MET gene amplification or EGFR mutation activate MET in lung cancers untreated with EGFR tyrosine kinase inhibitors

We analyzed MET protein and copy number in NSCLC with or without EGFR mutations untreated with EGFR tyrosine kinase inhibitors (TKIs). MET copy number was examined in 28 NSCLC and 4 human bronchial epithelial cell lines (HBEC) and 100 primary tumors using quantitative real‐time PCR. Positive results were confirmed by array comparative genomic hybridization and fluorescence in‐situ hybridization. Total and phospho‐MET protein expression was determined in 24 NSCLC and 2 HBEC cell lines using Western blot. EGFR mutations were examined for exon 19 deletions, T790M, and L858R. Knockdown of EGFR with siRNA was performed to examine the relation between EGFR and MET activation. High‐level MET amplification was observed in 3 of 28 NSCLC cell lines and in 2 of 100 primary lung tumors that had not been treated with EGFR‐TKIs. MET protein was highly expressed and phosphorylated in all the 3 cell lines with high MET amplification. In contrast, 6 NSCLC cell lines showed phospho‐MET among 21 NSCLC cell lines without MET amplification (p = 0.042). Furthermore, those 6 cell lines harboring phospho‐MET expression without MET amplification were all EGFR mutant (p = 0.0039). siRNA‐mediated knockdown of EGFR abolished phospho‐MET expression in examined 3 EGFR mutant cell lines of which MET gene copy number was not amplified. By contrast, phospho‐MET expression in 2 cell lines with amplified MET gene was not down‐regulated by knockdown of EGFR. Our results indicated that MET amplification was present in untreated NSCLC and EGFR mutation or MET amplification activated MET protein in NSCLC. © 2008 Wiley‐Liss, Inc.     

Combined inhibition of IGFR enhances the effects of gefitinib in H1650: a lung cancer cell line with EGFR mutation and primary resistance to EGFR-TK inhibitors

Purpose

H1650 non-small cell lung cancer (NSCLC) cells display primary resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) although they have a deletion mutation on exon 19 of the EGFR gene. We investigated the effect of inhibition of both insulin-like growth factor receptor (IGFR) and EGFR signaling considering that IGFR signaling pathway has been implicated in the development and progression with therapeutic resistance of various cancers including lung cancer.

Methods

Three human NSCLC cell lines with an EGFR mutation of PC-9, HCC827 and H1650 were used for experiment. Cell viability and proliferative activity were assessed by MTT and three-dimensional culture assay. Combination index was obtained by CalcuSyn software. The change of EGFR- and IGFR-related signals was evaluated by western blots.

Results

H1650 cells were 1,000 times more resistant to gefitinib and erlotinib than HCC827 and PC-9 cells possessing the same EGFR mutation. Phosphatase and tensin homolog loss and sustained phosphorylation of Akt in spite of treatment with gefitinib were evident only in H1650 cells. Interestingly, IGFR phosphorylation was decreased by gefitinib in HCC827 and PC-9 cells while being maintained in H1650 cells. Combined treatment with the IGFR inhibitors α-IR3 and AG1024 enhanced gefitinib-induced growth inhibition and apoptosis, and down-regulated phosphorylation of Akt, EGFR and IGFR.

Conclusion

Combined inhibition of IGFR signaling enhances the growth inhibitory and apoptosis-inducing effects of gefitinib, suggesting that this approach could be useful to overcome the primary resistance to EGFR-TKIs in lung cancer.     

Effects of Src inhibitors on cell growth and epidermal growth factor receptor and MET signaling in gefitinib-resistant non-small cell lung cancer cells with acquired MET amplification

The efficacy of epidermal growth factor receptor (EGFR)–tyrosine kinase inhibitors such as gefitinib and erlotinib in non-small cell lung cancer (NSCLC) is often limited by the emergence of drug resistance conferred either by a secondary T790M mutation of EGFR or by acquired amplification of the MET gene. We now show that the extent of activation of the tyrosine kinase Src is markedly increased in gefitinib-resistant NSCLC (HCC827 GR) cells with MET amplification compared with that in the gefitinib-sensitive parental (HCC827) cells. In contrast, the extent of Src activation did not differ between gefitinib-resistant NSCLC (PC9/ZD) cells harboring the T790M mutation of EGFR and the corresponding gefitinib-sensitive parental (PC9) cells. This activation of Src in HCC827 GR cells was largely abolished by the MET-TKI PHA-665752 but was only partially inhibited by gefitinib, suggesting that Src activation is more dependent on MET signaling than on EGFR signaling in gefitinib-resistant NSCLC cells with MET amplification. Src inhibitors blocked Akt and Erk signaling pathways, resulting in both suppression of cell growth and induction of apoptosis, in HCC827 GR cells as effectively as did the combination of gefitinib and PHA-665752. Furthermore, Src inhibitor dasatinib inhibited tumor growth in HCC827 GR xenografts to a significantly greater extent than did treatment with gefitinib alone. These results provide a rationale for clinical targeting of Src in gefitinib-resistant NSCLC with MET amplification. ( Cancer Sci 2009)     

MicroRNA-214 regulates the acquired resistance to gefitinib via the PTEN/AKT pathway in EGFR-mutant cell lines

Patients with non-small cell lung cancer (NSCLC) who have activating epidermal growth factor receptor (EGFR) mutations derive clinical benefit from treatment with EGFR-tyrosine kinase inhibitors ((EGFR-TKIs)- namely gefitinib and erlotinib. However, these patients eventually develop resistance to EGFR-TKIs. Despite the fact that this acquired resistance may be the result of a secondary mutation in the EGFR gene, such as T790M or amplification of the MET proto-oncogene, there are other mechanisms which need to be explored. MicroRNAs (miRs) are a class of small non-coding RNAs that play pivotal roles in tumorigenesis, tumor progression and chemo-resistance. In this study, we firstly successfully established a gefitinib resistant cell line-HCC827/GR, by exposing normal HCC827 cells (an NSCLC cell line with a 746E-750A in-frame deletion of EGFR gene) to increasing concentrations of gefitinib. Then, we found that miR-214 was significantly up-regulated in HCC827/ GR. We also showed that miR-214 and PTEN were inversely expressed in HCC827/GR. Knockdown of miR-214 altered the expression of PTEN and p-AKT and re-sensitized HCC827/GR to gefitinib. Taken together, miR-214 may regulate the acquired resistance to gefitinib in HCC827 via PTEN/AKT signaling pathway. Suppression of miR-214 may thus reverse the acquired resistance to EGFR-TKIs therapy.     

Preclinical Evaluation of MET Inhibitor INC-280 With or Without the Epidermal Growth Factor Receptor Inhibitor Erlotinib in Non–Small-Cell Lung Cancer

BackgroundAlthough the epidermal growth factor receptor (EGFR) inhibitor erlotinib is initially effective in non–small-cell lung cancer (NSCLC) patients with tumors harboring activating mutations of EGFR, most subsequently develop acquired resistance. One recognized resistance mechanism occurs through activation of bypass signaling via the hepatocyte growth factor (HGF)-MET pathway. INC-280 is a small molecule kinase inhibitor of MET. We sought to demonstrate the activity of INC-280 on select NSCLC cell lines both as a single agent and in combination with erlotinib using exogenous HGF to simulate MET up-regulation.MethodsFour NSCLC cell lines (HCC827, PC9, H1666, and H358) were treated with either single-agent INC-280 or in combination with erlotinib with or without HGF. The activity of the drug treatments was measured by cell viability assays. Immunoblotting was used to monitor expression of EGFR/pEGFR, MET/pMET, GAB1/pGAB1, AKT/pAKT, and ERK/pERK as well as markers of apoptosis (PARP and capase-3 cleavage) in H1666, HCC827, and PC9.ResultsAs a single agent, INC-280 showed minimal cytotoxicity despite potent inhibition of MET kinase activity at concentrations as low as 10 nM. Addition of HGF prevented erlotinib-induced cell death. The addition of INC280 to HGF-mediated erlotinib-resistant models restored erlotinib sensitivity for all cell lines tested, associated with cleavage of both PARP and caspase-3. In these models, INC-280 treatment was sufficient to restore erlotinib-induced inhibition of MET, GAB1, AKT, and ERK in the presence of HGF.ConclusionAlthough the MET inhibitor INC-280 alone had no discernible effect on cell growth, it was able to restore sensitivity to erlotinib and promote apoptosis in NSCLC models rendered erlotinib resistant by HGF. These data provide a preclinical rationale for an ongoing phase 1 clinical trial of erlotinib plus INC-280 in EGFR-mutated NSCLC.     

Asian Thoracic Oncology Research Group (ATORG) Expert Consensus Statement on MET Alterations in NSCLC: Diagnostic and Therapeutic Considerations

Non-small cell lung cancer (NSCLC) is a heterogeneous disease, with many oncogenic driver mutations, including de novo mutations in the Mesenchymal Epithelial Transition (MET) gene (specifically in Exon 14 [ex14]), that lead to tumourigenesis. Acquired alterations in the MET gene, specifically MET amplification is also associated with the development of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance in patients with EGFR-mutant NSCLC. Although MET has become an actionable biomarker with the availability of MET-specific inhibitors in selected countries, there is differential accessibility to diagnostic platforms and targeted therapies across countries in Asia-Pacific (APAC).The Asian Thoracic Oncology Research Group (ATORG), an interdisciplinary group of experts from Australia, Hong Kong, Japan, Korea, Mainland China, Malaysia, the Philippines, Singapore, Taiwan, Thailand and Vietnam, discussed testing for MET alterations and considerations for using MET-specific inhibitors at a consensus meeting in January 2022, and in subsequent offline consultation.Consensus recommendations are provided by the ATORG group to address the unmet need for standardised approaches to diagnosing MET alterations in NSCLC and for using these therapies. MET inhibitors may be considered for first-line or second or subsequent lines of treatment for patients with advanced and metastatic NSCLC harbouring MET ex14 skipping mutations; MET ex14 testing is preferred within multi-gene panels for detecting targetable driver mutations in NSCLC. For patients with EGFR-mutant NSCLC and MET amplification leading to EGFR TKI resistance, enrolment in combination trials of EGFR TKIs and MET inhibitors is encouraged.     

MET increased gene copy number and primary resistance to gefitinib therapy in non-small-cell lung cancer patients

Background: MET amplification has been detected in 20% of non-small-celllung cancer patients (NSCLC) with epidermal growth factor receptor(EGFR) mutations progressing after an initial response to tyrosinekinase inhibitor (TKI) therapy. Patients and methods: We analyzed MET gene copy number usingFISH in two related NSCLC cell lines, one sensitive (HCC827)and one resistant (HCC827 GR6) to gefitinib therapy and in twodifferent NSCLC patient populations: 24 never smokers or EGFRFISH-positive patients treated with gefitinib (ONCOBELL cohort)and 182 surgically resected NSCLC not exposed to anti-EGFR agents. Results: HCC827 GR6-resistant cell line displayed MET amplification,with a mean MET copy number >12, while sensitive HCC827 cellline had a mean MET copy number of 4. In the ONCOBELL cohort,no patient had gene amplification and MET gene copy number wasnot associated with outcome to gefitinib therapy. Among thesurgically resected patients, MET was amplified in 12 cases(7.3%) and only four (2.4%) had a higher MET copy number thanthe resistant HCC827 GR6 cell line. Conclusions: MET gene amplification is a rare event in patientswith advanced NSCLC. The development of anti-MET therapeuticstrategies should be focused on patients with acquired EGFR-TKIresistance. Key words: EGFR, gefitinib, MET, non-small cell lung cancer, tyrosine kinase inhibitor Received for publication March 30, 2008. Revision received August 4, 2008. Accepted for publication August 18, 2008.     

Design and Rationale for a Phase II,Randomized, Open-Label,Two-Cohort Multicenter Interventional Study of Osimertinib with or Without Savolitinib in De Novo MET Aberrant,EGFR-Mutant Patients with Advanced Non-Small-Cell Lung Cancer: The FLOWERS Trial

IntroductionEpidermal growth factor receptor (EGFR) mutations are well-known genetic alterations in advanced non-small cell lung cancer (NSCLC) which are associated with remarkable survival benefits from first-line treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKIs). However, around 30% of patients exhibit primary resistance to EGFR-TKIs therapy. Co-existing MET amplification/over-expression has showed shorter time to progression on EGFR-TKI monotherapy. Osimertinib (TAGRISSO, AZD9291) has been recommended in EGFR-mutant advanced NSCLC patients as first-line treatment. Savolitinib (AZD6094, HMPL-504) is a highly selective MET-TKI which has demonstrated anti-tumor activity in various cancers with MET alterations.MethodsThis FLOWERS study, a phase II, randomized, open-label, 2-cohort multicenter trial aimed to evaluate the efficacy and safety of osimertinib with or without savolitinib as first-line therapy in patients with de novo MET amplified/over-expressed, EGFR-mutant positive, locally advanced or metastatic NSCLC. Approximately 44 patients will be randomized to receive osimertinib (80 mg once daily) monotherapy or osimertinib (80 mg once daily) and savolitinib (300 mg twice daily) combination therapy; patients in osimertinib monotherapy cohort confirmed as MET positive (MET-amplified/over-expressed) after disease progression will have the opportunity to receive the cross-over combination therapy as second-line treatment. Primary endpoint will be objective response rate. Key secondary endpoints will be progression-free survival, duration of response, disease control rate, overall survival, safety and tolerability.ConclusionThe results of the study will provide better perspectives on the efficacy and safety of EGFR-TKI plus MET-TKI combination therapy (osimertinib plus savolitinib) in patients with de novo MET-amplified/over-expressed, EGFR-mutant positive, treatment naïve, advanced NSCLC and offer a meaningful guidance in clinical practice (NCT05163249).     

Oncogene swap as a novel mechanism of acquired resistance to epidermal growth factor receptor‐tyrosine kinase inhibitor in lung cancer

Mutant selective epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs), such as rociletinib and AZD9291, are effective for tumors with T790M secondary mutation that become refractory to first‐generation EGFR‐TKI. However, acquired resistance to these prospective drugs is anticipated considering the high adaptability of cancer cells and the mechanisms remain largely obscure. Here, CNX‐2006 (tool compound of rociletinib) resistant sublines were established by chronic exposure of HCC827EPR cells harboring exon 19 deletion and T790M to CNX‐2006. Through the analyses of these resistant subclones, we identified two resistant mechanisms accompanied by MET amplification. One was bypass signaling by MET amplification in addition to T790M, which was inhibited by the combination of CNX‐2006 and MET‐TKI. Another was loss of amplified EGFR mutant allele including T790M while acquiring MET amplification. Interestingly, MET‐TKI alone was able to overcome this resistance, suggesting that oncogenic dependence completely shifted from EGFR to MET. We propose describing this phenomenon as an “oncogene swap.” Furthermore, we analyzed multiple lesions from a patient who died of acquired resistance to gefitinib, then found a clinical example of an oncogene swap in which the EGFR mutation was lost and a MET gene copy was gained. In conclusion, an “oncogene swap” from EGFR to MET is a novel resistant mechanism to the EGFR‐TKI. This novel mechanism should be considered in order to avoid futile inhibition of the original oncogene.     

MicroRNA-34a overcomes HGF-mediated gefitinib resistance in EGFR mutant lung cancer cells partly by targeting MET

In non-small-cell lung cancer (NSCLC) that harbours an activating epidermal growth factor receptor (EGFR) mutation, over-expression of hepatocyte growth factor (HGF) is an important mechanism involved in the acquired resistance to EGFR-tyrosine kinase inhibitors (TKIs) by restoring activity of the PI3K/Akt pathway via phosphorylation of MET. In our study, we found that the forced expression of miR-34a inhibited cell growth and induced apoptosis partly by targeting MET in HGF-induced gefitinib-resistant HCC827 and PC-9 cells. Furthermore, dramatic tumour regression was observed in the miR-34a plus gefitinib group in HGF-induced gefitinib resistant mouse xenograft models. This study demonstrates for the first time that miR-34a rescues HGF-induced gefitinib resistance in EGFR mutant NSCLC cells.     

Impact of bevacizumab in combination with erlotinib on EGFR‐mutated non–small cell lung cancer xenograft models with T790M mutation or MET amplification

Erlotinib (ERL), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, shows notable efficacy against non–small cell lung cancer (NSCLC) harboring EGFR mutations. Bevacizumab (BEV), a humanized monoclonal antibody to vascular endothelial cell growth factor (VEGF), in combination with ERL (BEV+ERL) significantly extended progression‐free survival in patients with EGFR‐mutated NSCLC compared with ERL alone. However, the efficacy of BEV+ERL against EGFR‐mutated NSCLC harboring T790M mutation or MET amplification, is unclear. Here, we examined the antitumor activity of BEV+ERL in four xenograft models of EGFR‐mutated NSCLC (three harboring ERL resistance mutations). In the HCC827 models (exon 19 deletion: DEL), ERL significantly inhibited tumor growth by blocking EGFR signal transduction. Although there was no difference between ERL and BEV+ERL in maximum tumor growth inhibition, BEV+ERL significantly suppressed tumor regrowth during a drug‐cessation period. In the HCC827‐EPR model (DEL+T790M) and HCC827‐vTR model (DEL+MET amplification), ERL reduced EGFR signal transduction and showed less pronounced but still significant tumor growth inhibition than in the HCC827 model. In these models, tumor growth inhibition was significantly stronger with BEV+ERL than with each single agent. In the NCI‐H1975 model (L858R+T790M), ERL did not inhibit growth or EGFR signal transduction, and BEV+ERL did not inhibit growth more than BEV. BEV alone significantly decreased microvessel density in each tumor. In conclusion, addition of BEV to ERL did not enhance antitumor activity in primarily ERL‐resistant tumors with T790M mutation; however, BEV+ERL enhanced antitumor activity in T790M mutation‐ or MET amplification‐positive tumors as long as their growth remained significantly suppressed by ERL.     

PEBP4 enhanced HCC827 cell proliferation and invasion ability and inhibited apoptosis

The purposes of this study were to investigate the effects of phosphatidylethanolamine-binding protein 4 (PEBP4) on the cell growth, proliferation, apoptosis, and invasion of non-small cell lung cancer (NSCLC) cells and to provide evidence for future treatment options for NSCLC. Western blot assays were performed to examine PEBP4 protein expression levels in NSCLC cell lines (HCC827, A549, NCI-H661, NCI-H292, and 95-D) and a normal human bronchial epithelial (HBE) cell line. A PEBP4 shRNA expression vector was constructed and transfected into HCC827 cells. Subsequently, the effects of PEBP4 on the cell viability, cell cycle distribution, apoptosis levels, and invasion properties of HCC827 cells were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, flow cytometry analyses, and transwell invasion assays. In addition, the effects of PEBP4 on the expression of proteins including cyclin D1, p53, Bcl-2, MMP-2, and MMP-9 were investigated. PEBP4 was highly expressed in lung cancer cells (HCC827, A549, NCI-H661, NCI-H292, and 95-D), but its expression was low in HBE cells. Cell viability, cell proliferation, and invasion of HCC827 cells in the PEBP4 knockdown group were significantly lower than that in the negative control and blank control groups (p?<?0.05), and there were no significant differences between the negative and blank control groups in terms of cell viability, cell proliferation, apoptosis, and invasion. In HCC827 cells, the expression levels of cyclin D1, Bcl-2, MMP-2, and MMP-9 in the PEBP4 knockdown group were significantly lower (p?<?0.05), and the expression of p53 protein was significantly higher than that in the negative and blank control groups (p?<?0.05). There were no significant differences between the negative and blank control groups in the expression levels of cyclin D1, p53, Bcl-2, MMP-2, and MMP-9. In conclusion, PEBP4 enhanced HCC827 cell proliferation and invasion ability and inhibited apoptosis. Decreased PEBP4 expression may play a role in the reduced invasion ability and increased apoptosis of the human NSCLC cell line HCC827.     

Heat stress induced,ligand-independent MET and EGFR signalling in hepatocellular carcinoma

Purpose: The aims of the present study were 2-fold: first, to test the hypothesis that heat stress induces MET and EGFR signalling in hepatocellular carcinoma (HCC) cells and inhibition of this signalling decreases HCC clonogenic survival; and second, to identify signalling pathways associated with heat stress induced MET signalling.

Materials and Methods: MET⁺ and EGFR⁺ HCC cells were pre-treated with inhibitors to MET, EGFR, PI3K/mTOR or vehicle and subjected to heat stress or control?±?HGF or EGF growth factors and assessed by colony formation assay, Western blotting and/or quantitative mass spectrometry. IACUC approved partial laser thermal or sham ablation was performed on orthotopic N1S1 and AS30D HCC tumours and liver/tumour assessed for phospho-MET and phospho-EGFR immunostaining.

Results: Heat-stress induced rapid MET and EGFR phosphorylation that is distinct from HGF or EGF in HCC cells and thermal ablation induced MET but not EGFR phosphorylation at the HCC tumour ablation margin. Inhibition of the MET and EGFR blocked both heat stress and growth factor induced MET and EGFR phosphorylation and inhibition of MET decreased HCC clonogenic survival following heat stress. Pathway analysis of quantitative phosphoproteomic data identified downstream pathways associated with heat stress induced MET signalling including AKT, ERK, Stat3 and JNK. However, inhibition of heat stress induced MET signalling did not block AKT signalling.

Conclusions: Heat-stress induced MET and EGFR signalling is distinct from growth factor mediated signalling in HCC cells and MET inhibition enhances heat stress induced HCC cell killing via a PI3K/AKT/mTOR-independent mechanism.     

Acquired resistance to cetuximab is mediated by increased PTEN instability and leads cross-resistance to gefitinib in HCC827 NSCLC cells

EGFR inhibitors, including the small-molecule tyrosine kinase inhibitors such as gefitinib, and the monoclonal antibodies directed at the receptor such as cetuximab, have demonstrated promising effects in non-small cell lung cancer (NSCLC). In this study, we generated cetuximab-resistant cell lines (HCC827-CR) from HCC827 NSCLC cells to investigate acquired resistance mechanisms to cetuximab. In HCC827-CR cells, Akt was hyperactivated and its activity was persistent upon cetuximab treatment. Blockade of PI3K/Akt activity restored cetuximab sensitivity in HCC827-CR cells. Further investigation revealed that increased PTEN instability mediates constitutive Akt activation. By 1 μM proteosomal inhibitor, MG-132, PTEN protein levels were restored and Akt activity was dramatically reduced. Overexpression of PTEN by transfection could not restore cetuximab sensitivity in HCC827-CR because overexpressed PTEN was degraded rapidly (∼72 h). The increased PTEN instability was confirmed by the treatment of HCC827-CR with a protein synthesis inhibitor, cycloheximide. In the presence of cycloheximide, overexpressed PTEN was degraded more rapidly (∼12 h) in HCC827-CR cells. Interestingly, HCC827-CR cells also revealed de novo resistance to gefitinib. Inhibition of PI3K/Akt signaling pathway restored sensitivity to gefitinib in HCC827-CR cells. Taken together, these data show that PTEN instability-mediated constitutive Akt activation is involved in acquired resistance mechanisms to cetuximab and also induces de novo resistance to gefitinib. Importantly, these findings suggest emergence of cross-resistance between two agents as a potential serious problem in the clinical setting.     

人非小细胞肺癌细胞EGFR基因启动子甲基化与吉非替尼敏感性之间的相关性研究

目的 探讨EGFR基因启动子甲基化水平与人非小细胞肺癌（NSCLC）细胞株对吉非替尼敏感性之间的相关性。方法 用不同浓度吉非替尼分别作用于NSCLC细胞株HCC827、H1650、H1975、H358、H1299、A549后,CCK-8法检测细胞增殖抑制率,DNA直接测序法、实时荧光定量PCR法、免疫印迹法和甲基化特异性PCR法分别检测上述NSCLC细胞株EGFR基因突变、EGFR mRNA、EGFR蛋白表达和启动子区甲基化状态。结果 CCK-8法检测结果显示,19外显子缺失突变的HCC827细胞对吉非替尼最敏感,而同为19外显子缺失突变的H1650细胞对吉非替尼不敏感;野生型的H358细胞对吉非替尼中度敏感,其敏感性甚至超过19外显子突变的H1650细胞,而同为EGFR野生型的H1299、A549细胞对吉非替尼敏感性较差。吉非替尼处理72h后,HCC827细胞与H358细胞相比、HCC827细胞和H358细胞与其他4株细胞相比,IC50值均有显著性差异（P＜0.05）。HCC827细胞EGFR启动子为未甲基化状态,其EGFR蛋白和mRNA表达最高;H358细胞为部分甲基化,其EGFR蛋白和mRNA为中等表达;其他4个细胞株均为高甲基化状态,EGFR蛋白和mRNA呈低表达;HCC827细胞的EGFR表达水平较H358细胞高,HCC827和H358细胞的EGFR表达较其他4个细胞株高,差异均有统计学意义（P＜0.05）。结论 EGFR基因启动子区高甲基化可能下调EGFR基因的表达水平,从而降低NSCLC对吉非替尼的敏感性;对该基因的甲基化检测可能对预测吉非替尼治疗NSCLC疗效有一定的临床指导意义。     

Capmatinib for the treatment of non-small cell lung cancer

Introduction: Activation of the MET pathway through MET amplifications or mutations is present in 3–4% of stage IV non-squamous non-small cell lung cancers (NSCLC). High MET amplifications and exon 14 skipping mutations are associated with poor prognosis: new treatments are needed for these patients. Capmatinib is a highly selective, potent small-molecule MET inhibitor with antitumor activity in NSCLC in vitro and in vivo.

Areas covered: This article provides an overview of the capmatinib clinical development program in NSCLC, both as monotherapy in NSCLC with a dysregulated MET pathway, and in combination with epidermal growth factor receptor (EGFR) inhibitor therapy in EGFR-mutant NSCLC with MET-based acquired resistance to previous EGFR inhibition.

Expert opinion: In the GEOMETRY Mono-1 study, treatment with capmatinib resulted in high response rates in stage IV NSCLC with MET exon 14 skipping mutations, particularly in first line, supporting testing for this biomarker at the time of diagnosis. Durable responses have been reported and results in MET-amplified NSCLC are eagerly anticipated. In EGFR-mutant NSCLC, notable responses have been observed in combination with an EGFR-tyrosine kinase inhibitor (TKI) in case of acquired resistance to EGFR-TKIs based on high MET amplification.     

TRAIL induces apoptosis in oral squamous carcinoma cells: a crosstalk with oncogenic Ras regulated cell surface expression of death receptor 5

TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis through its death receptors (DRs) 4 and/or 5 expressed on the surface of target cells. The selectivity of TRAIL towards cancer cells has promoted clinical evaluation of recombinant human TRAIL (rhTRAIL) and its agonistic antibodies in treating several major human cancers including colon and non-Hodgkin''s lymphoma. However, little is known about their ability in killing oral squamous cell carcinoma (OSCC) cells. In this study, we tested the apoptotic responses of a panel of seven human OSCC cell lines (HN31, HN30, HN12, HN6, HN4, Cal27, and OSCC3) to rhTRAIL and monoclonal antibodies against DR4 or DR5. We found that rhTRAIL is a potent inducer of apoptosis in most of the oral cancer cell lines tested both in vitro and in vivo. We also showed that DR5 was expressed on the surface of the tested cell lines which correlated with the cellular susceptibility to apoptosis induced by rhTRAIL and anti-DR5 antibody. By contrast, little or no DR4 was detected on the surface of OSCC3 and HN6 cells rendering cellular resistance to DR4 antibody and a reduced sensitivity to rhTRAIL. Notably, the overall TRAIL sensitivity correlated well with the levels of endogenous active Ras in the cell lines tested. Expression of a constitutively active Ras mutant (RasV12) in OSCC3 cells selectively upregulated surface expression of DR5, but not DR4, and restored TRAIL sensitivity. Our findings could have implications for the use of TRAIL receptor targeted therapies in the treatment of human OSCC tumors particularly the ones harboring constitutively active Ras mutant.     

Response to dual blockade of epidermal growth factor receptor (EGFR) and cycloxygenase-2 in nonsmall cell lung cancer may be dependent on the EGFR mutational status of the tumor

BACKGROUND: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have demonstrated clinical benefit in patients with nonsmall cell lung cancer (NSCLC), particularly those with tumors that have EGFR-TK domain mutations. Moreover, the EGFR and cyclooxygenase (COX)-2 pathways are known to enhance the procarcinogenic effects of each other in different tumor types. Therefore, it was hypothesized that tumor EGFR mutation status may influence the effectiveness of simultaneous EGFR and COX-2 inhibition in patients with NSCLC. METHODS: Three NSCLC cell lines with varying EGFR mutation status and sensitivities to EGFR-TKIs were selected: H3255 (L858R), H1650 (del E746-A750), and H1781 (wild-type EGFR). Cells were treated with erlotinib, gefitinib, or celecoxib alone, and the combination of both EGFR-TKI inhibitors with celecoxib. Cell survival and apoptosis was assessed and correlated with the expression of COX-2, EGFR, pEGFR, Akt, pAkt, expression, and derived prostaglandin E2 (PGE(2)). RESULTS: Celecoxib by itself was found to have no effects on cell growth or apoptosis in any of the cell lines. Erlotinib and gefitinib inhibited cell growth and induced apoptosis in both mutant cell lines and did so in H1781 cells at 10-fold higher concentrations. Celecoxib when added to erlotinib or gefitinib significantly enhanced the antiproliferative and proapoptotic effects in both mutant cell lines but had no additional effects in H1781 cells. Greater down-regulation of COX-2, EGFR, pEGFR, Akt, pAkt, and PGE(2) was found when H3255 cells were treated with the combination compared with any of the single agents alone. CONCLUSIONS: The results of the current study demonstrate that the effectiveness of the addition of celecoxib to an EGFR-TKI is significantly greater in NSCLC cells with EGFR mutations, which is likely due to more complete inhibition of both pathways.     

Differential roles of trans-phosphorylated EGFR, HER2, HER3, and RET as heterodimerisation partners of MET in lung cancer with MET amplification

Background:

MET is a receptor tyrosine kinase (RTK) whose gene is amplified in various tumour types. We investigated the roles and mechanisms of RTK heterodimerisation in lung cancer with MET amplification.

Methods:

With the use of an RTK array, we identified phosphorylated RTKs in lung cancer cells with MET amplification. We examined the roles and mechanisms of action of these RTKs with immunoprecipitation, annexin V binding, and cell migration assays.

Results:

We identified epidermal growth factor receptor (EGFR), human EGFR (HER)2, HER3, and RET in addition to MET as highly phosphorylated RTKs in lung cancer cells with MET amplification. Immunoprecipitation revealed that EGFR, HER2, HER3, and RET each formed a heterodimer exclusively with MET and that these associations were markedly reduced in extent by treatment with a MET kinase inhibitor. RNA interference-mediated depletion of EGFR, HER2, or HER3 induced apoptosis in association with inhibition of AKT and ERK signalling pathways, whereas depletion of HER2 or RET inhibited both cell migration and STAT3 signalling.

Conclusion:

Our data suggest that heterodimers of MET with EGFR, HER2, HER3, or RET have differential roles in tumour development, and they provide new insight into the function of trans-phosphorylated RTKs as heterodimerisation partners of MET in lung cancer with MET amplification.