Genome-wide analysis of gefitinib-tolerant gene expression in single cell-derived clones
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ABSTRACT: Analysis of gene expression profiles for gefitinib-tolerant cells derived from single cells. The hypothesis tested here was that a small population of cancer cells contribute to tolerance status in a bulk of EGFR mutated lung cancer cells. Results provide important infromnation of the tolerance to the EGFR tyrosine kinase inhibitor, gefitinib, such as upregualtion of cancer-stemness related genes.
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:EGFR inhibitors (EGFRi) are effective against EGFR mutant lung cancers. The efficacy of these drugs however is mitigated by the outgrowth of resistant cells, most often driven by a secondary acquired mutation in EGFR, T790M. We recently demonstrated that T790M can arise de novo during treatment (Hata et al., Nature Medicine 2016); it follows that one potential therapeutic strategy to thwart resistance would be identifying and eliminating these cells (referred to as drug tolerant cells (DTCs)) prior to acquiring secondary mutations like T790M. We have developed DTCs to EGFRi in EGFR mutant lung cancer cell lines. Subsequent analyses of DTCs included RNA-seq, high-content microscopy, and protein translational assays. Based on these results, we tested the ability of MCL-1 BH3 mimetics to combine with EGFR inhibitors to eliminate DTCs and shrink EGFR mutant lung cancer tumors in vivo.
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:Gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), induces substantial clinical responses for non-small cell lung cancer (NSCLC) cells harboring EGFR activating mutations, but most of them invariably develop resistance. By generating a gefitinib resistance (PC9GR) from a human NSCLC-derived drug sensitive cell line (PC9), we studied differences of transcription dynamics between them by the aid of a computational decoupling of hidden regulatory signals from time course gene expression profiles. Given a collection of transcription factors (TFs) and their regulatory targets, the method captured temporally-synchronized shifts in evolving expression of target genes sharing each TF regulatory unit, and drew underlying regulatory signals. The analysis identified sterol regulatory element binding protein 1 (SREBP-1) as a key regulatory agent that facilitates the maintenance of drug tolerance, involving transcription controls of a G1-specific cyclin dependent kinase inhibitor whose expression was specifically elevated in PC9, but in turn, reduced in PC9GR Gefitinib-resistance cell line (PC9GR) was established derived from lung adenocarcinoma cell line PC9. PC9 cells and PC9GR cells were treated with the four different conditions, control (No treatment), EGF-treatment, gefitinib-treatment, and both gefitinib and EGF-treatment. In each condition, the gene expression was measured at 26 time points during 24 hrs.
Project description:Using a kinome-centred CRISPR/Cas9 genetic screen, we identify here that inhibition of the epidermal growth factor receptor (EGFR) is synthetic lethal with lenvatinib in liver cancer cells. We found that the combination of the EGFR inhibitor gefitinib and lenvatinib displays potent anti-proliferative effect in HCC cell lines that express EGFR in vitro and of xenografted HCC cell lines or patient-derived HCC tumours in mice. Herre, we analyzed the different transcriptome profiling of HCC cells treated with DMSO, lenvatinib, gefitinib, and lenvatinib plus gefitinib by RNA-sequencing.
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:Microarrays were used to analyze differential gene expression and to help determine the efficacy of Iressa (gefitinib), a tyrosine kinase inhibitor, on endometrial cancer cells. Type I Ishikawa H and type II Hec50co endometrial carcinoma cells both express EGFR and sEGFR, but differ markedly in their responsiveness to the EGFR inhibitor gefitinib. This difference is paralleled by differences in the expression of sEGFR and EGFR, as well as in their transcriptional response following treatment with either EGFor gefitinib. The small cluster of differently regulated genes reported here in these type I vs. type II endometrial cancer-derived cell lines may identify candidate biomarkers useful for predicting sensitivity to EGFR blockade. Type I (Ishikawa H cells) and type II (Hec50co) derived endometrial carcinomas, were dosed with either EGF(epidermal growth factor) or Iressa (gefitinib) for 12 or 24 hours and gene expression was examined.
Project description:Even after osimertinib is administered to lung adenocarcinomas with EGFR mutations, there are a few cells which survive, and these tolerant cells are considered to be the source of later recurrence. We used microarray analysis to explore the mechanism behind the tolerance to osimertinib in lung adenocarcinoma cells with EGFR mutations.
Project description:Even after osimertinib is administered to lung adenocarcinomas with EGFR mutations, there are a few cells which survive, and these tolerant cells are considered to be the source of later recurrence. We used microarray analysis to explore the mechanism behind the tolerance to osimertinib in lung adenocarcinoma cells with EGFR mutations.