Oncogenic epidermal growth factor receptor mutants with tandem duplication: gene structure and effects on receptor function

Ciesielski, Michael; Fenstermaker, Robert A.

doi:10.1038/sj.onc.1203409

Cited by 39 publications

(35 citation statements)

References 28 publications

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“…Slow-migrating forms of EGFR were previously reported in glioma cells as being caused by tandem duplication of exons in the extracellular or intracellular domains of EGFR (18)(19)(20)(21)(22). To determine whether the slow-migrating form of EGFR in DiFi5 cells may reflect EGFR mutation such as tandem duplication of exons, we used reverse transcription-PCR to amplify EGFR cDNA from DiFi and DiFi5 cells with three individual pairs of primers that collectively cover the whole range of EGFR-coding sequences.…”

Section: Resultsmentioning

confidence: 99%

Epidermal Growth Factor Receptor (EGFR) Ubiquitination as a Mechanism of Acquired Resistance Escaping Treatment by the Anti-EGFR Monoclonal Antibody Cetuximab

et al. 2007

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Cetuximab is an epidermal growth factor receptor (EGFR)-blocking antibody that has been approved for treatment of patients with metastatic colorectal cancer. In this study, we investigated biochemical changes in signaling pathways of a cetuximab-resistant subline of DiFi colorectal cancer cells (DiFi5) that was developed by exposing the parental sensitive cells to subeffective doses of cetuximab over an extended period of time. Compared with parental DiFi cells that express high levels of EGFR and in which cetuximab induces apoptosis, the cetuximab-resistant DiFi5 cells showed markedly lower protein levels of EGFR, an increased association of EGFR with Cbl, and an increased ubiquitination of EGFR. DiFi5 cells also had a markedly higher level of Src-Y416 phosphorylation both at baseline and on EGF stimulation. Although EGFR levels were low, DiFi5 cells responded to EGF stimulation with robust phosphorylation of EGFR on Y845 and strong phosphorylation of Akt and extracellular signalregulated kinase, comparable to those of parental cells. Most importantly, inhibition of Src kinase activity with PP2 reversed the resistance of DiFi5 cells to cetuximab-induced apoptosis without affecting the levels of EGFR in the cells. Our results indicate that colorectal cancer cells may develop acquired resistance to cetuximab via altering EGFR levels through promotion of EGFR ubiquitination and degradation and using Src kinase-mediated cell signaling to bypass their dependency on EGFR for cell growth and survival. [Cancer Res 2007;67(17):8240-7]

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Section: Resultsmentioning

confidence: 99%

Epidermal Growth Factor Receptor (EGFR) Ubiquitination as a Mechanism of Acquired Resistance Escaping Treatment by the Anti-EGFR Monoclonal Antibody Cetuximab

et al. 2007

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“…Tandem duplications of EGFR might also account for our results. These have been reported for EGFR (31), for other regions on 7q (32), and for loci on other chromosomes 7 in glioma cell lines. FISH may not be able to detect this type of genetic aberration because the extra copy could be juxtaposed to its parent, and Southern blotting may not be sensitive enough to detect the copy number difference.…”

Section: Genetic Classification Of Glioblastoma Multiformementioning

confidence: 99%

Array Comparative Genomic Hybridization Identifies Genetic Subgroups in Grade 4 Human Astrocytoma

Misra

Pellarin

Nigro

et al. 2005

Clinical Cancer Research

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Alterations of DNA copy number are believed to be important indicators of tumor progression in human astrocytoma. We used an array of bacterial artificial chromosomes to map relative DNA copy number in 50 primary glioblastoma multiforme tumors at f1.4-Mb resolution.We identified 33 candidate sites for amplification and homozygous deletion in these tumors.We identified three major genetic subgroups within these glioblastoma multiforme tumors: tumors with chromosome 7 gain and chromosome10 loss, tumors with only chromosome 10 loss in the absence of chromosome 7 gain, and tumors without copy number change in chromosomes 7 or 10. The significance of these genetic groups to therapeutics needs further study.Studies suggest that astrocytic brain tumor behavior is related to genetic defects acquired over the course of tumor development (1 -6). It is believed that understanding these genetic defects will help predict response to treatment and patient outcome. Previous studies using comparative genomic hybridization (CGH; refs. 7, 8) related radiation sensitivity of tumors and patients' survival to copy number aberrations (CNA) on several chromosomes (2, 3). This report describes CNAs identified on a bacterial artificial chromosome (BAC) array of f1.4-Mb resolution [array comparative genomic hybridization (aCGH)], in a set of 50 glioblastoma multiforme (GBM).We compared specificity and sensitivity of the chromosome CGH and aCGH techniques by comparing array and chromosome CGH in the same tumor samples, by counting fluorescence in situ hybridization (FISH) signals generated by BAC clones used in the array, and by using real-time quantitative PCR. The results obtained by aCGH confirmed other characterizations of the GBM genome.Analysis of high-resolution aCGH data identified genetic subgroups in this set of GBM and loci for candidate oncogenes and tumor suppressor genes, many of which were previously unknown. We expect that future higher resolution studies of these regions will lead to identification of target genes for therapy. Materials and MethodsCell lines. The cell lines used in this study (SF767, SF126, U343MG, and SF210) were obtained from the Tissue Bank at the Brain Tumor Research Center, University of California, San Francisco (UCSF; ref. 9). Cells were cultured in minimal essential medium with Earle's buffered salt solution supplemented with 10% FCS and nonessential amino acids.Tumor DNA. Fifty tumor samples (GBM, grade 4 astrocytoma) were obtained from the tissue bank at the Brain Tumor Research Center, UCSF. All patients signed consent for specimens to be used for research purposes. The samples were originally chosen for evaluation using chromosome CGH (8). Tumor specimens were snap-frozen in liquid nitrogen immediately after resection and stored at À80jC. Tumors were diagnosed by the Division of Neuropathology at UCSF according to WHO classification (10). Sections on both sides contiguous to the processed specimens were histologically assessed to determine the percentage of tumor cells present in the ...

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“…by guest www.bloodjournal.org From and duration of intracellular signaling events to attain the appropriate cellular response. [27][28][29] Lack or gain of function can lead to uncontrolled cell growth and malignancies. 30 Although negative regulatory mechanisms have been extensively studied for several different membrane receptors, the mechanisms of regulation of activated c-Mpl after Tpo stimulation, in particular those of receptor degradation, are largely unknown.…”

Section: Discussionmentioning

confidence: 99%

Ubiquitination and degradation of the thrombopoietin receptor c-Mpl

Saur

Sangkhae

Geddis

et al. 2010

Blood

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Regulation of growth factor and cytokine signaling is essential for maintaining physiologic numbers of circulating hematopoietic cells. Thrombopoietin (Tpo), acting through its receptor c-Mpl, is required for hematopoietic stem cell maintenance and megakaryopoiesis. Therefore, the negative regulation of Tpo signaling is critical in many aspects of hematopoiesis. In this study, we determine the mechanisms of c-Mpl degradation in the negative regulation of Tpo signaling. We found that, after Tpo stimulation, c-Mpl is degraded by both the lysosomal and proteasomal pathways and c-Mpl is rapidly ubiquitinated. Using site-directed mutagenesis, we were able to determine that c-Mpl is ubiquitinated on both of its intracellular lysine (K) residues (K 553 and K 573 ). By mutating these residues to arginine, ubiquitination and degradation were significantly reduced and caused hyperproliferation in cell lines expressing these mutated receptors. Using short interfering RNA and dominant negative overexpression, we also found that c-Cbl, which is activated by Tpo, acts as an E3 ubiquitin ligase in the ubiquitination of c-Mpl. Our findings identify a previously unknown negative regulatory pathway for Tpo signaling that may significantly impact our understanding of the mechanisms affecting the growth and differentiation of hematopoietic stem cells and megakaryocytes. (Blood. 2010;115: 1254-1263) IntroductionHematopoiesis is tightly regulated by several cytokines and growth factors to ensure that numbers of circulating blood cells remain constant under normal conditions. In many hematologic disorders, cytokine and growth factor signaling is dysfunctional, resulting in the overproduction or underproduction of 1 or more blood cell lineages. Thrombopoietin (Tpo) is a hematopoietic cytokine that, via its receptor c-Mpl, supports hematopoietic stem cell maintenance and proliferation and is the primary regulator of megakaryopoiesis. 1,2 Absence of Tpo signaling results in thrombocytopenia, reduced numbers of transplantable stem cells, and eventually aplastic anemia in humans. [3][4][5] Conversely, excessive Tpo signaling, usually due to mutations in c-Mpl or its secondary signaling proteins, results in hyperproliferation of numerous cell lineages, causing myeloproliferative syndromes. 6-8 Therefore, the control of Tpo-mediated signaling is critical in maintaining physiologic numbers of circulating blood cells.Protein phosphatases, suppressors of cytokine signaling (SOCS) proteins, and inhibitory intracellular mediators are all mechanisms that contribute to the negative regulation of cytokine signaling. [9][10][11][12] However, the process of receptor internalization and degradation is one of the quickest and most effective ways in which activated receptors are negatively regulated. We recently demonstrated a mechanism for Tpo-stimulated c-Mpl internalization, through the interaction of adaptor protein 2 with YRRL motifs located at Y 521 and Y 591 in the c-Mpl intracellular domain; elimination of these sites significantly reduced degrad...

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Oncogenic epidermal growth factor receptor mutants with tandem duplication: gene structure and effects on receptor function

Cited by 39 publications

References 28 publications

Epidermal Growth Factor Receptor (EGFR) Ubiquitination as a Mechanism of Acquired Resistance Escaping Treatment by the Anti-EGFR Monoclonal Antibody Cetuximab

Epidermal Growth Factor Receptor (EGFR) Ubiquitination as a Mechanism of Acquired Resistance Escaping Treatment by the Anti-EGFR Monoclonal Antibody Cetuximab

Array Comparative Genomic Hybridization Identifies Genetic Subgroups in Grade 4 Human Astrocytoma

Ubiquitination and degradation of the thrombopoietin receptor c-Mpl

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