Gene expression profiling of generated Erlotinib-resistant (ER) cell lines
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ABSTRACT: Targeted therapies have provided advantages to cancer patients, but these therapies are limited by differential responses and developed resistance. To discover mechanisms of resistance, we developed drug-resistant cell lines and have their gene expressions profiled and compared. HCC827 cells, which were initially sensitive to Erlotinib treatment, are subjected to Erlotinib treatment. Two clonal populations that resistant to Erlotinib resulted.
Project description:The non-small cell lung cancer (NSCLC) cell line HCC827 harbors an activating EGFR mutation (exon 19 deletion) that confers sensitivity to the FDA-approved EGFR inhibitor erlotinib. By applying the ClonTracer barcoding system, we were able to show the presence of pre-existing sub-populations in HCC827 that contribute to erlotinib resistance. Prior studies implicated that MET amplification confers resistance to erlotinib in this cell line. Therefore we examined the effects of the c-Met inhibitor crizotinib on the barcoded HCC827 population when treated either sequentially or simultaneously with both inhibitors. Despite the significant reduction in barcode complexity, the erlotinib/crizotinib combination treatment failed to eradicate all of the resistant clones implying the presence of an erlotinib/crizotinib dual resistant subpopulation. We performed transcriptome profiling (RNA-seq) to elucidate the potential resistance mechanisms of the dual resistant subpopulation in comparison to vehicle-treated or single agent erlotinib-resistant HCC827 cell populations as controls. mRNA profiling of the subpopulations of human NSCLC cell line HCC827 that contribute to EGFR inhibitor erlotinib and MET inhibitor crizotinib resistance
Project description:We were interested in characterizing the transcriptional changes that occur on a genome-wide scale following treatment of EGFR-mutant lung cancer cells with targeted therapies. HCC827 human lung cancer cells harboring an amplified EGFR allele with an activating in frame deletion of 15 nucleotides in exon 19 were treated in triplicate with 1uM erlotinib (EGFR inhibitor), AZD-6244 (MEK inhibitor) or BEZ-235 (PI3-Kinase/mTOR inhibitor) for 6 hours, followed by total mRNA isolation and whole transcriptome analysis using Affymetrix U133 Plus 2.0 expression arrays.
Project description:The non-small cell lung cancer (NSCLC) cell line HCC827 harbors an activating EGFR mutation (exon 19 deletion) that confers sensitivity to the FDA-approved EGFR inhibitor erlotinib. By applying the ClonTracer barcoding system, we were able to show the presence of pre-existing sub-populations in HCC827 that contribute to erlotinib resistance. Prior studies implicated that MET amplification confers resistance to erlotinib in this cell line. Therefore we examined the effects of the c-Met inhibitor crizotinib on the barcoded HCC827 population when treated either sequentially or simultaneously with both inhibitors. Despite the significant reduction in barcode complexity, the erlotinib/crizotinib combination treatment failed to eradicate all of the resistant clones implying the presence of an erlotinib/crizotinib dual resistant subpopulation. We performed transcriptome profiling (RNA-seq) to elucidate the potential resistance mechanisms of the dual resistant subpopulation in comparison to vehicle-treated or single agent erlotinib-resistant HCC827 cell populations as controls.
Project description:Given the recent reports on the role of AXL in mediating resistance to EGFR-targeted therapy, we generated cell line models of Erlotinib-resistance to investigate the effect of AXL inhibitors on EGFR TKI resistance. For this, EGFR-mutant PC9 cells were passed through a persister bottleneck by applying strong drug selection pressure to generate drug-tolerant erlotinib persister cells. We created four Erlotinib-resistant clones from one parental population; S1-34, S2-10, S2-17 and S2-30. Whole exome and RNA sequencing analyses were performed to probe the differences in Erlotinib-resistance mechanisms present in these persister-derived Erlotinib-resistant cells.
Project description:We characterized the gene expression profile of Epithelial Growth Factor Receptor (EGFR) inhibitor (Erlotinib)-sensitive and resistant human NSCLC cell lines. Total RNA was extracted from the cell lines and expression profiles were studied by Agilent microarray analysis. Wide changes in gene expression profiles occur in the Erlotinib-resistant cell lines when compared with their parental cell lines (HCC827 and HCC4006).
Project description:This study aims to identify new genes and pathways associated with erlotinib sensitivity in order to develop novel therapeutic strategies. Here, we induced artificial knock-out (KO) mutations in erlotinib-resistant human lung cancer cells (NCI-H820) using a genome-scale CRISPR-Cas9 sgRNA library to screen for genes involved in erlotinib susceptibility.
Project description:Microarray expression analysis to identify global changes in transcription in response to RAF inhibition. Genes under RAF control were identified in a panel of BRAFV600E tumor cells, following the short-term inhibition of RAF using a pan-RAF kinase inhibitor, PLX4032 (Plexxikon). For comparison with changes in gene expression in response to MEK inhibition using PD0325901 (Pfizer), the following array data was referenced: (http://www.ncbi.nlm.nih.gov/geo/ (accession no. GSE10086)). Cell lines growing in culture (n=5) were treated with the RAF inhibitor PLX4032 (250nM or 1000nM) or vehicle alone (0.1% DMSO) as control, for eight hours.
Project description:Resistance to tyrosine kinase inhibitors (TKIs) presents a growing challenge in the development of therapeutic targets for cancers such as triple negative breast cancer (TNBC), where conventional therapies are ineffective for combatting systemic disease. Due to increased expression, the receptor tyrosine kinases EGFR (epidermal growth factor receptor) and c-Met are potential targets for treatment. However, targeted anti-EGFR and anti-c-Met therapies have faced mixed results in clinical trials due to acquired resistance. We hypothesize that the upregulation of kinases within the EGFR and c-Met signaling axes contribute to the development of acquired erlotinib and cabozantinib resistance. To test this, we developed two separate models for cabozantinib and erlotinib resistance using the MDA-MB-231 and MDA-MB-468 cell lines, respectively. With a SILAC (Stable Isotope Labeling of Amino acids in Cell Cul-ture)-labeled quantitative mass spectrometry proteomics approach, we assessed the effects of erlotinib- or cabozantinib resistance on the phosphoproteome, proteome and kinome. Using this integrated proteomics approach, we identified several potential kinase mediators of cabozan-tinib-resistance and confirmed the contribution of AKT1 to erlotinib-resistance in TNBC resistant cell lines.
Project description:The non-small cell lung carcinoma (NSCLC) PC9 cell line is an established preclinical model for tyrosine kinase inhibitors. To be able to better understand the differences in response between individual cells, we performed treatment of PC9 cells grown in cell culture with etoposide, erlotinib and its combination with crizotinib, followed by Drop-seq. The addition of crizotinib was guided by our previous data that an erlotinib-resistant drug population may be sensitive to crizotinib. To better understand the common events in drug resistance, we compared the resistant cell populations arising from the treatment with etoposide and from the treatment with erlotinib. The results of our study will address emerging drug resistance that limits clinical usefulness of conventional and targeted strategies, particularly in NSCLC.