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Functional analysis of receptor tyrosine kinase mutations in lung cancer identifies oncogenic extracellular domain mutations of ERBB2.

ABSTRACT: We assessed somatic alleles of six receptor tyrosine kinase genes mutated in lung adenocarcinoma for oncogenic activity. Five of these genes failed to score in transformation assays; however, novel recurring extracellular domain mutations of the receptor tyrosine kinase gene ERBB2 were potently oncogenic. These ERBB2 extracellular domain mutants were activated by two distinct mechanisms, characterized by elevated C-terminal tail phosphorylation or by covalent dimerization mediated by intermolecular disulfide bond formation. These distinct mechanisms of receptor activation converged upon tyrosine phosphorylation of cellular proteins, impacting cell motility. Survival of Ba/F3 cells transformed to IL-3 independence by the ERBB2 extracellular domain mutants was abrogated by treatment with small-molecule inhibitors of ERBB2, raising the possibility that patients harboring such mutations could benefit from ERBB2-directed therapy.

SUBMITTER: Greulich H

PROVIDER: S-EPMC3437859 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Functional analysis of receptor tyrosine kinase mutations in lung cancer identifies oncogenic extracellular domain mutations of ERBB2.

Greulich Heidi H Kaplan Bethany B Mertins Philipp P Chen Tzu-Hsiu TH Tanaka Kumiko E KE Yun Cai-Hong CH Zhang Xiaohong X Lee Se-Hoon SH Cho Jeonghee J Ambrogio Lauren L Liao Rachel R Imielinski Marcin M Banerji Shantanu S Berger Alice H AH Lawrence Michael S MS Zhang Jinghui J Pho Nam H NH Walker Sarah R SR Winckler Wendy W Getz Gad G Frank David D Hahn William C WC Eck Michael J MJ Mani D R DR Jaffe Jacob D JD Carr Steven A SA Wong Kwok-Kin KK Meyerson Matthew M

Proceedings of the National Academy of Sciences of the United States of America 20120820 36

We assessed somatic alleles of six receptor tyrosine kinase genes mutated in lung adenocarcinoma for oncogenic activity. Five of these genes failed to score in transformation assays; however, novel recurring extracellular domain mutations of the receptor tyrosine kinase gene ERBB2 were potently oncogenic. These ERBB2 extracellular domain mutants were activated by two distinct mechanisms, characterized by elevated C-terminal tail phosphorylation or by covalent dimerization mediated by intermolecu ...[more]

PMID: 22908275

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Project description:Most adult patients have a D816V mutation in phosphotransferase domain (PTD), we have described that half of the children carry mutations in extracellular domain (ECD). KIT-ECD versus IT-PTD-mutants were introduced into rodent Ba/F3, EML, Rat2 and human TF1 cells to investigate their biological effect. ECD- and PTD-mutants also displayed distinct whole-genome transcriptional profiles in EML cells. We observed differences in their signaling properties: they both activated STAT pathways, whereas AKT pathway was only activated by ECD-mutants. Consistently, AKT inhibitor suppressed ECD-mutant-dependent proliferation, clonogenicity and erythroid differentiation. Expression of myristoylated AKT restored erythroid differentiation in EMLPTD cells, suggesting the differential role of AKT in those mutants. Overall, our study implied different pathogenesis of pediatric versus adult mastocytosis, which might explain their diverse phenotypes. Each EML cell line (Del417-419insY mutant and D816V mutant) was cultured with or without 250 ng/mL SCF for 48h. Gene expression profile analysis was performed using whole-genome microarrays. RNA expression profiling of cell lines was done with Affymetrix M430 2.0 mouse oligonucleotide microarrays containing 45.101 probe sets, representing 21.408 transcripts and variants including 17.482 well-characterized mouse genes. Preparation of cRNA, hybridizations, washes, detection and quantification were done as recommended by the supplier. For each sample, synthesis of the first-strand cDNA was done from 3 μg total RNA by T7-oligo(dT) priming, followed by second-strand cDNA synthesis. After purification, in vitro transcription associated with amplification generated cRNA-containing biotinylated pseudouridine. Biotinylated cRNA was purified, quantified and chemically fragmented (95°C for 35 min), then hybridized to microarrays in 200 μL hybridization buffer at 45°C for 16 h. Automated washes and staining with streptavidin-phycoerythrin were done as recommended. Signal amplification was done by biotinylated antistreptavidin antibody with goat-IgG blocking antibody. Scanning was done with Affymetrix GeneArray scanner and quantification with Affymetrix GCOS software.

Dataset Information

Functional analysis of receptor tyrosine kinase mutations in lung cancer identifies oncogenic extracellular domain mutations of ERBB2.

Publications

Functional analysis of receptor tyrosine kinase mutations in lung cancer identifies oncogenic extracellular domain mutations of ERBB2.

Similar Datasets

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