Gefitinib-sensitive Cell Line Research Articles

PPARα agonist fenofibrate relieves acquired resistance to gefitinib in non-small cell lung cancer by promoting apoptosis via PPARα/AMPK/AKT/FoxO1 pathway.

Recent studies show that intracellular accumulation of cholesterol leads to acquired resistance to gefitinib in non-small cell lung cancer (NSCLC) cells. In this study we investigated how to regulate the cholesterol levels in gefitinib-resistant NSCLC cells. We showed that intracellular cholesterol levels in gefitinib-resistant cell lines (PC-9/GR, H1975, H1650, and A549) were significantly higher than that in gefitinib-sensitive cell line (PC-9). Treatment with gefitinib (5 μM) significantly increased intracellular cholesterol levels in PC-9/GR, H1975, and H1650 cells. Gefitinib treatment downregulated the expression of PPARα, LXRα, and ABCA1, leading to dysregulation of cholesterol efflux pathway. We found that a lipid-lowering drug fenofibrate (20, 40 μM) dose-dependently increased the expression of PPARα, LXRα, and ABCA1, decreased the intracellular cholesterol levels, and enhanced the antiproliferative effects of gefitinib in PC-9/GR, H1975, and H1650 cells. We revealed that fenofibrate increased the gefitinib-induced apoptosis via regulating the key proteins involved in the intrinsic apoptosis pathway. In PC-9/GR, H1975 and H1650 cells, fenofibrate dose-dependently increased the expression of AMPK, FoxO1, and decreased the expression of AKT, which were remarkably weakened by knockdown of PPARα. In PC-9/GR cell xenograft mice, combined administration of gefitinib (25 mg · kg-1 · d-1) and fenofibrate (100 mg · kg-1 · d-1) caused remarkable inhibition on tumor growth as compared to treatment with either drug alone. All the results suggest that fenofibrate relieves acquired resistance to gefitinib in NSCLC by promoting apoptosis via regulating PPARα/AMPK/AKT/FoxO1 pathway. We propose that combination of gefitinib and fenofibrate is a potential strategy for overcoming the gefitinib resistance in NSCLC.

Identification of Hub Genes and Small Molecule Drugs Associated with Acquired Resistance to Gefitinib in Non-Small Cell Lung Cancer.

Targeting EGFR, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs), brings lights to the treatment of non-small cell lung cancer (NSCLC). Although T790M mutation responded as one of the main reasons of acquired resistance, still 15% of the resistance patients can't be explained by the known mechanisms. The purpose of this research was to identify some new mechanisms of gefitinib acquired resistance, and to predict small molecules drugs which may reverse drug resistance by integrated bioinformatics analysis. The GSE34228 data package containing the microarray data of acquired gefitinib-resistant cell line (PC9GR) and gefitinib-sensitive cell line (PC9) from the GEO database were downloaded, and gene co-expression networks by weighted gene co-expression network analysis (WGCNA) were constructed to identified key modules and key genes related to gefitinib resistance. Furthermore, the significantly differentially expressed genes (DEGs) between the two cell types were screened out, and a protein-protein interaction (PPI) network to obtain the key genes of DEGs was accordingly constructed. Through the above two methods, 4 hub genes, PI3, S100A8, AXL and PNPLA4 were mined as the most relevant to gefitinib resistance. Among them, PI3, S100A8 were down-regulated in PC9GR cell samples, while AXL, PNPLA4 were up-regulated. The gene set enrichment analysis (GSEA) for single gene showed that the four hub genes were mainly correlated with cell proliferation and cycle. Besides, small molecule drugs with the potential to overcome resistance, such as Emetine and cephaeline, were screened by CMap database. Consistent with this, in vitro experiments results have shown that emetine and cephaeline can increase the sensitivity of drug-resistant cells to gefitinib, and the mechanism may be related to the regulation of PI3 and S100A8. In conclusion, 4 hub genes were found to be related to the occurrence of gefitinib resistance in non-small cell lung cancer, and several small molecule drugs were screened out as potential therapeutic agents to overcome gefitinib resistance, which may lead a new way for the treatment of NSCLC of acquired resistance to gefitinib.

Cancer exosomal microRNAs from gefitinib-resistant lung cancer cells cause therapeutic resistance in gefitinib-sensitive cells.

Exosomes and their cargo microRNAs play a significant role in various biological processes in cancer. We hypothesized that microRNAs in exosomes secreted by gefitinib-resistant lung cancer cells might induce resistant phenotypes in otherwise gefitinib-sensitive lung cancer cells. We isolated exosomes generated by the gefitinib-resistant human lung adenocarcinoma cell line PS-9/ZD. PC-9, which is a gefitinib-sensitive cell line, was treated with the PC-9/ZD exosomes, and these PC-9 cells were analyzed for cell proliferation after treatment with gefitinib. miRNA arrays were analyzed in PC-9 and PC-9/ZD cells, and we isolated microRNAs that were expressed at elevated levels in PC-9/ZD cells. Furthermore, we transfected these microRNAs into PC-9 cells and analyzed the effects on the cells' sensitivity to gefitinib. Exosomes isolated from PC-9/ZD cells significantly increased the proliferation of PC-9 cells during gefitinib treatment. A microRNA array analysis showed that miR-564, miR-658, miR-3652, miR-3126-5p, miR-3682-3p and miR-6810-5p were significantly upregulated in PC-9/ZD cells. PC-9 cells transfected with miR-564 or miR-658 showed chemo-resistant phenotypes. Exosomal miR-564 and miR-658 derived from gefitinib-resistant lung cancer cells induce drug resistance in sensitive cells. Cell-to-cell interaction via exosomal microRNAs may be a novel mechanism and therapeutic target of resistance against gefitinib.

Abstract 4755: Clathrin mediated endocytosis as a potential therapeutic strategy for overcoming refractoriness of EGFR TKI

Abstract Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) have significantly improved on clinical outcomes in many non-small cell lung cancer (NSCLC) patients. However, therapeutic efficacy of EGFR TKIs is limited on patients who have gefitinib-sensitizing EGFR gene mutations (Exon 19del, L858R). Although, not as sensitive as patients who have these mutations, some patients who have wild type EGFR responded to EGFR TKIs. Therefore, we hypothesized that there are underlying mechanisms that cause most of wild type EGFR NSCLC patients to be refractory. In this study, we demonstrated that intractability of EGFR TKIs in wild type EGFR NSCLC was closely related to clathrin mediated endocytosis (CME), which offered a novel strategy for overcoming EGFR TKI resistance. At first, we classified wild type EGFR NSCLC cell lines into gefitinib sensitive cells and refractory cells according to the IC50 values of gefitinib. Then, we performed immunocytochemistry (ICC) and immunoprecipitation (IP) to examine EGFR levels in early endosome with gefitinib treatment in gefitinib sensitive and refractory cell lines. As a result, EGFR endocytosis proceeded in gefitinib refractory cell lines (SNU1327, H1703) despite of gefitinib treatment, but not in sensitive cell lines (H358, Calu-3). EGFR is known to be internalized through CME and non clathrin mediated pathway (NCE). Then, we treated CME inhibitor, Phenylarsine oxide (PAO), or NCE inhibitor, Filipin III, in gefitinib sensitive and refractory cell lines to vefity if there is any difference in dependency of two main endocytosis pathway. As the ICC and IP data shown, blocking NCE did not inhibit EGFR internalization but blocking CME inhibited EGFR endocytosis in gefitinib refractory cell lines. Interestingly, it was the opposite in the gefitinib sensitive cell lines. Furthermore, we observed that cell survival decreased significantly during CME inhibition and combination treatment with gefitinib in refractory cell lines, but not in sensitive cell lines. To enlighten the elusive pathway, we examined the ratio of EGFR recycling and degradation. As a result, we observed EGFR degradation during CME or NCE proceeded conditions in gefitinib sensitive cell lines. However, in gefitinib refractory cell lines, EGFR protein was accumulated in CME dependent manner. These results implied that CME led EGFR recycling and sustained gefitinib refractory cell lines. For further studies, we are confirming signal molecules activated through CME. In conclusion, our study provides the possibility of new scheme that clathrin-mediated endocytosis inhibition could be a potential therapeutic strategy for the limitation of EGFR TKI therapy. This study was supported by a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2013R1A1A2057538). Citation Format: Boyeon Kim, Young Soo Park, Jae Sook Sung, Jong Won Lee, Saet Byeol Lee, Yeul Hong Kim. Clathrin mediated endocytosis as a potential therapeutic strategy for overcoming refractoriness of EGFR TKI [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4755.

Abstract 384: Identification of a lung adenocarcinoma cell line addicted to ERBB4, an actionable target for cancer therapy

Abstract Cancer cells often survive by getting addicted to specific genes. In most cases, such genes function as an enzyme, especially kinases that are specifically activated in cancers by genetic aberrations; e.g., BCR-ABL fusion kinase found in chronic myelogenous leukemias and epidermal growth factor receptor (EGFR) with activating mutations found in non-small cell lung cancers (NSCLCs). These activated kinases would be an ideal target for cancer therapy and indeed imatinib and gefitinib displayed good clinical benefits to the patients in these diseases. However, only a limited population of cancer cells harbor such well-characterized driver genes which could be attacked by targeted drugs developed so far and there remain unmet medical needs to identify novel therapeutic targets and drugs for the rest of cancers. To this end, we exploited our original database of drug efficacy of >100 kinase inhibitors across a panel of 39 human cancer cell lines (JFCR39) to characterize each of the cell line in terms of susceptibility to these kinase inhibitors. Expectedly, cell lines harboring PIK3CA hotspot mutation and PTEN mutation were susceptible to PI3K and mTOR inhibitors, whereas those harboring KRAS or BRAF mutation to MEK inhibitors, respectively. Of note, we found four of the cells responding to a series of EGFR family tyrosine kinase inhibitors (TKIs) including gefitinib and lapatinib. Gefitinib exert selective effect on NSCLCs addicted to activated EGFR mutant; however, none of the four gefitinib-sensitive cell lines possessed such EGFR mutation. Of these, an NSCLC cell line NCI-H522 hardly expressed EGFR but overexpressed ERBB4 instead, while an ovarian cancer cell line SK-OV3 overexpressed ERBB2 as reported previously. Actually, treatment with a certain TKI in this class efficiently dephosphorylated ERBB4 in NCI-H522 along with inhibition of cell growth and suppression of downstream factor ribosomal S6 protein. Moreover, knockdown of ERBB family kinases revealed that cell growth was dependent on overexpressed ERBB4 in NCI-H522 cells. These results indicate that NCI-H522 cells exhibited susceptibility to EGFR family TKIs via inhibition of ERBB4. Of note, NCI-H522 cells exhibited hypersensitivity to three of these TKIs, sapitinib, PF-7775 and GW583340, as compared to gefitinib. Especially, GW583340 suppressed growth of NCI-H522 cells at the same or lower concentration than in HCC827 cells harboring an EGFR active mutation. Consistent with this observation, in vitro kinase assay revealed that sapitinib and PF-7775 exhibited ~10-fold higher ERBB4-inhibitory effect than gefitinib did while GW583340 displayed selective inhibition on ERBB4, with ~10-fold higher activity than that on activated EGFR. In conclusion, we identified NCI-H522 as an ERBB4-dependent cell line, and found ERBB4 as a novel therapeutic cancer target that could be attacked by ERBB4-directing TKIs. Citation Format: Noritaka Tanaka, Kanami Yamazaki, Yuko Uno, Yoshimi Ohashi, Yumiko Nishimura, Masaaki Sawa, Shingo Dan. Identification of a lung adenocarcinoma cell line addicted to ERBB4, an actionable target for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 384.

High cholesterol in lipid rafts reduces the sensitivity to EGFR-TKI therapy in non-small cell lung cancer.

Overcoming EGFR-TKI resistant which has the initial enthusiasm over substantial clinical responses is a formidable challenge on nowadays. In this study, we showed that cholesterol level in lipid rafts in gefitinib resistant non-small cell lung cancer (NSCLC) cell lines was remarkably higher than gefitinib sensitive cell line, and depletion of cholesterol increased gefitinib sensitivity. Furthermore, cholesterol-depleted enhanced gefitinib inhibit phosphorylation of EGFR, Akt-1, MEK1/2, and ERK1/2 and these were reversed in cholesterol add-back experiments. Gefitinib resistant cell lines showed high affinity of gefitinib and EGFR when cholesterol was depleted. Therefore, targeting cholesterol combined with EGFR-TKI is potentially a novel therapeutic strategy for gefitinib resistant treatment.

MiR-19a contributes to gefitinib resistance and epithelial mesenchymal transition in non-small cell lung cancer cells by targeting c-Met

Gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is used as a first-line treatment for advanced non-small cell lung cancer (NSCLC). However, most NSCLC patients inevitably develop gefitinib resistance, and the mechanisms underlying this resistance are not fully understood. In this study, we show that miR-19a is significantly down-regulated in gefitinib-resistant NSCLC cell lines compared with gefitinib-sensitive cell lines. In addition, the down-regulation of miR-19a suppressed the expression of epithelial markers but induced the expression levels of mesenchymal markers. A mechanistic analysis revealed that miR-19a regulated c-Met expression by directly targeting the c-Met 3′UTR. Overexpression of miR-19a decreased c-Met expression and re-sensitized gefitinib-resistant NSCLC cells in vitro and in vivo. Consistent with the in vitro findings, the miR-19a serum level was significantly decreased in NSCLC patients with acquired gefitinib resistance compared with the level observed prior to the acquisition of resistance in each patient, indicating that miR-19a expression may be a valuable biomarker for the prediction of acquired gefitinib resistance in a clinical setting. Our data demonstrate that the miR-19a/c-Met pathway plays a critical role in acquired resistance to gefitinib and that the manipulation of miR-19a might provide a therapeutic strategy for overcoming acquired gefitinib resistance.

Dihydroartemisinin enhances the sensitivity of gefitinib in non-small cell lung cancer cells by inhibiting STAT3

Lung cancer is the most frequent cause of cancer death, and non-small cell lung cancer (NSCLC) is the most common subtype. EGFR (epidermal growth factor receptor) mutations are found in about 50% of Asian patients with lung adenocarcinoma and confer sensitivity to EGFR tyrosine kinase inhibitor (EGFR-TKI). Progression free survival with TKI treatment is 9 to 12 months, and median survival time is about 20–30 months. However, acquired resistance to TKI unfailingly occurs, in most cases due to the acquisition of secondary mutations in the EGFR . Many studies have shown that bypass pathway activation ( c-MET or Her-2 amplification) and the activation of the downstream pathways (PI3K/AKT, PTEN, ERK/MAPK, STAT3) can lead to acquired resistance to EGFR-TKI. In addition, transformation from NSCLC to SCLC or conferred epithelial to mesenchymal transition has also been identified as mechanisms of acquired resistance to EGFR-TKI. The need to overcome both innate and acquired resistance has been a major therapeutic challenge. Our research group aim to explore the natural compounds or their derivatives which can overcome the acquired resistance to EGFR-TKI from the library of traditional Chinese medicine monomer and related derivatives. Fortunately, we found that dihydroartemisinin (DHA) had a synergistic inhibition effect with gefitinib (first-generation EGFR-TKI) in non-small cell lung cancer cells. The combined index (CI) was 0.751 in gefitinib-sensitive cell line PC-9 cells and 0.766 in gefitinib-resistant cell line H1975 cells, respectively. DHA is a semi-synthetic derivative and main active metabolite of the artemisinin, a natural product isolated from a Chinese medicinal herb ( Artemisia annua Linn.). It is one of first-line antimalarial drugs. Moreover, DHA has been shown to exert antibacterial, antiviral and therapeutic effects on lupus erythematosus. In addition to these efficacies, evidence from epidemiological, pharmacological and case control studies has suggested that DHA possesses antitumor activity and selective cytotoxicity to various malignancies. Notably, its low toxicity to host is the major incentive for developing the compound as an anticancer agent. Our present study also investigated the mechanisms by which DHA repressed cell growth in NSCLC cells, alone or in combination with EGFR-TKI. We found that DHA could inhibit phosphorylation of STAT3 (Tyr705) in non-small cell lung cancer cells. When combined with gefitinib, DHA also could inhibit gefitinib-activated STAT3 and does not affect its TKI activity. Many studies have shown that STAT3 is persistently tyrosine-phosphorylated or activated in NSCLC (cell lines and primary tumors), leading to acquired resistance to EGFR-TKI. In summary, DHA can be used to overcome acquired resistance to TKIs in EGFR-mutant lung adenocarcinoma due to its STAT3 inhibitory activity and low toxicity.

Abstract 5551: Anti-tumorigenic function of polyphenolic compounds on gefitinib resistant lung cancer

Abstract EGFR-mutant non-small cell lung cancer (NSCLC) is initially very sensitive to EGFR tyrosine kinase inhibitors (TKIs). Even with the remarkable effect of EGFR TKIs, patients eventually acquire resistance to gefitinib or elortinib, leading to a clinical challenge for overcoming TKI resistance. Some polyphenolic compounds are known as potential anti-tumorigenic compounds, such as resveratrol, curcumin or EGCG. However the function of these natural compounds in resistant NSCLC remains to be elucidated. Here, we identified a polyphenolic compound (named as compound A) from screening a set of natural compounds for anti-cancer effects on NSCLC. We found that compound A is a potential anticancer agent both in vivo and in vitro. Moreover, the growth inhibition of compound A turns out to be selective for cancer cells. Next, we further evaluated the growth inhibitory effect of compound A in two sets of NSCLC panels which are either sensitive or resistant to gefitinib treatment. Interestingly, the cell lines which are resistant to gefitinib are highly sensitive to compound A treatment, whereas the gefitinib-sensitive (GS) cell lines are not responsive to the compound A treatment. In gefitinib resistant (GR) cells, compound A increases expression of autophagy markers, p62 and LC3, which might result in the growth inhibition of the GR panel of cells but not in the GS cell panel. Moreover, we profiled the metabolic genes in the GR and GS cell lines. Surprisingly, compound A treatment significantly reduces the expression of a subset of enzymes associated with Warburg effect, which includes GLUT1, LDHA, PDK1, HK1, and HK2. As a result, compound A decreases Warburg effect, subsequently suppressing cell proliferation, especially of the GR cell lines. Taken together, compound A decreases Warburg effect and up-regulates autophagy process that may contribute to tumor suppression function of compound A in gefitinib-resistant cancer. Citation Format: Ai N. H. Phan, Yangsik Jeong. Anti-tumorigenic function of polyphenolic compounds on gefitinib resistant lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5551. doi:10.1158/1538-7445.AM2015-5551

Gefitinib-Resistance Is Related to BIM Expression in Non-Small Cell Lung Cancer Cell Lines

Recent evidence indicates that both the phosphatidylinositol 3-kinase (PI3K)/AKT and the MEK/ERK pathways are strictly regulated by epidermal growth factor receptor in non-small cell lung cancer (NSCLC) that responds to Gefitinib. Gefitinib resistance is partly owing to the activation of two major downstream signaling pathways PI3K/AKT or MEK/ERK. In this study, we found that in Gefitinib-sensitive cell lines, Gefitinib could induce tumor cell apoptosis via upregulation of a proapoptotic protein BIM. Small interfering RNA results showed that silencing of BIM could alleviate apoptosis induced by Gefitinib. We adopted a combination of PI3K inhibitor (LY294002) and MEK inhibitor (U0126) against Gefitinib resistance in cell lines. As expected, the combination substantially induced apoptosis and restored the sensitivity to Gefitinib by increasing the expression of BIM. Our studies provided a theoretical basis for overcoming drug resistance in NSCLC via combination therapy.

Abstract 625: Transcription factor slug conferring resistance To EGFR tyrosine kinase inhibitor gefitinib

Abstract Purpose: Somatic EGFR mutations of non-small cell lung cancer (NSCLC) are reported to be associated with clinical responsiveness to tyrosine kinase inhibitor gefitinib. Despite the dramatic responses to gefitinib, most patients ultimately have a relapse. The underlying mechanism of the drug resistance is not well clear. Recently, many studies have shown that epithelial to mesenchymal transition (EMT) is involved in resistance to gefitinib in EGFR. Zinc finger transcription factor slug is one of the EMT regulators. In this study, we will investigate whether the slug plays a role in the gefitinib resistance. Materials and Methods: PC9/IR (gefitinib-resistant cell line) was isolated from PC9 (gefitinib-sensitive cell line) after long-term treatment of gefitinib. To identify the factors involved in gefitinib resistance, we analyzed the expression levels of EMT regulators (Slug, Snail, Twist, Zeb-1) in PC9/IR and PC9 cells using quantitative real time PCR and western blot. To investigate the association of Slug and gefitinib-induced resistance, we also engineered to stably express Slug and short hairpin RNA targeting Slug transcript in PC9 and PC9/IR, respectively. Results: The findings showed that mRNA and protein levels of Slug obviously increased in PC9/IR comparing with PC9 and expression of other EMT regulators Snail, Twist, Zeb-1 were similar between these two cell lines. After gefitinib treatment, PC9/IR expressing shRNA against Slug underwent apoptosis and activation of the intrinsic apoptosis pathway. Ectopic expression of Slug protected PC9 cells from undergoing apoptosis in response to gefitinib treatment. Knockdown the expression of slug reversed gefitinib resistance of PC9/IR cells by restoring Bim expression. Conclusions: These results suggest that expression of slug may contribute to resistance to gefitinib in PC9/IR and may be a useful target for treatment of resistance to EGFR tyrosine kinase inhibitor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 625.

PTEN inactivation in lung cancer cells and the effect of its recovery on treatment with epidermal growth factor receptor tyrosine kinase inhibitors

To understand the mechanisms of PTEN inactivation, which is reported to be involved in tumor progression and drug resistance in lung cancer, we analyzed the expression levels of PTEN at mRNA and protein levels, along with the genetic and epigenetic status of the PTEN gene, in a panel of lung cancer cell lines. Western blot analysis showed that six out of 25 (24%) cell lines displayed low expression of PTEN protein. The level of PTEN mRNA correlated well with corresponding protein expression in each of these six cell lines. In two of the six cell lines genomic analysis revealed homozygous deletions of the PTEN gene. Another two of the six cell lines displayed hypermethylation of the PTEN gene promoter assessed by methylation-specific PCR. The levels of PTEN mRNA and protein expression in PC9/f9 and PC9/f14 cells, which are gefitinib-resistant derivatives of the gefitinib-sensitive cell line, PC9, were reduced compared to the parental line. After treatment with the demethylating agent 5-aza-2'deoxycytidine (5-AZA) and the histone deacetyltransferase (HDAC) inhibitor Trichostatin A (TSA), the expression levels of PTEN mRNA and protein in these four cell lines (PC9/f9, PC9/f14, PC10 and PC14) were actually restored. In summary, reduction in PTEN protein expression was regulated by histone deacetylation and hypermethylation of the gene promoter, as well as homozygous deletion. In addition, we demonstrated that the combination treatment of gefitinib and TSA induced significant growth inhibition in gefitinib-resistant PC9/f9 and PC9/f14 cells. These findings suggest that the combination of the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib with the demethylating agent 5-AZA and the HDAC inhibitor TSA may be a useful strategy for the treatment of some lung cancers.

The EGFR inhibitor gefitinib suppresses ligand-stimulated endocytosis of EGFR via the early/late endocytic pathway in non-small cell lung cancer cell lines

The drug gefitinib (Iressa), which is a specific inhibitor of EGFR tyrosine kinase, has been shown to suppress the activation of EGFR signaling for survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. A recent study demonstrated rapid down-regulation of ligand-induced EGFR in a gefitinib-sensitive cell line and inefficient down-regulation of EGFR in a gefitinib-resistant cell line in the exponential phase of growth; this implies that each cell type employs a different unknown down-regulation mechanism occurs. However, the mechanism of drug sensitivity to gefitinib remains unclear. In this study, to further substantiate the effect of gefitinib on the EGFR down-regulation pathway and to understand the detailed internalization mechanism of gefitinib-sensitive PC9 and gefitinib-resistant QG56 cell lines, we examined the internalization of Texas red-EGF in the absence or presence of gefitinib in both cell lines. The distribution of internalized Texas red-EGF, early endosomes, and late endosomes/lysosomes was then assessed by confocal immunofluorescence microscopy. Here, we provide novel evidence that efficient endocytosis of EGF-EGFR occurs via the endocytic pathway in the PC9 cells, because the internalized Texas red-EGF-positive small punctate vesicles were transported to the late endosomes/lysosomes and then degraded within the lysosomes after 60 min of internalization. Additionally, gefitinib exerted a strong inhibitory effect on the endocytosis of EGFR in PC9 cells, and the internalization rate of EGFR from the plasma membrane via the early endosomes to the late endosomes/lysosomes was considerably delayed. This indicates that gefitinib efficiently suppresses ligand-stimulated endocytosis of EGFR via the early/late endocytic pathway in PC9 cells. In contrast, the internalization rate of ligand-induced EGFR was not significantly changed by gefitinib in QG56 cells because even in the absence of gefitinib, internalized EGFR accumulation was noted in the early and late endosomes after 60 min of internalization instead of its delivery to the lysosomes in QG56 cells. This suggests that the endocytic machinery of EGFR might be basically impaired at the level of the early/late endosomes. Taken together, this is the first report demonstrating that the suppressive effect of gefitinib on the endocytosis of EGFR is much stronger with PC9 cells than QG56 cells. Thus, impairment in some steps of the EGF-EGFR traffic out of early endosomes toward the late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines.

Monitoring Tumor Glucose Utilization by Positron Emission Tomography for the Prediction of Treatment Response to Epidermal Growth Factor Receptor Kinase Inhibitors

The mechanisms underlying the sensitivity of non-small cell lung cancer to epidermal growth factor receptor (EGFR) kinase inhibitors are complex, and there are no established markers to accurately predict treatment outcome in individual patients. We investigated whether tumors responding to EGFR inhibitors can be identified by measuring treatment-induced changes in glucose utilization by positron emission tomography with the glucose analogue fluorodeoxyglucose (FDG-PET). We studied a panel of cell lines with a spectrum of sensitivity to EGFR kinase inhibitors. After incubation with the EGFR kinase inhibitor gefitinib for various time points, FDG uptake, glucose transport rates, and hexokinase activity were determined. FDG uptake in vivo was assessed by microPET imaging of tumor xenografts in mice. In gefitinib-sensitive cell lines, there was a dramatic decrease in FDG uptake as early as 2 hours after treatment. Immunoblots showed the translocation of glucose transporters (GLUT3) from the plasma membrane to the cytosol; glucose transport rates were reduced 2.6-fold at this time. There was also a modest reduction of hexokinase activity. These metabolic alterations preceded changes in cell cycle distribution, thymidine uptake, and apoptosis. MicroPET studies showed an up to 55% decrease of tumor FDG uptake in sensitive xenografts within 48 hours. In contrast, gefitinib-resistant cells exhibited no measurable changes in FDG uptake, either in cell culture or in vivo. Glucose metabolic activity closely reflects response to gefitinib therapy. FDG-PET may be a valuable clinical predictor, early in the course of treatment, for therapeutic responses to EGFR kinase inhibitors.

Decreased internalisation of ErbB1 mutants in lung cancer is linked with a mechanism conferring sensitivity to gefitinib

A majority of gefitinib (IRESSA)-responsive tumours in non-small cell lung cancer have been found to carry mutations in ErbB1. Previously, it has been observed that internalisation-deficient ErbB1 receptors are strong drivers of oncogenesis. Using a computational model of ErbB1 trafficking and signalling, it is found that a deficiency in ErbB1 internalisation is sufficient to explain the observed signalling phenotype of these gefitinib-responsive ErbB1 mutants in lung cancer cell lines. Experimental tests confirm that gefitinib-sensitive cell lines with and without ErbB1 mutations exhibit markedly slower internalisation rates than gefitinib-insensitive cell lines. Moreover, the computational model demonstrates that reduced ErbB1 internalisation rates are mechanistically linked to upregulated AKT signalling. Experimentally it is confirmed that impaired internalisation of ErbB1 is associated with increased AKT activity, which can be blocked by gefitinib. On the basis of these experimental and computational results, it is surmised that gefitinib sensitivity is a marker of a reliance on AKT signalling for cell survival that may be brought about by impaired ErbB1 internalisation.

Defining gefitinib sensitivity/resistance in non-small cell lung cancer (NSCLC) cell lines by affymetrix gene arrays

7027 Background: Gefitinib (Iressa) and other EGFR inhibitors are active in 12–19% of NSCLC patients previously treated with chemotherapy. Currently, there are no selection criteria for determining which NSCLC patients will benefit from this treatment. EGFR expression does not predict Gefitinib sensitivity. Using a panel of NSCLC cell lines with varying sensitivity to Gefitinib we defined a panel of biomarkers that predict sensitivity or resistance. Methods: Gefitinib sensitivity was determined in 18 NSCLC cell lines using MTT assays. Cell lines were classified as Gefitinib sensitive (IC50<1μM), resistant (IC50>10μM) or intermediate sensitivity (IC50 1–10μM). Oligonucleotide microarray (Affymetrix HGU 133A, 22,000 genes) analysis was performed on 10 cell lines. Three distinct filtration and normalization algorithms were used to process the expression data and a list of genes was generated that was both statistically significant (unadjusted p=0.001 cutoff) and corrected for occurrence of false positives. This approach was used in combination with 5 distinct machine learning algorithms to build a test set for predictor genes that was successful in 100% of test cases. The best discriminators (>3 fold difference in expression between sensitive and resistant cell lines) were selected for quantitative real-time RT-PCR. Results: A list of 144 genes was generated from the Affymetric array analysis. By using the mathematical algorithm 16 candidate genes were selected for RT-PCR. Thirteen of the 16 genes were verified to discriminate between sensitive and resistant cell lines by quantitative real-time RT-PCR. Conclusions: Based on NSCLC cell line studies it was possible to identify 13 different genes, which strongly discriminated Gefitinib sensitive cell lines from the resistant ones. This biomarker panel may be of significant value for selecting NSCLC patients for Gefitinib treatment. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Amgen; AstraZeneca Allos Therapeutics; AstraZeneca Eli Lilly

Defining gefitinib sensitivity/resistance in non-small cell lung cancer (NSCLC) cell lines by affymetrix gene arrays

7027 Background: Gefitinib (Iressa) and other EGFR inhibitors are active in 12–19% of NSCLC patients previously treated with chemotherapy. Currently, there are no selection criteria for determining which NSCLC patients will benefit from this treatment. EGFR expression does not predict Gefitinib sensitivity. Using a panel of NSCLC cell lines with varying sensitivity to Gefitinib we defined a panel of biomarkers that predict sensitivity or resistance. Methods: Gefitinib sensitivity was determined in 18 NSCLC cell lines using MTT assays. Cell lines were classified as Gefitinib sensitive (IC50<1μM), resistant (IC50>10μM) or intermediate sensitivity (IC50 1–10μM). Oligonucleotide microarray (Affymetrix HGU 133A, 22,000 genes) analysis was performed on 10 cell lines. Three distinct filtration and normalization algorithms were used to process the expression data and a list of genes was generated that was both statistically significant (unadjusted p=0.001 cutoff) and corrected for occurrence of false positives. This approach was used in combination with 5 distinct machine learning algorithms to build a test set for predictor genes that was successful in 100% of test cases. The best discriminators (>3 fold difference in expression between sensitive and resistant cell lines) were selected for quantitative real-time RT-PCR. Results: A list of 144 genes was generated from the Affymetric array analysis. By using the mathematical algorithm 16 candidate genes were selected for RT-PCR. Thirteen of the 16 genes were verified to discriminate between sensitive and resistant cell lines by quantitative real-time RT-PCR. Conclusions: Based on NSCLC cell line studies it was possible to identify 13 different genes, which strongly discriminated Gefitinib sensitive cell lines from the resistant ones. This biomarker panel may be of significant value for selecting NSCLC patients for Gefitinib treatment. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Amgen; AstraZeneca Allos Therapeutics; AstraZeneca Eli Lilly