Introduction
Lung cancer is the leading cause of cancer-related death in many countries, including the China [
1]. Non-small cell lung cancer (NSCLC) accounts for up to 80% of all lung cancer cases; patients typically present with advanced disease at the time of diagnosis. The prognosis of patients with advanced lung cancer remains poor, and recent studies show that conventional therapies may have reached a therapeutic plateau as evidenced by the 5-year survival rate for NSCLCs, which remains at 15% [
2,
3]. The EGF receptor tyrosine kinase inhibitors (EGFR-TKIs) gefitinib and erlotinib have shown marked therapeutic effects against NSCLCs with
EGFR activating mutations, such as exon 19 deletions and L858R point mutations [
4]. Almost all tumors, however, acquire resistance to EGFR-TKIs after varying periods of time. Common mechanisms for acquired resistance include emergence of an
EGFR gatekeeper mutation (T790M) and
MET gene amplification [
5,
6]. In addition,
PIK3CA mutations as well as
KRAS mutations have been found to contribute to EGFR-TKIs resistance in a subpopulation of tumors [
7,
8]. The limited therapeutic options currently available for patients with advanced lung cancer create a pressing need to identify new therapeutic strategy.
Selumetinib (AZD6244) is an oral, non-ATP competitive inhibitor and highly specific for extracellular signal-regulated kinase (ERK) kinase (MEK)1/2, a key enzyme in the RAS-RAF-MEK-ERK pathway. AZD6244 had minimal effects on the p38, c-Jun-NH
2-kinase, PI3K, and MEK5/ERK5 pathways and is currently in phase II clinical trial in
KRAS-mutant NSCLC [
9,
10].
In vivo, AZD6244 could inhibit the tumor growth in HT-29 xenograft model, which is a colorectal tumor model carrying a
BRAF mutation, at a dose of 100 mg/kg and the tumor growth inhibition of AZD6244 is better than gemcitabine [
11]. However, the inhibition of MEK signaling alone may not be sufficient in patients with gefitinib-resistant NSCLC, and negative feedback mechanisms in PI3K pathway may be problematic when it is used alone [
12]. By contrast, combined blockade of both pathways was able to overcome the reciprocal pathway activation induced by inhibitor-mediated release of negative feedback loops and resulted in a significant tumor growth inhibition. Thus, coinhibition of both pathways has shown use in reducing tumor growth in a variety of xenograft models [
13,
14], and clinical trials of such combinations are under way in adults.
BEZ235 is an orally available dual inhibitor of PI3K and mTOR that is being evaluated in phase I/II trials [
15]. With the aim of developing effective therapeutic strategy for treatment gefitinib-resistant NSCLCs, we have initially evaluated the antitumor activity of AZD6244 alone or combination with BEZ235 in a panel of three human NSCLC cell lines, which were selected according to their different mutation status for
EGFR- T790M
, MET and
KRAS/
PIK3CA genes. We hypothesized that targeting the MEK pathway in combination with selective inhibitors of PI3K/mTOR signaling, could overcome gefitinib-resistant NSCLC and enhance the antitumor efficacy.
Methods
Reagents
AZD6244 and BEZ235 were purchased from Sellech Chemicals (Houston, TX, USA), all drugs were dissolved in sterile dimethylsulfoxide (DMSO) and a 10 mM working solution was prepared and stored in aliquots at -22°C. Working concentrations were diluted in culture medium just before each experiment. RPMI1640 media and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). Fibronectin and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO, USA). Phospho-AKT (Ser473, p-AKT), phospho-S6 (Ser240/244, p-S6), phospho-4E-BP1 (Ser 65, p-4E-BP1), phospho-ERK1/2 (Thr202/Tyr204, p-ERK1/2), phospho-MEK1/2 (Ser217/221, p-MEK1/2), AKT, S6, 4E-BP1, MEK1/2 and ERK1/2 antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). CD31 and Ki-67 antibodies for IHC were purchased from Cell Signaling Technology (Danvers, MA, USA). All other chemicals used in this study were of analytical reagent grade.
Cell lines
The NCI-H1975
EGFR T790M mutation [
16], NCI-H460
KRAS/
PIK3CA mutation and NCI-H1993
MET amplification [
17,
18] human NSCLC cell lines were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). The cells were cultured in RPMI1640 medium supplemented with 10% FBS, 100 mg/L streptomycin, 100 IU/mL penicillin and 0.03% L-glutamine (Hyclone, Logan, UT, USA) and maintained at 37°C with 5% CO
2 in a humidified atmosphere.
Cell viability assay
Cell viability was measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] dye reduction method. Tumor cells (1 × 104 cells/100 mL/well) in RPMI1640 medium with 10% FBS were plated into 96-well plates and cultured with indicated compounds for 72 h, followed by the addition of 50 mL of MTT solution (2 mg/mL; Sigma, St. Louis, MO) to each well and further incubation for 2 h. The medium was removed, and the dark blue crystals in each well were dissolved in 100 mL dimethyl sulfoxide. The absorbance of the wells was measured with a microplate reader at test and reference wavelengths of 490 nm, respectively. Percent growth was reported relative to untreated controls. Each experiment contained at least triplicate samples and was performed at least three times.
Efficacy study in vivo
BALB/C nude mice (female, 6-7 weeks old) were obtained from Vital River (Beijing, China). Mice were maintained under super pathogen-free conditions and housed in barrier facilities on a 12-h light/dark cycle, with food and water ad libitum. All animal experiments were performed in accordance with protocols approved by the Shandong University Experimental Animal Care and Use Committee. Mice were injected subcutaneous (s.c.) with 5 × 10
6 NCI-H1993, NCI-H1975 and NCI-H460 cells that had been resuspended in 200 μL of matrigel (BD Biosciences, Milan, IT). AZD6244, solubilized in a methocel/polysorbate buffer, was injected by oral gavage twice daily at the dose of 25 mg/kg for 3 weeks [
19]. BEZ235, was reconstituted in NMP (1-methyl-2 pyrrolidone) and PEG300, and injected by oral gavage once daily at the dose of 20 mg/kg for 3 weeks [
20]. When the mean volumes of tumors were between 150 and 200 mm
3, mice were randomly divided in four groups (ten mice per group). The tumor volume and body weight in each group were balanced. The animals were ear-punched for identification during the study. Two orthogonal diameters of the tumor are measured with digital vernier calipers and individual animal weights were weighed and recorded twice a week. Tumor volume (TV) are measured and recorded during treatment period by the formula: TV = Length × Width
2/2. Growth inhibition from the start of treatment was assessed by comparison of the differences in tumor volume between control and treated groups. Tumor growth inhibition T/C ratio is calculated by the following equations: T/C ratio = (V
t - V
0)
Compound treated/(V
t - V
0)
Control × 100 %.
Western blot analysis
The expressions of, p-ERK1/2, p-AKT, p-S6, p-MEK1/2 and p-4E-BP1 in tumor tissues were examined by Western Blotting. Fresh tumors in each group were resected after last treatment with AZD6244 and/or BEZ235 for 2 h on Day 21 of the efficacy study. Tumor tissues were lysed by lysis buffer (50 mM HEPES [pH 7.4], 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA [pH 8.0], 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride 1 mM sodium orthovanadate, 10 μg/mL aprotinin, 50 μg/mL leupeptin, and 1 μg/mL pepstatin A). The resected tumor samples were homogenized with lysis buffer containing 25 mM b-glycerophosphate and 0.5% (v / v) phosphatase inhibitor cocktail 2 (Sigma-Aldrich, St. Louis, MO, USA) at 4°C. Cellular debris was removed by centrifugation at 17 860 g for 20 min at 4°C. Aliquots of the supernatants containing 5–20 μg of protein were subjected to SDS-PAGE under reducing conditions. The protein concentration of the supernatant was determined by Bio-Rad protein assay reagent (Bio-Rad, CA, USA). Equal amounts of protein were separated by sodium dodecyl sulfate/polyacrylamidegel electrophoresis (SDS/PAGE) on 10% gels, blotted on polyvinylidene difluoride (PVDF), and probed with p-ERK1/2, p-AKT, p-S6, p-MEK1/2, p-4E-BP1, MMP2, MMP9, ERK1/2, AKT, S6, MEK1/2 and 4E-BP1 rabbit monoclonal antibody and subsequently with goatanti-rabbit (HRP), and detected by chemiluminescence. To measure protein loading, antibodies directed against β-actin were used.
Immunohistochemical analysis
Fresh tumors in each group were resected after last treatment with AZD6244 and/or BEZ235 for 2 h on Day 21 of the efficacy study, fixing in formalin and embedding the tumor tissue. Cutting and mounting the section. Immunocytochemical analysis was performed according to the method described on the commercial kits to examine the expressions of CD31 and Ki-67 (Cell Signaling Technology, Danvers, MA, USA).
Caspase activity assay
The apoptotic markers, activity of caspase-3,-8 and -9 were measured by using caspase colorimetric protease kits (Abnova, Walnut, CA, USA). Fresh tumors in each group were resected after last treatment with AZD6244 and/or BEZ235 for 2 h on Day 21 of the efficacy study, and then tumor lysis containing 200 μg of protein was incubated with 5 μL of 4 mM p NA-conjugated substrate (DEVD-p NA, IETD-p NA and LEHD-p NA) at 37°C for 2 h. The amount of p NA released was measured at 405 nm using a microplate reader.
Statistical analysis of the data
All results and data were confirmed in at least three separate experiments. Data are expressed as means ± SD, and were analyzed by ANOVA using Statistics Package for Social Science (SPSS) software (version 13.0; SPSS, Chicago, IL, USA). Test article can be demonstrated as an effective compound until T/C ratio ≤ 42% and a value of P < 0.05 was indicated to be statistically significance on tumor volume calculation.
Discussion
Although advances have been made in cancer treatment with the development of selective molecular targeted therapies, several relevant issues for their optimal and effective use remain unsolved. Recent studies have demonstrated that the EGFR-TKI gefitinib and erlotinib are associated with a high response rate and prolong progression-free survival in patients with EGFR mutant lung cancer [
22]. Responders to these agents, however, later relapse after acquiring EGFR-TKI resistance, making it urgent to develop novel therapeutic agents that can overcome acquired resistance to EGFR-TKI. Current clinical approaches to combat resistance in lung adenocarcinoma include irreversible and mutant-selective inhibitors of EGFR, combination of cetuximab and afatinib [
23] and combination of an EGFR inhibitor with a drug targeting a resistance pathway, such as the combination of gefitinib and a MET inhibitor [
24]. However, alternative RTK pathways that are activated following EGFR inhibition are another area for investigation. These alternative pathways may bypass or evade inhibition of EGFR signaling, thereby enabling combinations of agents to simultaneously attack multiple molecular targets for cancer growth inhibition. One potential solution to overcome multiple mechanisms of resistance is to target downstream pathways. In this study, we show that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor is effective against tumor cell lines refractory to gefitinib by three different mechanisms: an
EGFR gatekeeper T790M mutation,
MET amplification, and
KRAS/PIK3CA mutation. To our knowledge, this is the first report of the effects of MEK TKI with PI3K/mTOR TKI therapy in gefitinib-resistant models of NSCLC.
MEK is a potentially relevant molecular therapeutic target since it is the activated downstream of the axis RAS/RAF proteins and in turn activates ERK to induce cell proliferation. Therefore, several selective MEK inhibitors have been developed [
25] and more than ten MEK inhibitors have entered early clinical trial evaluation [
26]. Unfortunately, clinical activity as single agent has been rarely observed with MEK inhibitors in gefitinib-resistant NSCLC patients [
27]. The benefit of blocking an individual pathway has been largely limited by the presence of a compensatory feedback loop between PI3K and MEK. For example, inhibition of the MEK pathway results in activation of the PI3K pathway [
28], and PI3K activation mediates resistance to MEK inhibition [
29]. In our study, in order to circumvent this compensatory feedback, concurrent blockade of the two pathways has been tested, and synergy in antitumor effects was detected, providing the rationale for phase I clinical trials. Moreover, early signs of clinical benefit have been reported in advanced cancer by a retrospective analysis on patients receiving agents that target both pathways [
30].
Gatekeeper mutations, the T790M mutation in
EGFR associated with resistance to gefitinib [
31], are common mechanisms by which tumor cells acquire resistance to molecularly targeted drugs. Although irreversible EGFR-TKIs, including BIBW2992, have been developed to overcome T790M-mediated resistance to gefitinib [
32], recent clinical trials have failed to show that monotherapy with irreversible EGFR-TKIs has benefits in patients with NSCLC refractory to gefitinib [
33]. This may be due, at least in part, to the low selectivity of this class of compounds to wild-type and mutant
EGFR. In addition, because HGF overexpression was frequently observed in tumors with the gatekeeper T790M mutation [
34], monotherapy with a mutant-selective EGFR-TKI may not be sufficient to inhibit the growth of tumors with acquired resistance to gefitinib. Our findings suggest that the combination of a MEK-TKI and a PIK3/mTOR-TKI may be effective in controlling these resistant tumors.
Many
KRAS-mutant cancer cells have been shown to be sensitive to MEK inhibitors, [
35] and
KRAS mutations can be detected in up to 30% of lung cancers, dependent upon histology and ethnicity [
36‐
38], suggesting that a subset of lung cancers would likely be highly sensitive to AZD6244. Our finding that AZD6244 was effective in one distinct
KRAS mutant human lung cancer NCI-H460 models supports and validates this hypothesis. Although monotherapy with AZD6244 resulted in antitumor and some antiangiogenic effects in all of our lung cancer models, the antitumor effects were more apparent in the NCI-H1993 lung adenocarcinoma model. The increased antitumor efficacy observed in this model is associated with differences in the inhibitory effect of p-AKT signaling pathway in NCI-H1975 and NCI-H460 lung tumors. However, additional studies are needed to elucidate this phenomenon.
In this study, we evaluated therapy directed against MEK and PI3K/mTOR in distinct gefitinib-resistant NSCLC xenograft models. MEK or PI3K/mTOR inhibition resulted in antitumor effects for these gefitinib-resistance NSCLC models by blocking key intracellular pathways controlling cell proliferation and survival as demonstrated both in vitro and in vivo. Surprisingly, PI3K/mTOR inhibition by BEZ235 also suppressed lung tumor angiogenesis and targeted both MEK and PI3K/mTOR activation in lung tumors, resulting in substantial antiangiogenic effects, which may due to the significantly reduced expression of MMP-9 in tumors. We concluded that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor was effective in suppressing the growth of gefitinib-resistant tumors caused by EGFR T790M mutation, MET amplification, and KRAS/PIK3CA mutation. These findings represent a promising strategy for the treatment of gefitinib-resistant NSCLC and provide a strong basis for the design of clinical trials for this purpose.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YQQ conceived and designed the experiments. XXW, YHY, YY and HL performed the experiments. XXW, YHY and DDM analyzed the data. XXW, YHY and HL wrote the paper. YQQ supervised the whole experimental work and revised the manuscript. All authors read and approved the manuscript.