination treatment to the same or greater extent thanpanobinostat treatment alone (Fig. 5B and C). Phospho-AKT

(Ser473) levels were significantly repressed in both cell lineswith both panobinostat and combination treatment at 18 and24 hours. Analysis of phospho-p44/42-MAPK-Tyr204/Thr202shows suppression with combination treatment in the H630 atboth 18 and 24 hours but only at 24 hours postcombinationtreatment in the LoVo cells. Interestingly, in the LoVo cells,panobinostat treatment appears to cause an increase in thelevels of phospho-p44/42-MAPK-Tyr204/Thr202 at 18 and

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Figure 5. Significant suppression of HER family and downstream signaling molecules following combination treatment with panobinostat (LBH589) andlapatinib (LAP). H630 and LoVo cells were treated with LBH589 and LAP for 18 and 24 hours and analyzed for mRNA and protein expression of HERfamily and downstream signaling. A, mRNA expression was determined for ERBB1, ERBB2, CCND1, NF-kB1, and IRAK1. Histogram bars representthe mean � SD (n ¼ 2 analyzed in triplicate). GAPDH expression was used to normalize mRNA expression. Statistical significance was determined byStudent's t test of treated samples when compared to controls, *, P < 0.05; **, P < 0.01; ***, P < 0.001. H630 (B) and LoVo (C) cell lines were evaluated for theeffects of treatment on HER pathway proteins by Western blot analysis. b-Tubulin controlled for loading.






24 hours over control levels that is abrogated by combinationtreatment (Fig. 5C).

Panobinostat downregulates HER3, a known resistancemarker to EGFR/HER2-targeted therapy

It has been shown that the increased expression andactivation of HER3, the preferred dimerization partner forHER2, represents a mechanism of resistance to lapatinibtreatment (38, 39). However, HER3 expression in CRC remainsto be firmly established (40). Basal HER3 mRNA and proteinexpression was determined for the 6 cell lines utilized

throughout this study. All cell lines had detectable expressionof ERBB3 mRNA (Fig. 6A, left). HER3 protein expression wasalso detectable in all cell lines, with a 15-fold range ofexpression from the highest; HT29 to the lowest; RKO(Fig. 6A, right).

We subsequently investigated the effects of panobinostaton HER3 mRNA and protein expression in H630 andLoVo CRC cells. Both cell lines showed a dose-dependentdecrease in ERBB3 mRNA expression with panobinostattreatment (Fig. 6B, left). In addition both cell lines showeda significant downregulation of HER3 protein following

HER3

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Figure 6. Combined panobinostat (LBH589) and lapatinib (LAP) treatment downregulates HER3 mRNA and protein in CRC cell lines. A, basal HER3mRNA and protein levels were determined in 6 CRC cell lines. Histogram represents mean � SD (n ¼ 2 analyzed in triplicate). Protein expression wasdetermined byWestern blotting. Densitometeric analysis was carried out using Scion Image and normalized to b-tubulin to control for loading. Relative proteinexpression is normalized to HCT116. B, H630 and LoVo cells were treated with LBH589 for 24 hours and HER3 mRNA and protein analyzed. Statisticalsignificance was determined by Student's t test of treated samples when compared to respective control, *, P < 0.05; **, P < 0.01; ***, P < 0.001. C, theeffects of the LAP plus LBH589 at 24 hours on HER3 mRNA and protein expression was analyzed as above.

LaBonte et al.





panobinostat treatment (Fig. 6B, right). Interestingly,whereas EGFR and HER2 could be potently downregulatedby the HSP90 inhibitor 17-AAG, HER3 protein expressionremained unchanged suggesting that the mechanism ofdownregulation of HER3 is transcriptional (SupplementaryFig. S5).To evaluate the effects of combination treatment on HER3

status, H630 and LoVo cells were treated with 3 mmol/Llapatinib and 10 and 15 nmol/L panobinostat alone and incombination for 24 hours and HER3 mRNA and proteinanalyzed. Lapatinib treatment did not modulate HER3 mRNAor protein expression at 24 hours in either cell line. However,combination treatment resulted in a significantly greaterdecrease in ERBB3 mRNA and protein compared to panobi-nostat alone (Fig. 6C, left). These data indicate that panobino-stat may be effective at disrupting the potential role of HER3as a mechanism of resistance to HER-targeted therapy.

Discussion

EGFR is overexpressed in 60% to 80% of CRC and is reportedto play an important role in promoting the growth andprogression of CRC. On the basis of EGFR's key role inpromoting the survival and progression of CRC and theemerging evidence that HDACi downregulate receptor TKexpression, the effect of targeting EGFR/HER2 in combinationwith HDAC inhibition in CRC cells was evaluated.Combining lapatinib with panobinostat resulted in synergis-

tic decreases in CRC cell proliferation, colony formation andrapidly induced apoptosis. Mechanistic evaluation of this com-bination showed that there was a significant enhancement ofDNA damage (analyzed by gH2A.X), increased activation ofcaspase-8 and PARP cleavage, decreased ERBB1 (EGFR) andERBB2 (HER2) mRNA and protein expression and decreasedphosphorylation of AKT –(Ser139) and MAPK-Thr202/Thy204.When tested in vivo, lapatinib plus panobinostat at therapeu-tically relevant doses showed significantly delayed tumorgrowth and a reduction in TV in the LoVo CRC xenograft.One of the most interesting observations is that CRC cell

lines with a wide range of expression of EGFR and/or HER2showed a highly significant sensitization to panobinostat as aresult of lapatinib treatment. These data would stronglysuggest that inhibition of HER-mediated signaling overcomesa key survival or resistance mechanism to HDACi treatment.On the basis of the modest growth inhibition and lack ofapoptosis observed with lapatinib treatment alone at lowerdoses (3 mmol/L), it seems plausible that cell survival andproliferation following HDACi treatment may be dependenton signaling from EGFR and/or HER2. We also confirmed apotential role for PI3K (phosphoinositide 3-kinase) and MAPKsignaling in mediating response to panobinostat treatment bycombinations using specific inhibitors of MEK (MAP/ERKkinase) and PI3K and observing synergistic growth inhibitoryeffects (Supplementary Fig. S4). Moreover, several cell linesutilized in this study possess activating mutations in RAS, RAF,and/or PIK3CA and yet synergistic drug interactions to varyingextents were observed in all cell lines. Interestingly, a recentreport showed that lapatinib was effective at suppressing

signaling via PI3K/AKT even in the presence of activatingPI3Kmutations providing an explanation for our observationsin PI3K mutant CRC cells (38). It would appear that conco-mitant treatment with lapatinib may reduce the cells ability totolerate and survive the cytotoxic effects of panobinostat. Thishypothesis is indeed supported by observations in CRC whereEGFR-targeted therapies have had their greatest impact andshowed the most promising efficacy in combination withother structurally and functionally diverse cytotoxic agents.A number of clinical studies reported that cetuximab resen-sitized irinotecan-refractory mCRC patients to irinotecan,implicating EGFR as the mode of resistance in a subset ofthese patients (7, 41). In addition, cetuximab and panitumu-mab have both improved clinical outcome in combinationwith oxaliplatin and irinotecan-containing regimens in KRASwild-type patients in randomized trials (42, 43). The clinicaldata therefore support the role of EGFR in mediating cyto-toxicity induced by chemotherapeutic agents.

The complex interplay between EGFR and the other mem-bers of ErbB receptor family members directly influences thesusceptibility of tumor cells to EGFR-targeted agents. Wetherefore evaluated the influence of HDACi treatment aloneand in combination with lapatinib on the mRNA and proteinexpression of HER2 and HER3. We observed very clear cell-linespecific effects on mRNA expression in response to panobino-stat treatment. Downregulation of EGFR and HER2 mRNAwas not a consistent observation throughout all the celllines and induction of EGFR mRNA was noted in some celllines. However, in all cell lines analyzed, downregulation ofEGFR and HER2 protein expression was observed at concen-trations where no downregulation (and even induction) ofmature mRNA transcript was observed. These data stronglyimply the disruption of EGFR and HER2 protein stabilityinduced by HDACi. In addition, It is likely that the induction ofmRNAobserved in some cell lines represents the activation of aregulatory or feedbackmechanism in an attempt to reestablishprotein expression following HDACi treatment.

The expression levels and functional roles ofHER2 andHER3in CRC are not well established or described. Our previousstudies and others have reported that a subset of CRC cells doexpress HER2 (33, 44, 45). This is of particular importance asHER2 is reportedly the preferred dimerization partner for othermembers of the HER family including EGFR and can intensifyreceptor-initiated signaling events (46). A recent study eval-uated pertuzumab, an antibody that inhibits HER2 dimeriza-tion, in CRC cell line and xenograft models. The authorsreported that pertuzumab showed growth inhibitory activityas a single agent and enhanced activity when combined withthe EGFR TKI erlotinib providing evidence supporting theinhibition of both EGFR and HER2 simultaneously in CRC(47). We also report that HER3 is expressed to varying extentsin our CRC models and that panobinostat is effective atdownregulating its expression at both the mRNA and proteinlevel. This is of particular importance given the reported role ofHER3 in mediating resistance to HER TKI therapy (48).

The mechanistic basis for the observed synergy betweenlapatinib and panobinostat combination appears to bemultifactorial. We provide the first report indicating that






panobinostat can downregulate both EGFR and HER2 pro-tein expression in a dose-dependent manner in a panel ofCRC cell lines. We also show that HSP90 inhibition incombination with lapatinib treatment shows a synergisticinteraction in our CRC models (Supplementary Fig. S5A)clearly suggesting a role for EGFR/HER2 signaling in med-iating the recovery from the unfolded protein response.Interestingly, studies evaluating HDACi treatment in addi-tional tumor types have reported significant differences inthe regulation of HER-family members and the cytotoxicitythat results. HDACi treatment appears to preferentiallydownregulate mutant EGFR in NSCLC when compared tothe wild-type protein as a result of increased dependence onHSP90 (19, 20). In addition, HER2-amplified breast cancercells appear to show increased sensitivity to HDACi treat-ment as a result of rapid depletion of HER2 followingentinostat treatment. Tumors with specific HER-familymolecular alterations, such as tyrosine kinase mutationand gene amplification, have significantly differently clin-icopathologic characteristics associated with these altera-tions and are treated with distinctly different therapeuticstrategies in the clinic. However, with CRC, it is well estab-lished that EGFR mutation is an extremely rare event andonly a small subset possess EGFR amplification (49).Response to EGFR-targeted therapy and EGFR expressiondo not correlate well in CRC (even in the context of KRASmutation) and confirmed reports of significant diseaseresponse has been observed in CRC patients whose tumorsexpress little to no EGFR as detected by immunohistochem-istry. The results of the current study would indicate thatEGFR and HER2 are substrates for HSP90 in the context ofCRC.

In our CRC models, HSP90 inhibition potently downregu-lated EGFR and HER2 but did not downregulate HER3 proteinand in fact induced ERBB2 and ERBB3 mRNA suggesting thepresence of regulatory mechanisms possibly as a result ofHER2 and EGFR protein loss (Supplementary Fig S5B andC). Induction of ErbB3 mRNA in response to HDACi treatmenthas been reported in NSCLC although the expression ofHER3 protein in this specific study was not evaluated (50)However, panobinostat induced dose-dependent down-regulation of HER3 at the mRNA and protein level. In thecurrent study, we noted that panobinostat can downregulateERBB1 and ERBB2 gene expression at clinically achievableconcentrations in the majority of the CRC cell lines we tested

indicating that panobinostat also elicits transcriptional effects,either through disruption of the gene transcriptional machin-ery, or possibly via an mRNA destabilization mechanism, inaddition to the disruption of HSP90 function. The potentialimportance of the transcriptional component was furthersuggested by the combination of lapatinib with the HDACientinostat. Entinostat is a Class I specific HDACi and does nottarget HDAC6 which is reported to regulate HSP90 function.The combination of lapatinib with entinostat showed additiveand synergistic effects in both the H630 and LoVo cell lines.However, entinostat is a structurally different HDACi belong-ing to the benzamide class and it is not yet clear whetherentinostat can influence protein stability via non-HSP90mechanisms. Currently, further analysis is underway to eval-uate both the contributory mechanisms to this transcriptionaleffect and additional potential contributingmechanisms to thedrug interaction that may include reactive oxygen species(ROS) and the potential role of EGFR and HER2 signalingin regulating the cellular response to ROS. The model thatthese data propose is one where lapatinib inhibits ligand-initiated signaling from EGFR and HER2, whereas panobino-stat simultaneously induces disruption of EGFR, HER2, andHER3 translational disruption (Supplementary Fig. S6). Thecombined loss of receptor-initiated signaling and the down-regulation of EGFR and HER2 expression and inhibition ofreplenishment through transcriptional and posttranslationalmechanisms result in rapid cell cycle arrest and apoptosis.These observations strongly support further clinical evalua-tion of HER-targeted therapies in combination with HDACiin the treatment of CRC.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This research was funded in part by award number P30CA014089 from theNational Cancer Institute. The content is solely the responsibility of the authorsand does not necessarily represent the official views of the National CancerInstitute or the National Institutes of Health.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 6, 2010; revised January 10, 2011; accepted March 12, 2011;published OnlineFirst April 4, 2011.

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LaBonte et al.





Published OnlineFirst April 4, 2011.Cancer Res Melissa J. LaBonte, Peter M. Wilson, Will Fazzone, et al. Inhibitor Panobinostat in Colorectal Cancer ModelsEnhances the Antitumor Activity of the Histone Deacetylase The Dual EGFR/HER2 Inhibitor Lapatinib Synergistically

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The Dual EGFR/HER2 Inhibitor Lapatinib Synergistically ... · 06/05/2011 · Melissa J. LaBonte 1, Peter M. Wilson , Will Fazzone1, Jared Russell2, Stan G. Louie2, Anthony El-Khoueiry