Microarray expression profile of long noncoding RNAs in EGFR-TKI resistance of lung adenocarcinoma
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ABSTRACT: Lung adenocarcinoma cells harboring epidermal growth factor receptor (EGFR) mutations are sensitive to EGFR tyrosine kinase inhibitors (TKIs). Prolonged cancer treatment will induce the development of acquired resistance to EGFR TKI. To gain insight into the molecular mechanisms of EGFR-TKIs resistance, we generate EGFR-TKI-resistant HCC827-8-1 cells to be analyzed by microarray with their parental HCC827cells. gefitinib resistant HCC827-8-1 cells with three replications; gefitinib-sensitive HCC827 cells with three replications
Project description:In this study, we explored the mechanisms of hypoxia-induced EGFR TKI resistance in non-small cell lung cancer (NSCLC) harbored activating EGFR mutation. The NSCLC cell lines were exposed to normorxia or 1% oxygen for 4 weeks, and then we tested EGFR TKI sensitivity in normoxic and hypoxic NSCLC cell lines. In this microarray experiment, we used normoxic HCC827 and hypoxia-induced gefitinib resistant clones, C2-3 and C2-10. Those clones were selected with gefitinib treatment after the HCC827 were exposed to 1% oxygen for 4 weeks, and the HCC827 C2-3 and C2-10 clones were selected at random for this study.
Project description:EGFR mediates stabilities of a wide spectrum of proteins. Treatment of the cells with EGFR tyrosine kinase inhibitors (EGFR-TKIs) leads to degradation of these proteins. To define the proteins and corresponding pathways of EGFR-stabilized proteins, we use mass spectrum to quantify the protein levels in the untreated control HCC827 cells and the cells treated with erlotinib or gefitinib.
Project description:Despite initial and often dramatic responses of epidermal growth factor receptor (EGFR)-addicted lung tumors to the EGFR-specific tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, nearly all develop resistance and relapse. To explore novel mechanisms mediating acquired resistance, we employed non-small-cell lung cancer (NSCLC) cell lines bearing activating mutations in EGFR and rendered them resistant to EGFR-specific TKIs through chronic adaptation in tissue culture. In addition to previously observed resistance mechanisms including EGFR-T790M 'gate-keeper' mutations and MET amplification, a subset of the seven chronically adapted NSCLC cell lines including HCC4006, HCC2279 and H1650 cells exhibited marked induction of fibroblast growth factor (FGF) 2 and FGF receptor 1 (FGFR1) mRNA and protein. Also, adaptation to EGFR-specific TKIs was accompanied by an epithelial to mesenchymal transition (EMT) as assessed by changes in CDH1, VIM, ZEB1 and ZEB2 expression and altered growth properties in Matrigel. In adapted cell lines exhibiting increased FGF2 and FGFR1 expression, measures of growth and signaling, but not EMT, were blocked by FGFR-specific TKIs, an FGF-ligand trap and FGFR1 silencing with RNAi. In parental HCC4006 cells, cell growth was strongly inhibited by gefitinib, although drug-resistant clones progress within 10 days. Combined treatment with gefitinib and AZD4547, an FGFR-specific TKI, prevented the outgrowth of drug-resistant clones. Thus, induction of FGF2 and FGFR1 following chronic adaptation to EGFR-specific TKIs provides a novel autocrine receptor tyrosine kinase-driven bypass pathway in a subset of lung cancer cell lines that are initially sensitive to EGFR-specific TKIs. The findings support FGFR-specific TKIs as potentially valuable additions to existing targeted therapeutic strategies with EGFR-specific TKIs to prevent or delay acquired resistance in EGFR-driven NSCLC. Examination of mRNA levels in DMSO and gefitinib-resistant cultures of HCC4006 and HCC827. Each group has two replicates.
Project description:Analysis of gefitinib short-term resistance at gene expression level. The hyposthesis tested in the present study was that short-term resistance towards gefitinib in NSCLC cells influences pathways that associates with resistance towards EGFR-TKI treatment. Results provide important information of the response of EGFR mutant NSCLC cells to gefitinib and also to resistance towards gefitinib resistance, up-or down-regulated specific resistance pathways and cellular functions. Total RNA obtained from HCC827 cell line (n=3), co-cultured HCC827 (with MRC-5 cells)(n=3), gefitinib treated (0.5µM) HCC827 (n=3), and co-cultured (MRC-5) + gefitinib treated HCC827 cells (n=3) for 48h after gefitinib treatment
Project description:Previous study has demonstrated that HCC827/gef cells are resistant to gefitinib-induced aspoptosis. To investigate the regulators contributed to gefitinib resistance in lung cancer, we analyzed the gene expression profiles between HCC827 andHCC827/gef cells. Reduced IGFBP7 in TKI-resistant cells reversed resistance to EGFR-TKIs, and increased EGFR-TKI-induced apoptosis through up-regulation of BIM and activation of caspases. Suppression of IGFBP7 attenuated phosphorylation of IGF-IR and downstream AKT in TKI-resistant cells.
Project description:Purpose: Multiple mechanims have been proposed that lead to reduced effectiveness of EGFR tyrosine kinase inhibitors (TKIs) in lung cancer and yet resistance to osimertinib and gefitinib still remains a challenge in the clinic. The goals of this study are to identify key genes contributing to tolerance and resistance to EGFR inhibition. Methods: mRNA profiles of gefitinib and osimertinib tolerant cells in PC9 and HCC827 cells were generated by deep sequencing using Illumina. In addition, mRNA profiles of cells (AALE, PC9 and HCC827) overexpressing with miR-147b or miR-21 and mRNA profiles of cells (H1975 and PC9ER) with miR-147b and miR-21 knocking down were generated by deep sequencing. The mappable reads were aligned to the human transcripts using Bowtie2 and gene abundance was estimated using RSEM. Results: Upregulation of miR-147b and miR-21 expression is related to tolerance and resistance to gefitinib and osimertinib in lung cancer. The signaling pathways of transcripts by knocking down miR-147b or miR-21 in resistant cells (H1975 and PC9ER) and by overexpressing miR-147b or miR-21 in both sensistive cells (HCC827 and PC9) and immortalized lung epithelial cells (AALE) are consistent with the key signaling pathways shown in tolerant cells to gefitinib and osimertinib in HCC827 and PC9 cells (HCC827GTR/OTR vs HCC827 and PC9GTR/OTR vs PC9). Conclusions: Our work identifies key signaling pathways that mediate EGFR-TKI tolerance and resistance in lung cancer. Our study provides potential targets to improve the efficacy of EGFR-TKIs therapy in cancer pagtients.
Project description:Lung adenocarcinoma cells harboring epidermal growth factor receptor (EGFR) mutations are sensitive to EGFR tyrosine kinase inhibitors (TKIs). Prolonged cancer treatment will induce the development of acquired resistance to EGFR TKI. To gain insight into the molecular mechanisms of EGFR-TKIs resistance, we generate EGFR-TKI-resistant HCC827-8-1 cells to be analyzed by microarray with their parental HCC827cells.
Project description:Analysis of gefitinib short-term resistance at gene expression level. The hyposthesis tested in the present study was that short-term resistance towards gefitinib in NSCLC cells influences pathways that associates with resistance towards EGFR-TKI treatment. Results provide important information of the response of EGFR mutant NSCLC cells to gefitinib and also to resistance towards gefitinib resistance, up-or down-regulated specific resistance pathways and cellular functions. Total RNA obtained from PC9 cell line (n=3), co-cultured PC9 (with MRC-5 cells)(n=3), gefitinib treated (0.5µM) PC9 (n=3), and co-cultured (MRC-5) + gefitinib treated PC9 cells (n=3) for 48h after gefitinib treatment
Project description:Purpose: To characterize microRNA signatures for tolerance, persistence and resistance to EGFR tyrosine kinase inhibitors (TKIs) in human lung cancer. Methods: microRNA profiles of gefitinib- and osimertinib-tolerant cells in PC9 and HCC827 cells were generated by deep microRNA sequencing using Illumina. In addition, microRNA profiles of PC9 subpopulations cells with characterizations of persistence and resistance to gefitinib were generated by deep microRNA sequencing. The mappable reads were aligned to the human genome and miRbase using Bowtie. Results: We identified a specific microRNA profile distinguishing tolerance, persistence and resistance to gefitinib or osimertinib from parental human lung cancer cells with mutated EGFR. The expressions of those microRNAs in lung cancer cells were validated by qRT-PCR. Functionally, knocking down top-upregulated microRNAs reduced the tolerance, persistence and resistance to gefitinib or osimertinib in those tolerant and resistant cells. Conversely, overexpression of those microRNAs enhanced the tolerance and resistance to EGFR inhibition in cells sensitive to gefitinib and osimertinib. Conclusions: Our work identifies a panel of microRNAs that mediate EGFR-TKI tolerance and resistance in lung cancer. Our study provides potential non-coding targets to improve the efficacy of EGFR-TKIs therapy in cancer pagtients.
Project description:About 10% of all NSCLC patients respond to gefitnib treatment and all of these patients will acquire resistance to the EGFR TKI. We used microarray to look at global gene expression changes in untreated cells vs gefitinib treated cells to identify key characters for the acquisition of resistance. NSCLC cells, H322c, were cultured 4 days in media containing 1?M gefitinib or 0.1% DMSO as a control. On day 4, RNA was extracted and submitted for microarray hybridization.