her2 as therapeutic target for overcoming atp-binding...

Therapeutic Discovery

HER2 As Therapeutic Target for Overcoming ATP-BindingCassette Transporter–Mediated Chemoresistance in SmallCell Lung Cancer

Toshiyuki Minami1, Takashi Kijima1, Yasushi Otani1, Satoshi Kohmo1, Ryo Takahashi1, Izumi Nagatomo1,Haruhiko Hirata1, Mayumi Suzuki1, Koji Inoue1, Yoshito Takeda1, Hiroshi Kida1,Isao Tachibana1, and Atsushi Kumanogoh1,2

AbstractSmall cell lung cancer (SCLC) easily acquires multidrug resistance after successful initial therapy. Over-

expression ofATP-binding cassette (ABC) transporters is important for themultidrug resistance.Among them,

ABCB1 and ABCG2 are known to be upregulated in chemoresistant SCLC cells. We found that human

epidermal growth factor receptor 2 (HER2) expressions are also upregulated in chemoresistant SBC-3/ETP,

SBC-3/SN-38, and SBC-3/CDDP cells, comparedwith chemosensitive SBC-3 cells. Lapatinib, a tyrosine kinase

inhibitor of HER2, could not suppress proliferation of these HER2-positive SCLC cells alone but successfully

restored chemosensitivity to etoposide and SN-38with a clinically applicable concentration. The reversal effect

of lapatinib was thought to be caused by inhibition of drug efflux pump functions of ABC transporters,

although lapatinib itself has been reported to be a substrate for them.Moreover, knocking down ofHER2 by an

short interfering RNAweakened the effect of lapatinib on ABCB1, indicating the involvement of HER2 in the

inhibitory mechanisms. Notably, we showed that caveolin-1 and Src play key roles in modulating ABCB1

function viaHER2 inactivation. In SBC-3/ETP cells, dephosphorylation of HER2 by lapatinib activates Src and

successively leads to increased caveolin-1 phosphorylation. Through this process, caveolin-1 dissociates from

HER2 and strengthens association with ABCB1, and finally impairs the pump functions. Furthermore, we

showed that treatment by lapatinib in combination with etoposide or irinotecan significantly suppresses the

growth of subcutaneous SBC-3/ETP and SBC-3/SN-38 tumors inmice, respectively. Collectively, these results

indicate that combination therapy with lapatinib and cytotoxic agents could conquer ABC transporter–

mediated chemoresistance especially in HER2-positive SCLC. Mol Cancer Ther; 11(4); 830–41. �2012 AACR.

IntroductionLung cancer is the leading cause of cancer-related

deaths worldwide. Approximately 15% of all histologictypes consist of small cell lung cancer (SCLC), the typebearing poorest outcome. The extreme aggressiveness ofSCLC is due to its early and widespread metastases anddevelopment of multidrug resistance (MDR) to chemo-therapy (1, 2). The current front-line standard chemother-apy regimen for SCLC, etoposide or irinotecan plus cis-

platin, is active inmost SCLC cases, but the disease recursshortly after the first successful treatment with MDRphenotype (2, 3). In recent years, molecular target therapyhas brought about a breakthrough and progress in thetreatment of non-SCLC. For instance, gefitinib and erlo-tinib, selective tyrosine kinase inhibitors (TKI) againsthuman epidermal growth factor receptor 1 (HER1), alsoknown as EGF receptor (EGFR), have presented survivalbenefit especially in EGFR-activating mutation–positivenon-SCLC (4, 5). They specifically and strongly blockEGFR-mediated proliferation and survival signals inEGFR mutation–positive cells (6). However, no favorabletherapeutic strategy has been established in recurrentSCLC to date. Development of novel strategy to overcomeMDR that confers significant survival benefit is urgentlydesired in SCLC.

It is well known that ATP-binding cassette (ABC)transporters play a crucial role in MDR of SCLC. Amongthem,ABCsubfamilyBmember 1 (ABCB1), alsoknownasP-glycoprotein (Pgp), and ABC subfamily G member 2(ABCG2), also known as breast cancer resistance protein(BCRP), have been reported in SCLC to cause resistanceagainst 2 key chemotherapeutic drugs, etoposide and

Authors' Affiliations: 1Department of Respiratory Medicine, Allergy andRheumatic Diseases, Osaka University Graduate School of Medicine; and2Department of Immunopathology, Immunology Frontier Research Center,Osaka University, Osaka, Japan

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

Corresponding Author: Takashi Kijima, Department of Respiratory Med-icine, Allergy and Rheumatic Diseases, Osaka University Graduate Schoolof Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3833; Fax: 81-6-6879-3839; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-11-0884

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 11(4) April 2012830

on July 17, 2019. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst March 2, 2012; DOI: 10.1158/1535-7163.MCT-11-0884

http://mct.aacrjournals.org/

irinotecan, respectively. EGFR-TKIs, gefitinib and erloti-nib, have been shown to inhibit ABCB1 and ABCG2functions and contribute to reverse MDR in breast cancercells and SCLC cells, regardless of EGFR expression. Thedirect functional inhibition of ABC transporters by com-petitive binding of EGFR-TKIs to their ATP-binding sitesis the most conceivable inhibitory mechanism (7–9).HER2, also known as ErbB2, belongs to the HER family

of receptor tyrosine kinases (RTK). It can transduce cel-lular proliferative and survival signals as a homodimerwithout ligand stimulation or a heterodimer with anotherHER family members formed by ligand stimulation (10).In breast cancer, HER2 is overexpressed in about 30% ofcases and its overexpression correlateswith poor outcome(11, 12). Lapatinib, a dual TKI of both EGFR and HER2, isclinically applicable in combination with capecitabine intrastuzumab-refractory patients withHER2-positivemet-astatic breast cancer (13). Moreover, like EGFR-TKIs,lapatinib has also been reported to inhibit the functionsof ABC transporters in breast cancer cells (14). In SCLC,HER2 expression is also reported to be an independentnegative prognostic factor of extensive disease (15–17).However, no studies have yet been conducted to assesswhether HER2 can be a therapeutic target in SCLC. Fromthese views, lapatinib is expected to not only induceapoptosis of HER2-positive chemoresistant SCLC cellsbut also restore chemosensitivity of ABC transporter–positive MDR SCLC.In the present study, we investigated the therapeutic

potential of lapatinib toward MDR SCLC. We haveshown that lapatinib can restore the chemosensitivity ofHER2- and ABC transporter–positive chemoresistantSCLC cells by dysfunctioning ABC transporters withina clinically applicable concentration. We also have eluci-dated the roles of Src and caveolin-1 in the signal trans-duction between the inhibition of HER2 and the dysfunc-tion of ABCB1.

Materials and MethodsCell lines and cell cultureSCLC cell lines (SBC-1 and SBC-2) were obtained from

the Japanese Collection of Research Bioresources, Tokyo,Japan. SBC-3 (18), its chemoresistant sublines SBC-3/CDDP (19), SBC-3/ETP (20), and SBC-3/SN-38 (21), andSBC-5 (22) were kindly provided by Dr. K. Kiura(Okayama University, Okayama, Japan). SCLC cell linesNCI-H69, NCI-N231, NCI-H446, a non-SCLC cell lineHCC827, and a breast carcinoma cell line SK-BR-3 werepurchased from American Type Culture Collection. Che-moresistant sublines H69/CDDP (23) and H69/VP (24)were obtained from National Cancer Center, Tokyo,Japan. OC-10, Smk, and CADO LC6 were provided byOsaka Medical Center for Cancer and CardiovascularDiseases, Osaka, Japan. OS-1, OS2RA, and OS3R5 wereestablished in our laboratory and their biologic propertieswere previously characterized (25). All of lung cancer celllines and SK-BR-3 were maintained in RPMI-1640 and

McCoy’s 5A medium, respectively, supplemented with10% heat-inactivated FBS, penicillin (100 units/mL), andstreptomycin (100mg/mL).All cell lineswere periodicallyauthenticated by morphologic inspection and tested neg-ative every 6 months for mycoplasma contaminationusing PCR ELISA Kit (Roche Applied Science).

AntibodiesMouse monoclonal antibodies (mAb) against HER2

(191924), ABCB1 (JSB-1), and phospho-tyrosine (PY99)were purchased from R&D Systems, Abcam, and SantaCruz Biotechnology, respectively. Rabbit mAbs againstcaveolin-1 (D46G3) and phospho-HER2 (6B12) were com-mercially available from Cell Signaling Technology. Wealso obtained rabbit polyclonal Abs (pAb) against HER2,phospho-caveolin-1 (Tyr 14), phospho-Src (Tyr 416),phospho-Src (Tyr 527), and Src from Cell Signaling Tech-nology; ABCB1 and ABCG2 from GeneTex; and EGFRfrom Santa Cruz Biotechnology. Goat pAb against actinwas also purchased from Santa Cruz Biotechnology.Mouse IgG2bk isotype control (Ancell) and horseradishperoxidase–conjugated goat antirabbit or antimouse IgG(BioRad) were also used.

ReagentsNippon Kayaku Co. supplied us with cisplatin

(CDDP; Cl2H6N2Pt) and etoposide (VP-16; C29H32O13).Irinotecan (CPT-11; C33H36N4O6) and its active form,SN-38 (C22H20N2O5), were provided by Yakult Co.Lapatinib (C29H26ClFN4O4S.2C7H8O3S) and gefitinib(C21H22ClFN4O3) were purchased from BioVision andSanta Cruz Biotechnology, respectively. Structures ofthese chemicals are shown in Fig. 1. Rhodamine 123,Hoechst 33342, and Cell Counting Kit-8 (CCK-8) werepurchased from Sigma Chemical, Invitrogen, andDojindo, respectively.

Flow cytometryCells (2� 105)were incubatedwith 0.5mg ofmouse anti-

humanHER2mAbor isotype controlmouse IgG2bk for 30minutes at 4�C followed by labeling with fluoresceinisothiocyanate (FITC)-conjugated goat antimouse IgG(Invitrogen). Stained cells were analyzed by FACSort(Becton Dickinson).

Immunoprecipitation and immunoblottingCells were untreated or treated with lapatinib and then

lysed in lysis buffer. For immunoprecipitation, after pre-clearingwith 20 mL protein G sepharose bead (GEHealth-care) slurry, whole-cell lysates were incubated with anti-human caveolin-1 or ABCB1 Ab (diluted 1:100 to 1:200)overnight at 4�C with gentle agitation. The immunopre-cipitates were washed 3 times in lysis buffer and dena-tured before carrying out SDS-PAGE. For immunoblot-ting, whole-cell lysates or immunoprecipitates were sep-arated in a 5% to 20% gradient gel (Wako) by SDS-PAGE,thereafter transferred to polyvinylidene difluoride mem-branes. The membranes were incubated with the proper

HER2 Involvement in ABC Transporter–Mediated MDR in SCLC

www.aacrjournals.org Mol Cancer Ther; 11(4) April 2012 831




primary Abs (diluted 1:250 to 1:500) overnight at 4�Cfollowed by appropriate horseradish peroxidase–conju-gated secondary Abs (diluted 1:5,000 to 1:10,000) for1 hour at room temperature. Immunoreactive bandswere visualized using a chemiluminescent techniquewith ECL Plus Western Blotting Detection Reagents (GEHealthcare).

Drug sensitivity assayCells (5 � 103 cells per well) were plated onto 96-well

tissue culture–treated plates (Corning Costar) and treatedwith serially diluted cytotoxic compounds in serum con-taining medium for 72 hours. The relative number ofviable cells was quantified using CCK-8 following theinstruction manual (26).

ABC transporter function analysisTo assess ABCB1- and ABCG2-inhibitory effect of lapa-

tinib, flow cytometric analysis using rhodamine 123 andHoechst 33342 was conducted as previously described(27, 28). Briefly, cells (1� 106/mL)were treatedwith 0.1, 1,or 10 mmol/L of lapatinib for 15 minutes, then rhodamineorHoechstwas added tofinal concentration of 0.1mmol/Lor 4 mg/mL, respectively. After incubation for 1 hour at37�C for staining, intracellular dye accumulations wereanalyzed by FACSort or FACSAria (Becton Dickinson).

HER2 knockdown analysisCells were transfected with either 30 pmol/L of short

interferingRNA(siRNA) against humanERBB2 (SHF27B-0846) or control RNAs (S30C-0126; B-Bridge International)

using Lipofectamine RNAiMAX (Invitrogen). After 48-hour incubation, cells were used for experiments.

Treatment of chemoresistant SCLC xenograftsMale, 6- to 8-week-old, BALB/cA Jcl nu/nu mice were

obtained from CLEA Japan. SBC-3/ETP cells (2 � 107) orSBC-3/SN-38 cells (1� 107) were injected subcutaneouslyin the flank of mice. When the tumor volume reachedapproximately 400 to 800 mm3, mice were randomizedinto the following 4 groups: (A) vehicle; (B) chemother-apy; (C) lapatinib; and (D) chemotherapy plus lapatinib.

Treatment schedules were as follows: etoposide oririnotecan was intraperitoneally administered at a doseof 20 mg/kg to mice bearing SBC-3/ETP xenograft ondays 1 to 3 or SBC-3/SN-38 xenograft on days 1, 5, and 9,respectively. Lapatinib was orally given at a dose of 100mg/kg 1 hour before chemotherapy. Tumor volume (V)was calculated according to the following equation: V(mm3) ¼ length � (width)2/2.

Statistical analysisAll the studies for statistical evaluationwere conducted

in triplicate in each experiment and repeated at least 3times. Mean� SD values were calculated and differenceswere evaluated by 2-sided Student t test. P < 0.05 wasconsidered statistically significant.

ResultsHER2 is preferentially expressed in Japanese SCLC

Because HER2 has been reported to be a negativeprognostic factor in ED-SCLC (15–17), we consider HER2as a key target molecule in treating SCLC. We firstassessed the expression of HER2 in SCLC cell lines byfluorescence-activated cell sorting (FACS) and immuno-blotting (Fig. 2A and B). Consistent with the previousfindings, the expression ofHER2wasdetected in none of 3SCLC cell lines (0%) of Caucasian origin (H69, H446, andN231). In contrast, 6 of 10 cell lines (60%) of Japaneseorigin (SBC-3, SBC-5, OS3R5, Smk, OC-10, and CADOLC6) expressed various amount of HER2. The higherfrequency of HER2 expression in SCLC cell lines fromJapanese patients suggests the ethnic difference betweenJapanese and Caucasians.

HER2 is upregulated in SBC-3–derivedchemoresistant cells

We next examined whether cytotoxic chemotherapyaffects HER2 expression by comparing chemosensitiveH69 and SBC-3 cells with chemoresistant SCLC cells,H69/CDDP, H69/VP, SBC-3/CDDP, SBC-3/ETP, andSBC-3/SN-38. These chemoresistant SCLC cells wereestablished from their parental cells by long-term expo-sure to respective cytotoxic agents. The expression ofHER2 was upregulated in all SBC-3–derived chemoresis-tant cells compared with that of parental cells but notdetected in any of H69 and H69-derived chemoresistantcells (Fig. 2C and D). This finding raises a possibility that

Lapatinib

Etoposide (VP-16)

Irinotecan (CPT-11)

Gefitinib

Cisplatin (CDDP)

SN-38

Figure 1. Chemical structures of lapatinib, gefitinib, etoposide (VP-16),cisplatin (CDDP), irinotecan (CPT-11), and SN-38.

Minami et al.

Mol Cancer Ther; 11(4) April 2012 Molecular Cancer Therapeutics832




upregulation of HER2 is involved in MDR mechanism ofSCLC.

Lapatinib restores chemosensitivity in ABCtransporter–positive SCLC cellsWe then investigated whether HER2 could be a direct

therapeutic target for SCLC. The cytotoxic effects of lapa-tinib on SCLC cells were examined using a CCK-8 cyto-toxicity assay. A phase I pharmacokinetic study hasshown that peak plasma concentration of lapatinib is2.43 mg/mL (¼ 2.58 mmol/L; ref. 29). Consistently, lapa-tinib alone could not affect the viability of HER2-positiveSCLC cells up to this concentration (Supplementary Fig.S1). Next, we examined the cytotoxic effects of combina-tion therapy with lapatinib and cytotoxic agents. Lapati-

nib at 1 mmol/L, which is a clinically achievable concen-tration, reversed drug resistance in HER2-positive cells,considerably in SBC-3/ETP (fold reversal ¼ 10.2; P ¼0.0076) and almost completely in SBC-3/SN-38 (foldreversal ¼ 15.5; P ¼ 0.0024), respectively. HER2-negativeH69/VP cells were also restored the sensitivity to etopo-side slightly by lapatinib (fold reversal ¼ 3.3; P ¼ 0.0175).On the other hand, 1 mmol/L of lapatinib could notenhance cytotoxicity of chemotherapeutic drugs in paren-tal cells and also could not reverse chemoresistancein cisplatin-resistant cells (Fig. 3A and SupplementaryTable S 1).

Because lapatinib has been reported to inhibit ABCtransporter functions and restore chemosensitivityin breast carcinoma (14), we evaluated the expression

Figure 2. HER2 is preferentiallyexpressed in Japanese SCLC andupregulated in SBC-3–derivedchemoresistant cells. A and B, HER2expression in 13 SCLC cell lines(H69, H446, andN231 are Caucasianorigin. SBC-1, SBC-2, SBC-3, SBC-5, OS-1, OS2RA, OS3R5, Smk, OC-10, and CADO LC6 are Japaneseorigin). C andD, comparison ofHER2expression between parental SBC-3and chemoresistant SBC-3 sublines.In FACSanalysis (A andC), cellswerestained with 5 mg/mL of either anti-HER2 mouse mAb or isotype-matched control antibody andpresented as histogram of HER2-stained cells (black shaded) overlaidwith control cells (solid line). Inimmunoblotting analysis (B and D),30 mg of total protein from whole-celllysate was separated by SDS-PAGEand transferred onto polyvinylidenedifluoride membranes.Immunodetection of HER2 and actinwas conducted using anti-HER2mouse mAb (diluted 1:500) and anti-actin goat pAb (diluted 1:800),respectively. Actin was used as aninternal control. Each experimentwas carried out at least twice andrepresentative results are shown.

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Figure 3. Lapatinib restores chemosensitivity in ABC transporter–positive SCLC cells. A, after cells were treatedwith serially diluted concentration of indicateddrugs for 72 hours with or without 1 mmol/L of lapatinib, relative number of viable cells was quantified by CCK-8 assay. Experiments were repeatedat least 3 times in triplicates. Points, themean percentage of viable cells; bars, SD. B, expression of ABCB1 and ABCG2 in parental and chemoresistant SCLCcells. Each lane contains 30 mg of total protein from whole-cell lysate. Anti-ABCB1 rabbit pAb (diluted 1:500) and ABCG2 rabbit pAb (diluted 1:500)were used for immunodetection. C, expression status of ABCB1andABCG2during lapatinib treatment. After cells were treatedwith various concentrations oflapatinib up to 72 hours, ABCB1 in SBC-3/ETP and ABCG2 in SBC-3/SN-38 cells were examined by immunoblotting as mentioned above and areshown with actin as internal control.

Minami et al.





of ABCB1 and ABCG2 in chemoresistant SCLC cellsby immunoblotting. As shown in Fig. 3B, overexpres-sion of ABC transporters was detected exclusively inthe cells, ABCB1 in H69/VP and SBC-3/ETP cells, andABCG2 in SBC-3/SN-38 cells, in which lapatinibcould restore the chemosensitivity. In contrast, nodetectable ABC transporters were expressed in H69/CDDP and SBC-3/CDDP cells in which lapatinib didnot show the reversal effect. We also confirmed thatlapatinib does not downregulate the expression ofABCB1 and ABCG2 up to 72 hours (Fig. 3C). Theseresults imply that lapatinib reverses the chemoresis-tance by inhibiting ABC transporter functions withoutaffecting their expression, and the reversal effectin ABCB1-positive cells has a dependency on HER2expression.

Lapatinib suppresses ABCB1 functions throughHER2 inactivationBecause lapatinib restored the sensitivity to etopo-

side of ABCB1-positive cells more effectively in HER2-positive SBC-3/ETP cells than in HER2-negativeH69/VP cells (Fig. 3A), we investigated whether HER2is involved in the lapatinib-mediated inhibition ofABCB1 functions. We then conducted a rhodamine 123flow cytometric assay to examine whether lapatinib in-hibits the efflux pump function of ABCB1 differentiallydepending on HER2 expression status. Intracellularaccumulation of rhodamine was less in etoposide-resis-tant SBC-3/ETP and H69/VP cells than the respectiveparental SBC-3 and H69 cells without lapatinib treat-ment. However, lapatinib increased the accumulationof rhodamine in both SBC-3/ETP and H69/VP cells butnot in their parental cells (Fig. 4A and B). Notably, thisaccumulation was observed even at a lower concentra-tion of lapatinib (1 mmol/L) in HER2-positive SBC-3/ETP cells. In contrast, in HER2-negative H69/VP cells,the accumulation was observed only with a higherconcentration (10 mmol/L), which is far beyond themaximum plasma concentration in humans. BecauseSBC-3/ETP cells express not only HER2 but also EGFR(Supplementary Fig. S2), we tested whether gefitinib, aselective EGFR-TKI, could exert similar effects. Gefitinibcould accumulate rhodamine in SBC-3/ETP cells onlywith a higher concentration (10 mmol/L) but not with amaximum clinical plasma concentration in humans(<1.0 mmol/L; ref. 30; Fig. 4C).To determine the involvement of HER2 in the func-

tional regulation of ABCB1 by lapatinib, we knockeddown HER2 in SBC-3/ETP cells using siRNA technique.FACS and immunoblotting analysis confirmed that theexpression of HER2 was successfully suppressed,whereas no change was seen in the expression of ABCB1(Supplementary Fig. S3). Lapatinib-induced increase inaccumulation of rhodamine was considerably attenuat-ed in HER2 knocked down SBC-3/ETP cells (Fig. 4D).The values of mean fluorescence intensity of intracel-lular rhodamine in cells transfected with control siRNA

versus HER2-targeting siRNA at various concentrationof lapatinib were 5.4 versus 2.1 at 0.1 mmol/L, 75 versus48 at 1 mmol/L, and 201 versus 127 at 10 mmol/L,respectively. These results indicate that lapatinib cansuppress ABCB1 functions partly through inactivationof HER2.

Caveolin-1 and Src are involved in the regulation ofABCB1 functions by HER2

Caveolin-1 is a major component and an integralmembrane protein of caveolae, which is known tointeract with ABCB1 (31). In addition, overexpressionof Src is reported to induce caveolin-1 phosphoryla-tion and also negatively modulate ABCB1 functions(32). We therefore focused on caveolin-1 and Src toexplore the mechanisms involved in lapatinib-mediat-ed suppression of ABCB1 functions in HER2-positiveSCLC cells. In SBC-3/ETP cells, HER2 was depho-sphorylated by lapatinib, whereas Src phosphorylationlevels were decreased at the 527th tyrosine residuein the C-terminal tail and increased at the 416th tyro-sine residue in the activation loop. Moreover, caveolin-1 was further phosphorylated at the 14th tyrosineresidue. Finally, Src enzyme activity was upregulatedalong with inactivation of HER2 by lapatinib, whichmight successively bring about further phosphoryla-tion of caveolin-1 and lead to the functional suppres-sion of ABCB1 (Fig. 5A).

Immunoprecipitation study showed that caveolin-1primarily associated with HER2. However, once SBC-3/ETP cells were exposed to lapatinib, interactionbetween caveolin-1 and HER2 was abated, whereas inter-action between caveolin-1 and ABCB1 was enhancedalong with phosphorylation of caveolin-1. In contrast,direct association of HER2 with ABCB1 was not detectedregardless of lapatinib treatment (Fig. 5B). Lapatinib-mediated molecular events involved in the suppressionof ABCB1 function via inactivation of HER2 are schema-tized in Fig. 5C.

Lapatinib suppresses ABCG2 functionsindependently of HER2 inactivation

We further investigated whether lapatinib inhibitsABCG2 functions directly or by way of HER2 inactiva-tion in ABCG2-positive SBC-3/SN-38 cells. IntracellularHoechst accumulation was dramatically increased bylapatinib dose dependently and reached plateau at1 mmol/L in SBC-3/SN-38 cells but not in ABCG2-negative SBC-3 cells, indicating that treatment with 1mmol/L of lapatinib completely inhibited ABCG2 func-tions in SBC-3/SN-38 cells comparable with the paren-tal SBC-3 cells (Fig. 6A). This inhibitory effect on ABCG2functions, unlike in case of ABCB1, was hardly everaffected by knocking down of HER2 (Fig. 6B). Thesefindings suggest that lapatinib inhibits ABCG2 func-tions independently of HER2 inactivation, which isdifferent from the HER2-dependent inhibitory mecha-nism of ABCB1.






Combination therapy with lapatinib and cytotoxicdrugs suppresses the growth of ABC transporter–positive SCLC tumors in vivo

To examine whether combination therapy of lapatinibwith cytotoxic agents could reverse MDR in vivo, weestablished SBC-3/ETPandSBC-3/SN-38 xenograftmod-els and subsequently treated them with or without lapa-tinib. There were no significant differences in tumor sizebetween animals treated with vehicle, cytotoxic agent(etoposide or irinotecan), or lapatinib alone. However, inboth xenografts, the lapatinib combination therapy witheither etoposide or irinotecan significantly inhibitedtumor growth compared with the treatment with onlyvehicle, cytotoxic agent, or lapatinib (P < 0.02 in SBC-3/

ETP xenograft, P < 0.0002 in SBC-3/SN-38 xenograft; Fig.7A and B). In addition, nomortality or serious decrease inbody weight was observed at the doses tested. Theseresults indicate that the combination therapy with lapa-tinib and cytotoxic agent is promising to overcome MDRSCLC in vivo.

DiscussionWe have investigated the participation of HER2 in

ABC transporter–mediated chemoresistance in SCLC,and a series of molecular mechanisms on how lapatinib-triggered HER2 inactivation leads to ABCB1 func-tional inhibition. As a result, we have presented thatclinically applicable low dose of lapatinib could reverse

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Figure 4. Lapatinib suppressesABCB1 functions partly throughHER2 inactivation. Cells weretreated with serially dilutedlapatinib for 15 minutes andthereafter exposed to 0.1 mmol/L ofrhodamine 123 for 1 hour at 37�C.Thereafter, intracellularaccumulation of rhodamine, as theinhibitory index of ABCB1functions, was analyzed byFACSort. A, intracellularrhodamine accumulation betweenHER2-positive ABCB1-negativeSBC-3 cells and HER2-positiveABCB1-positive SBC-3/ETP cellsin the presence (0.1, 1 or 10mmol/L)or absence of lapatinib. B,intracellular rhodamineaccumulation between HER2-negative ABCB1-negative H69cells and HER2-negative ABCB1-positive H69/VP cells in thepresence or absence of lapatinib.C, intracellular rhodamineaccumulation assay with EGFR-TKI, gefitinib in HER2-positiveEGFR-positive SBC-3/ETP cells.D, comparison of rhodamineaccumulation at each lapatinibconcentration between controlsiRNA-transfected SBC-3/ETPcells and HER2 siRNA-transfectedSBC-3/ETP cells. These flowcytometric assays were conductedat least twice with similar resultsand representative histograms areshown. RNAi, RNA interference.

Minami et al.





chemoresistance of SCLC cells that overexpress ABCtransporters and that the reversal effect by lapatinib ismore prominent in HER2-positive cells than in HER2-negative cells. These findings imply that combinationtherapyof lapatinibandcytotoxic agentwouldbeeffectivefor chemoresistant SCLC.Some studies have identified HER2 expression as a

negative prognostic factor of SCLC, especially in exten-

sive diseases in Caucasians (15, 16). HER2 overexpres-sion has been reported to be about 10% to 30% inWestern patients with SCLC (17) and 10% (1 of 11) ofSCLC cell lines established from Caucasian patients(33). However, here we have found that HER2 is expres-sed more frequently in SCLC cell lines of Japaneseorigin (60%) than Caucasian origin (0%), showing thepossibility of ethnic differences of HER2 expression in

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Figure 5. Caveolin-1 and Src are involved in the regulation of ABCB1 functions by HER2. A, SBC-3/ETP cells were treated with 1 mmol/L lapatinib up to 120minutes. Tyrosine (Tyr) phosphorylation and expression of HER2, Src, and caveolin-1 (Cav-1) in whole-cell lysates were examined by immunoblotting. Eachlane contains 30mgof total protein, and antibodydilution used todetect eachproteinwas as follows: anti-phospho-HER2 rabbitmAb (1:250), anti-HER2 rabbitpAb (1:500), anti-phospho-Src (Tyr 416 and Tyr 527) rabbit pAb (1:500), anti-Src rabbit pAb (1:500), anti-phospho-Cav-1 (1:250), and anti-Cav-1 (1:500). B,SBC-3 cells were treatedwith 1mmol/L lapatinib up to 120minutes, lysed in lysis buffer, and cell lysateswere immunoprecipitated (IP)with anti-ABCB1mousemAb (diluted 1:50) and anti-Cav-1 rabbit mAb (diluted 1:200). Immunoprecipitates were separated by SDS-PAGE and transferred onto polyvinylidenedifluoride membranes. Immunodetection of ABCB1, Cav-1, and phospho-tyrosine was done with anti-ABCB1 rabbit pAb (1:500), anti-Cav-1 rabbit mAb(1:500), and anti-phospho-tyrosinemousemAb (1:500), respectively. Representative blots from 3 independent experiments with similar results are shown. C,molecular events on how lapatinib (Lap) acts as an inhibitor of ABCB1, despite being its substrate, in the presence of etoposide (VP-16) are schematized. Inaddition to direct inhibition (light blue curved line), indirect inhibitory pathway (pink curved line) through suppressing HER2 signal transduction is presumed toexist on the findings in A and B. Lapatinib as a substrate for ABCB1 is not shown in this schema. IB, immunoblotting; P, phosphate.






SCLC. On the basis of the clinical evidence that EGFR-TKI–sensitive mutations are detected more frequentlyin East Asian patients with non-SCLC than in Cauca-sians (6, 34), we consider ethnic difference as an impor-tant factor for molecular targeting therapy. We alsoshowed that HER2 is upregulated after acquiringMDR phenotype in a HER2-positive SCLC cells. In thiscontext, it would be clinically important to determinewhether targeting HER2 therapy is effective in SCLC.

Targeting cell surface RTKs is an attractive strategyfor cancer treatment. In fact, several clinical trials tar-geting RTKs such as c-Kit and EGFR by TKIs have beenconducted in SCLC but eventuated in disappointingresults (35, 36). We here tested the antitumor activity

of targeting HER2 by lapatinib as monotherapy atclinically achievable concentration in vitro. However,we could not observe satisfactory effects (Supplemen-tary Fig. S1). A possible reason why inhibition of onespecific RTK was not effective for SCLC is that SCLCcells lack addiction to a specific RTK signaling pathway.Indeed, we have detected the presence of multipleRTKs, such as HER2, EGFR, insulin-like growth fac-tor-1 receptor, c-Kit, and c-Met, in each SCLC cell line(data not shown). SCLC cells thus probably use a varietyof growth factor/RTK signals to proliferate and survive.Therefore, additional effects apart from its direct inhib-itory effect on RTKs are needed for TKIs to exertexpected results in SCLC.

SB

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Figure 6. Lapatinib suppressesABCG2 functions independent ofHER2 inactivation. Cells weretreated with serially dilutedlapatinib for 15 minutes andthereafter exposed to 4 mg/mL ofHoechst 33342 for 1 hour at 37�C.Thereafter, intracellularaccumulation of Hoechst, as theinhibitory index of ABCG2functions was analyzed byFACSAria. A, comparison ofintracellular Hoechst accumulationbetween ABCG2-negative SBC-3cells and ABCG2-positive SBC-3/SN-38 cells in the presence (0.1 or1 mmol/L) or absence of lapatinib.B, comparison of intracellularHoechst accumulation betweencontrol siRNA–transfected SBC-3/SN-38 cells and HER2 siRNA–transfected SBC-3/SN-38 cells ateach concentration of lapatinib.These experiments were repeatedat least twice with similar results,and representative data are shown.RNAi, RNA interference.

Minami et al.





Recently, it has been reported that many anticancerTKIs are substrates for ABC transporters and also act asinhibitors of them to increase intracellular accumulationof coexisting other substrates such as cytotoxic drugs (37–39). HER-TKIs (e.g., gefitinib, erlotinib, and lapatinib)have been shown to have potentials to restore sensitivityin chemoresistant cancer cells by inhibiting ABC trans-porter functions (7–9, 14). These studies proposed thatHER-TKIs directly inhibit drug efflux pump functionsbecause the inhibitory effects were observed regardlessof HER expression. However, in our present study, theinhibitory effects of lapatinib on ABCB1 were observedwith a lower concentration in HER2-positive SBC-3/ETPcells than in HER2-negative H69/VP cells (Figs. 3Aand 4A andB), and the effectwas considerably attenuatedin HER2 knocked down SBC-3/ETP cells (Fig. 4D). Our

results suggest that lapatinib not only inhibits ABCB1functions directly but also indirectly through HER2 inac-tivation. Contrastively, inhibitory effect of lapatinib onABCG2 functions is far stronger than on ABCB1 and 1mmol/L of lapatinib is enough to overcomeABCG2-medi-ated MDR in SCLC (Figs. 3A and 6A and B).

We here have highlighted the involvement of HER2 inthe regulation of ABC transporter functions. This study isthe first to elucidate the mechanism behind lapatinib’sreversal of ABCB1-mediated MDR, partly through HER2inactivation. We focused on caveolin-1 as a moleculewhich intervenes between HER2 and ABCB1. Caveolin-1 is recognized as a negative regulator of EGFR andHER2(40, 41).Moreover, a recentwork showed that overexpres-sion of caveolin-1 decreased the transport activity ofABCB1 (31). In another study, downregulation of caveo-lin-1 by siRNA was shown to reduce the interaction withABCB1 and augment its activity (32). In HER2-positiveSBC-3/ETP cells, lapatinib suppresses HER2 tyrosinekinase activity, which causes caveolin-1 phosphorylationand dissociation from HER2, perhaps because cavelin-1no longer needs to downregulate HER2. On the contrary,Src enzyme activity is upregulated. This is a key phenom-enon to explain how interaction between caveolin-1 andABCB1 enhances to inhibit ABCB1 functions. Barakat andcolleagues reported that Src overexpression inducedcaveolin-1 phosphorylation and downregulated ABCB1functions through increasing interaction between caveo-lin-1 and ABCB1 in rat brain endothelial cells (32). Inaddition, Hawkins and colleagues showed that YEEIP,a Src kinase–activating peptide, induced caveolin-1 phos-phorylation and led to downregulate ABCB1 activity inrat brain capillaries (42). Our findings presented in Fig. 5support these observations. Namely, lapatinib treatmentinduces caveolin-1 phosphorylation through activatingSrc activity, then enhances interaction between caveo-lin-1 and ABCB1, and finally inhibits ABCB1 activity.Thus, we have crystallized the molecular regulation sys-tem between HER2 and ABCB1 and that is why relativelow concentration of lapatinib can restore chemosensitiv-ity in HER2-positive SCLC cells.

In the clinical setting, relapse with brain metastases isthe major problem in SCLC. Systemic chemotherapyalone is less effective for asymptomatic brain metastasesthan expected (43), for which blockade of drug pene-tration into the brain by the blood–brain barrier (BBB) isbelieved to be the main reason. ABCB1 is known to beabundantly expressed on the BBB (44) and excrete itssubstrates. Polli and colleagues showed that the con-centration of orally administered lapatinib in the brainwas far lower than in the plasma in rat because thiscompound served not only as a inhibitor but also as asubstrate of ABCB1 (38). They further showed thatABCB1 and ABCG2 on the BBB synergistically hinderedlapatinib from penetrating into the central nervoussystem (45). On the other hand, the BBB is thought tohave been already disrupted in advanced SCLC withradiologically recognizable brain metastases (46).

ASBC-3/ETP

BSBC-3/SN-38

Tu

mo

r vo

lum

e (×

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3 )T

um

or

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CPT-11 (n = 10)

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Figure 7. Combination therapy with lapatinib and cytotoxic drugssuppresses the growth of ABC transporter–positive SCLC tumors in vivo.SCLC cells were injected subcutaneously in the flank of athymic nudemice. When the tumor volume reached approximately 400 to 800 mm3,mice were randomized into 4 treatment groups (n ¼ 9–11 for each). A,etoposide (VP-16) was i.p. administered into mice bearing SBC-3/ETPtumors at a dose of 20 mg/kg on days 1 to 3. B, irinotecan was i.p.administered into mice bearing SBC-3/SN-38 tumors at a dose of20 mg/kg on days 1, 5, and 9. Lapatinib was orally given at a dose of100 mg/kg 1 hour before every cytotoxic drug administration. Tumorvolume (V) was calculated according to the following equation: V (mm3)¼length � (width)2/2. There was no significant difference (N.S.) in tumorsize between animals treated with vehicle, cytotoxic drug, or lapatinibalone. However, in both xenografts, lapatinib combination therapysignificantly suppressed the tumor growth compared to cytotoxic drugalone (P < 0.02 in A andP < 0.0002 in B). Points, themean tumor volumes;bars, SD; arrows, drug administration.






Moreover, Taskar and colleagues reported that the con-centration of lapatinib in metastatic brain tumors wasmarkedly higher than normal brain tissues in mice thatreceived intracardiac injection of breast cancer cells (47).Lapatinib, like other TKIs, serves as a substrate of ABCtransporters and is excreted from cells at lower concen-trations. However, it functions as an inhibitor of ABCtransporters at higher doses in turn (39). Therefore, inrelapsed SCLC, lapatinib is expected to penetrate intometastatic brain tumors and reach the concentrationenough to restore the chemosensitivity.

Collectively, our present study showed that an ethnicdifference might exist in HER2 expression in SCLC. Weare now conducting histologic study of HER2 status inJapanese patients with SCLC. We also proved that HER2is involved in the inhibitory effect of lapatinib on ABCtransporters. Lapatinib combination therapy is promisingto bring about better prognosis by overcoming MDRand is valuable for future clinical applications in patientswith SCLC who develop chemoresistance, especially inpatients with HER2-positive SCLC.

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

Authors' ContributionsConception and design: T. Minami, T. KijimaDevelopment of methodology: T. Minami, T. Kijima, R. TakahashiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Minami, T. KijimaAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): T. Minami, T. Kijima, Y. Otani, S. Kohmo,I. Nagatomo, H. Hirata, M. Suzuki, K. Inoue, Y. Takeda, H. Kida,I. Tachibana, A. KumanogohWriting, review, and/or revision of themanuscript: T.Minami, T. Kijima,Y. Otani, S. Kohmo, R. Takahashi, I. Nagatomo, H. Hirata, M. Suzuki, K.Inoue, Y. Takeda, H. Kida, I. Tachibana, A. KumanogohAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): T. Minami, T. Kijima, R. Takahashi,A. KumanogohStudy supervision: T. Kijima, A. Kumanogoh

Grant SupportThe study was supported by Grant-in-Aid for Scientific Research (C)

from the Ministry of Education, Culture, Sports, Science and Technology,Japan (21590993 to T. Kijima) and Funding Program for Next GenerationWorld-Leading Researchers (NEXT Program) and Special CoordinationFunds for Promoting Science and Technology, Japan (A. Kumanogoh).

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 November 2, 2011; revised February 15, 2012; acceptedFebruary 15, 2012; published OnlineFirst March 2, 2012.

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2012;11:830-841. Published OnlineFirst March 2, 2012.Mol Cancer Ther Toshiyuki Minami, Takashi Kijima, Yasushi Otani, et al.

Mediated Chemoresistance in Small Cell Lung Cancer−TransporterHER2 As Therapeutic Target for Overcoming ATP-Binding Cassette

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