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의학박사 학위논문
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
2015 년 2 월
서울대학교 대학원
의학과 내과학 전공
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 억제제인 17-DMAG 에 대한 ALK 유전자 전위가 있는 폐암 세포주의 내성 발현 기전에 관한 연구
지도교수 김 영 환
이 논문을 의학박사 학위논문으로 제출함
2014년 10월
서울대학교 대학원
의학과 내과학 전공
김 희 정
김희정의 의학박사 학위논문을 인준함
2014 년 12 월
위 원 장 (인)
부위원장 (인)
위 원 (인)
위 원 (인)
위 원 (인)
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 Rho123 18
Figure 5. P-gp silencing with siRNA 19
Figure 6. Cell viability after transfection with P-gp siRNA or
verapamil pretreatment 20
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 cells 25
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-RFLP 32
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.
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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).
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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.
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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).
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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.
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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.
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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
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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
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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
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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.
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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.
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국문초록
열충격단백질 90 (heat shock protein 90, Hsp90) 억제제는 역형성 림프종 인산화효소 (anaplastic lymphoma kinase, ALK) 재배열이 동반된 폐암 환자에서 좋은 치료 효과를 보이는 것으 로 알려져 있다. 이번 연구에서는 ALK 재배열이 있는 폐암 세 포주인 H3122와 H2228이 젤다나마이신 계열의 Hsp90 억제 제인 17-dimethylaminoethylamino-17-demethoxy- geldanamycin (17-DMAG) 에 대해 획득 내성을 가지는 기전 을 규명하고자 하였다.
각 폐암 세포주에 점차 17-DMAG 의 농도를 높여가면서 반 복적으로 노출시킴으로써 H3122/DR-1, H3122/DR-2, H2228/DR 와 같은 약제 내성 세포주를 확립하였다. MTT assay를 이용하여 세포 생존을 확인하였고, 관련 단백질과 mRNA의 발현은 western blot과 정량적 역전사 PCR 로 확인 하였다. P-당단백의 기능 평가는 로다민 123 처리 후 형광 활 성 세포 정렬 방법을 통해 분석하였다.
17-DMAG에 대한 내성 세포주는 다른 계열의 Hsp90 억제제 인 AUY 922 및 ALK 억제제에 대해서 교차 내성을 보이지 않
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았는데, 이는 ALK 경로에 대한 의존성을 유지하고 있는 것을 시사한다. 모든 내성 세포주에서 단백질 및 RNA 수준의 P-당 단백 (ABCB1/MDR1) 발현이 증가되어 있었으며, P-당단백의 기질에 해당하는 로다민 123의 유출 또한 늘어난 것으로 확인 되었다. P-당단백에 대한 si-RNA 형질 주입을 하거나 P-당단 백 억제제인 verapamil을 전 처치하였을 때, 모든 내성 세포주 에서 17-DMAG에 대한 약제 감수성을 회복하였다. 또한, P- 당단백이 과발현 됨으로써 paclitaxel에 대한 내성을 획득한 A549/PR과 H460/PR 세포주에서도 P-당단백 기능을 억제함 으로써 17-DMAG 의 세포 성장 저해 효과가 개선되는 것으로 나타났다. 이전에 17-DMAG 내성에 관여하는 것으로 보고된 바 있는 NAD(P)H: 퀴논 산화환원효소 1 (NQO1) 은 H3122/DR-1, H3122/DR-2 세포주에서 발현이 감소한 것으로 나타났으나 dicumarol 전 처치를 통해 NQO1을 억제하였을 때 약제 감수성에 영향을 미치지 않는 것으로 확인되었다.
결론적으로 ALK 재배열이 동반된 폐암 세포주에서 17- DMAG에 대한 내성을 획득하는 기전 중의 하나로 P-당단백의 과발현이 중요한 역할을 할 것으로 보인다.
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주요어 : 폐암, 역형성 림프종 인산화효소(anaplastic lymphoma kinase), 열충격단백질 90 (heat shock protein 90), 내성, P-당단백 (P-glycoprotein)
