Dataset Information

A quantitative phosphoproteomic analysis to study the dynamics of tyrosine phosphorylation in EGFR signaling abrogated by TKIs

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lung

DISEASE(S): Lung Cancer

SUBMITTER: Udayan Guha

LAB HEAD: Udayan Guha

PROVIDER: PXD004373 | Pride | 2017-03-29

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	11-18_3s_Erlot_pY_R1.raw	Raw
	11-18_3s_Erlot_pY_R1a.raw	Raw
	11-18_3s_Erlot_pY_R2.raw	Raw
	11-18_3s_Erlot_pY_R2a.raw	Raw
	11-18_3s_Erlotinib.zip	Other

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Publications

Quantitative Tyrosine Phosphoproteomics of Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor-treated Lung Adenocarcinoma Cells Reveals Potential Novel Biomarkers of Therapeutic Response.

Zhang Xu X Maity Tapan T Kashyap Manoj K MK Bansal Mukesh M Venugopalan Abhilash A Singh Sahib S Awasthi Shivangi S Marimuthu Arivusudar A Charles Jacob Harrys Kishore HK Belkina Natalya N Pitts Stephanie S Cultraro Constance M CM Gao Shaojian S Kirkali Guldal G Biswas Romi R Chaerkady Raghothama R Califano Andrea A Pandey Akhilesh A Guha Udayan U

Molecular & cellular proteomics : MCP 20170322 5

Mutations in the Epidermal growth factor receptor (EGFR) kinase domain, such as the L858R missense mutation and deletions spanning the conserved sequence <sup>747</sup>LREA<sup>750</sup>, are sensitive to tyrosine kinase inhibitors (TKIs). The gatekeeper site residue mutation, T790M accounts for around 60% of acquired resistance to EGFR TKIs. The first generation EGFR TKIs, erlotinib and gefitinib, and the second generation inhibitor, afatinib are FDA approved for initial treatment of EGFR mutat ...[more]

PMID: 28331001

Similar Datasets

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.

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-M-NM-:B (NF-M-NM-:B) 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-M-NM-:B-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. RNA was isolated from cells treated with 1 M-NM-<M erlotinib, 3 M-NM-<M or 5 M-NM-<M quinacrine or a combination of both for 6, 12, 24, or 48 h using the RNeasy Mini Kit (QIAGEN) according to the manufacturerM-bM-^@M-^Ys instructions. Microarray analysis was performed using the Affymetrix Human Gene 2.1 ST Array at the Gene Expression & Genotyping Core Facility at Case Comprehensive Cancer Center. Raw CEL files were pre-processed using the Affymetrix Expression Console Software 1.30 with Robust Multi-array Average (RMA) normalization (background correction, quantile normalization and log2 transformation). Probes were annotated using the HuGene 2.1 st hg19 probeset annotation files downloaded from the Affymetrix website.

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:Lung cancer continues to be the leading cause of cancer mortality worldwide. The treatment of lung cancer patients harboring mutant EGFR with orally administered EGFR TKIs has been a paradigm shift. Osimertinib and rociletinib are two 3rd generation irreversible EGFR TKIs targeting the EGFR T790M mutation. Osimertinib is the current standard care for patients with EGFR mutations due to increased efficacy, lower side effects, and enhanced brain penetrance. Unfortunately, all patients develop resistance to it. Genomic approaches have primarily been used to interrogate resistance mechanisms. Here, we have characterized the proteome and phosphoproteome of a series of isogenic EGFR mutant lung adenocarcinoma cell lines that are either sensitive or resistant to these drugs. To our knowledge, this is the most comprehensive proteomic dataset resource to date to investigate 3rd generation EGFR TKI resistance in lung adenocarcinoma. We have interrogated this unbiased global quantitative proteomic and phosphoproteomic dataset to uncover alterations in signaling pathways, and to reveal a proteomic signature of EMT and kinases / phosphatases with altered protein expression and phosphorylation in the TKI resistant cells. We validated the significant role of SHP2 in the activation of RAS/MAPK and PI3K/AKT signaling pathways. Furthermore, we performed anticorrelation analyses of this phosphoproteomic dataset with the published drug-induced P100 phosphoproteomic datasets from the Library of Integrated Network-Based Cellular Signatures (LINCS) program to predict drugs with the potential to overcome EGFR TKI resistance. We identified that dactolisib, a PI3K/mTOR inhibitor, in combination with osimertinib, may overcome osimertinib resistance both in vitro and in vivo. Introduction Lung cancer continues to be the leading cause of cancer mortality in the world (1). Many lung adenocarcinoma patients with activating epidermal growth factor receptor (EGFR) mutations initially respond dramaticlly to the first- or second-generation EGFR tyrosine kinase inhibitors (TKIs). However, they eventually develop resistance. The most common mechanism of acquired resistance is the EGFR T790M gatekeeper site residue mutation (2). Osimertinib, a third generation irreversible EGFR TKI has been approved by the FDA to treat patients harboring the EGFR T790M mutation who have developed resistance to first- and second- generation EGFR TKIs (3). Recently, osimertinib was also approved for the front-line treatment of patients harboring EGFR mutations (4). Rociletinib is another irreversible inhibitor targeting the EGFR T790M mutation, which has minimal activity against wild-type EGFR. Both drugs have therapeutic benefits and have demonstrated activity in tumors with T790M-mediated resistance to other EGFR tyrosine kinase inhibitors (5, 6). Further development of rociletinib was ceased in 2016 due to less than expected efficacy, poor brain penetration leading to tumor progression in brain tissues and off-target effects on IGFR activation leading to hyperglycemia (7, 8). Although 3rd-generation TKIs provide clinical benefit to most patients with EGFR mutations, some patients, demonstrating primary resistance, still do not respond to these inhibitors. Complete responses are rare, and all patients eventually develop resistance, suggesting primary and acquired resistance mechanisms decrease the efficacy of the drugs (9, 10). Genomic approaches have been used primarily to interrogate osimertinib resistance mechanisms (9, 11-15). Several mechanisms of osimertinib resistance have been identified (16), including novel second site EGFR mutations, activated bypass pathways such as MET amplification, HER2 amplification, RAS mutations, BRAF mutations, PIK3CA mutations, and novel fusion events (17). However, the resistance mechanism is complex and still not fully understood. Previously, we have used SILAC-based quantitative phosphoproteomics to identify the global dynamic modification which occur upon treatment of TKI-sensitive and -resistant lung adenocarcinoma cells with the 1st and 2nd generation EGFR TKIs, erlotinib and afatinib. Utilizing this strategy, we identified the targets of mutant EGFR signaling pathways responsible for TKI resistance, and possible off-target effects of the drugs (18, 19). In this study, we employed SILAC-based quantitative mass spectrometry to characterize alterations in the proteome and phosphoproteome which occur upon acquired resistance and sought to identify novel mechanisms of resistance to the third generation EGFR TKIs, osimertinib and rociletinib. To our knowledge, this is the most comprehensive 3rd generation EGFR TKI resistant proteome and phosphoproteome analysis resource available to date.