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의학박사 학 논문

Mechanism of resistance to

17-DMAG, an Hsp90 inhibitor,

in lung cancer cell lines

with ALK rearrangement

Hsp90 억제제인 17-DMAG 에 대한

ALK 유전자 전위가 있는 폐암 세포주의

내성 발현 기전에 관한 연구

2015년 2월

서울 학교 학원

의학과 내과학 공

김 희 정

Mechanism of resistance to

17-DMAG, an Hsp90 inhibitor,

in lung cancer cell lines

with ALK rearrangement

February 2015

Seoul National University

Internal Medicine

Hee Joung Kim

Hsp90 억제제 17-DMAG 에 대한

ALK 전 전 가 는 폐암

포주 내 발현 기전에 한 연

지도 수 김 영 환

문 학박사 학 문 로 제출함

2014 10월

울대학 대학원

학과 내과학 전공

김 희 정

김희정 학박사 학 문 함

2014 12월

원 ( )

부 원 ( )

원 ( )

원 ( )

원 ( )

Professor Chairman

Professor Vice Chairman

Professor

Professor

Professor

Mechanism of resistance to 17-DMAG,

an Hsp90 inhibitor,

in lung cancer cell lines

with ALK rearrangement

by

Hee Joung Kim

A Thesis Submitted to the Department of Medicine

in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy in Medical Science (Internal Medicine)

at the Seoul National University College of Medicine

December, 2014

Approved by thesis committee:

- i -

Abstract

Mechanism of resistance to 17-DMAG,

an Hsp90 inhibitor,

in lung cancer cell lines with ALK rearrangement

Hee Joung Kim

Department of Medicine (Internal Medicine)

The Graduate School

Seoul National University

Heat shock protein 90 (Hsp90) inhibitors have shown clinical activity against

lung cancer cells with rearrangement of the anaplastic lymphoma kinase

(ALK) gene. We investigated the mechanism of acquired resistance to

17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a

geldanamycin analog Hsp90 inhibitor, in H3122 and H2228 lung cancer cell

lines with ALK rearrangement.

Resistant cell lines (H3122/DR-1, H3122/DR-2, and H2228/DR) were

established by repeated exposure to increasing concentrations of 17-DMAG.

MTT assay, western blotting analysis and reverse transcription-polymerase

chain reaction were done to assess cytotoxicity, relative proteins and mRNA

- ii -

expression. Flow cytometry after incubation with rhodamine 123 (Rho123)

performed for determining the activity of P-glycoprotein (P-gp; ABCB1/

MDR1).

The resistant cells showed no cross-resistance to AUY922 or ALK

inhibitors, suggesting persistent ALK dependency of cells with acquired

resistance to 17-DMAG. Interestingly, all resistant cells showed the induction

of P-gp at the protein and RNA levels, which was associated with increased

efflux of the P-gp substrate Rho123. Transfection with siRNA directed against

P-gp or treatment with verapamil, an inhibitor of P-gp, restored sensitivity to

the drug in all cells with acquired resistance to 17-DMAG. Furthermore, we

observed that the growth-inhibitory effect of 17-DMAG decreased in A549/PR

and H460/PR cells generated to overexpress P-gp by long-term exposure to

paclitaxel and that these cell lines recovered the sensitivity to 17-DMAG

through inhibition of P-gp. Although the expression of NAD(P)H:quinone

oxidoreductase 1 (NQO1), previously known as one of the factors associated

with 17-DMAG resistance, decreased in H3122/DR-1 and H3122/DR-2 cells,

NQO1 inhibition by dicumarol did not affect the response of H3122 cells to

17-DMAG.

- iii -

Keywords : lung cancer, anaplastic lymphoma kinase, heat shock

protein 90, resistance, P-glycoprotein

Student number : 2009-30513

In summary, P-gp overexpression is one of the possible mechanisms of

acquired resistance to 17-DMAG in lung cancer cells with ALK

rearrangement.

- iv -

Contents

Abstract i

Contents iv

List of tables v

List of figures vi

List of abbreviations and symbols viii

1. Introduction 1

2. Materials and methods 5

3. Results 11

4. Discussion 35

5. References 40

Korean abstract 47

- v -

Table 1. Chemosensitivity of 17-DMAG-resistant cell lines 13

Table 2. Chemosensitivity of paclitaxel-resistant cell lines 24

List of Tables

- vi -

Figure 1.Establishment of acquired resistance to 17-DMAG in

H3122 and H2228 cells 12

Figure 2.Modulation of ALK signaling in parental and

17-DMAG–resistant cells 15

Figure 3.The induction of P-gp/MDR1 expression in 17-DMAG

–resistant cells 16

Figure 4. Flow cytometric patterns of parental and resistant

cells stained with Rho12318

Figure 5. P-gp silencing with siRNA 19

Figure 6. Cell viability after transfection with P-gp siRNA or

verapamil pretreatment20

Figure 7. P-gp inhibition by verapamil treatment 21

Figure 8. Establishment of acquired resistance to paclitaxel in

A549 and H460 cells 23

Figure 9. The induction of P-gp/MDR1 expression in

paclitaxel-resistant cells25

Figure 10. Restoration of paclitaxel sensitivity of resistant cells

by verapamil pretreatment 26

Figure 11. P-gp silencing with siRNA 27

List of Figures

- vii -

Figure 13.No cross-resistance relationship between 17-DMAG

and AUY922 29

Figure 14.NQO1 expression in the parental cells and cells with

acquired resistance 31

Figure 15.NQO1 expression in the parental cells and cells with

acquired resistance by PCR-RFLP32

Figure 16. Total RNA and mRNA levels of NQO1 33

Figure 17.Cell viability after treatment with dicumarol, a

selective inhibitor of NQO1 34

Figure 18.Effects of a combination of 17-DMAG and

rapamycin in parental and 17-DMAG-resistant cells 38

Figure 12. Cell viability after transfection with P-gp siRNA 28

- viii -

List of abbreviations and symbols

NSCLC: non-small cell lung cancer

EML4-ALK: echinoderm microtubule-associated protein-like 4–anaplastic

lymphoma kinase

EGFR: epidermal growth factor receptor

KRAS: Kirsten rat sarcoma 2 viral oncogene homolog

TKIs: tyrosine kinase inhibitors

FISH: fluorescence in situ hybridization

c-MET: mesenchymal-epithelial transition factor

Hsp: heat shock protein

17-DMAG: 17-Dimethylaminoethylamino-17-demethoxygeldanamycin

17-AAG: 17-allylamino-17-demethoxygeldanamycin

MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Rho123: rhodamine 123

SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis

P-gp: permeability glycoprotein

- ix -

FACS: flow cytometry

NQO1: NAD(P)H:quinone oxidoreductase 1

RT-PCR: Quantitative reverse transcription-polymerase chain reaction

MRP: multidrug resistance protein

MDR: multidrug resistance

GAPDH: glyceraldehyde-3-phosphate dehydrogenase

siRNA: small interfering RNA

DR: drug resistance

ATP: adenosine triphosphate

PR: paclitaxel resistance

IC50: half maximal inhibitory concentration

HSF: heat shock factor

RFLP: restriction fragment length polymorphism

- 1 -

1. Introduction

Targeted therapy using tyrosine kinase inhibitors against

oncogenic driver mutations in non-small cell lung cancer (NSCLC) has

been developed to enhance selective cytotoxic effects on tumor cells.

The echinoderm microtubule-associated protein-like 4–anaplastic

lymphoma kinase (EML4-ALK) fusion oncogene, a result of an

inversion within chromosome 2p, leads to constitutive kinase activity by

ALK dimerization (1), represents a major molecular target in lung

cancer. Although ALK-rearranged lung cancer has accounted for only

3–7% of NSCLC cases since it was first discovered in 2007, this variant

could represent more than 70,000 new cases worldwide annually.

Furthermore, the detection rates for genetic screening will be higher in

selected subgroups based on clinical features commonly associated with

ALK rearrangement, including a history of no smoking or light

smoking, adenocarcinoma histology, and wild-type epidermal growth

factor receptor (EGFR) and KRAS (2). ALK tyrosine kinase inhibitors

(TKIs) have been found to be remarkably effective in the treatment of a

subset of NSCLC patients harboring ALK rearrangement, as confirmed

by fluorescence in situ hybridization (FISH) (3).

Crizotinib is an orally administered multitargeted small-

molecule tyrosine kinase inhibitor that inhibits mesenchymal epithelial

- 2 -

transition growth factor (c-MET) as well as ALK phosphorylation. It is

recommended as a first-line treatment option for patients with locally

advanced or metastatic NSCLC with ALK gene rearrangement (4).

Compared to standard chemotherapy, crizotinib treatment afforded

superior progression-free survival (7.7 months vs. 3.0 months) and

objective response rate (65% vs. 20%) in patients with previously

treated, advanced NSCLC with ALK rearrangement (5). However,

despite the successful initial response, most patients eventually develop

acquired resistance to crizotinib (6, 7), similar to the development of

acquired resistance to EGFR-TKIs. There are multiple mechanisms of

drug resistance, including various acquired mutations that hamper drug

binding, oncogenic bypass via activation of EGFR or c-kit (7, 8), and

epithelial-mesenchymal transition (9). Ceritinib, a second-generation

ALK inhibitor, has demonstrated efficacy in patients resistant to

crizotinib as well as crizotinib-naive patients (10) and has already been

approved by the US Food and Drug Administration for use in patients

who have progressed on or are intolerant of crizotinib. Other second-

generation ALK inhibitors such as CH5424802, AP26113, ASP3026,

X-396, and TSR-011 are undergoing phase I or phase II clinical trials

(11). In addition, heat shock protein 90 (Hsp90) inhibitors have been

suggested as therapeutic options to overcome drug resistance, on the

basis of their anti-tumor activity in preclinical models of ALK-driven

- 3 -

lung cancer (12, 13) and small-scale clinical trials on ALK-positive lung

cancers (14).

Hsp90 is a molecular chaperone that plays an important role in

the modification and stabilization of a variety of proteins that have been

implicated in tumor cell proliferation and survival. Both EGFR and

EML4–ALK fusion protein, which are known to be major oncogenic

drivers in NSCLC, have been shown to be client proteins for Hsp90 (12,

15, 16). Therefore, Hsp90 could be an alternative therapeutic target

instead of direct kinase inhibition in ALK-driven lung cancer. In vivo

and in vitro studies have demonstrated that treatment with Hsp90

inhibitors such as 17-Dimethylaminoethylamino-17-demethoxy

geldanamycin (17-DMAG), ganetespib (STA-9090), and IPI-504

reduced protein levels of the ALK fusion, enhanced cell death, led to

tumor regression, and prolonged survival in xenograft models (13, 15,

16). Anti-tumor activity has also been observed in phase I and phase II

clinical trials of ganetespib and IPI-504 (14, 17), and clinical trials are

underway on a number of Hsp90 inhibitors, both as monotherapies and

in combination with ALK TKIs for ALK-positive lung cancer.

17-DMAG is a water-soluble analog of geldanamycin that can

be orally administered. It has excellent bioavailability and has shown

more potent anti-tumor activity than that of 17-allylamino-17-

demethoxygeldanamycin (17-AAG) (18). 17-DMAG has shown an anti-

- 4 -

proliferative effect on the growth of NSCLC cell lines that are resistant

to EGFR-TKIs (19) and a synergistic effect with radiation against

NSCLC cell lines (20). Clarification of the resistance mechanisms

relevant to ALK-positive NSCLC is important for finding ways to

overcome multidrug resistance. We have established a series of ALK-

positive lung cancer cell lines by selecting cells in increasing

concentrations of 17-DMAG and have investigated the mechanism of

resistance to 17-DMAG.

- 5 -

2. Materials and Methods

2.1. Cell culture and reagents

The H2228 cell line was purchased from the American Type

Culture Collection (Rockville, MD). The H3122 cell line was a gift

from Adi F. Gazdar (UT Southwestern, Dallas, TX). Cells were cultured

in 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL

streptomycin (Invitrogen, Carlsbad, CA) at 37°C in an atmosphere with

5% CO2. Crizotinib, TAE-684, 17-DMAG, AUY-922, and verapamil

hydrochloride were obtained from Selleck Chemicals Co. Ltd (Houston,

TX). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT) solution, 3,3’-methylene-bis(4-hydorxycoumarin) (dicumarol),

and rhodamine 123 (Rho123) were purchased from Sigma (St. Louis,

MO).

2.2. Establishment of 17-DMAG resistance in H3122

and H2228 cells

17-DMAG–resistant cells were developed by chronic, repeated

exposure to 17-DMAG. Over a period of 6 months, the cells were

continuously exposed to increasing concentrations of the drug in

culture, and the surviving cells were cloned. Cloned cells could survive

- 6 -

exposure to >50 nM 17-DMAG. In all studies, the resistant cells were

cultured in a drug-free medium for >1 week to eliminate the effects of

17-DMAG.

2.3. MTT assay

Cells were seeded onto 96-well plates and incubated overnight

and then treated with their respective agents for an additional 72 h. Cell

viability was determined using the previously described MTT-based

method (21). Each assay consisted of 8 replicate wells and was repeated

at least three times. Data are expressed as the percentage survival of the

control, which was calculated using absorbance after correcting for

background noise.

2.4. Western blot analysis

Whole cell lysates were prepared using EBC lysis buffer (50

mM Tris-HCl [pH 8.0], 120 mM NaCl, 1% Triton X-100, 1 mM EDTA,

1 mM EGTA, 0.3 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium

orthovanadate, 0.5% NP40, and 5 U/mL aprotinin) and centrifuged.

Proteins were separated using SDS-PAGE and transferred to

polyvinylidene difluoride membranes (Invitrogen) for western blot

analysis. Membranes were probed with antibodies against p-ALK

- 7 -

(Tyr1604), ALK, p-Akt (Ser473), P-glycoprotein (P-gp) (all from Cell

Signaling Technology, Beverly, MA), Akt, p-Erk (Thr202/Tyr204), Erk,

HSF1, Hsp90, Hsp70, Hsp27, NAD(P)H:quinone oxidoreductase 1

(NQO1), and β-actin (all from Santa Cruz Biotechnology, Santa Cruz,

CA) as the primary antibody, and then membranes were treated with

horseradish peroxidase-conjugated secondary antibody. All membranes

were developed using an enhanced chemiluminescence system (Thermo

Scientific, Rockford, IL).

2.5. Quantitative reverse transcription-polymerase

chain reaction (RT-PCR)

Total RNA isolation and cDNA synthesis were performed

using the RNA mini-kit protocol (Qiagen Inc., Valencia, CA) and

AccuPower RT mix reagent (Bioneer Corp., Daejeon, South Korea).

according to the manufacturer’s instructions. The oligonucleotide

sequences for amplification were as follows: forward primer 5′-

AGGCCTATTACCCCAGCAT-3′ and reverse primer 5′-

CGATCTTGGCGATGTTGATG-3′ for MRP1; forward primer 5′-

AATAGCACCGACTATCCA-3′ and reverse primer 5′-

GTGGGATAACCCAAGTTG-3′ for MRP2; forward primer 5′-

TGAGATCATCAGTGATACTAA-3′ and reverse primer 5′-

- 8 -

ATGCGGCTCTTGCGGAG-3′ for MRP3; forward primer 5′-

GTACATTAACATGATCTGGTC-3′ and reverse primer 5′-

CGTTCATCAGCTTGATCCGAT-3′ for MDR1; forward primer 5′-

AAGCAGTGCTTTCCATCA-3′ and reverse primer 5′-

TCCTGCCTGGAAGTTTAG-3′ for NQO1; forward primer 5′-

GCGAGAAGATGACCCAGATC-3′ and reverse primer 5′-

CCAGTGGTACGGCCAGAGG-3′ for β-actin; forward primer 5′-

GAGTCAACGGATTTGGTCGT-3′ and reverse primer 5′-

TTGATTTTGGAGGGATCTCG-3′ for glyceraldehyde-3-phosphate

dehydrogenase (GAPDH). PCR cycling conditions were as follows:

94°C for 60 s, primer annealing for 60 s, and elongation at 72°C, for a

total of 30–35 cycles (MRP1, MRP2, MRP3, MDR1, NQO1) or 25

cycles (β-actin, GAPDH). A final extension was terminated by a final

incubation at 72°C for 10 min. Annealing temperatures were 49°C for

MRP2, 55°C for β-actin and MDR1, 58°C for MRP1 and MRP3, and

60°C for NQO1 and GAPDH.

2.6. Rhodamine 123 efflux assay

Cells were incubated with or without 1 μM Rho123 for 1 h.

The cells were then washed twice in ice-cold medium and harvested

(Rho123 accumulation of cells) or incubated for 3 h in Rho123-free

- 9 -

medium. All samples were kept at 4°C until a cytometric analysis was

performed. Fluorescence of Rho123 was analyzed using a FACS calibur

flow cytometer and processed using Cell Quest Software (Becton-

Dickinson, Franklin Lakes, NJ). Rho123 efflux was measured by

counting cells in the M1 region of the plot and determined as the

percentage of cells in the M1 region of the plot.

2.7. Transfection of small interfering RNA

Small interfering RNA (siRNA) oligonucleotides specific to P-

gp and the siRNA control were purchased from Santa Cruz

Biotechnology. Introduction of siRNA was performed using

Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s

instructions. After transfection, the suppression of P-gp was determined

by western blot analysis. For the MTT assay, cells were seeded onto 96-

well plates after siRNA transfection, and then treated with the indicated

drugs for 72 h.

2.8. Detection of NQO1 polymorphism

DNA purification and detection of the gene polymorphism

were performed according to the previously reported methods (22).

Briefly, for the amplification of NQO1 gene fragment (230 bp), the

- 10 -

following pair of forward and reverse primers was used: 5′-

TCCTCAGAGTGGCATTCTGC-3′ and 5′-

TCTCCTCATCCTGTACCTCT-3′. The amplification was carried out

using AccuPower TagPCR PreMix (Bioneer Corp.). Each PCR mixture

contained forward and reverse primers (0.5 pmoL each) and 50 ng

genomic DNA at a final volume of 20 μL. PCR conditions consisted of

initial denaturing at 94°C for 5 min, 35 amplification cycles (95°C for

30 s, 58°C for 30 s, and 72°C for 30 s), and a final extension at 72°C for

5 min. For RFLP, the amplified fragments were digested with Hinf1

(Thermo Scientific) and analyzed by agarose gel electrophoresis. Wild-

type (Pro187Ser) NQO1 was identified by a 191-bp band while the

homozygous variant (Ser/Ser) and heterozygous variant (Pro/Ser)

displayed a 151-bp band and two other bands (191 bp and 151 bp),

respectively.

- 11 -

3. Results

3.1. Cells with acquired resistance to 17-DMAG are

sensitive to ALK inhibitors

17-DMAG-resistant sublines were derived from the parental

H3122 and H2228 cell lines by stepwise selection using increasing

concentrations of 17-DMAG, as described in the Materials and Methods

section. The resistant subline acquired two clones (H3122/DR-1 and

H3122/DR-2) from H3122 and only one clone (H2228/DR) from

H2228 because H2228 cells did not form colonies. As shown in Figure

1 and Table 1, all resistant cells showed about 10-fold higher resistance

to 17-DMAG than the parental cells, although H3122/DR cells acquired

a higher resistance than H2228/DR. Interestingly, 17-DMAG-resistant

cells showed no cross-resistance to ALK inhibitors or AUY922, a

potent, novel synthetic resorcinylic isoxazole amide inhibitor of Hsp90.

These results demonstrate that 17-DMAG–resistant cells maintain ALK

dependency.

The EML4-ALK fusion oncoprotein has been described as a

client protein of Hsp90, and Hsp90 inhibitors have been shown to lower

ALK levels in cells in culture and xenograft, leading to growth

inhibition (13, 15). To evaluate why the resistant cells demonstrated

sensitivity to AUY922, we examined the modulation of ALK signaling

- 12 -

Figure 1. Establishment of acquired resistance to 17-DMAG in

H3122 and H2228 cells

Cell viability and drug concentrations responsible for 50% growth

inhibition were determined using the MTT assay. Cells were treated

with 17-DMAG for 72 h. The values were calculated with data from at

least three independent experiments. Bars represent the standard

deviations.

- 13 -

Table 1. Chemosensitivity of 17-DMAG-resistant cell lines

Cells were treated with 17-DMAG, AUY922, crizotinib, and TAE-684

for 72 h. The values were calculated from growth curves obtained from

experiments performed in triplicate.

Drug IC50 values (mM, mean ± S.D.)

H3122 H3122/DR-1 H3122/DR-2

17-DMAG 0.03 (±0.1) 0.2 (±0.2) 0.9 (±1.5)

AUY922 0.03 (±0.1) 0.03 (±0.1) 0.04 (±0.2)

Crizotinib 0.5 (±0.3) 0.4 (±0.2) 0.4 (±0.1)

TAE-684 0.06 (±0.1) 0.05 (±0.1) 0.06 (±0.1)

Drug IC50 values (mM, mean ± S.D.)

H2228 H2228/DR

17-DMAG 0.23 (±0.2) 9.5 (±1.2)

AUY922 0.08 (±0.07) 0.07 (±0.1)

Crizotinib 0.1 (±0.5) 0.2 (±0.3)

TAE-684 0.08 (±0.1) 0.07 (±0.1)

- 14 -

using western blot analysis. In both parental and 17-DMAG–resistant

cells, AUY922 treatment was observed to suppress ALK activity, Akt

and Erk levels (Fig. 2). Unlike parental cells, all 17-DMAG–resistant

cells maintained ALK activity in the presence of 0.1 μM 17-DMAG,

indicating that the inhibitory effect of 17-DMAG on ALK signaling was

lower in all resistant cells than in parental cells. These observations may

not result from the reduction of drug binding affinity because certain

drug-resistance mutations were not detected at the Hsp90 N-terminal

domain containing the ATP-binding site (data not shown). Taken

together, these results indicate that the acquisition of 17-DMAG

resistance may be caused by failure to completely abolish ALK activity.

3.2. The induction of P-gp leads to 17-DMAG

resistance

Previous studies showed that the geldanamycin (17-AAG) and

ansamycin derivatives of Hsp90 inhibitors were inactive in P-gp and/or

MRP1 expressing cell lines (23-25). We examined the protein and

mRNA of major transporters involved in drug pumping. As shown in

Fig. 3, mRNA levels of multidrug resistance proteins (MRP1, 2, 3)

showed no difference between parental and resistance cells, but mRNA

- 15 -

Figure 2. Modulation of ALK signaling in parental and 17-DMAG–

resistant cells

Cells were treated with the indicated concentrations of 17-DMAG or

AUY922 for 6 h. The molecules of ALK-related signals were detected

by western blot analysis.

- 16 -

Figure 3. The induction of P-gp/MDR1 expression in 17-DMAG–

resistant cells

(A) Detection of MRPs and MDR1 mRNA was performed by

quantitative RT-PCR. The sizes of PCR products were 525 bp (MRP1),

1254 bp (MRP2), 828 bp (MRP3), 157 bp (MDR1), and 230 bp

(GAPDH). (B) P-gp expression was assessed using western blot

analysis.

- 17 -

and protein levels of P-gp were significantly increased in all resistant

cells. We next used Rho123 as a surrogate indicator to determine the

activity of P-gp. P-gp–mediated transport, indicated by intracellular

decrease of Rho123 fluorescence, was studied using flow cytometry.

Compared with parental cells (H3122 and H2228), a significant

decrease of intracellular Rho123 was observed in all resistant cells. The

mean percentage of Rho123 efflux (M1 region) was 6.5% (H3122),

37.3% (H3122/DR-1), 49.5% (H3122/DR-2), 12.5% (H2228), and

28.1% (H2228/DR) (Fig. 4). To determine whether the induction of P-

gp affects sensitivity to 17-DMAG, we used siRNA and inhibitor

(verapamil) to inhibit the expression or activity of P-gp. Verapamil is a

selective inhibitor of P-gp (26, 27). siRNA treatment effectively

suppressed P-gp expression (Fig. 5), and there were no significant

changes in the rate of proliferation with 5 μM verapamil in all cell lines

(data not shown). The inhibition of P-gp (suppression of protein

expression or reduction of activity) restored sensitivity to 17-DMAG in

all resistant cells (Fig. 6). Interestingly, H2228 cells showed some

increase in sensitivity to 17-DMAG through the inhibition of P-gp.

When resistant cells were pretreated with verapamil, the inhibition of

ALK signaling following 17-DMAG treatment was equal to that of the

parental cells (Fig. 7).

- 18 -

Figure 4. Flow cytometric patterns of parental and resistant cells

stained with Rho123

Cells were treated with 1 μM Rho123 for 1 h (gray peak) and then

incubated with Rho123-free media for 3 h (red peak). Rho123

fluorescence was analyzed by flow cytometry. Rho123 efflux was

measured by counting cells at the left of the dashed line of the plot (M1

region).

- 19 -

Figure 5. P-gp silencing with siRNA

Control and P-gp siRNA (100 nM) were introduced into parental or

resistant cells, and P-gp silencing was confirmed by western blot

analysis.

- 20 -

Figure 6. Cell viability after transfection with P-gp siRNA or

verapamil pretreatment

Cells were treated with the indicated concentrations of 17-DMAG after

transfection with anti-P-gp siRNA or pretreatment of 5 μM verapamil.

Cell viability was measured 72 h later using the MTT assay.

- 21 -

Figure 7. P-gp inhibition by verapamil treatment

Cells were pretreated with or without 5 μM verapamil and then treated

with the indicated concentrations of 17-DMAG for 6 h. The molecules

of ALK-related signals were detected by western blot analysis.

- 22 -

We also found that the induction of P-gp led to 17-DMAG

resistance in other cell lines. Paclitaxel-resistant cells were generated

using A549 and H460 cell lines. These resistant cells acquired about

100-fold higher resistance to paclitaxel than the parental cells (Fig. 8

and Table 2). Interestingly, induction of P-gp was observed in all

paclitaxel-resistant cells, although H460/PR cells showed higher P-gp

expression than A549/PR cells (Fig. 9). Cell survival assay showed that

pretreatment with verapamil completely overcame paclitaxel resistance

(Fig. 10). These results demonstrate that the induction of P-gp plays a

significant role in the acquisition of resistance to paclitaxel.

As expected, paclitaxel-resistant cells showed cross-resistance

to 17-DMAG, and the inhibition of P-gp restored sensitivity to 17-

DMAG in paclitaxel-resistant cells (Fig. 11 and 12). As with 17-

DMAG–resistant cells, the induction of P-gp did not affect sensitivity to

AUY922 (Fig. 13). These results further confirm that P-gp expression is

not associated with the efficacy of AUY922, but it plays a major role in

the mechanism of 17-DMAG resistance.

3.3. NQO1 expression was not associated with

acquired resistance to 17-DMAG

- 23 -

Figure 8. Establishment of acquired resistance to paclitaxel in A549

and H460 cells

Cell viability and drug concentrations responsible for 50% growth

inhibition were determined using the MTT assay. Cells were treated

with paclitaxel for 72 h. The values were calculated with data from at

least three independent experiments. Bars represent the standard

deviations

- 24 -

Table 2. Chemosensitivity of paclitaxel-resistant cell lines

Cells were treated with paclitaxel for 72 h. The values were calculated

from growth curves obtained from experiments performed in triplicate

Cell line IC50 values of paclitaxel (mM, mean ± S.D.)

A549 0.08 (±0.02)

A549/PR-1 18.3 (±2.5)

A549/PR-2 13.5 (±1.8)

H460 0.01 (±0.01)

H460/PR-1 71.1 (±10.5)

H460/PR-2 68.4 (±7.6)

- 25 -

Figure 9. The induction of P-gp/MDR1 expression in paclitaxel-

resistant cells

P-gp expression was analyzed using western blot analysis.

- 26 -

Figure 10. Restoration of paclitaxel sensitivity of resistant cells by

verapamil pretreatment

Cells were pretreated with or without 5 μM verapamil and then treated

with the indicated concentrations of paclitaxel. After 72 h, cell viability

was measured using the MTT assay.

- 27 -

Figure 11. P-gp silencing with siRNA

Control and P-gp siRNA (100 nM) were introduced into resistant cells,

and P-gp silencing was confirmed by western blot analysis.

- 28 -

Figure 12. Cell viability after transfection with P-gp siRNA or

verapamil pretreatment

Cells were treated with the indicated concentrations of 17-DMAG after

transfection with P-gp siRNA. Cell viability was measured 72 h later by

using the MTT assay.

- 29 -

Figure 13. No cross-resistance relationship between 17-DMAG and

AUY922

Cells were treated with the indicated concentrations of AUY922 after

transfection of P-gp siRNA. Cell viability was measured after 72 h

using the MTT assay.

- 30 -

Some papers suggested that NQO1 expression, induction of

other heat shock proteins (Hsp70 and Hsp27) or activation of heat shock

factor 1 (HSF1) led to resistance to Hsp90 inhibitors (28-31). We first

examined these factors on the basal protein level. NQO1 expression was

significantly decreased in H3122/DR-1 and H3122/DR-2 cells

compared to the H3122 parental cells, but H2228/DR cells showed no

difference in NQO1 expression compared to parental cells (Fig. 14). For

the other above-mentioned factors, no difference was observed between

parental and resistant cells. A previous study demonstrated that NQO1

protein levels are influenced by a single nucleotide polymorphism

(C609T) in the NQO1 gene (29). Polymorphism of the NQO1 gene was

not detected in H3122/DR-1 or H3122/DR-2 cells (Fig. 15). In addition,

H3122 parental and resistant cells showed similar levels of NQO1

mRNA expression (Fig. 16). To confirm the correlationship between

NQO1 expression and sensitivity to 17-DMAG, we treated cells with

dicumarol, a selective inhibitor of NQO1. Cytotoxic effect was not

observed in parental cells until dicumarol treatment at 20 μM (Fig.

17A). Cells were thus treated with 17-DMAG alone or in combination

with 20 µM dicumarol. All cells showed similar sensitivity to 17-

DMAG regardless of dicumarol treatment (Fig. 17B).

- 31 -

Figure 14. NQO1 expression in the parental cells and cells with

acquired resistance

Lysates from each cell line were subjected to western blot analysis. The

indicated antibodies were used to evaluate the level of proteins involved

in resistance to Hsp90 inhibitors.

- 32 -

Figure 15. NQO1 expression in the parental cells and cells with

acquired resistance by PCR-RFLP

NQO1 gene fragments were amplified (left panel) and digested by

HInf1 endonulcease (right panel).

- 33 -

Figure 16. Total RNA and mRNA levels of NQO1

The total RNA was isolated and the mRNA levels of NQO1 were

measured by quantitative RT-PCR.

- 34 -

Figure 17. Cell viability after treatment with dicumarol, a selective

inhibitor of NQO1

Cells were treated with the indicated concentrations of dicumarol (A) or

a combination of 17-DMAG with 20 μM dicumarol (B). Cell viability

was determined by using the MTT assay.

- 35 -

4. Discussion

To investigate the mechanism of resistance to 17-DMAG in

NSCLC with ALK rearrangement, two drug-resistant cell lines,

H3122/DR and H2228/DR, were established from the parental H3122

and H2228 cell lines by repeated exposure to 17-DMAG. H3122

expresses 117 kDa variant 1 of EML4-ALK, and H2228 expresses 90

kDa variant 3 of EML4-ALK (32). 17-DMAG–resistant cell lines did

not show cross-resistance to a structurally different Hsp90 inhibitor

(AUY922) or to the ALK tyrosine kinase inhibitors crizotinib and TAE-

684 (Table 1). The 17-DMAG IC50 values for the resistant H3122/DR-1,

H3122/DR-2, and H2228/DR cell lines were 7-, 30-, and 41-fold higher

than those of the parent H3122 and H2228 cell lines. For the treatment

of a distinct subset of NSCLC cases with ALK rearrangement, the

inhibition of ALK and downstream effector pathways is essential (33).

ALK signaling activity in parental cells was sufficiently suppressed by

17-DMAG and AUY922, while it was persisted in resistant cells, even

at higher 17-DMAG concentrations. Therefore, H3122/DR and

H2228/DR cells were both adequate models with which to determine

the mechanism of 17-DMAG resistance.

Quantitative RT-PCR analysis of mRNA expression of

multidrug-resistance (MDR)–related transporters revealed that

- 36 -

H3122/DR and H2228/DR cell lines overexpressed P-gp/MDR-1, a

permeability glycoprotein of the cell membrane that pumps a variety of

drugs out of cells. Western blot assay also demonstrated that P-gp

expression was increased in H3122/DR and H2228/DR cells compared

to parent cells. Rho123 efflux activity was also increased in resistant

cells compared to parent cells. Transfection with P-gp–specific siRNA

or pretreatment with verapamil, an inhibitor of P-gp, decreased P-gp

expression to levels comparable to those of the parental cells and

restored sensitivity to 17-DMAG. Therefore, it is concluded that P-gp

overexpression causes drug resistance in H3122 and H2228 cell lines.

These results suggest that H3122/DR and H2228/DR cells could

develop resistance to 17-DMAG by the acquisition of MDR-1. Several

studies have reported that a number of drugs, such as taxol,

doxorubicin, vincristine, VP-16, and cis-diamminedichloroplatinum (II),

increased P-gp expression after chronic exposure in lung cancer cell

lines and animal models (34-37). We confirmed similar resistance

patterns to paclitaxel in A549 and H460 cell lines. Clinical data in

patients with NSCLC also showed that MRP expression is associated

with poor prognosis; thus, MRP plays a role in multidrug resistance in

NSCLC (38).

Overexpression of P-gp is one of the principal causes of

treatment failure in cancer. Diverse attempts are being made to

- 37 -

overcome resistance via P-gp overexpression, but there is still no

definite solution without significant side effects (39). Both parental cell

lines, including H3122, H2228, A549, and H460, and cell lines resistant

to 17-DMAG or paclitaxel showed persistent sensitivity to AUY922, a

novel non-geldanamycin Hsp90 inhibitor. There was no cross-resistance

between 17-DMAG and AUY922, which are structurally different

Hsp90 inhibitors. An alternate way to overcome resistance is

combination therapy. In this study, we showed that the combination of

rapamycin, a mTOR inhibitor, and 17-DMAG induces tumor cell

growth inhibition additively (Fig 18). Therefore, other types of Hsp90

inhibitors or combination therapy with additional drugs, such as

targeted agents and third-generation P-gp inhibitors, would be

candidates for strategies to overcome multidrug-resistance.

NQO1 is a metabolizing enzyme that forms homodimers and

catalyzes quinones to hydroquinones. There are a few reports that low

NQO1 activity is associated with acquired resistance to Hsp90

inhibitors in breast and glioblastoma cell lines (23,29). However, some

clinical studies reported that NQO1 overexpression is associated with

poor prognosis in solid tumors, including breast, stomach, cervix, and

liver tumors (40-43). We did not found any differences in NQO1

protein or mRNA levels or NQO1 DNA polymorphism between parent

and resistant cell lines. In addition, there were no significant changes in

- 38 -

Figure 18. Effects of a combination of 17-DMAG and rapamycin in

parental and 17-DMAG-resistant cells

Cells were treated with the indicated concentrations of 17-DMAG,

rapamycin, or a combination of the two drugs for 72 h. Cell viability

was measured after 72 h by using the MTT assay.

- 39 -

cell viability after treatment with dicumarol, a selective inhibitor of

NQO1, in parent cells. These results imply that NQO1 depletion is

unlikely to develop as a mechanism of resistance to 17-DMAG.

The mechanisms of resistance to 17-DMAG are complicated;

however these studies demonstrate that continuous exposure of lung

cancer cells to 17-DMAG leads to overexpression of P-gp with resultant

development of 17-DMAG resistance. P-gp over-expression may

contribute to acquired resistance to 17-DMAG, an Hsp90 inhibitor, in

lung cancer cells with ALK rearrangement.

- 40 -

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- 47 -

문초

열충격단 질 90 (heat shock protein 90, Hsp90) 억 는

역형 림프 산화효 (anaplastic lymphoma kinase, ALK)

재 열 동 폐암 환 에 치료 효과 보 는 것

알 다. 연 에 는 ALK 재 열 는 폐암

포주 H3122 H2228 다나마 신 계열 Hsp90 억

17-dimethylaminoethylamino-17-demethoxy-

geldanamycin (17-DMAG) 에 해 획득 내 가지는

규 하고 하 다.

각 폐암 포주에 차 17-DMAG 도 여가

복 출시킴 H3122/DR-1, H3122/DR-2,

H2228/DR 같 약 내 포주 확립하 다. MTT

assay 하여 포 생 확 하 고, 단 질과

mRNA 현 western blot과 량 역 사 PCR 확

하 다. P-당단 능 평가는 다민 123 처리 후 형 활

포 통해 하 다.

17-DMAG에 한 내 포주는 다 계열 Hsp90 억

AUY 922 ALK 억 에 해 차 내 보 지 않

- 48 -

았는 , 는 ALK 경 에 한 지하고 는 것

시사한다. 든 내 포주에 단 질 RNA 수 P-당

단 (ABCB1/MDR1) 현 가 어 었 , P-당단

질에 해당하는 다민 123 출 또한 늘어난 것 확

었다. P-당단 에 한 si-RNA 형질 주 하거나 P-당단

억 verapamil 처치하 , 든 내 포주

에 17-DMAG에 한 약 감수 회복하 다. 또한, P-

당단 과 현 paclitaxel에 한 내 획득한

A549/PR과 H460/PR 포주에 도 P-당단 능 억 함

17-DMAG 포 해 효과가 개 는 것

나타났다. 에 17-DMAG 내 에 여하는 것 보고

는 NAD(P)H: 퀴 산화환원효 1 (NQO1)

H3122/DR-1, H3122/DR-2 포주에 현 감 한 것

나타났 나 dicumarol 처치 통해 NQO1 억 하

약 감수 에 향 미치지 않는 것 확 었다.

결 ALK 재 열 동 폐암 포주에 17-

DMAG에 한 내 획득하는 하나 P-당단

과 현 한 역할 할 것 보 다.

- 49 -

주 어 : 폐암, 역형 림프 산화효 (anaplastic

lymphoma kinase), 열충격단 질 90 (heat shock protein 90),

내 , P-당단 (P-glycoprotein)

학 : 2009-30513