Lung cancer is the leading cause of cancer-related deaths worldwide 1. Approximately 85% of patients are diagnosed with nonsmall cell lung cancer (NSCLC), most of which were advanced stages. Over the past decades, treatments for advanced NSCLC were confined to platinum-based chemotherapy, with an unsatisfactory response rate and a median overall survival of 30% and 10–11 months, respectively 2. Targeted therapies such as the epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) tyrosine kinase inhibitors (TKIs) such as crizotinib, ceritinib, and alectinib, and the monoclonal antibody against human vascular endothelial growth factor (VEGF), bevacizumab, have led to an increase in the response rate to 65–80% 3,4.
Alectinib, a second-generation and more potent ALK inhibitor, shows high efficacy in the treatment of EML4-ALK-positive NSCLC patients. Compared with crizotinib, alectinib showed longer progression-free survival (PFS) and lower toxicities in the first-line setting in previously untreated EML4-ALK-positive patients in the ALEX study. On the basis of an independent review, the median PFS was estimated to be 25.7 months. The superior efficacy, good brain permeability, and favorable tolerability support alectinib as a new first-line therapeutic option for NSCLC patients harboring the EML4-ALK fusion gene 5. Despite these clinical successes, patients inevitably develop acquired resistance irrespective of the initial good response. Several mechanisms have been identified, including secondary mutations, such as V1180L and I1171T 6, epithelial–mesenchymal transition (EMT) 7, histological transformation to small-cell lung cancer 8,9, and alternative signaling pathway activation 10. Isozaki et al. 10 reported that bypass track activation occurs more frequently than do secondary mutations and that the EGFR, c-MET, and IGF-1R bypass signaling pathway may be more relevant to drug resistance to alectinib. Alectinib can effectively block ALK activation to abrogate its downstream signaling through the phosphoinositide 3-kinase-protein kinase B (PI3K-AKT) and RAS-RAF extracellular signal-regulated kinase (ERK) pathways. However, elevated expression of active epidermal growth factor (EGF), c-Met, and IGF leads to activation of ligand-independent bypass proliferation signaling, conferring alectinib resistance to NSCLC. Chronic ALK inhibition was associated with enhanced IGF-1R signaling that triggers resistance 11.
Angiogenesis is essential for the progression of various types of solid tumors. Neovascularization is required for the growth of most solid tumors and facilitates the spread of tumor cells to secondary sites 12. Overexpression of VEGF and VEGFR is correlated with increased tumor growth rate, microvessel density, proliferation, tumor metastatic potentiality, and poor patient prognosis in a variety of malignancies 13. Therefore, inhibition of VEGFR signaling is an attractive therapeutic target in clinical practice, which has been identified by incremental data as single use or in combination with other antitumor regiments. VEGF-dependent signaling may be as an alternative survival pathway in drug-acquired resistance to EGFR-TKIs 13. A previous study showed that activation of EGFR by its ligands such as EGF or TGF-α results in increased VEGF expression 14. The ligands are the pivotal inducer of not only EGFR-TKI resistance but also angiogenesis in EGFR-mutant lung cancer. The most likely mechanism is associated with enhanced signaling by phosphatidylinositol 3-kinase, which synergizes with hypoxia to regulate VEGF 15. Furthermore, the hypoxia-inducible factors (HIF) signaling pathway was more likely to be activated in EML4-ALK lung cancer compared with EGFR, K-Ras, and B-Raf-mutated tumors, thus reinforcing the ALK–hypoxia angiogenesis axis. In addition, knockdown of HIF-1α or HIF-2α induces a decrease in VEGFA production and tumor vessel formation 16. Therefore, we hypothesized that the IGF-1R bypass signaling pathway triggering resistance may combine with the production of the VEGFA and simultaneously inhibit the driver signal (ALK) and angiogenesis (VEGF), which may be beneficial for controlling the progression of EML4-ALK gene fusion lung cancer with hIGF-triggered ALK-TKI resistance.
Apatinib (YN968D1) is a novel small-molecule tyrosine kinase inhibitor that selectively inhibits VEGFR2, c-Kit, Ret, and cellular Src (c-Src). On the basis of its high efficiency, apatinib was approved by the China Food and Drug Administration for the treatment of advanced gastric cancer and is being studied in phase II and III clinical trials in NSCLC, hepatocellular carcinoma, colorectal cancer, and breast cancer. It inhibits VEGF-mediated endothelial cell migration and proliferation, thus blocking new blood vessel formation in tumor tissue 17. Highly selective competition within the ATP-binding site of VEGFR2 blocks the downstream signal transduction and inhibits tumor angiogenesis 18. The 2012 American Society of Clinical Oncology annual meeting presented the data on the phase II study of apatinib in a third-line setting in patients with advanced nonsquamous and NSCLC, with a median PFS of 4.7 months for the apatinib group versus 1.9 months for the placebo arms. Apatinib represents a new treatment option for patients with advanced nonsquamous NSCLC. However, the exact antitumor effect in previous ALK-TKI responders required further exploration. Apatinib inhibits VEGFR2 activation, cell proliferation, and migration, suggesting its potential to overcome acquired resistance related to angiogenesis and its ability to enhance sensitivity. The aims of our study were to explore the antitumour effect of the combination of alectinib with apatinib in EML4-ALK-positive and ALK-TKI-resistant NSCLC cells.
Materials and methods
Alectinib was purchased from Selleckchem (Selleck, Texas, USA). Apatinib was kindly provided by Hengrui Pharmaceutical Co. Ltd (Jiangsu, China). Alectinib and apatinib were dissolved in dimethylsulfoxide (DMSO) with a final concentration of 103 and 104 µmol/l, respectively, and stored at −20°C. The stocks were diluted to the target concentration in complete medium, ensuring that the concentration of DMSO was less than 0.1%. We found that 0.1% DMSO did not affect the cell growth rate compared with 0% DMSO in lung cancer cells (data not shown). Recombinant hIGF-1 was produced in Escherichia coli by Cell Signaling Technology (#8917). Sterile citrate (20 mmol/l, pH: 3.0) was added to the hIGF-1 and solubilized for 30 min at room temperature with occasional gentle vortexing and stored in −20°C while avoiding repeated freeze–thaw cycles.
Alectinib-sensitive H3122 containing EML4-ALK variant 1 and H2228 containing EML4-ALK variant 3 cell lines were obtained from Kebai Biotechnology Co. Ltd (Nanjing, China). The cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI-1640, BI) supplemented with 10% fetal bovine serum (BI) and 1% penicillin–streptomycin at 37°C in 5% CO2 and 90% humidity.
Cell growth assay
H3122 and H2228 cells (3000 cells/well) were seeded into 96-well plates. After 24 h of incubation, the cells were treated with alectinib (0–3 µmol/l), apatinib (0–100 µmol/l), hIGF-1 (100 ng/ml), or a combination of these agents for 72 h in serum-containing medium. For the reversal assay, apatinib was added to the cytotoxicity assay at nontoxic concentrations. The viability was determined using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) assay according to the manufacturer’s instructions.
Flow cytometric analysis was used to detect apoptosis by examining altered plasma membrane phospholipid using the lipophilic dye Annexin V. The IC50 value of alectinib and a nontoxic concentration of apatinib (10 μmol/l) were used as the experiment concentrations. Briefly, the cells were treated with alectinib, hIGF-1 (100 ng/ml), and/or apatinib for 48 h, harvested by trypsin, washed twice with PBS, and resuspended at a density of 1×107 cells/ml. Thereafter, 5 μl of Annexin V-PE and 5 μl of propidium iodide were added to 100 μl of the cell suspension and incubated for 30 min at room temperature in the dark. Next, labeled cells were detected using a Cytomics FC 500 flow cytometer (BD FACSCalibur, New York, New York, USA).
Perpendicular and horizontal coordinates were outlined at intervals of 1.0 cm on the back of 6-well plates. H3122 cells were plated in 6-well plates at a density of 5×105 cells/well. Once the cells grew to 80% confluence, a vertical scratch was made on the cell layer. The cells were incubated in McCoy’s 5A medium with 5% CO2 and saturated humidity at 37°C. Cell migration was observed and photographed 24 h after wounding and compared with the 0 h images. The wound-healing area was calculated using Image Tool (Image-Pro Plus) using the following formula: wound-healing rate=(initial scratch width−final scratch width)/initial scratch width×100%.
H3122 cells were plated in 6-well plates at a density of 500 cells/well. After 72 h of incubation, the cells were treated with 100 ng/ml hIGF-1 or combined with 10 µmol/l apatinib. The numbers of cell colonies in the control and treatment groups were counted after 2 weeks and were stained with crystal violet. The experiment was conducted three times. The colony-forming efficiency was calculated using the following formula: colony-forming efficiency=(the number of colony-forming units/the number of inoculated cells)×100%.
H3122 and H2228 cells were incubated with hIGF-1 (100 ng/ml, 10 min) or in combination with alectinib (1 µmol/l, 6 h) and/or apatinib (10 µmol/l, 24 h). Whole-cell protein lysates with different treatments were harvested. The protein concentration was quantified using a BCA protein assay kit (Biyotime, Shanghai, China). Equal amounts of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes; the membrane was then blocked with 5% skim milk in tris-buffered saline with Tween for 2 h at room temperature and probed with the primary antibodies overnight at 4°C. After washing with tris-buffered saline with Tween, the membrane was incubated with the horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature and detected by enhanced chemiluminescence reagent (Biyuntian, Shanghai, China). Antibodies against AKT, p-AKT, mTOR, p-mTOR, ERK, p-ERK, signal transducer, and activator of transcription 3 (STAT3), P-STAT3, E-cadherin, vimentin, HIF-1a, VEGFR2, and P-VEGFR2 were purchased from Cell Signaling Technology (Cambridge, Massachusetts, USA). Horseradish peroxidase-conjugated secondary antibodies (mouse and rabbit) were purchased from Beyotime Biotechnology (Shanghai, China). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abcam, Cambridge Science Park, UK) was used as a loading control.
SPSS for Windows (version 16.0; SPSS Inc., Chicago, Illinois, USA) was used for the statistical analysis of the values, which were recorded as the mean±SD. Data were assessed by one-way analysis of variance and Student’s t-tests. P values less than 0.05 were considered statistically significant.
Effect of apatinib on EML4-ALK fusion gene-positive cells
H3122 cells (EML4-ALK E13;A20) and H2228 cells (EML4-ALK 6a/b;A20) were sensitive to apatinib at relatively high concentrations. Apatinib could not inhibit the proliferation of H3122 and H2228 cells at concentrations below 10 μmol/l, with IC50 values of 42.97 and 58.63 μmol/l, respectively (Fig. 1). We selected a nontoxic concentration of 10 μmol/l of apatinib for the reversal assay.
Apatinib reverses hIGF-1-induced alectinib resistance
The viability of the H3122 and H2228 cells was inhibited by alectinib in a dose-dependent manner (Fig. 2a and c), with an IC50 of 0.01737±0.17 and 0.1883±0.20 μmol/l, respectively. To assess whether ligand-induced activation of IGF-1R could influence the antiproliferative effects of the ALK blockade, we treated H3122 cells and H2228 cells with alectinib alone or in combination with hIGF-1. The addition of 100 ng/ml hIGF-1-induced resistance to the growth-inhibitory effects of alectinib, with an IC50 of 0.2618±0.03 and 3.012±0.11 μmol/l, respectively. The resistance index was 15.07 and 15.96, respectively. We next speculated whether apatinib could overcome alectinib resistance triggered by hIGF-1. We used a nontoxic concentration of 10 μmol/l of apatinib for the reversal assay. The growth of the H3122 and H2228 cells was suppressed synergistically by co-treatment with alectinib and apatinib. The reversing multiple was 4.3 and 7.2, respectively (Fig. 2b and d). The differences in sensitivity to alectinib between the co-administration group and the monotherapy groups in the IGF-1-triggered resistant cells were significant (P<0.05).
According to the flow cytometry assay, IGF-1 exposure in alectinib-sensitive cells tended to decrease the cell apoptosis rate. Alectinib and apatinib co-treatment synergistically promoted H3122 and H2228 cell apoptosis (Fig. 3). Taken together, these data suggest that apatinib resensitized cells resistant to alectinib by inhibiting their viability and promoting apoptosis.
Apatinib suppresses the migration and proliferation of hIGF-1-induced H3122 cells
Scratch and colony-formation assays were used to evaluate the effects of hIGF-1 and apatinib on the migration and proliferation of H3122 cells. In comparison with the blank group, cell migration was reduced in the apatinib plus hIGF-1 group and was increased in the hIGF-1 group (P<0.05). When stimulated with 100 ng/ml hIGF-1, the wound-healing rate and the colony-forming efficiency were increased. The rates were reduced significantly in combination with apatinib. The wound-healing rates were 80.79±1.28 versus 56.18±0.96% and the colony-forming efficiencies were 31.5±1.04 versus 16.5±0.17% in the hIGF-1 and combination groups, respectively (n=3, P<0.05; Fig. 4a and c). These data suggest that apatinib can weaken the migration and proliferation ability that were strengthened by hIGF-1 in H3122 cells.
The combination of alectinib and apatinib inhibited the PI3K/AKT/mTOR, RAS/RAF/ERK, and janus kinase/STAT3 signaling pathways, resulting in the suppression of drug resistance
The combination of apatinib with alectinib successfully reversed the resistance of H3122/IGF and H2228/IGF cells. To identify the molecular mechanisms of this reversal, we examined the effects of apatinib and alectinib on signal transduction in H3122/IGF and H2228/IGF cells by western blotting. We found that alectinib inhibited the phosphorylation of Akt and ERK1/2 in H3122 and H2228 cells. However, alectinib failed to inhibit the phosphorylation of Akt, Erk1/2, and STAT3 in the presence of IGF. IGF-1R phosphorylation was elevated in the hIGF-1-triggered alectinib-resistant cells, together with the upregulated expression of p-AKT, p-P70S6K, p-mTOR, p-ERK, and p-STAT3. The phosphorylation proteins were decreased in combination with apatinib. However, the drugs showed negligible inhibition of the total proteins (Fig. 5). Taken together, these results suggest that the IGF-1R pathway plays a role in maintaining downstream signaling in the presence of continuous ALK inhibition and may, therefore, be a mechanism by which cells evade the ALK blockade. One of the probable mechanisms by which apatinib suppresses drug resistance is the synergistic inhibition of the main oncogenic signaling pathway in H3122 cells.
Apatinib reverses the sensitivity of hIGF-1-triggered alectinib-resistant cells along with inhibition of the HIF-1a/VEGFR2 pathway
Chinese scholars have confirmed that apatinib may inhibit VEGF-VEGFR2-PI3K-AKT signaling and induce apoptosis in intrahepatic cholangiocarcinoma cells 19. Our studies indicated that exogenous IGF-1 showed intrinsically higher HIF-VEGF-VEGFR2 signaling intensity compared with those of alectinib-sensitive counterparts. In this study, the protein levels of the HIF-1a, VEGFR2, and p-VEGFR2 were increased with treatment with exogenous hIGF-1 in EML4-ALK fusion gene cell lines. With the combination of alectinib and apatinib, the HIF-VEGF-VEGFR2 signaling was inhibited (Fig. 5a). Taken together, these results indicate another possible mechanism of IGF-1-inducing resistance related to HIF-VEGF-VEGFR signaling and that apatinib could strengthen the ability of alectinib to suppress the main signaling pathways relative to tumorigenesis and neoangiogenesis in both alectinib-sensitive and IGF-1-induced resistant cells.
EMT status is associated with H3122 cell line sensitivity to Alectinib
As a major transcription factor, HIF1α plays a central role in hypoxic cellular responses, and this transcription factor is reportedly related to the EMT. Kim et al. 20 reported that the EMT mediates resistance to the first-generation ALK inhibitor crizotinib. Therefore, we hypothesized high expression of HIF-1a along with EMT in hIGF-1-triggered resistant cells. Our study showed that 100 ng/ml hIGF-1 treatment resulted in the decreased expression of E-cadherin and increased expression of vimentin, indicative of EMT. The expression of E-cadherin was increased and that of vimentin was decreased in the co-administration group compared with that in the other groups (Fig. 5a). These findings indicate that EMT status is associated with H3122 cell line sensitivity to alectinib and that EMT status may be a potentially useful marker for predicting the therapeutic effect.
The development of resistance to targeted therapy remains a major challenge in the treatment of various cancers. Overcoming resistance is an important endeavor that is necessary to increase the overall survival of cancer patients. The occurrence of drug resistance is often accompanied by increased VEGF expression 21. Suppressing tumorigenesis and neoangiogenesis simultaneously is a new strategy that has been shown to potentially improve the treatment efficacy and reverse of drug resistance by increasing permeability and inhibiting angiogenesis 22,23. A previous study showed that the combination of erlotinib and bevacizumab exerts synergistic effects to inhibit tumor growth and improve overall survival 24. The therapeutic target for both ALK and VEGFR may help to efficiently inhibit the activation of bypass pathways and overcome ALK inhibitor resistance. Apatinib is a potent angiogenesis inhibitor that targets VEGFR2, with an IC50 of 2 nmol/l. On the basis of the safety profile, apatinib can be used in combination with alectinib. The aim of the current study was to determine whether apatinib might be a useful compound for reversing drug resistance. We showed that the combined use of alectinib and apatinib could inhibit the growth of IGF-triggered ALK-TKI-resistant tumors containing the EML4-ALK fusion gene.
IGF-1R is overexpressed in tumor tissue and is correlated with treatment resistance and worse prognosis in many types of tumors 25,26. IGF-1R signaling has also been reported to mediate resistance to both first-generation and second-generation ALK inhibitors 11,27. IGF-1R has kinase activity that is activated when in combination with its ligands IGF-1 and IGF-2. A variety of tyrosine residues are then phosphorylated and the two main signaling pathways, PI3K/AKT, and mitogen-activated protein kinase (MAPK)/ERK, are activated 11. As a result, tumor cells unlimitedly proliferate and mitosis. Our study found that hIGF-1 treatment can induce resistance to alectinib. The resistance index was 15.07. At the molecular level, the phosphorylation of AKT, mTOR, P70S6K, ERK, and STAT3 was increased after induction with 100 ng/ml hIGF-1 for 10 min IGF-1R and EML4-ALK may share the same downstream of signaling pathways. Hence, the probable mechanism of IGF-1-triggered alectinib resistance was by means of the activation of bypass signaling. We also observed a cell-acquired EMT phenotype along with IGF-1R upregulation. In the co-administration group, E-cadherin expression was increased and vimentin expression was decreased. These data suggest that EMT may be a potentially useful marker to predict the therapeutic effect.
In addition, one of the findings of our study was that IGF-1-triggered resistance to alectinib was associated closely with angiogenesis. The rapid growth of cancer cells results in a hypoxic state, and microvascular pruning limits oxygen supply to the tumor, which activates the hypoxia-inducible transcription factors, HIF-1α and HIF-2α. Li et al. 28 reported IGF-1-induced lung cancer angiogenesis and Ma et al. 29 reported that the inhibition of the PI3K/Akt/VEGF signaling pathway by phosphatase and tensin homolog may suppress tumor angiogenesis in human pancreatic cancer cells. In this study, the expression of HIF-1a increased following treatment with IGF-1. This was accompanied by transcriptional upregulation of HIF targets such as VEGF, thereby promoting autocrine VEGF signaling. A previous study showed that EML4-ALK lung tumors had significantly higher levels of VEGFA compared with the levels in EGFR, K-Ras, and B-Raf-mutated tumors, thus reinforcing the ALK–hypoxia angiogenesis axis 16. In our study, following treatment with exogenous hIGF-1, the protein expressions of HIF-1a, VEGF, VEGFR2, and p-VEGFR2 increased. Therefore, the HIF-1a/VEGF/VEGFR2 signaling axis participated in the development of drug resistance, which was related closely to angiogenesis.
These observations indicated that IGF induces resistance to alectinib by multiple mechanisms. ALK and VEGF(R) share parallel and reciprocal downstream signaling and may have cross-talk. In addition, stabilized HIFs bind to the VEGF promoter and activate its transcription in a tumor hypoxic environment. Interestingly, hypoxia-induced activation of HIF is accompanied by the translational upregulation of PI3K-Akt, Ras-MAPK, and STAT3 signaling. IGF regulates the expression of VEGF through the MAPK and PI3K signaling pathways and at least two different transcription factors (STAT3 and HIFs) are involved. Bypass activation of ALK upregulated the expression of VEGF. Furthermore, our data showed that the combination of alectinib and apatinib could inhibit the downstream signal of AKT/mTOR, ERK, and STAT3 when the IGF-1R and VEGFR signaling pathways were activated by their relevant ligands. In our study, the ALK and VEGFR signaling pathways were closely linked. As a consequence, the combination of antiangiogenesis drugs effectively increased the therapeutic effect and reversed the resistance. Therefore, apatinib might be a promising alternative in combination with ALK-TKIs.
Synergistic antitumor activity has been shown between inhibitors of EGFR and VEGF or VEGFR pathways. Rechallenge with apatinib after acquired resistance to EGFR-TKI results in ORR and DCR of 25 and 100%, respectively. The median PFS was 4.60 months 22. The results showed that the combination of EGFR-TKI and antiangiogenic agents might be a treatment option for patients with acquired resistance to EGFR. Clinical studies of the combination of ALK-TKI with small-molecule inhibitors of VEGFR have not been reported. To our knowledge, this is the first report on the combined effects of ALK-TKI with the next-generation antiangiogenic drug, apatinib, in alectinib-resistant cell lines. These results contribute toward the understanding of the cross-talk between ALK and VEGFR in EML4-ALK fusion gene-positive NSCLC and their possible combined blockade in therapy. The results provide a strong basis for the design of clinical trials for this purpose. In addition, clinical trials are warranted to evaluate the efficacy and safety of the combination therapy in EML4-ALK rearrangement lung cancer patients and those with acquired resistance to ALK-TKI.
This work was supported by grants from the National Natural Science Foundation of China (81260357).
Conflicts of interest
There are no conflicts of interest.
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