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H1299 EGF and Iressa stimulation

ABSTRACT: Controlled activation of epidermal growth factor receptor (EGFR) is systematically guaranteed at the molecular level, however aberrant activation of EGFR is frequently found in cancer. Transcription induced by EGFR activation often involves coordinated expression of genes that positively and negatively regulate the original signaling pathway, therefore alterations in EGFR kinase activity may reflect changes in gene expression associated with the pathway. In this study, we investigated transcriptional changes following EGF stimulation with or without the EGFR kinase inhibitor Iressa in H1299 human non-small-cell lung cancer cells (parental H1299, H1299 cells which overexpress wild-type: EGFR-WT and mutant EGFR: L858R). Our results clearly showed differences in transcriptional activity in the absence or presence of EGFR kinase activity, and genes sharing the same molecular functions showed distinct expression dynamics. The results showed particular enrichment of EGFR/ErbB signaling-related genes in a differentially expressed gene set, and significant protein expression of MIG6/RALT(ERRFI1), an EGFR negative regulator, was confirmed in L858R. High MIG6 protein expression was correlated with basal EGFR phosphorylation and inversely correlated with EGF-induced ERK phosphorylation levels. Investigation of NCI-60 cell lines showed that ERRFI1 expression was correlated with EGFR expression regardless of tissue type. These results suggest that cells accumulate MIG6 as an inherent negative regulator to suppress excess EGFR activity when basal EGFR kinase activity is considerably high. Taken together, an EGFR mutation can cause transcriptional changes to accommodate the activation potency of the original signaling pathway at the cellular level.

ORGANISM(S): Homo sapiens

PROVIDER: GSE11729 | GEO | 2009/07/30

SECONDARY ACCESSION(S): PRJNA106031

REPOSITORIES: GEO

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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: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.

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.

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H1299 EGF and Iressa stimulation

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