Expression data from EGFR mutant transgenic mice
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE17373 | GEO | 2009/12/01
SECONDARY ACCESSION(S): PRJNA119103
REPOSITORIES: GEO
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Project description:We performed mRNA expression profiling of lung tumors from C/L858R, C/T790M, and C/L+T mice and from corresponding normal lung tissue. Experiment Overall Design: mRNA was extracted from pulverized lung samples using Trizol (Invitrogen) and then hybridized to MOE 430 2.0 chips (Affymetrix) using standard hybridization techniques.
2009-12-14 | E-GEOD-17373 | biostudies-arrayexpress
Project description:We previously reported that differential protein degradation of TKI-sensitive [L858R, del(E746-A750)] and resistant (T790M) epidermal growth factor receptor (EGFR) mutants upon erlotinib treatment correlates with drug sensitivity. However, the molecular mechanism remains unclear. We also reported SMAD ubiquitination regulatory factor 2 (SMURF2) as a stabilizer of EGFR in a ligase (E3) activity-dependent manner. Here, using in vitro and in vivo ubiquitination assays, mass spectrometry, and super-resolution microscopy, we show SMURF2-EGFR functional interaction is critical in receptor stability and TKI sensitivity. We found that L858R/T790M EGFR is a preferred substrate of SMURF2-UBCH5 (an E3-E2) complex-mediated K63-linked polyubiquitination, which preferentially stabilizes mutant receptor. We identified three lysine (K) residues (K721, 1037 and 1164) as the sites of ubiquitination and replacement of K to acetylation-mimicking asparagine (Q) at K1037 position in L858R/T790M background converts the stable protein sensitive to erlotinib-induced degradation. Using STochastic Optical Reconstruction Microscopy (STORM) imaging, we show that SMURF2 presence allows longer membrane retention of activated EGFR upon EGF treatment, whereas, siRNA-mediated SMURF2 knockdown fastens receptor endocytosis and lysosome enrichment. In an erlotinib-sensitive PC9 cells, SMURF2 overexpression increased EGFR levels with improved erlotinib tolerance, whereas, SMURF2 knockdown decreased EGFR steady state levels in NCI-H1975 and PC9-AR cells to overcome erlotinib and AZD-9291 resistance respectively. Additionally, disruption of the SMURF2-UBCH5 complex destabilized EGFR. Together, we propose that SMURF2-mediated preferential polyubiquitination of L858R/T790M EGFR may be competing with acetylation-mediated receptor internalization to provide enhanced receptor stability and that disruption of the E3-E2 complex may be an attractive alternate to overcome TKI resistance
2020-07-29 | PXD018324 | Pride
Project description:The purpose of this research is to study the cellular origins of EGFR-mutant lung cancers and their responses to therapies. We discovered that activation of EGFR T790M/L858R mutation in lung epithelial cells can drive lung cancers with alveolar or bronchiolar features, which can be originated from alveolar type 2 (AT2) cells or bronchioalveolar stem cells (BASCs), but not basal cells or club cells of the trachea. Crucially, the tumoroids with different cell-of-origins or epigenetic states had distinct drug vulnerabilities.
2021-09-18 | GSE180360 | GEO
Project description:Lung adenocarcinoma PC9 cells were engineered to not only contain the driving EGFR-mutations, but also EGFR T790M and EGFR C797S that provide resistance to EGFR inhibitors.
2021-07-19 | E-MTAB-9887 | biostudies-arrayexpress
Project description:Lung cancer is the leading cause of cancer death. Mutations in the kinase domain of EGFR, a predominant driver oncogene, such as L858R missense mutation and a series of deletions spanning the conserved sequence 747LREA750, are associated with sensitivity to tyrosine kinase inhibitors (TKIs). However, patients receiving EGFR-TKIs (gefitinib and erlotinib) develop drug-resistance due to a secondary mutation at the gatekeeper residue (T790M) in about 50-60% of cases, urging for new drug development. Afatinib, a FDA approved second-generation EGFR-TKI that was developed to circumvent T790M-mediated resistance, has not been very effective in clinical trials. In this study, we performed a global phosphoproteomic screen to identify targets that undergo mutant EGFR-dependent tyrosine phosphorylation and their modulation by erlotinib or afatinib. We undertook stable isotope labeling of amino acids in cell culture (SILAC), phosphopeptide enrichment, and quantitative mass spectrometry to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring L858R or L858R/T790M mutations and their modulation by erlotinib and afatinib inhibition. We identified and quantified 397, 429, 223, and 594 phosphotyrosine sites in H3255, 11-18,PC9, and H1975 cell lines that were grown in presence of FBS and in presence/absence of TKIs, respectively. These account for a total of 907 unique phospho-tyrosine sites in 496 proteins. Among them, 187 phosphotyrosine sites were found to be in 89 kinases, which may serve as intermediary regulatory kinases in EGFR signalling pathway. Further analysis indicated that in TKI-sensitive H3255 and 11-18 cells, there were 58/111 and 65/101 tyrosine sites that were hypophosphorylated in presence of erlotinib and afatinib, respectively. However, in TKI-resistant H1975 cells, 189 and 264 tyrosine sites were hypophosphorylated in presence of erlotinib and afatinib respectively, indicating that the afatinib-specific additional sites could be validated for identifying potentially new drug targets to counter TKI-resistance. Ingenuity pathway analysis (IPA) of proteins with altered phosphorylation sites demonstrated that several canonical pathways including ephrin receptor signalling and integrin signalling pathways were enriched, which may play important roles in cell growth and proliferation. However, upon EGF stimulation of serum starved H3255 cells in presence or absence of TKIs, 99 tyrosine sites that were hyperphosphorylated upon EGF stimulation were inhibited in presence of erlotinib or afatinib. But in H1975 cells treated with erlotinib, 48 of the above sites were either unchanged or were hyperphosphorylated. These sites include EGFR (Y1197/869/998), JAK1 (Y1034), FRK (Y497), GAB1 (Y657/689), MAPK1 (Y187), MAPK3 (Y204), MET (Y1252/1253). Furthermore, a total of 112 sites that were observed to be hypophosphorylated upon EGF stimulation in H1975 cells, were found to be hyperphosphorylated upon erlotinib inhibition. This could possibly be due to the activation of downstream phosphatases with EGF stimulation. We are now performing in-depth bioinformatic analysis and validation experiments using functional genomics to understand the role of targets of mutant EGFR signalling in lung cancer.
2017-03-29 | PXD004373 | Pride
Project description:Osimertinib, a third-generation EGFR-TKI, has applied to non-small cell lung cancer harboring activated EGFR mutation with or without T790M. However, the appearance of tumors resistant to osimertinib has been reported. We established and characterized osimertinib-resistant cells derived from NCI-H1975 cells harboring activating EGFR and T790M mutation.
2022-04-28 | GSE201608 | GEO
Project description:Non-small cell lung cancer (NSCLC) lung adenocarcinoma (LUAD) is a leading cause of death worldwide. Activating mutations in the tyrosine kinase domain of the oncogene epidermal growth factor receptor (EGFR) are responsible for ~10-50% of all LUAD cases. Although EGFR tyrosine kinase inhibitors (TKIs) have been effective in prolonging NSCLC patient survival and quality of life, acquired resistance mechanisms and disease progression are inevitable. Contemporary second- and third-line treatments, such as immunotherapy, remain ineffective for these patients, presenting a clear and unmet need for alternative or adjuvant therapeutics for the treatment of mutant EGFR positive NSCLC. Here we show that an anti-EGFR aptamer (EGFRapt) decreases viability of NSCLC cell lines harboring the L858R ± T790M mutation in EGFR but not cell lines harboring wild-type or exon 19 deletions. In a humanized xenograft mouse model, EGFRapt decreased tumor burden compared to controls when delivered intratumorally over multiple doses. To elucidate the mechanism by which EGFRapt exerts these effects, we examined kinase-dependent and kinase-independent mechanisms and found that it is cell line dependent, either inhibiting cellular proliferation or inducing cell death. Post hoc transcriptomics analysis comparing EGFRapt to control aptamer or vehicle validated these findings and provided additional mechanistic insights. Overall, these data establish that EGFRapt has direct anti-cancer activity in mutant EGFR positive NSCLC via targetable mechanisms that are independent of existing approaches and provide a foundation for further development of nucleic acid-based therapies that target EGFR.
2024-11-21 | GSE259407 | GEO
Project description:Erlotinib is a tyrosine kinase inhibitor (TKI) that is approved as a second-line monotherapy in patients with advanced non-small cell lung cancer (NSCLC). In these patients, erlotinib prolongs survival but its benefit remains modest since many tumors express wild-type EGF receptor (wtEGFR) lacking a TKI-sensitizing mutation, develop a second-site EGFR mutation, e.g., EGFR-L858R/T790M, or activate an alternate receptor tyrosine kinase, e.g., through MET amplification. To test potential drug combinations that could improve the efficacy of erlotinib, we combined erlotinib with quinacrine, which inhibits the FACT (facilitates chromatin transcription) complex that is required for nuclear factor-κB (NF-κB) transcriptional activity. In A549 (wtEGFR), H1975 (EGFR-L858R/T790M) and H1993 (MET amplification) NSCLC cells, the combination of erlotinib and quinacrine was highly synergistic, as quantified by Chou-Talalay combination indices. The combination inhibited colony formation, induced cell cycle arrest and apoptosis, and slowed xenograft tumor growth. Quinacrine decreased the level of active FACT subunit SSRP1 and suppressed NF-κB-dependent luciferase activity. Knockdown of SSRP1 decreased cell growth and sensitized cells to erlotinib. Transcriptomic profiling showed that quinacrine, alone or in combination with erlotinib, significantly affected cell cycle-related genes that contain binding sites for transcription factors that regulate SSRP1 target genes. As potential biomarkers of drug synergy, we identified genes that were more strongly suppressed by the combination than by either single treatment, and whose increased expression predicted poorer survival in lung adenocarcinoma patients. Combination of quinacrine and erlotinib is synergistic in NSCLC through suppression of FACT, resulting in inhibition of cell cycle progression.
2014-06-19 | GSE57422 | GEO
Project description:Mutations in the epidermal growth factor receptor (EGFR) kinase domain occur in 10-30% of lung adenocarcinoma. Leucine to arginine substitution at amino acid position 858 (L858R) accounts for around 50% of EGFR-tyrosine kinase inhibitor (TKI) sensitizing mutations. A second site mutation in the gatekeeper residue (T790M) accounts for around 60% of acquired resistance to EGFR-TKIs. We sought to identify the immediate direct and indirect targets of these mutant EGFRs in lung adenocarcinoma and their modulation by erlotinib, the first generation, and most widely used EGFR-directed TKI. We undertook stable isotope labeling of amino acids in cell culture (SILAC), phosphopeptide enrichment, and quantitative mass spectrometry to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring L858R or L858R/T790M mutations and their modulation by erlotinib inhibition. Phosphorylation at the majority of phosphosites identified exhibited no change upon either EGF stimulation or erlotinib inhibition. Only around 7% of phosphosites identified and quantified showed increased phosphorylation upon EGF stimulation in either cell line. However, while phosphorylation at 61% of these phosphosites decreased upon erlotinib inhibition in the TKI sensitive H3255 cells, only 24% of such sites exhibited decreased phosphorylation upon erlotinib inhibition in the TKI resistant H1975 cells. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not the resistant cells include EGFR, Insulin receptor, HGF, MAPK, mTOR, p70S6K and JAK/STAT signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutive phosphorylated in these lung adenocarcinoma cells, but phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinase families, AGC, CAMK and STE were significantly enriched and those of proline directed kinase families, CMGC and CK were significantly depleted among substrates that exhibit increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma.
2015-01-30 | PXD001101 | Pride
Project description:To identify gene expression changes associated with receptor tyrosine kinase (RTK) activation, we performed RNA-Seq analysis of murine pro-B BAF3 cells over-expressing the oncogenic form of EGFR (EGFR-L858R-T790M), FGFR (TEL-FGFR1 fusion), MET (TPR-MET fusion) or RET (CCDC6-RET fusion) genes. We reported the RTK-driven transcriptional reprogramming of metabolic genes.
2020-04-10 | GSE111292 | GEO