Abstract
Advanced lung cancer is one of the most lethal malignancies. Many anticancer agents have been developed for lung cancer with epidermal growth factor receptor (EGFR) mutations, but its prognosis remains extremely poor. The development of molecularly-targeted therapies is required for patients with lung cancer with secondary mutation of the EGFR gene. In this study, in order to assess the validity of heparin-binding EGF-like growth factor (HB-EGF) as a therapeutic target for lung cancer with EGFR mutation, we examined the antitumor effects of a specific inhibitor (cross-reacting material 197; CRM197) on lung cancer cells with EGFR mutation. HB-EGF was the most predominantly expressed EGFR ligand in lung cancer cells with EGFR mutation. CRM197 induced significant cell apoptosis and marked suppression of tumorigenicity in lung cancer cells with single or double mutation of EGFR. These results suggest that HB-EGF is a rational target for the treatment of lung cancer with EGFR mutation.
Lung cancer is the most common cancer and remains the leading cause of cancer-related deaths worldwide. In total, 85% of lung cancer cases are non-small cell lung cancer, (NSCLC) and 15% are small cell lung cancers (SCLC). More than half of patients who were diagnosed with NSCLC at an advanced stage have an extremely poor prognosis; median overall survival is less than 12 months and 5-year survival is less than 1% despite advances in chemotherapy (1, 2).
NSCLC encompasses the pathologically distinct adenocarcinoma, squamous cell carcinoma, and large cell carcinoma sub-types. Approximately 45% and 40% of patients with NSCLC are positive for epidermal growth factor receptor (EGFR) exon 19 deletions and exon 21 L858R mutations, respectively (3, 4). These mutations are predictive of treatment benefit from small molecule EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib, gefitinib, and afatinib. In addition, such sensitizing EGFR mutations occur in approximately 10% of Caucasian patients and more than 50% of Asian patients with NSCLC (5). However, most patients develop resistance to these small-molecule EGFR TKIs after the first 8 to 16 months (6). T790M in EGFR is an acquired resistance mutation in 60-70% of patients who initially respond to prior treatment with small-molecule EGFR TKIs (6). On the basis of these lines of evidence, the development of molecularly-targeted therapies is required to ameliorate the clinical prognosis in NSCLC with T790M mutation.
Heparin-binding EGF-like growth factor (HB-EGF) is an EGFR ligand (7-9), and is initially synthesized as a transmembrane protein, similarly to other members of the EGF family of growth factors (7-9) Previously, we reported that RNA interference of proHB-EGF (HBEGF) gene, or the addition of cross-reacting material 197 (CRM197), a specific HB-EGF inhibitor, resulted in significant apoptosis of cancer cells harboring HB-EGF expression, indicating that HB-EGF could be a valid target for cancer therapy in ovarian, breast, bladder, and gastric cancer, among others (10-12). However, there was no enhancement of expression of any EGFR ligand in lung cancer without mutations. It is therefore plausible that not only EGFR, but also EGFR ligands, could be considered as therapeutic targets in lung cancer with EGFR mutation.
In order to investigate the antitumor effect of CRM197 in lung cancer with EGFR mutation, we examined the expression of EGFR ligands, cell apoptosis and tumorigenicity following gefitinib or CRM197 treatment of lung cancer cells with and without EGFR mutations.
Materials and Methods
Reagents and antibodies. Cross-reacting material 197 (CRM197) and gefitinib were kindly provided by Professor Eisuke Mekada (Department of Cell Biology, Osaka University, Osaka, Japan) and by AstraZeneca K.K. (Osaka, Japan), respectively.
Cell culture. NCI-H460. A549, NIC-H441, RERF-LC-A1, PC-14, and NCI-H1975 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). NCI-H460. A549, and NIC-H441 cells have no EGFR mutations, whereas RERF-LC-A1, PC-14, and NCI-H1975 have mutations in EGFR at L858R, exon 19 deletion (746-750), and L858R plus T790M, respectively. All cell lines were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS; ICN Biomedicals, Irvine, CA, USA), 100 U/ml of penicillin G and 100 μg/ml of streptomycin (Invitrogen Corp., Carlsbad, CA, USA) in a humidified atmosphere of 5% CO2 at 37°C.
Real-time quantitative polymerase chain reaction (PCR) analysis of EGFR ligands. RNA extraction and cDNA synthesis were performed using TRIzol and SuperScript II reverse transcriptase (Invitrogen Corp.), respectively, according to the manufacturer's protocols. The primer and probe sequences for EGF and epigen were as follows: EGF forward primer, 50-CTT TGC CTT GCT CTG TCA CAG T-30; EGF reverse primer, 50-AAT ACC TGA CAC CCT TAT GAC AAA TTT-30; EGF probe, 50-AAG TCA GCC AGA GCA GGG CTG TTA AAC TCT-30; epigen forward primer, 50-TCT ATC TTT TAT TCA ACG CAA TGA CA-30; epigen reverse primer, 50-GGG CTG TGA TTG GAG GTG TT-30; epigen probe, 50-ACT GAC CGA AGA GGC AGC CGT GAC T-30. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected with Assays-on-Demand primer and probe sets Hs99999905_m1 (Applied Biosystems, Foster City, CA, USA). The procedures used for TaqMan quantitative real-time PCR analyses, as well as the sequences of the oligonucleotide primer pairs, TaqMan probes for HB-EGF, amphiregulin, transforming growth factor alpha, epiregulin and betacellulin,and the calculation for mRNA expression index (EI), have been described previously (13). All experiments were carried out independently three times.
Cell apoptosis assay. Extracellular matrix components were removed by detaching cells with trypsin-EDTA and then allowing them to recover for 30 min in RPMI-640 with 10% FBS (14). In order to assess the pharmacological effects of gefitinib or CRM197 on cell apoptosis, after rinsing with serum-free medium, cells were incubated in fresh serum-free medium at 37°C for 30 min before seeding (1×106) on polylysine-coated 6-cm dishes. Samples were incubated with serum-free RPMI 1640 at 37°C for 1 h to allow complete adherence of cells, and then incubated with serum-free RPMI-1640 plus different concentrations of gefitinib or 1 μg/ml of CRM197 at 37°C for 48 h. After these procedures, cells were harvested, pooled, and fixed with 4% paraformaldehyde at 4°C for 30 min and resuspended in 70% ethanol at −20°C for 30 min. After washing in phosphate-buffered saline (PBS), the cells were incubated with terminal deoxynucleotidyl transferase (TdT) reaction reagent (MEBSTAIN Apoptosis Kit Direct; MBL Co. Ltd., Nagoya, Japan) for 1 h at 37°C, according to the manufacturer's instructions. TdT-mediated 2’-deoxyuridine,-5’-triphosphate (dUTP) nick end labeling (TUNEL)-positive cells were quantified as apoptotic cells by flow cytometric analysis using a FACScalibur (Becton–Dickinson, Franklin Lakes, NJ, USA).
Tumor growth in nude mice. Subconfluent cell cultures were detached from plates with trypsin-EDTA. A total volume of 250 μl containing 5×106 cells suspended in serum-free RPMI-1640 was injected into female BALB/c nu/nu mice at 5 weeks of age (Charles River Laboratories). Injected mice were examined every week for tumor apparition. Tumor volume was calculated as described previously (15). In order to assess the effect of inhibition by CRM197, CRM197 dissolved in 1 ml of 20 mM HEPES and 0.15 M NaCl (pH 7.2) (1 mg) was injected intraperitoneally into tumor-bearing mice. One week after subcutaneous injection of RERF-LC-A1 and NCI-H1975 cells, 1 mg/kg of CRM197 was administered daily for 10 consecutive days during the course of the treatment. The animal protocol was approved by the guidelines of Animal Care of Fukuoka University (Approval No. 1106482) and Ethics Committee. Animals were observed on a daily bases. Humane endpoints were defined as a loss of more than 10% of body mass, a tumor greater than 20 mm, or inability to ambulate or rise for food and water. If animals reached these endpoints they were euthanized by exsanguination. Animal surgery and euthanasia using decapitation were performed under inhalation (isoflurane) anesthesia, and all efforts were made to minimize suffering.
Statistical analysis. Data were analyzed using the Mann–Whitney U-test. A value of p<0.05 was considered statistically significant. Data for many of the experiments were analyzed using Tukey HSD test; statistical significance was also set at p<0.05.
Results
Characterization of lung cancer cells. In order to identify molecular features in lung cancer, we examined the expression of each EGFR ligand in several lung cancer cell lines using RT-PCR. Lung cancer cells with EGFR mutation (RERF-LC-A1, PC-14 and NCI-H1975) had markedly higher expression of each EGFR ligand, compared to lung cancer cell lines without EGFR mutation (NCI-H460. A549, and NCIH441) (Figure 1). In lung cancer cells with EGFR mutation, HBEGF exhibited the highest expression of the EGFR ligands examined. These results suggest that lung cancer cells with EGFR mutation have characteristics of enhanced expression of EGFR and EGFR ligands.
Response to gefitinib. With the aim of investigating the response of lung cancer cells to gefitinib, we evaluated the apoptotic rate in each cell line after incubation with different concentrations of gefitinib. NCI-H460. A549, and NCI-H1975 cells exhibited little response to gefitinib, even at high concentrations. In RERF-LC-A1 cells, a significant dose-dependent increase in apoptosis was observed following treatment with gefitinib (Figure 2). These results suggest that lung cancer cells with EGFR mutation at L858R, such as RERF-LC-A1, are remarkably sensitive to gefitinib, and that lung cancer cells with double mutations at L858R and T790M, such as NCI-H1975, are relatively insensitive to gefitinib.
Characterization of lung cancer cells. The mRNA expression indices of epidermal growth factor receptor (EGFR) ligands in lung cancer cell lines harboring wild-type or mutant EGFR were determined by real-time polymerase chain reaction. Data represent the means±SEM of the mRNA expression indices for each EGFR ligand. HB-EGF: Heparin-binding EGF-like growth factor; TGFα: transforming growth factor alpha.
In vitro and in vivo response to CRM197 in lung cancer cell lines with EGFR mutation. We assessed the antitumor effects of CRM197 on lung cancer cell lines with EGFR mutation by analyzing cell apoptosis in vitro and tumorigenicity in vivo. RERF-LC-A1 and NCI-H1975 cells exhibited apoptotic rates of 4.43% and 7.46%, respectively, following treatment with CRM197 (Figure 3). In addition, tumor formation by RERF-LC-A1 cells was completely suppressed, and that by NCI-H1975 was also markedly inhibited (Figure 4). These results suggested that HB-EGF is a rational target for lung cancer with single and double mutations of EGFR, and that CRM197 is a potential molecularly-targeted agent for lung cancer with secondary mutation of T790M EGFR.
Response of lung cancer cells to gefitinib. Apoptosis rates in lung cancer cells after treatment with different concentrations of gefitinib. NCI-H460 and A549, no epidermal growth factor receptor (EGFR) mutation; RERF-LC-A1, single EGFR mutation; NCI-H1975, double EGFR mutation. Data represent the means±SEM of apoptosis rates in lung cancer cell lines. *p<0.05 versus NCI-H460 cells. **p<0.05 versus A549 cells.
In vitro response to cross-reacting material 197 (CRM197). The antitumor effect of CRM197 against lung cancer cell lines with epidermal growth factor receptor (EGFR) mutation in vitro. RERF-LC-A1 (A) and NCI-H1975 (B) cells demonstrated apoptotic rates of 4.43% and 7.46%, respectively, after CRM197 treatment. d-UTP: 2’-Deoxyuridine 5’-triphosphate; FITC: fluorescein sothiocyanate isomer-I.
Discussion
The expression of EGFR ligands, especially HB-EGF, was remarkably enhanced in lung cancer cell lines with EGFR mutation compared to those without EGFR mutation. HB-EGF was identified as a promising therapeutic target in lung cancer with single as well as double mutations of EGFR.
The tyrosine kinase function of EGFR is encoded by exons 18-24, in which the majority of patients with NSCLC patients with EGFR defects have mutations (6). Following the occurrence of mutations in exons 18-24, signaling between EGFR and its downstream molecules may be continuously activated, and may lead to enhancement of the autocrine amplification loop between EGFR and its ligands. Therefore, it is plausible that the markedly activated signal of the EGFR pathway may induce the expression of EGFR ligands in lung cancer with EGFR mutation. In mouse fibroblasts, the transfection of EGFR, human EGFR2 (HER2), or AKT up-regulates the expression of HB-EGF, accompanied by activation of downstream EGFR pathways (16). Resistance to EGFR TKIs is inevitable because of a variety of mechanisms including the secondary mutation of EGFR (T790M), aberrations in the downstream EGFR pathways such as KRAS mutation and loss of PTEN, and the activation of alternative pathways (17, 18). According to these lines of evidence, the predominant expression of HB-EGF in lung cancer with EGFR mutation might be linked to the activation of EGFR signaling.
In vivo response to cross-reacting material 197 (CRM197). Weekly alterations in growth of tumor derived from RERF-LC-A1 (A) and NCI-H1975 (B) cells in NOD/SCID mice (n=10). CRM197 (1 mg/day) was injected intraperitoneally every day for 10 days once tumor volume exceeded 100 mm3. Arrows indicate the days on which CRM197 was administered. Data represent the means±SEM of tumor volumes. *p<0.05 versus control mice without CRM197 treatment.
The majority of patients with lung cancer with EGFR mutations who are treated with first-generation TKIs such as gefitinib or erlotinib acquire secondary EGFR mutation (T790M) as a resistance mechanism to these TKIs within 8 to 16 months (6). Novel molecularly-targeted agents against lung cancer with EGFRT790M have been developed as third-generation TKIs (19, 20). However, it is likely that resistance to these third-generation TKIs may emerge as a result of further additional EGFR mutations. Cetuximab, a monoclonal antibody to EGFR, has been used predominantly in combination with EGFR TKIs against TKI-resistant tumors. In 126 patients with NSCLC whose disease had progressed during treatment with erlotinib or gefitinib, the combination of afatinib and cetuximab demonstrated little difference in progression-free survival between patients with and without EGFRT790M (21). In addition, the combination of erlotinib and cetuximab indicated no clinical response in patients with resistance to erlotinib in a phase I/II study (22). In principle, cetuximab binds to the extracellular domain of EGFR and prevents ligand-dependent receptor activation. However, cetuximab can block the homodimerization of EGFR but not the heterodimerization of EGFR and other HER receptors, hence cetuximab does not suppress AKT signaling in cancer cells (16). Conversely, the blockage of HB-EGF can inhibit both HB-EGF-dependent homodimerization and heterodimerization, demonstrating that an inhibitor of HB-EGF suppresses AKT signaling as well as ERK. CRM197, an inhibitor of HB-EGF, and an anticancer agent with a high molecular weight, was shown to be clinically safe and effective for patients with ovarian cancer (23). The combination of TKIs and CRM197 may be a potential treatment for patients with NSCLC with EGFR mutation.
In conclusion, HB-EGF may also be a rational target for lung cancer with resistance to TKIs because of EGFR downstream aberrations. CRM197 might be expected to improve prognosis in patients with NSCLC with EGFR mutation.
Acknowledgements
This work was supported in part by a Grant-in-Aid for challenging Exploratory Research (no. 26670731), Scientific Research (B) (no. 26293362), and fund from the Central Research Institute of Fukuoka University; The Center for Advanced Molecular Medicine, Fukuoka University from the Ministry of Education, Culture, Sports, Science and Technology (Tokyo, Japan); a Grant-in-Aid from the Kakihara Science and Technology Foundation (Fukuoka, Japan), and Kyowa Hakko Kirin Co. Ltd. (Tokyo, Japan) to S. Miyamoto.
Footnotes
↵* These Authors contributed equally to this study.
This article is freely accessible online.
Conflicts of Interest
The Authors have no potential conflicts of interest in regard to this study.
- Received May 2, 2017.
- Revision received May 29, 2017.
- Accepted May 30, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
