Hepatocyte Growth Factor Reduces Sensitivity To The Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor, Gefitinib,in Lung Adenocarcinoma Cells Harboring Wild-TypeEGFR

BACKGROUND AND OBJECTIVENon-small cell lung cancers (NSCLC) account for about 85% of lung cancers. Among patients with NSCLC, more than 65% present with locally advanced or metastatic disease [1].Unfortunately, less than 15% of patients with lung cancer survive more than five years. Over the past decade, the identification of specific genetic alterations, such as oncogenic drivers, in lung cancers has lead to a new periodof targeted therapy [2]. Targeting oncogenic drivers, such as epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), has brought the most encouraging improvements in lung cancer treatment. Clinical trials of targeted therapy, such as with EGFR-tyrosine kinase inhibitor (EGFR-TKI) therapy in EGFR mutant NSCLC and crizotinibtherapy in ALKrearranged NSCLC,have demonstrated major improvements in treatment response, quality of life, and progression-free survival compared to chemotherapy[3-5].EGFR-TKIs, such as erlotinib, gefitinib, and afatinib, are established as initial standard therapies [6-9]. These treatments are particularly effective against NSCLCs harboring activating mutations inEGFR, such asthe exon 19 deletion and L858R mutation. Activating mutations in EGFR are observed in up to 50% of lung adenocarcinomas in Asians and approximately 10% of Caucasians with NSCLC [10]. Although most patients with EGFR mutations initially respond to TKItherapy, almost all develop acquired resistance. Therefore, intrinsic and acquired resistance havebecome serious barriersto the outcomes of patients treated with these reagents.Many of the EGFR-TKIresistant mechanisms have been revealed. Recent studies using new generation EGFR-TKIs show good efficacy inresistant tumors with theEGFRT790Mgatekeeper mutation, which accounts for approximately 50% of resistant tumors [11,12]. Previously, we have reported that hepatocyte growth factor (HGF), the ligand of the MET receptor, induces resistance to gefitinibor new generation EGFR-TKIs in EGFR mutant lung adenocarcinomas through the MET/Gabl/PI3K/Akt pathway, without involvement of ErbB3[13,14], although ErbB3was critical inMETamplification-induced gefitinib resistance[15]. We also found that the MET inhibitor, E7050, successfully overcame HGF-induced resistance to EGFR-TKIs [16,17].For most patients with advanced lung cancer harboringwild-typeEGFR, chemotherapy is the prior choice [18]. However, resistance to chemotherapeutic reagents usually develops over time and limits the clinical benefit from chemotherapy. Although the effect of EGFR-TKIsin NSCLC patients with wild-type EGFR remains controversial, some clinical data have shown survival benefit derived fromEGFR-TKI treatment for patients previously treated with chemotherapy but still developed disease progression[19-22]. Therefore, several guidelines recommend EGFR-TKIs as an option of second-line treatments for NSCLC patients with wild-type EGFR [23]. Considering the nearly unavoidable resistance to EGFR-TKIs, we propose that a resistancemechanism may also exist in wild-typeEGFR lung cancer. If the potential resistance can be identifiedprior toEGFR-TKI therapy, this specific group of patients may benefit more from EGFR-TKIs.Since HGF/MET was previously identified as playing a critical role in the resistancemechanism of EGFR-TKIs in EGFR mutant NSCLC, we investigated whether HGFalso influenced EGFR-TKI sensitivityin lung adenocarcinoma cells harboringwild-type EGFR. We found that HGF significantly reduced sensitivityto gefitinib through the PI3K/Aktand MAPK pathways in lung cancer cells with wild-type EGFR. A MET inhibitor, PHA-665752, completely restored the sensitivity to EGFR-TKI. The results of this study suggest that combined use of EGFR-TKI and MET inhibitors or inhibitionof downstream signaling molecules, such as PI3K or MEK, might be a better second or third-line strategyfor a group ofpatients withadvanced lung cancerharboringwild-type EGFR.Methods1. To detect the sensitivity of EGFR wild-type lung adenocarcinoma cells to EGFR-TKIGefitinib sensitivitywas examined inthe lung cancer cell lines,H358 and A549, which expresswild-typeEGFR.Both cell lines express amutant KRAS genethatisknown as a marker of low sensitivity to EGFR inhibition and chemotherapy[24].Based on MTT manual, A549 and H358’s cell proliferation were detected under the different concentrations of EGFR-TKI (gefitinib and erlotinib).To detect wild-type EGFR lung adenocarcinoma cells’ sensitivity to EGFR-TKI after HGF treatedCells were treated by HGF 30 minutes in advance, added with different concentrations of EGFR-TKI gefitinib, and cultured another more 72 hours. Detect the changes of H358 and A549’s sensitivity to gefitinib in the presence of HGF, according to MTT protocol. Detect whether the effect of HGF can be abrogated by pretreatment with an anti-HGF neutralizing antibody or a small molecule met inhibitor PHA-665752, or not.Examined the expression of EGFR-wild cell with MET, EGFR, ErbB3After extracting whole cell protein lysates of A549 and H358, detect the protein concentration using a bicinchoninic acid protein assay kit. Equivalent amounts of total protein were resolved via SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were then blocked using 5% nonfat milk for 1 hour at room temperature, followed by incubation in the primary antibodies overnight at 4℃. After washing with TBST, the membranes were incubated in HRP-conjugated secondary antibodies for 1 hour at room temperature. The immunoreactive bands were visualized using an ECL western blot substrate.2. Examined the expression of EGFR-wild cell with MET, EGFR, ErbB3 and downstream protein after treated with gefitinib or HGF or MET inhibitorThe protocol is just like the upper shown.3. To decect the connection among met, EGFR, and ErbB3Using immunoprecipitation instructions, we examine the relationship among met, EGFR, and ErbB3.4. To detect the possibility of restoring sensitivity to gefitinib after specific down-regulation of MET and ErbB3Transfection of siRNA was performed using Lipofectamine 2000 reagent according to the manufacturer’s protocol. Duplexed Stealth RNAi (Invitrogen) targeted to MET and ErbB3 and Stealth RNAi Negative Control Low GC Duplex#3 (Invitrogen) were used for the RNA interference (RNAi) assay. One day before transfection aliquots of 2×104 tumor cells in 400 μL of antibiotic-free medium were plated into 24-well plates. After incubation for 24h, the cells were transfected with small interfering RNA (siRNA; 50 μmol) or scramble RNA using Lipofectamine 2000 (1μL) in accordance with the manufacturer’s instructions. After 24 h incubation, the cells were washed with PBS and plated into 96-well plates. Cell proliferation in the presence or absence of gefitinib (Iμmol/L) and/or recombinant human HGF (20 ng/nL)was measured with MTT as previously described. And at the same time, detect the protein expression of MET, EGFR, ErbB3 and downstream protein.5. To detect the change of gefitinib sensitivity in EGFR wild-type cell lines after the up-regulation of HGFEGFR-wild lung adenocarcinoma cells were infected with HGF, using the same protocol as upper shown to stably express HGF. After transfection, detect HGF expression by using human HGF ELISA test kit. And detect the gefitinb sensitivity among the transfected cells before and after by MTT protocol.6. To detect the function both PI3K/Akt and MAPK signaling pathways to gefitinib resistance in lung cancer cells harboring wild-type EGFRDetect the phosphorylation of Akt and the proliferation of wild-type EGFR A549 cells and EGFR-mutant PC-9 cells after using the PI3K inhibitors, GSK2126458 andPF04691502 and the MEK inhibitors, AZD-8330 andTAK-733.The method to detect protein and proliferation is just same as above.7. Statistical analysisAll data are expressed as the mean±SDs of experiments repeated at least three times. Significant differences between the means were measured by a two-tailed unpaired Student t test or one-way ANOVA. P<0.05 was considered statistically significant.Conclusion:1. HGF reduces sensitivity to gefitinib in lung adenocarcinoma cells harboring wild-type EGFR.2. HGF reduces sensitivity to gefitinib by directly restoring the phosphorylation of Akt and ERK1/2.3. Specific down-regulation of MET, but not ErbB3, restored gefitinib sensitivity by inhibiting HGF induced-phosphorylation of Akt and ERK1/2.4. HGF derived from tumor cells reduces sensitivity to gefitinib in lung adenocarcinoma cells harboring wild-type EGFR.5. Both PI3K/Akt and MAPK signaling pathways are essential in lung cancer cells harboring wild-type EGFR.