Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain-1

<p><b>Copyright information:</b></p><p>Taken from "Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain"</p><p>http://www.molecular-cancer.com/content/6/1/56</p><p>Molecular Cancer 2007;6():56-56.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2064929.</p><p></p> by immunofluorescence to detect phosphorylated Akt (pAkt). Phosphorylation of endogenous Akt was only detected in cells expressing the Del746-bearing protein. B. A similar analysis was carried out to detect phosphorylated ERK (pERK). Only cells expressing YFP-EGFR-ICD Del746 contained detectable levels of endogenous pERK. Exposure time is indicated inside the panels. DNA was counterstained with Hoechst. C. Images (400X) illustrate two morphological characteristics of Akt phosphorylation in cells expressing YFP-EGFR-ICD Del746. On one hand, pAkt showed a preferential localization to membrane ruffles, and often accumulated at the tip of lamellipodial protrusions (arrowhead). On the other hand, cells expressing high (cell#1) or low (cell#2) levels of YFP-EGFR-ICD Del746, often contained similar levels of pAkt

Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain-2

<p><b>Copyright information:</b></p><p>Taken from "Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain"</p><p>http://www.molecular-cancer.com/content/6/1/56</p><p>Molecular Cancer 2007;6():56-56.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2064929.</p><p></p> endogenous phosphorylated Akt (red). Cells were treated for 20 hours with the indicated concentration of erlotinib. The different response of each EGFR variant to erlotinib treatment is readily visualized by immunoflourescence. YFP-EGFR-ICD wild type does not induce Akt phosphorylation, and relocates into thick cytoplasmic fibrils at 10 μM erlotinib. One thousand-fold lower concentration of the drug (10 nM) inhibited Del746-induced Akt phosphorylation, and caused fibrilar relocation of the ectopic protein. The double mutant Del746/T790M did not form fibrils and induced Akt phosphorylation even in the presence of 10 μM erlotinib. . Images show that YFP-EGFR-ICD Del746 (green) remains phosphorylated at Y1092 (red) after relocating into fibrils in the presence of erlotinib

Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain-0

<p><b>Copyright information:</b></p><p>Taken from "Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain"</p><p>http://www.molecular-cancer.com/content/6/1/56</p><p>Molecular Cancer 2007;6():56-56.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2064929.</p><p></p>he signal peptide. YFP-EGFR-ICD contains the tyrosine kinase (TK) domain and part of the regulatory region (Reg), but lacks the extracellular and juxtamembrane (JM) domains. . Expression of YFP-EGFR-ICD Del746 induces morphological changes in MCF-7 cells. Unlike cells transfected with wild type (WT) YFP-EGFR-ICD, MCF-7 cells expressing YFP-EGFR-ICD Del746 frequently show long lamellipodial protrusions (arrowheads). Using immunofluorescence, increased autophosphorylation of YFP-EGFR-ICD Del746 at tyrosine residues Y869 (left set of panels) and Y1092 (right set of panels) can be detected. Phosphorylation is virtually undetectable in cells expressing YFP-EGFR-ICD WT. Images were taken using 160 × magnification and the exposure time indicated inside the panels. The fluorescent signal was consistently brighter using the anti-pY1092 antibody (note the shorter exposure time used). . Semi-quantitative comparison of YFP-EGFR-ICD autophosphorylation level using computer-assisted image analysis. Images of several transfected cells (400 × magnification) were taken using 40 ms (YFP) or 160 ms (AF-594) exposure times. The fluorescence intensity in the green and the red channels was measured within a cytoplasmic area (YFP signal and AF-594 signal), and within an area outside the cells (background). In the graph, the intensity of the YFP and AF-594 fluorophores for each cell was plotted against each other using Excel, and the best-fitting trend lines (highest R) were added. At similar expression levels (YFP intensity), the level of pY1092 is higher for YFP-EGFR-ICD bearing the Del746 mutation (white squares) than for the wild type protein (circles). The V948R mutation (open triangles) virtually abrogated autophosphorylation. The experiment was repeated twice with similar results. Graph shows the data from one experiment. au: arbitrary units

Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain-4

<p><b>Copyright information:</b></p><p>Taken from "Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain"</p><p>http://www.molecular-cancer.com/content/6/1/56</p><p>Molecular Cancer 2007;6():56-56.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2064929.</p><p></p>he signal peptide. YFP-EGFR-ICD contains the tyrosine kinase (TK) domain and part of the regulatory region (Reg), but lacks the extracellular and juxtamembrane (JM) domains. . Expression of YFP-EGFR-ICD Del746 induces morphological changes in MCF-7 cells. Unlike cells transfected with wild type (WT) YFP-EGFR-ICD, MCF-7 cells expressing YFP-EGFR-ICD Del746 frequently show long lamellipodial protrusions (arrowheads). Using immunofluorescence, increased autophosphorylation of YFP-EGFR-ICD Del746 at tyrosine residues Y869 (left set of panels) and Y1092 (right set of panels) can be detected. Phosphorylation is virtually undetectable in cells expressing YFP-EGFR-ICD WT. Images were taken using 160 × magnification and the exposure time indicated inside the panels. The fluorescent signal was consistently brighter using the anti-pY1092 antibody (note the shorter exposure time used). . Semi-quantitative comparison of YFP-EGFR-ICD autophosphorylation level using computer-assisted image analysis. Images of several transfected cells (400 × magnification) were taken using 40 ms (YFP) or 160 ms (AF-594) exposure times. The fluorescence intensity in the green and the red channels was measured within a cytoplasmic area (YFP signal and AF-594 signal), and within an area outside the cells (background). In the graph, the intensity of the YFP and AF-594 fluorophores for each cell was plotted against each other using Excel, and the best-fitting trend lines (highest R) were added. At similar expression levels (YFP intensity), the level of pY1092 is higher for YFP-EGFR-ICD bearing the Del746 mutation (white squares) than for the wild type protein (circles). The V948R mutation (open triangles) virtually abrogated autophosphorylation. The experiment was repeated twice with similar results. Graph shows the data from one experiment. au: arbitrary units

Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain-3

<p><b>Copyright information:</b></p><p>Taken from "Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain"</p><p>http://www.molecular-cancer.com/content/6/1/56</p><p>Molecular Cancer 2007;6():56-56.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2064929.</p><p></p>ers). . Kinase activity and erlotinib sensitivity of different exon 20 mutations. Graph shows that autophosphorylation levels are lower for T790M (white diamonds) than for S768I (white squares) or Ins770SVD (black triangles). Low expression levels hampered the accurate evaluation of Ins774HV (black circles). Images show that YFP-EGFR T790M did not effectively induce phosphorylation of endogenous Akt in MCF-7 cells, and did not relocate into fibrils upon erlotinib treatment. S768I-induced pAkt was inhibited by 100 nM erlotinib and the ectopic protein relocated into fibrils at 1 μM. The phosphorylation of Akt induced by exon 20 insertions was only inhibited at 10 μM erlotinib. This drug concentration also induced relocation of YFP-EGFR-ICD Ins770SVD into fibrils. . Kinase activity and erlotinib sensitivity of different exon 21 mutations. Graph shows that the common L858R mutation confers higher autophosphorylation levels to YFP-EGFR-ICD than P848L and A859T. Images show that, unlike L858R, these uncommon exon 21 mutants did not induce phosphorylation of endogenous Akt. Erlotinib blocked L858R-induced pAkt at 10 nM, and caused relocation of the ectopic protein into fibrils at 100 nM. Both effects were readily abrogated by the TKI-resistant mutation T790M. In all cases, data corresponding to one experiment are shown. Each EGFR mutant was tested at least twice with similar results

Summary of Complete Genomics sequencing results.

<p>+ Novel: not previously described in dbSNP131; ^IINDEL group includes insertion, deletion and substitutions.</p

Whole Genome and Transcriptome Sequencing of a B3 Thymoma

<div><p>Molecular pathology of thymomas is poorly understood. Genomic aberrations are frequently identified in tumors but no extensive sequencing has been reported in thymomas. Here we present the first comprehensive view of a B3 thymoma at whole genome and transcriptome levels. A 55-year-old Caucasian female underwent complete resection of a stage IVA B3 thymoma. RNA and DNA were extracted from a snap frozen tumor sample with a fraction of cancer cells over 80%. We performed array comparative genomic hybridization using Agilent platform, transcriptome sequencing using HiSeq 2000 (Illumina) and whole genome sequencing using Complete Genomics Inc platform. Whole genome sequencing determined, in tumor and normal, the sequence of both alleles in more than 95% of the reference genome (NCBI Build 37). Copy number (CN) aberrations were comparable with those previously described for B3 thymomas, with CN gain of chromosome 1q, 5, 7 and X and CN loss of 3p, 6, 11q42.2-qter and q13. One translocation t(11;X) was identified by whole genome sequencing and confirmed by PCR and Sanger sequencing. Ten single nucleotide variations (SNVs) and 2 insertion/deletions (INDELs) were identified; these mutations resulted in non-synonymous amino acid changes or affected splicing sites. The lack of common cancer-associated mutations in this patient suggests that thymomas may evolve through mechanisms distinctive from other tumor types, and supports the rationale for additional high-throughput sequencing screens to better understand the somatic genetic architecture of thymoma.</p> </div

Prediction of mutation effect on protein function.

<p>A coding sequence mutation can damage or preserve the function of the related protein. Several bioinformatics algorithms have been developed to predict the effect of mutations. We used 2 independent methods based on different assumptions. SIFT prediction is based on the degree of conservation of amino acid residues in sequence alignments derived from closely related species. Polyphen2 prediction is based on various sequence and structure-based features of the substitution site.</p

Candidate structural variations identified by complete genome sequencing.

<p>Structural variations such as large deletions, insertions inversions or translocations were predicted using whole genome sequencing data. Structural variations were hypothesized in the presence of a junction sequence: a hybrid sequence composed of two non-adjacent fragment of DNA. In the table are reported the candidate structural variations of this B3 thymoma: the extremity position of the 2 joint sequences and the strand of orientation. Among these candidates only the translocation t(11;X) was confirmed by PCR.</p

Features of the B3 thymoma.

<p>(A) Preoperative Chest CT-scan showing a mediastinal mass protruding in the left hemithorax. (B) Haematoxylin and Eosin staining of postoperative tumor section showing the lobular aspects typical of B3 thymomas, the almost complete absence of intratumoral thymocytes and the presence of cancer cells characterized by polygonal shape and round nuclei. (C) Whole-genome sequencing results showing (1) Sanger sequencing-confirmed SNVs and INDELs, (2) reference genomic coordinates, (3) copy number gain (green) and loss (red), and (4) structural variations.</p