Background
Targeted therapy using tyrosine kinase inhibitors against oncogenic driver mutations in non-small cell lung cancer (NSCLC) has been developed to enhance selective cytotoxicity against tumor cells. The echinoderm microtubule-associated protein-like 4 - anaplastic lymphoma kinase gene (EML4-ALK) fusion oncoprotein, which arises from an inversion within chromosome 2p and results in constitutive kinase activity by dimerization of ALK, represents a major molecular target in lung cancer [
1]. Although, ALK-rearranged lung cancer accounts for only 3-7 % of NSCLC since its discovery in 2007, this population could represent more than 70,000 new cases worldwide annually. Furthermore, the detection rates are higher in the selected subgroup for genetic screening based on clinical features commonly associated with ALK-rearrangement, including never or light smoking history, adenocarcinoma histology, and wild-type epidermal growth factor receptor (EGFR) and KRAS status [
2].
Crizotinib is an oral-administered multitargeted small molecule tyrosine kinase inhibitor, which inhibits mesenchymal epithelial transition growth factor (c-MET) as well as ALK phosphorylation that is recommended as a first-line treatment option for patients with locally advanced or metastatic NSCLC who have the ALK gene rearrangement [
3]. Crizotinib shows superiority over standard chemotherapy in progression-free survival (7.7 vs. 3.0 mo) and objective response rate (65 % vs. 20 %) in patients with previously treated, advanced NSCLC with ALK rearrangement [
4]. However, despite the successful initial response, most patients inevitably encounter the development of acquired resistance while being treated with crizotinib [
5,
6] similar to EGFR tyrosine kinase inhibitors (TKIs). There are multiple resistance mechanisms such as various acquired mutations, which hamper drug binding, oncogenic bypass through EGFR or c-KIT activation [
5,
7], and induction of the epithelial-mesenchymal transition [
8]. Ceritinib, a second-generation ALK inhibitor, is effective in patients resistant to crizotinib as well as crizotinib-naive patients and is approved by the US Food and Drug Administration for patients who have tumor progression or are intolerant of crizotinib [
9]. The other second-generation ALK inhibitors such as CH5424802, AP26113, ASP3026, X-396, and TSR-011 are undergoing phase I or II clinical trials [
10]. In addition, heat shock protein (HSP) 90 inhibitors are suggested as therapeutic options to overcome resistance on the basis of anti-tumor activity in preclinical models of ALK-driven lung cancer [
11,
12] and small-scale clinical trials on ALK-positive lung cancers [
13].
Hsp90 is a molecular chaperone that plays an important role in the modification and stabilization of a variety of proteins implicated in tumor cell proliferation and survival. Both EGFR and EML4–ALK fusions, which are known to be major oncogenic drivers in NSCLC, are client proteins for Hsp90 [
14,
15,
11]. Therefore, Hsp90 could be an alternative therapeutic target instead of direct kinase inhibition in ALK-driven lung cancer.
In vivo and
in vitro studies demonstrated that treatment with Hsp90 inhibitors such as 17-DMAG, ganetespib (STA-9090), or IPI-504 reduced protein levels of the ALK fusion protein, enhanced cell death, led to tumor regression, and prolonged survival of xenograft models [
14,
15,
12]. Antitumor activity also has been observed in phase I and II clinical trials with ganetespib or IPI-504 [
16,
13], and a number of Hsp90 inhibitors - both as monotherapies and in combination with ALK tyrosine kinase inhibitors - are undergoing clinical trials for ALK-positive lung cancer patients.
Although many studies have identified resistance factors associated with ALK inhibitors, the mechanisms of resistance to Hsp90 inhibitors are poorly understood. Clarification of the resistance mechanisms relevant to ALK-positive lung cancer may be important to find ways to overcome drug resistance. In this study, we generated resistant cells by treating ALK-positive cells with increasing concentrations of 17-DMAG, and investigated the mechanism of their resistance.
Methods
Cell culture and reagents
The human NSCLC cell line H2228, A549 and H460 were 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) supplemented with 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). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution, 3,3’-methylene-bis(4-hydorxycoumarin) (dicumarol), and Rho123 were purchased from Sigma-Aldrich (St. Louis, MO).
Establishment of 17-DMAG or paclitaxel resistance in NSCLC cells
Cells resistant to 17-DMAG or paclitaxel were developed by chronic, repeated exposure to each drug. Over a period of 6 months, cells were continuously exposed to increasing concentrations of the drug in culture and the surviving cells were cloned. These cells could survive exposure >50 nM of 17-DMAG or >100 nM of paclitaxel. In all studies, resistant cells were cultured in drug-free medium for >1 week to eliminate the effects of 17-DMAG or paclitaxel.
MTT assay
Cells were seeded onto 96-well plates and incubated overnight, and then treated with their respective agents for an additional 3 days. Cell viability was determined using the previously described MTT-based method [
17]. Each assay consisted of eight replicate wells and was repeated at least three times. Data were expressed as the percent survival of the control, which was calculated using absorbance after correcting for background noise.
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 % NP-40, and 5 U/mL aprotinin) and centrifuged. Proteins were separated using SDS-PAGE and transferred to PVDF membranes (Invitrogen) for western blot analysis. Membranes were probed with antibodies against p-ALK (Tyr1604), ALK, p-Akt (Ser473), P-gp (all from Cell Signaling Technology, Beverly, MA), Akt, p-Erk (Thr202/Tyr204), Erk, HSF1, Hsp90, Hsp70, Hsp27, NQO1, and β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA) as the first 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).
Detection of NQO1 polymorphism
DNA purification and detection of the gene polymorphism were performed according to the previously reported methods [
18]. Briefly, for the amplification of the
NQO1 gene fragment (230 bp), a pair of forward and reverse primers were as follows; 5’-TCCTCAGAGTGGCATTCTGC-3’ and 5’-TCTCCTCATCCTGTACCTCT-3’. The amplification was carried out by using AccuPower TagPCR PreMix (Bioneer Corp., Daejeon, Korea). Each PCR mixture contained forward and reverse primers (each 0.5 pmoL) and 50 ng of genomic DNA in 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 restriction fragment length polymorphism (RFLP), the amplified fragments were digested with Hinf1 (Thermo Scientific) and analyzed on agarose gel electrophoresis. The wild-type (Pro187Ser) allele of
NQO1 was identified by a 191 bp band while the homozygous variant (Ser/Ser) and the heterozygous variant (Pro/Ser) displayed only a 151 bp band and two bands (191 bp and 151 bp), respectively.
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, according to the manufacturer’s instructions (Bioneer Corp., Daejeon, Korea). The oligonucleotide sequences for amplification were as follows: forward primer 5’-AAGCAGTGCTTTCCATCA-3’ and reverse primer 5’-TCCTGCCTGGAAGTTTAG-3’ for NQO1; 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’-ATGCGGCTCTTGCGGAG-3’ for MRP3; forward primer 5’-GTACATTAACATGATCTGGTC-3’ and reverse primer 5’-CGTTCATCAGCTTGATCCGAT-3’ for MDR1; 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 glyceraldehydes-3-phosphate dehydrogenase (GAPDH). PCR cycling conditions were as follows: 94 °C for 60 s and primer annealing for 60 s, elongation at 72 °C, for a total of 30–35 cycles (NQO1, MRP1, MRP2, MRP3, MDR1) and 25 cycles (β-actin, GAPDH), respectively. 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.
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 medium. All samples were kept at 4 °C until cytometric analysis was performed. Fluorescence of Rho123 was analyzed on a FACScalibur flow cytometer and processed by Cell Quest Software (BD Bioscience, San Jose, CA). Rho123 efflux was measured by counting cells in the M1 region of the plot and calculated as the percentage of cells in the M1 region of the plot.
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.
Discussion
In our present study, we established three drug-resistant cell lines to investigate the mechanisms of acquired resistance to 17-DMAG in lung cancer with ALK rearrangement. We show from our findings that induction of P-gp expression is the main mechanism of resistance. In addition, we extend this finding to acquired resistance to paclitaxel.
The compound 17-DMAG is the first water-soluble analog of 17-AAG, has excellent bioavailability, and is quantitatively metabolized much less than is 17-AAG [
28]. Thus, the mechanisms of acquired resistance to these drugs may be similar. NQO1 is a homodimeric metabolic enzyme that catalyzes the conversion of quinones to hydroquinones and has an important role in sensitivity to 17-AAG [
29,
21]. Loss or low activity of NQO1, by the reduction of its mRNA or the emergence of inactivating polymorphisms in the
NQO1 gene, leads to resistance to 17-AAG in pancreatic cancer cells and glioblastoma cell lines [
30,
21]. NQO1 expression is reduced in H3122/DR-1 and −2 cells, but not in H2228/DR cells. Nor do we find any differences in mRNA levels or DNA polymorphisms in
NQO1 between the parental and resistant cell lines. In addition, there are no significant changes in cell survival after treatment of the parental cells with dicumarol. These results suggest that NQO1 depletion is an unlikely resistance mechanism to 17-DMAG in cells with ALK rearrangement.
Hsp90 is a chaperone of client proteins relevant in NSCLC pathogenesis, including ALK and EGFR [
31]. The inhibition of Hsp90 simultaneously disrupts these oncogenic signaling pathways, and consequently, cancer cell proliferation is inhibited by the induction of apoptosis or cell cycle arrest. Hsp90 inhibitors may be used to combat resistance to tyrosine kinase inhibitors (EGFR-TKIs and ALK inhibitors) regardless of secondary mutations [
32,
12]. Hsp90 inhibitors inhibit the downstream effector pathways by controlling ALK through its degradation. We also observe that Hsp90 inhibitors sufficiently suppress the ALK signaling pathway in parental cells, but all 17-DMAG-resistant cells require higher concentrations of 17-DMAG to inhibit these pathways. Interestingly, resistant cells do not show cross-resistance to a different kind of Hsp90 inhibitor, AUY922, or to ALK tyrosine kinase inhibitors, crizotinib and TAE-684. These results imply that the resistant cells are still dependent on ALK signaling, and that acquisition of resistance to 17-DMAG may be caused by low intracellular 17-DMAG concentrations.
P-glycoprotein and multidrug resistance proteins (MRPs), ATP-binding cassette (ABC)-superfamily multidrug efflux pumps are responsible for some cases of chemoresistance. Expression of these pumps reduces cellular accumulation of cytostatic agents due to active efflux of these substrates [
33‐
36]. The mRNA, protein, and activity of only one MRP family member
P-gp is significantly induced in all 17-DMAG-resistant cells. Although verapamil pretreatment restores sensitivity to 17-DMAG in all resistant cells, a
P-gp-specific siRNA was also used because verapamil can inhibit all MRP drug efflux pump proteins including P-gp. Similar to resistant cells, the inhibition of P-gp in the parental line H2228 enhances the sensitivity of cells to 17-DMAG, but not in the H3122 line. The baseline P-gp expression in the H2228 line may contribute to its slight resistance to 17-DMAG compared to H3122 cells. Therefore, we suggest that the induction of P-gp is associated with the primary or acquired resistance to 17-DMAG in cells with ALK rearrangement.
Induction of P-gp also leads to 17-DMAG resistance in other resistant cells. A number of drugs, such as taxol, doxorubicin, vincristine, VP-16, and cis-diamminedichloroplatinum (II), increase P-gp expression in lung cancer cell lines and animal models after chronic exposure [
37‐
40]. Consistent with previous studies, we also detected induction of P-gp in cells with acquired resistance to paclitaxel. These resistant cells show cross-resistance to 17-DMAG, whilst the inhibition of P-gp restores the sensitivity to paclitaxel and 17-DMAG. Clinical evaluation of Hsp90 inhibitors, as single agents and in combination with various chemotherapy-agents, is currently in progress. Our findings suggest that P-gp expression should be considered in preclinical and clinical evaluation.
Overexpression of P-gp that recognizes a wide variety of chemotherapeutic agents and pumps them out of the cell is one of the principal causes of treatment failure in cancer. Diverse attempts are being made to overcome resistance via P-gp overexpression, although significant side effects remain a concern [
41]. The four 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. Consistent with our current results, previous studies have shown that AUY922 has effectiveness independent of P-gp expression [
42,
21]. Thus, the treatment with new Hsp90 inhibitors may help overcome the acquired resistance to 17-DMAG caused by P-gp expression. A second alternative way to overcome resistance is through combination therapy; many drugs are known to inhibit the activity of P-gp [
43‐
45]. We find that combined treatment with 17-DMAG and rapamycin overcomes drug resistance in 17-DMAG-resistant cells (Additional file
1). Previous studies have demonstrated rapamycin as a P-gp inhibitor [
46,
47], and rapamycin is already approved for clinical use. Other types of Hsp90 inhibitors or a combination with additional therapeutic drugs, such as new P-gp inhibitors, are candidate strategies to overcome 17-DMAG-resistance caused by P-gp expression.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
HJK, JCL and RJK designed the research. HJK, YJC, IJB and JEL performed the research. HJK and YJC carried out most of the studies, including MTT, Western Blots and RT-PCR assays. IJB and JEL helped in the transient transfections and Rhodamine 123 efflux assay. YWK, CMC and KYL provided discussion and advice. JCL and RJK wrote the paper. All authors read and approved the final manuscript.