EGFR-mutant Lung Adenocarcinoma Cell Research Articles

Abstract 3361: EGFR signaling regulates aerobic glycolysis in EGFR-mutated lung adenocarcinoma

Abstract Recent accumulating evidences suggest that genetic mutations in tumor cells cause several unique metabolic phenotypes that are critical for cancerous cell proliferation. Mutations in the tyrosine kinase epidermal growth factor receptor (EGFR) induce oncogenic addiction in lung adenocarcinoma (LAD). However, the linkage between oncogenic mutated EGFR and cancer metabolism has not yet been clearly elucidated. In this study, we measured extracellular lactate production, glucose consumption and the glucose-induced extracellular acidification rate (ECAR) after the inhibition of EGFR signaling in LAD cell culture. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolomic techniques, the major intracellular metabolites in EGFR-mutated LAD cells were quantified in the absence or presence of the tyrosine kinase inhibitor (TKIs) erlotinib. The molecular profile regulating the metabolic enzymes at the transcriptional and post-translational levels was characterized by RT-PCR, Western blotting and flow cytometry. We demonstrated that EGFR signaling was required for lactate production, glucose consumption and glucose-induced ECAR, indicating that EGFR signaling promoted the Warburg effect in LAD cells. Moreover, comprehensive metabolomic analysis revealed that the levels of key intermediate metabolites in glycolysis and the pentose phosphate pathway (PPP) were decreased after treatment with EGFR inhibitors. The glucose transport carried out by GLUT1 and GLUT3 was the most rapid and critically regulated function of glucose metabolism linked to EGFR signaling, although the expression of MYC-dependent glycolytic genes and the phosphorylation of pyruvate kinase muscle (PKM2) were down-regulated by EGFR inhibition at a later time point. A modest down-regulation of both total and membrane-bound GLUT3 was also observed after EGFR inhibition. Despite an equivalent total amount of GLUT1 in LAD cells, the levels of membrane-bound GLUT1 was decreased by EGFR-TKI treatment, suggesting that EGFR signaling controlled GLUT1 function through post-translational modification. We concluded that EGFR signaling regulated aerobic glycolysis in EGFR-mutated LAD cells. Our data suggests that the glycolytic pathway plays a central role in the pathogenesis of EGFR-mutated lung cancer. This regulatory pathway could be an attractive target for the development of targeted therapies to better treat patients with EGFR-mutated LAD. Citation Format: Hideki Makinoshima, Masahiro Takita, Shingo Matsumoto, Atsushi Yagishita, Satoshi Owada, Hiroyasu Esumi, Katsuya Tsuchihara. EGFR signaling regulates aerobic glycolysis in EGFR-mutated lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3361. doi:10.1158/1538-7445.AM2014-3361

Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Global Metabolic Pathways in EGFR-mutated Lung Adenocarcinoma

Genetic mutations in tumor cells cause several unique metabolic phenotypes that are critical for cancer cell proliferation. Mutations in the tyrosine kinase epidermal growth factor receptor (EGFR) induce oncogenic addiction in lung adenocarcinoma (LAD). However, the linkage between oncogenic mutated EGFR and cancer cell metabolism has not yet been clearly elucidated. Here we show that EGFR signaling plays an important role in aerobic glycolysis in EGFR-mutated LAD cells. EGFR-tyrosine kinase inhibitors (TKIs) decreased lactate production, glucose consumption, and the glucose-induced extracellular acidification rate (ECAR), indicating that EGFR signaling maintained aerobic glycolysis in LAD cells. Metabolomic analysis revealed that metabolites in the glycolysis, pentose phosphate pathway (PPP), pyrimidine biosynthesis, and redox metabolism were significantly decreased after treatment of LAD cells with EGFRTKI. On a molecular basis, the glucose transport carried out by glucose transporter 3 (GLUT3) was downregulated in TKI-sensitive LAD cells. Moreover, EGFR signaling activated carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), which catalyzes the first step in de novo pyrimidine synthesis. We conclude that EGFR signaling regulates the global metabolic pathway in EGFR-mutated LAD cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, which is an attractive target for the development of more effective targeted therapies to treat patients with EGFR-mutated LAD.

SOX2 expression is an early event in a murine model of EGFR mutant lung cancer and promotes proliferation of a subset of EGFR mutant lung adenocarcinoma cell lines

ObjectivesPrimary and acquired resistance to EGFR TKIs in EGFR mutant lung cancer occurs primarily through secondary mutations in EGFR or Met amplification. Drug resistance can also be mediated by expression of pluripotency transcription factors, such as OCT4, SOX2 and NANOG that decrease terminal differentiation. In this study, we investigated the expression and role of SOX2 in model systems of EGFR mutant tumors. Materials and methodsImmunoblotting or immunohistochemistry was used to assess expression of pluripotency transcription factors in lungs of transgenic mice or in human NSCLC cell lines. Expression of SOX2 was reduced by shRNA knockdown, and response to erlotinib and cellular proliferation were assessed. Results and conclusionInduction of mutant EGFR in transgenic CCSP-rtTA/TetO-EGFRL858R/T790M mice correlated with increased OCT4 and SOX2 expression in lung tissue prior to tumor development. Established lung tumors retained SOX2 expression. To assess a role for SOX2 in tumorigenesis, a panel of NSCLC cell lines with activating EGFR mutations was assessed for SOX2 expression. Two of six cell lines with mutant EGFR showed detectable SOX2 levels, suggesting SOX2 expression did not correlate with EGFR mutation status. To assess the role of SOX2 in these cell lines, HCC827 and H1975 cells were infected with lentivirus containing SOX2 shRNA. Knockdown of SOX2 decreased proliferation in both cell lines and increased sensitivity to erlotinib in HCC827 cells. Because constitutive activation of the PI3K/Akt pathway is associated with EGFR TKI resistance, cells were treated with PI3K/AKT inhibitors and expression of SOX2 was examined. PI3K/Akt inhibitors decreased SOX2 expression in a time-dependent manner. These data suggest targeting SOX2 may provide therapeutic benefit in the subset of EGFR-mutant tumors with high constitutive levels of SOX2, and that until more direct means of inhibiting SOX2 are developed, PI3K/Akt inhibitors might be useful to inhibit SOX2 in EGFR TKI resistant tumors.

Enhanced autophagy is required for survival in EGFR-independent EGFR-mutant lung adenocarcinoma cells

Lung cancers harboring epidermal growth factor receptor (EGFR) mutations depend on constitutive activation of the kinase for survival. Although most EGFR-mutant lung cancers are sensitive to EGFR tyrosine kinase inhibitors (TKIs) and shrink in response to treatment, acquired resistance to TKI therapy is common. We demonstrate here that two EGFR-mutated lung adenocarcinoma cell lines, HCC827 and HCC4006, contain a subpopulation of cells that have undergone epithelial-to-mesenchymal transition and survive independent of activated EGFR. These EGFR-independent cancer cells, herein termed gefitinib-resistant (GR) cells, demonstrate higher levels of basal autophagy than their parental cells and thrive under hypoxic, reduced-serum conditions in vitro; this somewhat simulates the hypoxic environment common to cancerous tissues. We show that depletion of the essential autophagy gene, ATG5, by small interfering RNA (siRNA) or chloroquine, an autophagy inhibitor, markedly reduces GR cell viability under hypoxic conditions. Moreover, we show a significant elevation in caspase activity in GR cells following knockdown of ATG5. These results suggest that GR cells can evade apoptosis and survive in hostile, hypoxic environments with constant autophagic flux. We also show the presence of autophagosomes in some cancer cells from patient samples, even in untreated EGFR-mutant lung cancer tissue samples. Together, our results indicate that autophagy inhibitors alone or in combination with EGFR TKIs may be an effective approach for the treatment of EGFR-mutant lung cancers, where basal autophagy of some cancer cells is upregulated.

Antitumor Impact ofp14ARFon Gefitinib-Resistant Non–Small Cell Lung Cancers

Activation of the epidermal growth factor receptor (EGFR) has been observed in many malignant tumors and its constitutive signal transduction facilitates the proliferation of tumors. EGFR-tyrosine kinase inhibitors, such as gefitinib, are widely used as a molecular-targeting agent for the inactivation of EGFR signaling and show considerable therapeutic effect in non-small cell lung cancers harboring activating EGFR mutations. However, prolonged treatment inevitably produces tumors with additional gefitinib-resistant mutations in EGFR, which is a critical issue for current therapeutics. We aimed to characterize the distinct molecular response to gefitinib between the drug-resistant and drug-sensitive lung adenocarcinoma cells in order to learn about therapeutics based on the molecular information. From the quantitative PCR analysis, we found a specific increase in p14(ARF) expression in gefitinib-sensitive lung adenocarcinoma clones, which was absent in gefitinib-resistant clones. Moreover, mitochondria-targeted p14(ARF) triggered the most augmented apoptosis in both clones. We identified the amino acid residues spanning from 38 to 65 as a functional core of mitochondrial p14(ARF) (p14 38-65 a.a.), which reduced the mitochondrial membrane potential and caused caspase-9 activation. The synthesized peptide covering the p14 38-65 a.a. induced growth suppression of the gefitinib-resistant clones without affecting nonneoplastic cells. Notably, transduction of the minimized dose of the p14 38-65 peptide restored the response to gefitinib like that in the sensitive clones. These findings suggest that the region of p14(ARF) 38-65 a.a. is critical in the pharmacologic action of gefitinib against EGFR-mutated lung adenocarcinoma cells and has potential utility in the therapeutics of gefitinib-resistant cancers.

Abstract 4631: Pharmacologic inhibition of NF-kappaB overcomes de novo resistance to erlotinib in models of EGFR-mutant lung adenocarcinoma.

Abstract Treatment of patients with lung cancers that harbor activating mutations in the kinase domain of EGFR (e.g. L858R, in frame Exon19 deletion), which account for 15-20% of lung adenocarcinomas, with the EGFR tyrosine kinase inhibitor (TKI) erlotinib, causes tumor regression in many patients. However, the magnitude of tumor regression is variable and responses are short-lived with a median duration of 9-12 months. Thus inhibition of mutant EGFR activity by treatment with erlotinib results in incomplete responses and is not curative in the majority of EGFR-mutant lung cancer patients. While mechanisms of acquired resistance to EGFR TKI treatment are well defined, mechanisms of de novo resistance, or incomplete initial response to EGFR TKI therapy, remain uncharacterized. We recently identified activation of the NF-kB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) signaling pathway as a key mediator of de novo resistance to EGFR TKI therapy in cell lines and tumor xenograft models of lung cancer. Furthermore, patients with EGFR-mutant lung cancers that demonstrate elevated IkB (NF-kB inhibitor) expression, and consequently low NF-kB activity, exhibit improved responses to EGFR TKIs compared to patients with low IkB levels. NF-kB is a family of transcription factors that regulates numerous biological processes, including cell proliferation and apoptosis, as well as a critical mediator of both innate and adaptive immune cell activity. We hypothesized that pharmacologic inhibition of NF-kB activity would enhance the efficacy of EGFR TKI in EGFR-mutant lung cancer. Using a novel rel (reticuloendotheliosis) inhibitor of NF-kB, PBS-1086 (rel∼MD, Inc.), we found that treatment of human lung cancer cells and tumor xenografts that harbor an EGFRL858R activating mutation (11-18), yet are EGFR TKI resistant, with erlotinib in combination with PBS-1086 induces tumor cell apoptosis and tumor regression. Erlotinib + PBS-1086 treatment results in decreased binding of the NF-kB subunit RelA to the IL6 promoter and a significant decrease in IL6 mRNA expression. IL6 has previously been shown to promote EGFR-mutant lung adenocarcinoma cell proliferation and survival, and its downregulation by PBS-1086 represents a possible mechanism of action of the compound. Using CC10-rtTA; Tet-Op-EGFR-mutant transgenic mouse models of lung adenocarcinoma, we have shown that treatment of mice with PBS-1086 enhances erlotinib-induced tumor regression. We believe that these results will guide future clinical trials of NF-kB inhibitors in combination with erlotinib to improve outcomes in selected EGFR-mutant lung cancer patients. Citation Format: Collin M. Blakely, Victor Olivas, Jie Zhang, Trever G. Bivona. Pharmacologic inhibition of NF-kappaB overcomes de novo resistance to erlotinib in models of EGFR-mutant lung adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4631. doi:10.1158/1538-7445.AM2013-4631

NF-κB signaling is activated and confers resistance to apoptosis in three-dimensionally cultured EGFR-mutant lung adenocarcinoma cells

Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells in suspension undergo apoptosis to a greater extent than adherent cells in a monolayer when EGFR autophosphorylation is inhibited by EGFR tyrosine kinase inhibitors (TKIs). This suggests that cell adhesion to a culture dish may activate an anti-apoptotic signaling pathway other than the EGFR pathway. Since the microenvironment of cells cultured in a monolayer are substantially different to that of cells existing in three-dimension (3D) in vivo, we assessed whether two EGFR-mutant lung adenocarcinoma cell lines, HCC827 and H1975, were more resistant to EGFR TKI-induced apoptosis when cultured in a 3D extracellular matrix (ECM) as compared with in suspension. The ECM-adherent EGFR-mutant cells in 3D were significantly less sensitive to treatment with WZ4002, an EGFR TKI, than the suspended cells. Further, a marked degradation of IκBα, the inhibitor of nuclear factor (NF)-κB, was observed only in the 3D-cultured cells, leading to an increase in the activation of NF-κB. Moreover, the inhibition of NF-κB with pharmacological inhibitors enhanced EGFR TKI-induced apoptosis in 3D-cultured EGFR-mutant cells. These results suggest that inhibition of NF-κB signaling would render ECM-adherent EGFR-mutant lung adenocarcinoma cells in vivo more susceptible to EGFR TKI-induced cell death.

Abstract 1891: NF-κB signaling plays a certain role in apoptosis resistance in 3D-cultured EGFR-mutant lung adenocarcinoma cells

Abstract It is generally thought that suspended tumor cells show reduced proliferative activity and are more resistant to conventional chemotherapy compared with adherent tumor cells. Two epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cell lines, HCC827 (E746-A750 deletion) and H1975 (L858R + T790M), cultured in suspension, however, undergo apoptosis much more easily than these two cell lines cultured in conventional monolayer dishes, i.e. anchorage dependent conditions, when the kinase activity of EGFR is completely inhibited by treatment with EGFR tyrosine kinase inhibitors (TKIs) regardless of suspension or monolayer culture conditions. These findings indicate that attached EGFR-mutant lung adenocarcinoma cells appear less dependent on constitutive EGFR signaling for survival than detached cells, probably because integrin ligation enhances cell survival in adherent cells. However, the microenvironment of cells in monolayer culture conditions is considerably different from that of cells in vivo. We thus tested whether EGFR-mutated lung adenocarcinoma cells in three dimensional (3D) conditions using Matrigel resist EGFR TKIs-induced apoptosis compared with suspended cells. We uncovered three major findings in this study as follows: 1) both HCC827 cells and H1975 cells cultured in 3D conditions were significantly less sensitive to EGFR TKIs-induced apoptosis than suspended cells. 2) Marked degradation of IαBβ, the inhibitor of NF-κB pathway, was observed only in 3D-cultured EGFR-mutated lung adenocarcinoma cells, resulting in NF-κB signaling activation. 3) TPCA-1, a potent IKKα inhibitor, can enhance the levels of apoptosis in 3D-cultured EGFR-mutant cells treated with EGFR TKIs. These results suggest that combined EGFR and NF-κB inhibition is required to induce apoptosis effectively in EGFR-mutated lung adenocarcinoma cells in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1891. doi:1538-7445.AM2012-1891

Abstract 129: miR-145 reduces tumor growth by regulating EGFR in human lung adenocarcinoma but not in Erlotinib-resistant cells

Abstract OBJECTIVE: Recent studies suggested that lung adenocarcinomas in never-smokers have characteristics distinct from those in smokers. miRNAs are important modulators in cellular pathways. In this study, we investigate the expression profile of miRNAs in lung adenocarcinomas from nonsmokers and identify their target genes. Our in vitro analyses also assess the miRNA expression associated with erlotinib therapy. METHODS: Lung adenocarcinoma and adjacent normal lung parenchyma were obtained from nonsmokers. Microarray analysis was preformed and the profiling results were validated by qRT-PCR. Transfected cell viability assays were applied to determine the effects of candidate miRNAs on lung cancer cells. Systemic bioinformatic analysis was performed to investigate the potential targets of miRNA in lung cancer. The associations of miRNA and its targeted genes’ expressions were analyzed by miRNA and mRNA microarray profiling in tumor specimens from patients with lung adenocarcinoma. Expressions of miRNA and its gene targets in lung cancer cells were determined by qRT-PCR and validated by Western blot analysis. RESULTS: Comparing paired lung adenocarcinoma tissue with corresponding noncancerous tissues, miR-126*, -145, -21, -182, -183, and -210 were found to be the most differentially expressed miRNAs. An obvious inhibition of cell growth was observed in the EGFR mutant lung adenocarcinoma cells after transfection of pre-miR-145. Our study further identified EGFR and NUTD1 as potential targets of miR-145. The mRNA expressions of EGFR and NUDT1 were significantly downregulated by 60% and 50% respectively after miR-145 transfection. The enforced expression of miR-145 in three lung adenocarcinoma cell lines led to a decrease (2.5-, 5-, and 2-fold) of EGFR protein and the abolishing of NUDT1 protein expressions. In vitro analysis showed that miR-145 can gradually inhibit cell proliferation on transfected lung adenocarcinoma cells over three time points (24, 48, and 72 hours). It was also noteworthy that miR-145 expression and EGFR status correlated with sensitivity to erlotinib. The 3 cell lines treated with erlotinib showing increased expression levels of miR-145 were all mutant EGFR, but not in the 4 lung cancer cell lines with wild-type EGFR or T790M point mutation. DISCUSSION: This study has identified several miRNAs that may play a role in carcinogenesis of lung adenocarcinomas in nonsmokers. Our results also show that restoration of miR-145 inhibits cancer cell growth in EGFR mutant lung adenocarcinoma by targeting EGFR and NUDT1. We further reveal that miR-145 may reduce tumor growth by regulating EGFR in human lung adenocarcinoma but not in erlotinib-resistant cells. Our findings provide new insight into the complex regulating pathway comprising miR-145 and EGFR. Understanding this important pathway may lead to new therapeutic strategies for lung adenocarcinoma in nonsmokers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 129. doi:1538-7445.AM2012-129

WZ4002, a third-generation EGFR inhibitor, can overcome anoikis resistance in EGFR-mutant lung adenocarcinomas more efficiently than Src inhibitors

Src has a role in the anoikis resistance in lung adenocarcinomas. We focused on two epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cell lines, HCC827 (E746-A750 deletion) and H1975 (L858R+T790M), in suspension to elucidate whether suspended lung adenocarcinoma cells are eradicated by long-term treatment with Src tyrosine kinase inhibitors (TKIs). We also examined metastasis-positive lymph nodes from 16 EGFR-mutant lung adenocarcinoma patients for immunohistochemical expression of mutant-specific EGFR. Almost all suspended HCC827 cells underwent apoptosis after 144 h of combination treatment with AZD0530, trichostatin A (TSA), and ABT-263, whereas many suspended H1975 cells survived the treatment. AZD0530 is a Src TKI, TSA is a histone deacetylase inhibitor, and ABT-263 is a Bcl-2 inhibitor. During the therapy, the phosphorylation of EGFR decreased in HCC827 cells and remained stable in H1975 cells. The phosphorylated EGFR of Src TKI-resistant H1975 cells, as well as HCC827 cells, was completely suppressed by the third generation EGFR TKI, WZ4002. Consequently, both the suspended cell lines were almost completely eradicated within 144 h, with the combined therapy of WZ4002, ABT-263, and TSA. Interestingly, treated suspended cells underwent apoptosis to a greater extent than did adherent cells. Intrasinus floating lung adenocarcinoma cells in the lymph nodes expressed a mutant-specific EGFR. These findings suggest that suspended EGFR-mutant lung adenocarcinoma cells depend significantly more on EGFR activation for survival than attached cells do. The tumor cells circulating in vessels, which express mutant-specific EGFR, would be highly susceptible to the combination therapy of WZ4002, ABT-263, and TSA.

High Expression Levels of Total IGF-1R and Sensitivity of NSCLC Cells In Vitro to an Anti-IGF-1R Antibody (R1507)

BackgroundThe IGF receptor type 1 (IGF-1R) pathway is frequently deregulated in human tumors and has become a target of interest for anti-cancer therapy.Methodology/Principal FindingsWe used a panel of 22 non-small cell lung cancer (NSCLC) cell lines to investigate predictive biomarkers of response to R1507, a fully-humanized anti-IGF-1R monoclonal antibody (Ab; Roche). 5 lines were moderately sensitive (25–50% growth inhibition) to R1507 alone. While levels of phospho-IGF-1R did not correlate with drug sensitivity, 4 out of 5 sensitive lines displayed high levels of total IGF-1R versus 1 out of 17 resistant lines (p = 0.003, Fisher's Exact). Sensitive lines also harbored higher copy numbers of IGF-1R as assessed by independent SNP array analysis. Addition of erlotinib or paclitaxel to R1507 led to further growth inhibition in sensitive but not resistant lines. In one EGFR mutant lung adenocarcinoma cell line (11–18), R1507 and erlotinib co-treatment induced apoptosis, whereas treatment with either drug alone induced only cell cycle arrest. Apoptosis was mediated, in part, by the survival-related AKT pathway. Additionally, immunohistochemical (IHC) staining of total IGF-1R with an anti-total IGF-1R Ab (G11;Ventana) was performed on tissue microarrays (TMAs) containing 270 independent NSCLC tumor samples. Staining intensity was scored on a scale of 0 to 3+. 39.3% of tumors showed medium to high IGF-1R IHC staining (scores of 2+ or 3+, respectively), while 16.7% had scores of 3+.Conclusions/SignificanceIn NSCLC cell lines, high levels of total IGF-1R are associated with moderate sensitivity to R1507. These results suggest a possible enrichment strategy for clinical trials with anti-IGF-1R therapy.

Expression levels of total IGF-1R and sensitivity of NSCLC cells in vitro to an anti-IGF-1R antibody (R1507)

8095 Background: The IGF-1R (IGF receptor type 1) pathway is frequently deregulated in human tumors and has become a target of interest for anti-cancer therapy. We investigated predictive biomarkers of response to an anti-IGF-1R antibody (Ab) in vitro in NSCLC. Methods: We examined the growth inhibitory effects of R1507, a fully-humanized IgG1 anti-IGF-1R monoclonal Ab (Roche), against a panel of 22 NSCLC cell lines using CellTiter Blue assays. Phospho-receptor tyrosine kinase (pRTK) arrays and ELISAs were used to determine the status of IGF-1R and other RTKs. SNP arrays were used to determine IGF-1R copy number. Immunohistochemical (IHC) staining of total IGF-1R was performed with G11 (an anti-total IGF-1R Ab; Ventana) on a tissue microarray (TMA) containing 77 independent NSCLC tumor samples. Staining intensity was scored on a scale of 0 to 3+ by a pathologist (JF). Results: 5 of 22 NSCLC cell lines were moderately sensitive (25–50% growth inhibition) to R1507 alone. ELISA and pRTK array analysis demonstrated that pIGF-1R levels in the presence or absence of serum did not correlate with drug sensitivity. However, 4 of 5 sensitive lines displayed high levels of total IGF-1R vs 1/17 resistant lines (p=0.003 Fisher's Exact). SNP array analysis showed that sensitive lines also harbor relatively higher copy numbers of IGF-1R. There was no correlation with EGFR/KRAS mutational status. 48% of TMA NSCLC tumors had scores of 2+ or greater, while 5% were scored as 3+. Addition of erlotinib or paclitaxel to R1507 led to further growth inhibition in sensitive but not resistant lines. In one EGFR mutant lung adenocarcinoma cell line, R1507 and erlotinib co-treatment induced apoptosis, whereas treatment with either drug alone induced only cell cycle arrest. Apoptosis was mediated, in part, by the survival-related AKT pathway, as pAKT was significantly downregulated by R1507 but not erlotinib. Conclusions: In NSCLC cell lines, high levels of total IGF-1R are associated with moderate sensitivity to R1507. These results suggest a possible enrichment strategy for clinical trials with anti-IGF-1R therapy. [Table: see text]

MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib.

In human lung adenocarcinomas harboring EGFR mutations, a second-site point mutation that substitutes methionine for threonine at position 790 (T790M) is associated with approximately half of cases of acquired resistance to the EGFR kinase inhibitors, gefitinib and erlotinib. To identify other potential mechanisms that contribute to disease progression, we used array-based comparative genomic hybridization (aCGH) to compare genomic profiles of EGFR mutant tumors from untreated patients with those from patients with acquired resistance. Among three loci demonstrating recurrent copy number alterations (CNAs) specific to the acquired resistance set, one contained the MET proto-oncogene. Collectively, analysis of tumor samples from multiple independent patient cohorts revealed that MET was amplified in tumors from 9 of 43 (21%) patients with acquired resistance but in only two tumors from 62 untreated patients (3%) (P = 0.007, Fisher's Exact test). Among 10 resistant tumors from the nine patients with MET amplification, 4 also harbored the EGFR(T790M) mutation. We also found that an existing EGFR mutant lung adenocarcinoma cell line, NCI-H820, harbors MET amplification in addition to a drug-sensitive EGFR mutation and the T790M change. Growth inhibition studies demonstrate that these cells are resistant to both erlotinib and an irreversible EGFR inhibitor (CL-387,785) but sensitive to a multikinase inhibitor (XL880) with potent activity against MET. Taken together, these data suggest that MET amplification occurs independently of EGFR(T790M) mutations and that MET may be a clinically relevant therapeutic target for some patients with acquired resistance to gefitinib or erlotinib.