SynergisticAntitumorActivityofCetuximabandNamitecanin ......2014/01/25 · inhibit EGFR: tyrosine...

Cancer Therapy: Preclinical

SynergisticAntitumorActivityofCetuximabandNamitecan inHuman Squamous Cell Carcinoma Models Relies onCooperative Inhibition of EGFR Expression and Depends onHigh EGFR Gene Copy Number

Michelandrea De Cesare1, Calogero Lauricella2, Silvio Marco Veronese2, Denis Cominetti1, Claudio Pisano3,Franco Zunino1, Nadia Zaffaroni1, and Valentina Zuco1

AbstractPurpose:Despite the frequent overexpression of epidermal growth factor receptor (EGFR) in squamous

cell carcinoma (SCC), the efficacy of cetuximab alone is limited. Given the marked activity of namitecan, a

hydrophilic camptothecin, against SCC models, the present study was performed to explore the efficacy of

the cetuximab–namitecan combination in a panel of SCC models.

Experimental Design: We examined the antiproliferative and antitumor activities of the cetuximab–

namitecan combination in four SCC models characterized by a different EGFR gene copy number/EGFR

protein level.We also assessed the effects of the combinationonEGFR expression at bothmRNAandprotein

levels and investigated the molecular basis of the interaction between the two agents.

Results: Cetuximab and namitecan exhibited synergistic effects, resulting in potentiation of cell growth

inhibition and, most importantly, enhanced therapeutic efficacy, with high cure rates in three SCC models

characterized by high EGFR gene copy number, without increasing toxicity. The synergistic antitumor effect

was also observed with the cetuximab–irinotecan combination. At the molecular level, the two agents

produced a cooperative effect resulting in complete downregulation of EGFR. Interestingly, when singly

administered, the camptothecin was able to strongly decrease EGFR expression mainly by transcriptional

inhibition.

Conclusions: Our results (i) demonstrate a marked efficacy of the cetuximab–namitecan combi-

nation, which reflects a complete abrogation of EGFR expression as a critical determinant of the

therapeutic improvement, in SCC preclinical models, and (ii) suggest EGFR gene copy number as a

possible marker to be used for patient selection in the clinical setting. Clin Cancer Res; 1–12. �2013

AACR.

IntroductionThe epidermal growth factor receptor (EGFR), a member

of the ErbB receptor tyrosine kinase family, plays an essen-

tial role in the regulation of important cellular processessuch as proliferation, survival, adhesion, migration, anddifferentiation (1, 2) as well as in DNA damage response(3, 4). Abnormal activation of EGFR through differentmechanisms, including overexpression, mutations, andautocrine ligand production (5), has been implicated as acrucial regulator of the pathogenesis process and progres-sion in a variety of human cancers (2, 6–8). There seem tobedistinct mechanisms for EGFR activation in different tumortypes. Specifically, in squamous cell carcinoma (SCC) of thecervix, head and neck, and lung, EGFRmutations are absentor detectable in small percentages of cases (<5%; refs. 9–12). In such tumors, a more frequent mechanism of EGFRactivation is the amplification/increased copy number ofthe gene (13), leading to protein overexpression. Genomicgain in the EGFR gene was consistently associated withadverse clinical outcome in patients with cervical (9), lung(14), and head and neck (15) SCC.

EGFR is a validated therapeutic target in many humancancers. Currently, there are two therapeutic approaches to

Authors' Affiliations: 1Molecular Pharmacology Unit, Fondazione IRCCSIstituto Nazionale per lo Studio e la Cura dei Tumori; 2Molecular PathologyUnit, Ospedale Niguarda Ca' Grande, Milan; and 3Sigma-Tau S.p.A.,Pomezia, Italy

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

N. Zaffaroni and V. Zuco contributed equally to this work.

Current address for C. Pisano: Biogem, Research Institute Gaetano Sal-vatore, Via Camporeale, Area PIP, 83031 Ariano Irpino, Italy.

CorrespondingAuthor:ValentinaZuco, Fondazione IRCCS IstitutoNazio-nale per lo Studio e la Cura dei Tumori, Via Venezian 1, 20133 Milan, Italy.Phone: 39-0223902239; Fax: þ39-0223902267; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-13-1684

�2013 American Association for Cancer Research.

ClinicalCancer

Research

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Published OnlineFirst December 10, 2013; DOI: 10.1158/1078-0432.CCR-13-1684

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inhibit EGFR: tyrosine kinase inhibitors (TKI) and anti-EGFR monoclonal antibodies (moAb). TKIs target theintracellular tyrosine kinase domain of the receptor bycompeting for the ATP-binding pocket, thus inhibitingphosphorylation of the receptor and its downstream targets.MoAbs, such as cetuximab, disrupt the EGFR signalingpathway through inhibition of ligand binding and induc-tion of receptor internalization followed by degradation inlysosomes (16). In addition, cetuximab binding to theextracellular EGFR domain promotes the activation of anti-body-dependent cellular cytotoxicity (17). Despite the roleof EGFR in the malignant phenotype, therapy with EGFRtargeting agents only exhibited limited efficacy. Indeed, noclinical responses were reported following cetuximabmonotherapy in cervical SCC (18) and a marginal efficacyof the moAb was observed in patients with head and neckSCC with metastatic disease (13% response rate; ref. 19).However, cetuximab is the first targeted therapeutic agent toshow a significant improvement in the overall survival forpatients with locally advanced head and neck SCC whenused in combination with radiation (20) or for recurrent ormetastatic disease when used in combination with chemo-therapy (21).

We recently reported that namitecan, a novel hydyophiliccamptothecin analog, exhibited antitumor activity in a largepanel of human tumor models (22, 23) and an excellentactivity, superior to that of irinotecan, in the treatment ofSCC models (23). Because EGFR expression has beenimplicated as a determinant of response to radiation andto chemotherapy (24–26), the present studywas performedto explore the therapeutic potential of cetuximab–namite-can combination against four SCCmodels characterized bydifferent levels of EGFR expression and to elucidate the roleof EGFR in response to the namitecan-containing therapy.For comparative purpose, we also assessed the effect ofcetuximab–irinotecan combination. The results providedevidence of a synergistic antitumor activity of cetuximab–

topoisomerase I inhibitor (namitecan or irinotecan) com-bination, with high cure rates, which was related to thelevel of EGFR protein expression/EGFR gene copy numberof the tumor model. Ex vivo and cellular studies demon-strated that the main mechanism of such a synergisticinteraction was the cooperative inhibition of EGFR expres-sion induced by the two agents. Specifically, the EGFRdownregulation induced by namitecan and SN-38 seemsto rely primarily on a marked and persistent transcriptionaldownregulation of gene expression. The early activation ofp38 mitogen-activated protein kinase (MAPK), which inturn phosphorylates EGFR at Ser1046/1047, thereby induc-ing its degradation, also contributed to topoisomerase Iinhibitor–induced EGFR inhibition.

Materials and MethodsCell lines

Three human SCC lines derived from skin (A431) anduterine cervix (Caski and SiHa) and a topotecan-resistantsubline of A431 (A431/topotecan; ref. 27) were used in thestudy. All the cell lines were cultured in RPMI-1640 media(Lonza) supplemented with 10% FBS and grown in ahumidified incubator with 5% CO2 at 37

�C. Cell lines areperiodically monitored for DNA profile of short tandemrepeats analysis by the AmpFISTR Identifiler PCR amplifi-cation kit (Applied Biosystems).

Determination of c-MET and EGFR gene copy numbervariations

Genomic DNA was extracted using the iPrep PurificationInstrument with the iPrep ChargeSwitch Forensic Kit (Invi-trogen) according to the manufacturer’s protocol, and rel-ative concentration was quantified using the Infinite 200NanoQuant Spectrophotometer (Tecan).

MET and EGFR gene copy number variations wereassessed using real-time PCR TaqMan CopyNumber Assays(Applied Biosystems), using RNase P gene as endogenouscontrol. Furthermore, MAD1L1 and CFTR genes, beinglocated respectively on the p and q-arm of chromosome7, were also used to exclude the presence of chromosome 7polysomy. The assays were performed using the AppliedBiosystems ViiA 7 Real-Time PCR System (Applied Biosys-tems) according to themanufacturer’s protocol. Copy num-ber variations of the target geneswere determined as relativequantification (RQ) based on the DDCt method and usingcontrol samples as calibrators with the ABI SDS software 1.1(Applied Biosystems).

Mutational status analysisMutation status of KRAS, BRAF, KRAS, NRAS, PIK3CA,

and EGFR genes was determined as detailed in Supplemen-tary Methods.

DrugsFor in vitro studies, namitecan (Sigma-Tau) and SN-38

and SB203580 and SB202190 (Sigma Chemical Company)were initially dissolved in dimethyl sulfoxide and thendiluted in sterile saline before use. Cetuximab (Erbitux;

Translational RelevanceThe epidermal growth factor receptor (EGFR) is a

validated therapeutic target in many human cancers. Inspite of the frequent EGFR overexpression in squamouscell carcinoma (SCC), the anti-EGFR antibody cetuxi-mab, as a single agent, exhibits marginal efficacy. Ourstudy shows that a combination including cetuximaband a topoisomerase I inhibitor, namitecan or irinote-can, produced synergistic effects, resulting in completeregression of SCC preclinical models characterized byhigh EGFR gene copy number. The result was achieved atwell-tolerated doses, thus indicating a good therapeuticindex. The study also provides a molecular basis for arational combination to be exploited for possible ther-apeutic applications in the clinical setting, and suggestsEGFR gene copy number as a possible marker for patientselection.

De Cesare et al.

Clin Cancer Res; 2014 Clinical Cancer ResearchOF2




Merck Serono)was diluted in sterile saline before use. For invivo studies, namitecan was dissolved using a magneticstirrer in sodium lactate buffer (50 mmol/L) adjusted topH 4.0 with the addition of hydrochloric acid. Irinotecanwas dissolved in sterile distilled water. Both camptothecinswere administered i.v. in a volume of 10mL/kg. Cetuximabwas ready to use andwas delivered intraperitoneally (i.p.) atthe dose of 0.2 mL/mouse.

Growth inhibition studyThe antiproliferative activity was evaluated after 72 hours

of drug exposure by cell counting (27).Drug concentrationsable to inhibit cell proliferation by 50% (IC50) and 20%(IC20) were calculated from dose–response curves.

Antitumor activity studyTo generate tumor xenografts, exponentially growing

cells (A431 and A431/topotecan, 107 cells/mouse; SiHa2.5 � 107 cells/mouse, Caski 107 cells/mouse) were s.c.injected into the mice flanks. For antitumor activitystudies, groups of four/five mice bearing tumorimplanted in both flanks were used. Tumor fragmentswere implanted on day 0, and tumor growth was followedby biweekly measurements of tumor diameters with aVernier caliper. Tumor volume (TV) was calculatedaccording to the formula TV (mm3) ¼ d2 � D/2, in whichd and D are the shortest and the longest diameter, respec-tively. Treatment started 5 to 13 days after implant, whenthe tumors were just palpable, but established (TV ¼ 80–90 mm3). Namitecan, irinotecan, and cetuximab wereadministered every fourth day for four times. Cetuximabwas given 1 hour after each administration of thecamptothecin.The efficacy of the drug treatment was assessed as (i) TV

inhibition percentage (TVI%) in treated versus controlmice, calculated as TVI%¼ 100� [(mean TV treated/meanTV control) � 100]; (ii) complete responses (CR), i.e.complete disappearance of the tumors for at least 10 days.The toxicity of the drug treatment was determined as bodyweight loss and lethal toxicity. Deaths occurring in treatedmice before the death of the first control mouse wereascribed to toxic effects.

Antibodies and Western blot analysisThe antibodies used in the studywere anti-EGFR (Upstate

Biotechnology); anti-vinculin (Sigma); anti-phospho EGFR(Ser1046/47), anti-phospho EGFR (Tyr1045), anti-phos-pho p38 MAPK (Thr180/Tyr182; Cell Signaling Technolo-gy); anti-p38 MAPK and anti-c-Cbl (Santa Cruz Biotech-nology). Western blot analysis was carried out as describedpreviously (27) and as detailed in SupplementaryMethods.

Quantitative reverse-transcription PCRTotal RNA was isolated from SCC cell lines using the

RNAqueous-4PCR Kit (Ambion Europe Ltd.), according tothe manufacturer’s instructions. EGFR mRNA expressionwas assessed by quantitative reverse-transcription-PCR(qRT-PCR) as detailed in Supplementary Methods.

Immunofluorescence stainingImmunofluorescence staining of EGFR and c-Cbl was

carried out as detailed in Supplementary Methods.

Data analysesIn in vitro growth inhibition studies, the type of drug

interaction was evaluated by the Chou and Talalay’s (28)method using the CalcuSyn software (Biosoft). Accordinglyto it, combination index (CI) value (CI < 1, ¼ 1, and >1)indicated synergism, additive effect, and antagonism,respectively. Synergism is further refined by CalcuSyn assynergism (CI ¼ 0.3–0.7) and strong synergism (CI ¼ 0.1–0.3). In antitumor activity studies, the Student t test and theFisher exact test (two-tailed) were used for statistical com-parison of TVs and complete responses, respectively, inmice.

ResultsThe panel of gynecologic SCC cell lines used in the study

was characterized by different levels of EGFR expression atboth protein (Fig. 1A) and mRNA (Fig. 1B) level. Specifi-cally, strong EGFR overexpression was found in the A431cell line and, although to a lesser extent, in its topotecan-resistant subline A431/topotecan,whichwas paralleled by amarked gain in EGFR gene copy number (Fig. 1B). Con-versely, moderate or almost negligible EGFR protein levelswere present in Caski and SiHa cell lines, respectively, inagreement with the small, although different increases inEGFR gene copy number (Fig. 1C). In addition, mutationalanalysis indicated that all cell lines harbored wild-typeEGFR, KRAS, NRAS, BRAF, and PI3KCA genes, with theonly exception of Caski cells in which a PI3KCA exon 9–activating mutation (p.E545K; ref. 29) was present (Fig.1C). Finally, a similar MET copy number was observed inthe different cell lines (Fig. 1C).

The namitecan–cetuximab combination inducedsynergistic antitumor effects in SCC models as afunction of EGFR gene copy number

Given the hypersensitivity of SCC to namitecan (23), inthe study we used a suboptimal dose of the drug (10mg/kg,i.e., 1 of 3 of the maximum tolerated dose) to allow acomparison of single-drug treatment and combination ofnamitecan with the anti-EGFR antibody, cetuximab. Irino-tecanwasused as reference compoundat 17mg/kg (1of 3ofthe maximum tolerated dose). Under such treatment con-ditions, in the A431 model namitecan still produced asignificant tumor growth inhibition (84%)with an appreci-able number of complete tumor regressions (3 of 8). Cetux-imab (1 mg/mouse) produced a good antitumor effectwithout evidence of complete tumor regression. The efficacyof the combination was impressive, because all animalsexhibited complete tumor response with no evidence ofdisease at the end of the experiment (90 days after thelast treatment; Fig. 2; Table 1). In an independent experi-ment, single-agent therapy with irinotecan resulted in asignificant tumor growth inhibition (78%) with evidenceof complete response in one tumor. Cetuximab showed a

Synergistic Antitumor Activity of Cetuximab and Namitecan

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good antitumor activity and induced 3 of 8 completeresponse. Combined cetuximab–irinotecan treatmentresulted in complete tumor regression in all treated animalswithout any evidence of tumor regrowth until the end ofexperiment (90 days after the last treatment; Table 1; Sup-plementary Fig. S1).

The A431/topotecan subline, which was highly resis-tant to topotecan (27), was still responsive to namitecanat the low dose level, producing appreciable tumorgrowth inhibition but without complete tumor regres-sion. Surprisingly, in spite of a reduced expression of thetarget EGFR, cetuximab was very effective in the control oftumor growth, resulting in 99% TVI and 6 of 8 completeresponses. The addition of cetuximab to treatment withnamitecan resulted in complete regression of all tumors,with no evidence of tumor regrowth in 8 of 8 animals atthe end of the experiment (Fig. 2, Table 1). Irinotecanproduced a 72% TVI without evidence of complete tumorregression. 8 of 8 animals treated with the combinationirinotecan and cetuximab experienced complete tumorregression (Table 1 and Supplementary Fig. S1). Com-plete responses were observed in all animals treated withcetuximab alone or in combination with irinotecan with-

out disease manifestation until the end of treatment(Table 1 and Supplementary Fig. S1).

The Caski model, which expresses a lower level of EGFR,was still responsive to both namitecan and cetuximab.Again, the combination of the two agents resulted inincreased efficacy, as evidenced by complete regression ofall treated tumors (Fig. 2, Table 1). The combination ofirinotecan with cetuximab was less effective than the com-bination containing namitecan, at least in terms of com-plete response rate (Table 1 and Supplementary Fig. S1).This findingwould suggest that the level of EGFR expressionis more critical for irinotecan to achieve synergisticinteraction.

The SiHamodel, which is characterizedby a very low levelof EGFR expression, exhibited a lower responsiveness toboth namitecan and cetuximab than other models. Whenanimalswere treatedwith the combination, only a slight notstatistically significant increase in efficacywas observed, andno animal experienced tumor regression (Fig. 2, Table 1). Itis important to emphasize that the curative efficacy of thecetuximab–namitecan (or irinotecan) combination wasachieved at well-tolerated doses of each agent withoutevidence of appreciable toxicity (Table 1).

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Figure 1. Biochemical andmolecular characteristics of SCCcell lines. A, EGFR proteinexpression levels. Western blotanalysis was performed on lysatesfrom SSC cells. Cropped blots arereported. Full-length gels areincluded in SupplementaryFig. S7. B, EGFR mRNA levels ofexpression. Real-time PCRanalysis of levels of EGFR in SSCcells. C, summary of EGFR, METgene amplification, and of EGFR,KRAS, BRAF, NRAS, and PI3KCAmutational status. Determination ofrelative EGFR or MET gene copynumber by quantitative real-timePCR was performed as describedin Materials and Methods. DNAsequence analyses were carriedout as described in Materials andMethods and SupplementaryMethods.

De Cesare et al.





In vitro growth inhibition studies on the same SCCmodels showed a variable cellular sensitivity to cetuximab,with IC50 values ranging from 1.47 to 931 mg/mL (Supple-mentary Fig. S1A), which was directly correlated to EGFRprotein expression/EGFR gene copy number of the tumorcell line. Conversely, no appreciable differences wereobserved in the sensitivity of the four cell lines to namitecan(Supplementary Fig. S2A). In combination studies, a syn-ergistic interaction between the effects of cetuximab andnamitecan, as determined by the Chou and Talalay method(28), was observed in A431, A431/topotecan, and Caskicells (Supplementary Fig. S2B). The extent of the synergisticeffects was related to the level of EGFR protein expression/EGFR gene copy number of the cell line. Consistent with invivo data, only an additive effect of the two agents incombination was found in the SiHa cells expressing thelowest EGFR level (Supplementary Fig. S2B).

Cetuximab and namitecan cooperate in inhibitingEGFR expressionIn the search for possible molecular determinants of the

cetuximab–namitecan synergistic interaction,we investigat-ed therapy-induced changes in the expression levels of

EGFR in A431, A431/topotecan, and SiHa xenografts. West-ern blot results showed that not only cetuximab but alsonamitecan, when singly administered, induced a markedreduction of EGFR levels, and that an almost completeabrogation of the protein expression was observed in alltumors exposed to the combined treatment (Fig. 3A).

Cellular studies were carried out to elucidate the mech-anism through which namitecan inhibited EGFR expres-sion. Consistent with in vivo findings, a 24-hour exposure tonamitecan induced a dose-dependent decrease in EGFRexpression in the different cell lines. In addition, resultsobtained in cells exposed to the drug combination con-firmed a cooperative effect of namitecan and cetuximab insuppressing EGFR protein expression (Fig. 3B). Similarresults were obtained when cells were treated with equimo-lar concentrations of SN-38 alone or in combination withcetuximab (Supplementary Fig. S3A).

Because it has been recently reported that topoisomer-ase I inhibition can trigger a transcriptional stress andconsequently interfere with translation of specific genes,such as hypoxia-inducible factor-1a (HIF-1a), in humancancer cells (30), we assessed the ability of namitecan tomodulate EGFR mRNA levels in treated cells. qRT-PCR

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Figure 2. Antitumor activity ofnamitecan, cetuximab alone or incombination against four SSCxenografts. Namitecan (10 mg/kg)and cetuximab (1 mg/mouse) wereadministrated i.v. or i.p.,respectively, with an intermittenttreatment schedule (every fourthday for four times). The indicatedratios refer to the number ofregrowing tumors/total treatedtumors. *, control untreatedtumors; ~, cetuximab; D,namitecan; *, namitecan pluscetuximab. Results of threeexperiments are shown.






results showed that, in all cell lines, EGFR mRNA levelswere decreased at 24 hours after treatment with namite-can. Conversely, no appreciable interference with EGFRmRNA levels was observed in cells exposed to cetuximabalone. In addition, the extent of the namitecan-inducedreduction of EGFR mRNA levels was not significantlymodified in the combination with cetuximab (Fig. 3C).As shown in Supplementary Fig. S3B, SN-38 was able toinhibit EGFR mRNA expression at a comparable extent.Overall, such data indicate that namitecan (or SN-38)-mediated topoisomerase I inhibition leads to EGFRmRNA downregulation.

Because the namitecan-induced effect on EGFR mRNAexpression levels could not justify the almost completeabrogation of EGFR protein observed in treated SCC cellsat 24 hours, we assessed the treatment-induced interferencewith EGFR cellular localization in A431 cells to definewhether the decline of EGFR protein abundance was par-alleled by an increased internalization and consequentubiquitination of EGFR (Fig. 4A). As expected, EGFR wasinternalized after treatment with cetuximab (31). Interest-ingly, also namitecan showed the ability to induce EGFRinternalization (Fig. 4A). Because it is known that the

process of EGFR degradation is dependent on the abilityof the E3 ubiquitin ligase c-Cbl to bind the receptor to itsphosphorylated Tyr1045 residue (32), we examinedwheth-er, following drug-induced internalization, EGFR coloca-lizes with the c-Cbl protein in A431 cells. Unlike the well-characterized EGF-induced formation of EGFR–c-Cbl com-plexes (Supplementary Fig. S4), cetuximab and namitecaninduced translocation of EGFR into intracellular vesicles,which did not colocalize with the c-Cbl protein (Fig. 4B), inaccordance with recent evidence indicating that cetuximabinduces internalization and subsequent ubiquitination ofEGFR, recruiting an E3 ligase distinct from c-Cbl (31, 33).Our fluorescence microscopy findings were further corrob-orated by immune coprecipitation assay results showingthat namitecan and cetuximab did not induce EGFR bind-ing to c-Cbl in spite of an increased EGFR ubiquitination(data not shown).

Because recent reports showed that some anticancerdrugs cause EGFR degradation via phosphorylation atSer1046/1047 residues by p38 MAPK in different tumorcell lines (32, 34, 35), and taking into account that irino-tecan has been shown to activate p38MAPK inHCT116 celllines (36), we examined the effects induced by namitecan

Table 1. Activity of i.v. namitecan, 10 mg/kg, or irinotecan, 17 mg/kg, and i.p. cetuximab, 1 mg/mouse,every fourth day for four times on human SCC xenografted in nude mice

Tumor Drug TVI%a CRb BWL%c (day)

A431 Namitecan 88 3/8 1Cetuximab 76 0/8 0Namitecan þ cetuximab 100 8/8� 0Irinotecan 78 1/8 5Cetuximab 92 3/8 0Irinotecan þ cetuximab 100 8/8

��3

A431/topotecan Namitecan 55 0/8 5Cetuximab 99 6/8 4Namitecan þ cetuximab 100 8/8

��5

Irinotecan 72 0/8 2Cetuximab 100 8/8 0Irinotecan þ cetuximab 100 8/8

��3

Caski Namitecan 97 4/8 5Cetuximab 93 0/8 0Namitecan þ cetuximab 100 8/8� 1Irinotecan 76 1/12 0Cetuximab 78 0/12 0Irinotecan þ cetuximab 97 2/12 2

SiHa Namitecan 69 1/10 2Cetuximab 63 0/10 2Namitecan þ cetuximab 86 0/10 1

NOTE: Tumor fragments were implanted on both flanks at day 0. Treatment started when mean tumor volume was 80 to 90 mm3.aTumor volume inhibition percentage in treated over control mice, determined 1 week after the last treatment.bComplete responses, i.e., disappearance of tumor lasting at least 10 days.cBody weight loss (BWL) percentage induced by treatment; the highest change is reported. No toxic death was observed.�, P < 0.05; ��, P < 0.01 by the Fisher exact test versus namitecan- and irinotecan-treated mice.��, P < 0.001 by the Fisher exact test versus irinotecan-treated mice.

De Cesare et al.





on the expression of the active phosphorylated form of p38MAPK in A431 cells (Fig. 5). The activation of p38 MAPKwas appreciable starting from a 1-hour exposure to nami-tecan (alone or in association with cetuximab) and stillpresent, at comparable levels, at 24 hours. In parallel, anincreased EGFR phosphorylation at Ser1046/1047 wasfound in cells exposed to namitecan, which reached itsmaximum at 4 hours (Fig. 5A). In contrast, a negligibleeffect on EFGR phosphorylation at Tyr1045 was observedfollowing namitecan exposure (data not shown), accordingto the lack of colocalization of internalized EGFR and c-Cbl(Fig. 4B). Comparable effects on p38 MAPK activation andEGFR phopshorylation at ser 1046/47 were observed when

A431 cells were exposed to SN-38 (Supplementary Fig.S5A).

To better understand the role of activated p38 MAPK innamitecan-induced EGFR downmodulation, we used twospecific inhibitors SB203580 (37) and SB202190 (36) inA431 and Caski cells. A 2-hour treatment of cells withSB203580 (10 mmol/L) or with SB202190 (5 mmol/L)before a 24-hour exposure to namitecan, alone or inassociation with cetuximab, strongly reduced EGFR phos-phorylation at Ser1046/47 in A431 cells (Fig. 5B andSupplementary Fig. S5B and S5C). In addition, pretreat-ment with SB203580 or SB202190, which did not affectby itself EGFR expression levels, partially restored EGFR

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Figure 3. A, EGFR proteinexpression in tumor xenograftstreated with cetuximab andnamitecan alone or in combination.A431, A431/topotecan (TPT), andSiHa tumor samples were frozenand then extracted 24 hours afterthe second administration ofnamitecan (30 or 10 mg/kg) andcetuximab (1 mg/mouse) alone orin combination. B, EGFR proteinexpression levels in cell lines. SCCcells were lysed after 24 hours oftreatment with differentconcentrations of namitecan(mmol/L) and with a cetuximabconcentration correspondingto IC20 values for each cell line(A431, 0.5 mg/mL; A431/topotecan, 50 mg/mL; Caski andSiHa, 100 mg/mL). Exposure periodof membrane to film has beenincreased in SiHa to highlight thedifferences in EGFR expressionlevels between control and treatedsamples. Cropped blots arereported. Full-length gels areincluded in Supplementary data(Supplementary Fig. S7). Toimprove the clarity of the SiHablots, membrane film has beendownsized and souncroppedblotscould not be presented inSupplementary Fig. S7. C, EGFRmRNA of expression levels in SCCcell lines. Real-time PCR analysisof levels of EGFR in cells treatedwith cetuximab or ST1968 alone orin combination for 24 hoursharvested as in A.






expression in namitecan-treated cells (Fig. 5B and Sup-plementary Fig. S5B and S5C). Similarly, pretreatmentwith p38 MAPK inhibitors was able to partially restoreEGFR protein expression in A431 cells exposed to SN-38for 24 hours (Supplementary Fig. S5B and S5C). Such aprotective effect against namitecan-induced EGFR degra-dation was even more pronounced in Caski cells (Fig.5C). Conversely, no appreciable effect of SB203580 andSB202190 pretreatment on cetuximab-induced EGFRdownmodulation was found in either cell line (Fig. 5Band C and Supplementary Fig. S5B and S5C). Takentogether, our findings support that namitecan and SN-38 promote the early activation of p38 MAPK, which in

turn phosphorylates EGFR at Ser1046/47, thus inducingits degradation.

However, the contribution of drug-induced p38 MAPKactivation to the overall inhibition of EGFR expression andconsequently to the cetuximab–topoisomerase I inhibitorsynergistic interaction seems to be limited. In fact, in A431cells, a 48-hour exposure to namitecan (or SN-38) alone orin association with cetuximab was able to induce a decreasein EGFR mRNA levels of about 90% (Fig. 5D), which wasaccompanied by a complete abrogation of EGFR proteinexpression (Fig. 5E). Due to the lack of substrate availableto be phosphorylated by p38 MAPK, pretreatment withthe inhibitor SB203580 failed to rescue EGFR protein

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Figure 4. A, immunofluorescenceanalysis of EGFR localization.A431 cells were treated withcetuximab (0.5 mg/mL) andnamitecan (0.01 mmol/L) for 4hours and then fixed byparaformaldehyde. Cells werestained with EGFR (green signal),and nuclei were counterstainedwith Hoechst 33342 (blue signal).Bar, 10 mm. Representative datafrom two to three independentexperiments are shown. B,immunofluorescence analysis ofEGFR and c-Cbl distribution incontrol and cetuximab-,namitecan-, or namitecan pluscetuximab-treated A431 cells.Cells were exposed to cetuximab(0.5 mg/mL) and namitecan(0.01 mmol/L) for 4 hours. Afterfixation in paraformaldehyde, cellswere stained for EGFR (greensignal) and c-Cbl (red signal).Bar, 10mm. Representative datafrom two to three independentexperiments are shown.

De Cesare et al.





expression in camptothecin-treated cells (Fig. 5E). In addi-tion, the pretreatment with p38 inhibitor SB203580 failedto impair namitecan–cetuximab synergistic interaction(Supplementary Fig. S6).

DiscussionAlthough single-agent therapy with cetuximab shows a

limited efficacy in the treatment of SCC (18), an improvedbenefit is expected when it is combined with conventional

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Figure 5. A, levels of EGFRphosphorylation at Ser1046/47 andof p38MAPKphosphorylation. A431 cellswere exposed to cetuximab (0.5mg/mL) or namitecan(0.01 mmol/L) for 1, 2, 4, and 24 hours, and protein extracts were then harvested and examined. Vinculin was used as loading control. Cropped blots arereported. Full-length gels are included in Supplementary Fig. S7. B and C, effect of p38 MAPK inhibition on EGFR phosphorylation. A431 (B) and Caski(C) cells were pretreated with 10 mmol/L SB203580 for 2 hours and then treated with cetuximab (0.5 mg/mL, A431 cells; 100 mg/mL, Caski cells) ornamitecan (0.01 mmol/L) alone or in combination for 24 hours. An antibody to vinculin was used to control for protein loading. For phosphorylated EGFR(Ser1046/47), a higher film exposure compared with Fig. 4A was shown to highlight the differences in expression levels between SB203580-pretreated andnot-pretreated samples. Cropped blots are reported. Full-length gels are included in Supplementary Fig. S7. D, EGFR mRNA of expression levels inA431 cells. Real-time PCR analysis of EGFRmRNA levels in A431 cells treated with cetuximab (0.5 mg/mL) or namitecan (0.01 mmol/L) or SN-38 (0.01 mmol/L)alone or in combination for 48 hours. E, effects of p38 MAPK inhibition on levels of EGFR phosphorylation and on EGFR protein expression. A431cells were pretreated with SB203580 for 2 hours and then treated with cetuximab (0.5 mg/mL) or namitecan (0.01 mmol/L) or SN-38 (0.01 mmol/L) alone or incombination for 48 hours. Cells were lysed and protein extracts were then examined. Vinculin was used as loading control. For phosphorylated EGFR(ser1046/47) and EGFR blots, exposure period of membrane to film has been increased to highlight the complete abrogation of EGFR protein expression.Consequently, the higher film exposure caused the drop of the EGFR protein modulation in cetuximab-treated compared with untreated cells. Cropped blotsare reported. Full-length gels are included in Supplementary Fig. S7.






cytotoxic agents (21, 38). In this context, novel rationalstrategies to improve the efficacy of combination treatmentsand to reduce drug-induced side effects are needed.

Our study provides evidence that cetuximab, when usedin combinationwith namitecan, induced a synergistic effectthat resulted in potentiation of cell growth inhibition in 3ofthe 4 SCC cell lines used in the study. The most interestingobservation was the enhanced therapeutic efficacy seen inthe treatment of SCC xenografts, in which the combinationproduced a curative effect in most treated animals. Consis-tent with in vitro findings, a synergistic effect of the combi-nation was appreciable in tumor models characterized by ahigh EGFR gene copy number. In fact, only the SiHa tumor,which carries the lowest number of EGFR gene copies, didnot exhibit a significant therapeutic benefit by the combi-nation treatment, thus suggesting a marginal impact of thereceptor tyrosine kinase in the growth of the tumor. Asynergistic antitumor effect was also observed when cetux-imab was combined with irinotecan only in the two highlyEGFR expressing tumor models. The substantial improve-ment with the combined therapy was achieved without anincrease in toxicity, because well-tolerated doses of eachagent were used. SCC is known to be responsive to nami-tecan, but curative efficacy requires treatment with maxi-mum tolerated doses, which are associated with significanttoxicity (22, 23).

The present study also provides valuable information onthe molecular/cellular bases of the synergistic drug interac-tion. Specifically, the combination of cetuximab and nami-tecan produced a cooperative effect resulting in a completedownregulation of EGFR. Although the effect of cetuximabon EGFR protein levels has been already described (31), theability of topoisomerase I inhibitors to downregulate EGFRexpression is somewhat unexpected. In a previous study, Liuand colleagues reported the ability of irinotecan to upre-gulate the EGFR pathway (39). However, the authorsshowed an increase in the phosphorylation of EGFR (atTyr 1068) in the absence of significant changes in total EGFRprotein expression in gastric cancer cells after 16 hours oftreatment with irinotecan in presence of EGF stimulation.Because no time-course experimentswere carried out, we donot know whether the increase of EGFR phosphorylationwas followed by a reduced expression of EGFR protein atlater time points, as observed in our study in A431 cells (Fig.5, Supplementary Fig. S5). Indeed, namitecan- or SN-38–treated cells showed an appreciable increase of EGFR phos-phorylation at ser1046/47 after 4 hours, which was accom-panied by EGFR downregulation at 24 hours. Due todifferent times of analysis and culture conditions (�EGF),results from the two studies are not directly comparable.

We herein demonstrate that namitecan (or SN-38)-induced EGFR downregulation was mainly the result oftranscriptional inhibition of gene expression. Indeed, drugtreatment appreciably decreased EGFRmRNA levels in SCCcells in a time-dependent manner with the maximuminhibition (about 90%) being observed at 48 hours. Theeffects of camptothecin-mediated topoisomerase I inhibi-tion at the transcription level have recently been investiga-

ted by Baranello and colleagues (30) in human cancer cellsfor another gene (e.g., HIF1-a). The study showed thattopoisomerase I inhibition can trigger a transcriptionalstress resulting in an impaired balance of mRNAs andantisense transcripts that consequently affect HIF1-aexpression (30).

Because, as expected, cetuximab did not affect EGFRmRNA levels, and the partial abrogation of EGFR mRNAlevels by namitecan, or SN-38, we observed at early timepoints could not explain the complete degradation of EGFRprotein observed in combination-treated cells, additionalmechanisms had to be considered.

There is accumulating evidence that EGFR downregula-tion involves internalization and subsequent degradationof the activated receptor in lysosomes (32, 40, 41). In thepresent study, we found that downregulation of EGFRinduced by namitecan as well as by cetuximab treatmentcorrelated with the internalization and increased ubiquiti-nation of EGFR, which occurred independently of c-Cbl, anubiquitin ligase responsible for EGFR degradation after EGFbinding (5, 31, 33). It has previously been reported thatother drugs induce downregulation of EGFR withoutthe requirement of c-Cbl binding (34, 35, 41–44). Inparticular, epigallocatechin gallate and 17-N-allylamino-17-demethoxygeldanamycin cause EGFR downregulationvia phosphorylation at Ser1046/1047 by p38 MAPK indifferent tumor cell lines (42). Although phosphorylationof serine residues and of Tyr1045 is essential for EGF-induced receptor ubiquitination (32, 41), Oksvold andcolleagues (45) found that only the serine residues arecritical for EGFR internalization and degradation. Differentagents such as UV radiation (46) and cisplatin (47), as wellas oxidative stress (48), can induce internalization of theEGFR but not degradation. On the contrary, gemcitabinewas found to cause ligand-independent internalization anddegradation of EGFR (49).

Here, we report that both namitecan and SN-38 inducedan early p38MAPKactivation andEGFRphosphorylation atser 1046/47 in SCC cells, consistent with a recent observa-tion indicating that irinotecan treatment activates p38MAPK in HCT 116 cell lines (36). The role of p38 MAPKas an early event in EGFR downregulation was confirmedthrough inhibition of p38MAPK with the specific inhibitorSB203580 and SB202190, which by themselves did notaffect the EGFR levels but partially or almost completelyrestored EGFR expression in namitecan- and SN-38–treatedSSC cells at 24 hours, without affecting cetuximab-inducedEGFR downmodulation. At later time points when a com-plete abrogation of EGFR protein expression, as a conse-quence of the marked transcriptional downregulation ofthe gene, was observed, the relevance of p38 MAPK seemednegligible. Indeed, the pretreatment with p38MAPK inhib-itor SB203580 failed to rescue EGFR protein expressionin namitecan- and SN-38–treated cells. In addition, themarginal role of p38 MAPK in the drug synergistic interac-tion was confirmed by inability of SB203580 to impairthe synergism. Conversely, cell exposure to SB203580enhanced namitecan cytotoxic activity, in keeping with

De Cesare et al.





previous evidence reported by Paillas and colleagues (36),demonstrating a synergistic interaction between irinotecanand p38 MAPK inhibition in colon adenocarcinoma celllines.Overall, results from the present study clearly demon-

strate a synergistic antitumor activity of the namitecan–cetuximab combination. It is likely that the therapeuticpotential of this combination could be a common featureof other camptothecins, as observed with irinotecan. How-ever, although in cellular studies the synergistic effect wasdirectly related to the level of EGFR protein expression/EGFR gene copy number, in vivo the contribution of EGFRseemed to be more complex, because no close correlationwas found between the efficacy of the combination and thelevel of gene expression. The low efficacy of namitecan pluscetuximab against the SiHa model was consistent with acritical role of modulation of EGFR function in tumorresponse.Given the complexity of tumor biology, the single-agent

therapywith adrug targeting a single alteration is unlikely toachieve an effective and persistent control of tumor growth.To overcome the limitation of single-agent therapy, com-bination strategies have widely been used. A rational selec-tion of the drug combination is critical to optimize treat-ment efficacy. As shown in our study, knowledge of thebiologic context of various tumor types and of the mech-anism of drug action provides the basis for identifyingsynergistic interactions to be exploited in the clinical setting.Taking into account the good therapeutic index and theimpressive efficacy of the cetuximab–camptothecin combi-nation, our results may have obvious clinical implications.

The combination therapy, including cetuximabanda camp-tothecin, may be a promising approach for the therapy ofSCC, with special reference to patients with tumor charac-terized by a high EGFR gene copy number.

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

Authors' ContributionsConception and design: C. Pisano, F. Zunino, N. Zaffaroni, V. ZucoDevelopment ofmethodology:M.DeCesare, C. Lauricella, S.M. Veronese,D. Cominetti, V. ZucoAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): M. De Cesare, C. Lauricella, S.M. Veronese, D.Cominetti, V. ZucoAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):M. De Cesare, S.M. Veronese, F. Zunino,N. Zaffaroni, V. ZucoWriting, review, and/or revision of the manuscript: S.M. Veronese, F.Zunino, N. Zaffaroni, V. ZucoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): V. ZucoStudy supervision: F. Zunino, N. Zaffaroni

AcknowledgmentsThe authors thank Sigma-Tau S.p.A. for kindly providing namitecan.

Grant SupportThis work was supported in part by grants from Associazione Italiana per

la Ricerca sul Cancro and the Italian Health Ministry.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 19, 2013; revised November 28, 2013; accepted December3, 2013; published OnlineFirst December 10, 2013.

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De Cesare et al.





Published OnlineFirst December 10, 2013.Clin Cancer Res Michelandrea De Cesare, Calogero Lauricella, Silvio Marco Veronese, et al.

Gene Copy NumberEGFRHigh Cooperative Inhibition of EGFR Expression and Depends onin Human Squamous Cell Carcinoma Models Relies on Synergistic Antitumor Activity of Cetuximab and Namitecan

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