Gefitinib-sensitive Cell Research Articles

Exosome-derived circKIF20B suppresses gefitinib resistance and cell proliferation in non-small cell lung cancer

BackgroundThe gefitinib resistance mechanism in non-small cell lung cancer (NSCLC) remains unclear, albeit exosomal circular RNA (circRNA) is known to possibly play a vital role in it.MethodsWe employed high-throughput sequencing techniques to detect the expressions of exosomal circRNA both in gefitinib-resistant and gefitinib-sensitive cells in this study. The circKIF20B expression was determined in serum exosomes and tissues of patients by qRT-PCR. The structure, stability, and intracellular localization of circKIF20B were verified by Sanger sequencing, Ribonuclease R (RNase R)/actinomycin D (ACTD) treatments, and Fluorescence in situ hybridization (FISH). The functions of circKIF20B were investigated by 5-Ethynyl-20-deoxyuridine (EdU), flow cytometry, Cell Counting Kit-8 (CCK-8), oxygen consumption rate (OCR), and xenograft model. Co-culture experiments were performed to explore the potential ability of exosomal circKIF20B in treating gefitinib resistance. The downstream targets of circKIF20B were determined by luciferase assay, RNA pulldown, and RNA immunoprecipitation (RIP).ResultsWe found that circKIF20B was poorly expressed in the serum exosomes of gefitinib-resistant patients (n = 24) and the tumor tissues of patients with NSCLC (n = 85). CircKIF20B was negatively correlated with tumor size and tumor stage. Decreasing circKIF20B was found to promote gefitinib resistance by accelerating the cell cycle, inhibiting apoptosis, and enhancing mitochondrial oxidative phosphorylation (OXPHOS), whereas increasing circKIF20B was found to restore gefitinib sensitivity. Mechanistically, circKIF20B is bound to miR-615-3p for regulating the MEF2A and then altering the cell cycle, apoptosis, and mitochondrial OXPHOS. Overexpressing circKIF20B parental cells can restore sensitivity to gefitinib in the recipient cells by upregulating the exosomal circKIF20B expression.ConclusionsThis study revealed a novel mechanism of circKIF20B/miR-615-3p/MEF2A signaling axis involving progression of gefitinib resistance in NSCLC. Exosomal circKIF20B is expected to be an easily accessible and alternative liquid biopsy candidate and potential therapeutic target in gefitinib-resistant NSCLC.Graphical The schematic diagram of mechanism in this study. Exosomal circKIF20B inhibits gefitinib resistance and cell proliferation by arresting the cell cycle, promoting apoptosis, and reducing OXPHOS via circKIF20B/miR-615-3p/MEF2A axis in NSCLC.

Role of ABCA2 and its single nucleotide polymorphisms (4873T>A and 4879G>C) in the regulation of multi-drug resistance in oral squamous carcinoma cells

Lysosomal exocytosis is an essential cellular event for remodeling the extracellular matrix through secreting lysosomal enzymes and developing drug resistance. However, the detailed mechanism underlying the lysosomal exocytosis-driven acquisition of drug resistance is not completely understood. Genetic variations in gefitinib-sensitive (HSC3) and -resistant (HSC3/GR) oral squamous carcinoma cell lines were identified using whole-exome sequencing (WES). The physiological role of the ATP-binding cassette subfamily A member 2 (ABCA2) in gefitinib-induced lysosomal trafficking was evaluated in vitro, through overexpressing ABCA2 and its single nucleotide polymorphisms (SNPs). WES analysis showed that the 554 SNPs harboring 244 genes appeared to be differentially generated depending on gefitinib resistance. Among these genes, ABCA2 was enriched in 24 of 39 gene ontology terms. Two missense SNPs of ABCA2, 4873T > A (rs1831123356) and 4873T > A, were generated only in gefitinib-sensitive cells. Furthermore, HEK293 cells expressing the wild-type ABCA2 (WT ABCA2) acquired tolerance for gefitinib-induced cytotoxicity by increasing gefitinib sequestration in lysosomes and lysosomal exocytosis. Conversely, cells expressing each ABCA2 SNP exhibited lower efficacy in developing tolerance to gefitinib-induced responses than those expressing WT ABCA2. Notably, HSC3/GR cells were also tolerant to erlotinib and sunitinib but not osimertinib. Furthermore, tolerance for multiple tyrosine kinase inhibitors was attenuated by the deletion of ABCA2. These findings demonstrate that ABCA2 and its SNPs should be considered prominent targets for overcoming multi-drug resistance and enhancing the efficacy of chemotherapeutics.

Activation of the HSP27-AKT axis contributes to gefitinib resistance in non-small cell lung cancer cells independent of EGFR mutations

PurposeAlthough epidermal growth factor receptor (EGFR)-activating mutations in non-small cell lung cancer (NSCLC) usually show sensitivity to first-generation EGFR-tyrosine kinase inhibitors (TKIs), most patients relapse because of drug resistance. Heat shock protein 27 (HSP27) has been reported to be involved in the resistance of EGFR-TKIs, although the underlying mechanism is unclear. Here, we explore the mechanisms of HSP27-mediated EGFR TKI resistance and propose novel therapeutic strategies.MethodsTo determine the mechanism of HSP27 associated gefitinib resistance, differences were assessed using gefitinib-sensitive and -resistant NSCLC cell lines. In vivo xenograft experiments were conducted to elucidate the combinatorial effects of J2, a small molecule HSP27 inhibitor, and gefitinib. Analyses of human NSCLC tissues and PDX tissues were also used for comparison of HSP27 and phosphorylated AKT expression.ResultsLarge-scale cohort analysis of NSCLC cases revealed that HSP27 expression correlated well with the incidence of EGFR mutations and affected patient survival. Increased pAKT and HSP27 was observed in gefitinib-resistant cells compared with gefitinib-sensitive cells. Moreover, increased phosphorylation of HSP27 by gefitinib augmented its protein stability and potentiated its binding activity with pAKT, which resulted in increased gefitinib resistance. However, in gefitinib-sensitive cells, stronger binding activity between EGFR and HSP27 was observed. Moreover, these phenomena occurred regardless of EGFR mutation including secondary mutations, such as T790M. AKT knockdown switched HSP27-pAKT binding to HSP27-EGFR, which promoted gefitinib sensitivity in gefitinib-resistant cells. Functional inhibition of HSP27 yielded sensitization to gefitinib in gefitinib-resistant cells by inhibiting the interaction between HSP27 and pAKT.ConclusionsOur results indicate that combination of EGFR-TKIs with HSP27 inhibitors may represent a good strategy to overcome resistance to EGFR-TKIs, especially in cancers exhibiting AKT pathway activation.

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.

Long non-coding RNA CASC9 promotes gefitinib resistance in NSCLC by epigenetic repression of DUSP1

Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, has greatly affected clinical outcomes in non-small cell lung cancer (NSCLC) patients. The long noncoding RNAs (lncRNAs) are known to regulate tumorigenesis and cancer progression, but their contributions to NSCLC gefitinib resistance remain poorly understood. In this study, by analyzing the differentially expressed lncRNAs in gefitinib-resistant cells and gefitinib-sensitive cells in the National Institute of Health GEO dataset, we found that lncRNA CASC9 expression was upregulated, and this was also verified in resistant tissues. Gain and loss of function studies showed that CASC9 inhibition restored gefitinib sensitivity both in vitro and in vivo, whereas CASC9 overexpression promoted gefitinib resistance. Mechanistically, CASC9 repressed the tumor suppressor DUSP1 by recruiting histone methyltransferase EZH2, thereby increasing the resistance to gefitinib. Furthermore, ectopic expression of DUSP1 increased gefitinib sensitivity by inactivating the ERK pathway. Our results highlight the essential role of CASC9 in gefitinib resistance, suggesting that the CASC9/EZH2/DUSP1 axis might be a novel target for overcoming EGFR-TKI resistance in NSCLC.

Y-box binding protein 1 (YB-1) promotes gefitinib resistance in lung adenocarcinoma cells by activating AKT signaling and epithelial-mesenchymal transition through targeting major vault protein (MVP).

Gefitinib is a first-line treatment option for epidermal growth factor receptor (EGFR)-mutatedlung adenocarcinoma. However, most patients inevitably develop gefitinib resistance. The mechanism underlying this resistance is not fully understood. Y-box binding protein 1 (YB-1) has been reported to play a role in modulating drug sensitivity, but its role in gefitinib resistance is currently unknown. Here, we investigated the role of YB-1 in gefitinib resistance of lung adenocarcinoma. We determined the expression of YB-1, epithelial-mesenchymal transition (EMT) and AKT signaling markers, as well as the viability of lung adenocarcinoma cell lines bearing mutant (HCC827, PC-9) or wild-type (H1299) EGFR. We also evaluated PC-9 cell migration and invasion using transwell assays. The clinical importance of YB-1 and major vault protein (MVP) was evaluated using primary lung adenocarcinoma patient samples. We found that YB-1 was significantly upregulated in gefitinib-resistant lung adenocarcinoma cells compared to gefitinib-sensitive cells. YB-1 augmented gefitinib resistance by activating the AKT pathway and promoting EMT. Decreased migration and invasion was observed upon MVP silencing in YB-1-overexpressing PC-9 cells, as well as restored gefitinib sensitivity. A retrospective analysis of 85 patients with lung adenocarcinoma revealed that YB-1 levels were significantly increased in tyrosine kinase inhibitor (TKI)-resistant patients compared to those in TKI-sensitive patients, indicating that YB-1 may serve as a biomarker to clinically predict acquired gefitinib resistance. YB-1 activates AKT signaling and promotes EMT at least in part by directly activating MVP. Hence, targeting the YB-1/MVP axis may help to overcome gefitinib resistance in lung adenocarcinoma patients.

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.

Co-administration of 20(S)-protopanaxatriol (g-PPT) and EGFR-TKI overcomes EGFR-TKI resistance by decreasing SCD1 induced lipid accumulation in non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) patients with sensitive epidermal growth factor receptor (EGFR) mutations are successfully treated with EGFR tyrosine kinase inhibitors (EGFR-TKIs); however, resistance to treatment inevitably occurs. Given lipid metabolic reprogramming is widely known as a hallmark of cancer and intimately linked with EGFR-stimulated cancer growth. Activation of EGFR signal pathway increased monounsaturated fatty acids (MUFA) and lipid metabolism key enzyme Stearoyl-CoA Desaturase 1 (SCD1) expression. However the correlation between EGFR-TKI resistance and lipid metabolism remains to be determined.MethodsIn this study the differences in lipid synthesis between paired TKI-sensitive and TKI-resistant patient tissues and NSCLC cell lines were explored. Oleic acid (OA, a kind of MUFA, the SCD1 enzymatic product) was used to simulate a high lipid metabolic environment and detected the affection on the cytotoxic effect of TKIs (Gefitinib and osimertinib) in cell lines with EGFR-activating mutations. (20S)-Protopanaxatriol (g-PPT), an aglycone of ginsenosides, has been reported to be an effective lipid metabolism inhibitor, was used to inhibit lipid metabolism. Additionally, synergism in cytotoxic effects and signal pathway activation were evaluated using CCK-8 assays, Western blotting, flow cytometry, Edu assays, plate clone formation assays and immunofluorescence. Furthermore, two xenograft mouse models were used to verify the in vitro results.ResultsGefitinib-resistant cells have higher lipid droplet content and SCD1 expression than Gefitinib-sensitive cells in both NSCLC cell lines and patient tissues. Additionally oleic acid (OA, a kind of MUFA, the SCD1 enzymatic product) abrogates the cytotoxic effect of both Gefitinib and osimertinib in cell lines with EGFR-activating mutations. As a reported effective lipid metabolism inhibitor, g-PPT significantly inhibited the expression of SCD1 in lung adenocarcinoma cells, and then down-regulated the content of intracellular lipid droplets. Combined treatment with Gefitinib and g-PPT reverses the resistance to Gefitinib and inhibits the activation of p-EGFR and the downstream signaling pathways.ConclusionsOur findings uncover a link between lipid metabolic reprogramming and EGFR-TKI resistance, confirmed that combination target both EGFR and abnormal lipid metabolism maybe a promising therapy for EGFR-TKI resistance and highlighting the possibility of monitoring lipid accumulation in tumors for predicting drug resistance.

Glycerol kinase 5 confers gefitinib resistance through SREBP1/SCD1 signaling pathway

BackgroundDrug resistance is common in cancer chemotherapy. This study investigates the role of Glycerol kinase 5 (GK5) in mediating gefitinib resistance in NSCLC.MethodsThe exosomal mRNA of GK5 was detected using a tethered cationic lipoplex nanoparticle (TCLN) biochip. Real-time PCR and Western blot were used to examine the expression of GK5 mRNA and protein in gefitinib-sensitive and -resistant human lung adenocarcinoma cells. The cell counting kit-8, EdU assay, flow cytometry, and JC-1 dye were used to measure cell proliferation, cell cycle, and the mitochondrial membrane potential.ResultsWe found that the exosomal mRNA of GK5 in the plasma of patients with gefitinib-resistant adenocarcinoma was significantly higher compared with that of gefitinib-sensitive patients. The mRNA and protein levels of GK5 were significantly upregulated in gefitinib-resistant human lung adenocarcinoma PC9R and H1975 cells compared with gefitinib-sensitive PC9 cells. Silencing GK5 in PC9R cells induced mitochondrial damage, caspase activation, cell cycle arrest, and apoptosis via SREBP1/SCD1 signaling pathway.ConclusionsWe demonstrated that GK5 confers gefitinib resistance in lung cancer by inhibiting apoptosis and cell cycle arrest. GK5 could be a novel therapeutic target for treatment of NSCLC with resistance to EGFR tyrosine kinase inhibitors.

The long non-coding RNA SNHG5 regulates gefitinib resistance in lung adenocarcinoma cells by targetting miR-377/CASP1 axis.

Gefitinib resistance is one of the major obstacles for the treatment of lung adenocarcinoma (LAD). The present study aimed to investigate the effects of the long non-coding RNA (lncRNA), small nucleolar RNA host gene 5SNHG5 on gefitinib resistance in LAD and explore the underlying mechanisms. The quantitative real-time PCR (qRT-PCR) results showed that SNHG5 expression was significantly down-regulated in LAD patients with acquired gefitinib resistance and gefitinib resistant LAD cell lines. SNHG5 overexpression sensitized gefitinib resistant LAD cells to gefitinib treatment, while knockdown of SNHG5 rendered gefitinib sensitive LAD cells to gefitinib treatment. Bioinformatics analysis showed that SNHG5 exerted its function through interaction with miR-377, which was further confirmed by luciferase reporter assay in 293T cells. Overexpression of SNHG5 suppressed the expression of miR-377, while the knockdown of SNHG5 increased the miR-377 expression. MiR-377 expression was significantly up-regulated in LAD specimens with acquired gefitinib resistance and was negatively correlated with SNHG5 expression. In addition, CASP1 was predicted as a downstream target of miR-377. Overexpression of miR-377 suppressed the expression of CASP1 in PC9 cells and knockdown of miR-377 increased the CASP1 expression in PC9GR cells. In vitro functional assay showed that knockdown of CASP1 in SNHG5-overexpressed PC9GR cells abolished their gefitinib resistance. Overall, the present study demonstrated, for the first time, that the SNHG5/miR-377/CASP1 axis functions as an important role in LAD cells gefitinib resistance and potentially contributes to the improvement of LAD diagnosis and therapy.

Increased glutamine anabolism sensitizes non-small cell lung cancer to gefitinib treatment

To better understand the resistance mechanism of non-small cell lung cancers (NSCLCs) to gefitinib, the metabolic profiles of gefitinib-resistant A549 cells and gefitinib-sensitive PC-9 cells were analyzed with a metabolomics analytical platform. A549 and PC-9 cells exhibited significant differences in the levels of glutamine-related metabolites. After gefitinib treatment, the glutamine level decreased in A549 cells but showed no change in PC-9 cells. The glutamine consumed by A549 cells was used to generate ATP and glutathione (GSH). As glutamine utilization was suppressed in gefitinib-treated PC-9 cells, the resulting ATP shortage and ROS accumulation led to cell death. The difference in glutamine metabolism was caused by differential changes in the levels of glutamine synthetase (GS, encoded by glutamate-ammonia ligase (GLUL)). GLUL expression was upregulated in gefitinib-sensitive cells, but it was either absent from gefitinib-resistant cells or no significant change was observed in the gefitinib-treated cells. GLUL overexpression in A549 cells significant sensitized them to gefitinib and decreased their invasive capacity. Conversely, knockout GS in PC-9 cells reduced gefitinib sensitivity and enhanced metastasis. Furthermore, the continuous exposure of gefitinib-sensitive HCC827 cells to gefitinib created gefitinib-resistant (GR) HCC827 cells, which exhibited a GLUL deletion and resistance to gefitinib. Thus, GLUL plays a vital role in determining the sensitivity of NSCLCs to gefitinib. Elevated GS levels mediate increased glutamine anabolism, and this novel mechanism sensitizes NSCLCs to gefitinib. The inhibition of glutamine utilization may serve as a potential therapeutic strategy to overcome gefitinib resistance in the clinic.

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-181a contributes gefitinib resistance in non-small cell lung cancer cells by targeting GAS7

Tyrosine kinase inhibitors (TKIs) exert potent therapeutic efficacy in non-small cell lung cancers (NSCLC) harboring epidermal growth factor receptor (EGFR) activating mutations. However, a major impediment for the effective treatment is the development of drug resistance. Some evidence supports a role for miRNAs in modulating NSCLC TKIs resistance. Here we show that miR-181a is significantly up-regulated in gefitinib-resistant cells compared with gefitinib-sensitive cells. Upregulation of miR-181a caused resistance of gefitinib, whereas downregulation of miR-181a sensitized NSCLC cells to gefitinib. Furthermore, the miR-181a plasma levels were significantly increased in acquired gefitinib resistant NSCLC patients compared with the plasma levels prior to gefitinib treatment in each patient. Bioinformatics analysis and luciferase reporter assay showed that growth arrest-specific 7 (GAS7) was a direct target gene of miR-181a. A significant inverse correlation between the expression of miR-181a and GAS7 was identified in NSCLC tissues. Downregulation of GAS7 expression could antagonize gefitinib re-sensitivity in PC9GR mediated by knockdown of miR-181a via AKT/ERK pathways and epithelial-to-mesenchymal transition markers. Additionally, GAS7 expression was downregulated in a large cohort of NSCLC patients, and a high mRNA level of GAS7 was associated with improved overall survival. Collectively, our findings provide a novel basis for using miR-181a/GAS7-based therapeutic strategies to reverse gefitinib resistance in NSCLC.

PUB048 Down-Regulation of Shisa3 Is Associated with Gefitinib Resistance in Non-Small Cell Lung Cancer

The failure of targeted therapy due to the resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, is considered a major problem in the treatment of non-small cell lung cancer (NSCLC) patients. Shisa3, a novel tumor suppressor, is associated with prolonged survival through promoting β-catenin degradation in NSCLC. Nevertheless, the relationship between Shisa3 and EGFR-TKIs resistance has not been elucidated. The correlation of shisa3 and EGFR-TKI resistance were determined by comparison of gene expression profiles in NSCLC patient tumor samples with different therapeutic benefits for EGFR-TKIs. The effect of Shisa3 on drug sensitivity, invasion, and migration in NSCLC cells and on tumor growth and drug susceptibility in mice models were examined by knockdown and overexpression of Shisa3. The underlying mechanism of Shisa3 was explored by microarray and pathway analysis. Finally, the correlation of Shisa3 expression and clinical effect for EGFR-TKIs was also calculated. We identified that Shisa3 was highly expressed in the tumor tissues of patients with partial response (PR) in EGFR-TKIs treatment and is down-regulated in gefitinib-resistant (PC9-Gef, H1975, H1650) cells compared to gefitinib-sensitive (PC9, HCC827) cells. The down-regulation of Shisa3 could induce gefitinib resistance and enhance invasiveness through PI3K/AKT/mTOR signaling predicted by microarray analysis. Furthermore, the down-regulated expression of Shisa3 could inhibit the activation of autophagy. Rather, the up-regulation of shisa3 could enhance gefitinib sensitivity by inhibit PI3K/AKT/mTOR signaling and the activation of autophagy. In addition, our data confirmed that Shisa3 was positively associated with clinical effect for EGFR-TKIs in 20 NSCLC patients. Our results reveal that the prospective role of Shisa3 as a novel biomarker for acquired gefitinib resistance and a potential target for NSCLC treatment.

A chemical genetics approach to identify therapeutic vulnerabilities in Gefitinib resistant, EGFR T790M

Background: The first generation EGFR inhibitor, Gefitinib, has been approved for the treatment of non-small cell lung carcinoma (NSCLC) with activating EGFR mutations. However resistance to Gefitinib quickly develops and is mainly driven by the EGFR T790M mutation in the kinase domain. On the other hand, while EGFR was found to be overexpressed in the majority of HNSCC, clinical trials on the use of Gefitinib for treatment of HNSCC has been disappointing thus far. This is mainly due to the lack of activating mutations in the EGFR kinase domain of HNSCC patients, as well as treatment resistance. In this study, we sought to identify alternative therapeutic vulnerabilities in Gefitinib resistant HNSCC that are negative for EGFR T790M mutation. Methods: We developed a pair of isogenic Gefitinib sensitive (TM17S) and resistant (TM17R) cell lines through stepwise chronic treatment of the TM17S cell line with Gefitinib. The TM17S and TM17R cell lines were characterized for their mutational profile using POLARIS Xplora cancer panel and through gene expression studies. High-throughput chemical screen with two annotated compound libraries was carried out to identify alternative therapeutic vulnerabilities in the TM17R cell line. Results: While the TM17R cell line was derived to be resistant to Gefitinib, a first generation EGFR tyrosine kinase inhibitor (TKI), TM17R cells were found to be resistant to all 3 generations of EGFR TKIs. Comparison of the mutational profiles of TM17S and TM17R cells did not reveal obvious mutational drivers of resistance. Strikingly the EGFR T790M mutation was not detected in both TM17S and TM17R cells. We also observed a down regulation of EGFR gene and protein expression. All these could account for the resistance of TM17R cells to all 3 generations of EGFR TKIs, and suggests an alternative mechanism of resistance. Results from the high-throughput chemical screen revealed an alternative therapeutic vulnerability of TM17R cells to Aurora kinase inhibitors. This therapeutic vulnerability was also observed in 2 other Gefitinib sensitive and resistant pairs of HNSCC cell lines. Conclusions: In EGFR T790M negative cohorts, a possible mechanism of resistance to EGFR TKIs would involve the downregulation of EGFR. While this could lead to resistance to all 3 generations of EGFR TKIs, resistant cells developed sensitivity to Aurora kinase inhibitors instead. Comparison of the gene expression profiles of these cell lines with similar therapeutic vulnerabilities could potentially identify biomarkers of Gefitinib resistance in HNSCC. Legal entity responsible for the study: NA Funding: The Agency for Science, Technology and Research (A*STAR) of Singapore Disclosure: All authors have declared no conflicts of interest.

Abstract 3169: SerpinB2 enhances invadopodia-like structure protrusions and is down-regulated in acquired gefitinib-resistant non-small cell lung cancer cells

Abstract Non-small cell lung cancer (NSCLC) is a major type of lung cancer which accounts for approximately 80-85% of all lung cancers. The targeted therapies have significantly improved the survival of advanced NSCLC patients, but the failure of targeted therapy due to the resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, is now considered a major problem. SerpinB2 is a component of the urokinase plasminogen activator (uPA) system and has been recognized as a biomarker for the progression and metastasis of lung cancer. We found that SerpinB2 is down-regulated in gefitinib-resistant (H292-Gef) cells compared to gefitinib-sensitive (H292) cells. The low SerpinB2 levels in H292-Gef cells were also associated with an enhancement in invasiveness and increase in the length of invadopodia-like structures in the cells. The effect on invasiveness and gefitinib sensitivity was confirmed by knockdown and overexpression of SerpinB2. In addition, an antitumor agent yuanhuadine (YD) was used to test the possibility to overcome the resistance through the up-regulation of SerpinB2. YD effectively elevated SerpinB2 levels and suppressed invasive properties in H292-Gef cells. Collectively, these findings demonstrate the prospective role of SerpinB2 as a novel biomarker for acquired gefitinib resistance and a potential target for NSCLC treatment. Acknowledgement This study was funded by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1D1A1A02062012). Citation Format: Song Yi Bae, Donghwa Kim, Hyen Joo Park, Woong Sub Byun, Ji-Young Hong, Hye-Jung Lee, Sang Kook Lee. SerpinB2 enhances invadopodia-like structure protrusions and is down-regulated in acquired gefitinib-resistant non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3169. doi:10.1158/1538-7445.AM2017-3169

Abstract 5872: Stat3 feedback activation-induced PTGIS expression is associated with acquired resistance to EGFR-TKI in lung cancer

Abstract Targeted cancer therapies can effectively promote tumor regression in clinical responses. Eventually, most tumors develop resistance to these drugs. Stat3 activation has been suggested as one of the mechanisms that cause acquired resistance to EGFR-tyrosin kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC). However, the underlying molecular mechanisms are not well studied. In this study, we found that treatment of gefitinib (EGFR-TKI) in NSCLC cell lines harboring EGFR-TKI sensitive mutation induced feedback activation of Stat3 in a time- and dose-dependent manner. High levels of IL-6 were detected in the conditioned medium of gefitinib-treated cells by cytokine array and were confirmed by ELISA. We demonstrated that IL-6 treatment could induce Stat3 activation and conditioned medium treatment-induced Stat3 activation was suppressed by IL-6 neutralizing antibody. We also demonstrated that gefitinib treatment-induced IL-6 secretion could be inhibited by knockdown of Stat3 expression. Moreover, pharmacological inhibition and genetic inhibition of Stat3 activation increased cell cytotoxicity of gefitinib in both PC9 and HCC827 cells that harboring EGFR-TKI sensitive mutation. Our data suggested that gefitinib treatment could induce activation of IL-6/Stat3 signaling loop and modulate cell cytotoxicity of gefitinib. Microarray and Ingenuity Pathway Analysis (IPA) analysis revealed prostaglandin I2 synthase (PTGIS) as a downstream target gene of IL-6/Stat3 signaling and PTGIS was up-regulated by gefitinib treatment. The induction of PTGIS by gefitinib via Stat3 feedback activation was confirmed in vitro genetically and pharmacologically. Moreover, we showed that cotreatment with PTGIS inhibitor improves the efficacy of EGFR inhibition in PC9 and HCC827 cells. Importantly, high expression of PTGIS was found in gefitinib-resistant PC9 cells (PC9/gef) compared with gefitinib-sensitive PC9 cells. Targeting PTGIS was also effective in decreasing viability of cells with acquired resistance to gefitinib in PC9/gef cells. Taken together, our study indicated that Stat3 feedback activation-induced PTGIS expression participates in modulating gefitinib efficacy and Stat3/PTGIS inhibition could potentially overcome acquired resistance to gefitinib in NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Hsuan-Heng Yeh, Shang-Yin Wu, Chun-Hua Hung, Wu-Chou Su. Stat3 feedback activation-induced PTGIS expression is associated with acquired resistance to EGFR-TKI in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5872. doi:10.1158/1538-7445.AM2017-5872