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

<p>Abstract</p> <p>Background</p> <p>The presence of EGFR kinase domain mutations in a subset of NSCLC patients correlates with the response to treatment with the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Although most EGFR mutations detected are short deletions in exon 19 or the L858R point mutation in exon 21, more than 75 different EGFR kinase domain residues have been reported to be altered in NSCLC patients. The phenotypical consequences of different EGFR mutations may vary dramatically, but the majority of uncommon EGFR mutations have never been functionally evaluated.</p> <p>Results</p> <p>We demonstrate that the relative kinase activity and erlotinib sensitivity of different EGFR mutants can be readily evaluated using transfection of an YFP-tagged fragment of the EGFR intracellular domain (YFP-EGFR-ICD), followed by immunofluorescence microscopy analysis. Using this assay, we show that the exon 20 insertions Ins770SVD and Ins774HV confer increased kinase activity, but no erlotinib sensitivity. We also show that, in contrast to the common L858R mutation, the uncommon exon 21 point mutations P848L and A859T appear to behave like functionally silent polymorphisms.</p> <p>Conclusion</p> <p>The ability to rapidly obtain functional information on EGFR variants of unknown relevance using the YFP-EGFR-ICD assay might prove important in the future for the management of NSCLC patients bearing uncommon EGFR mutations. In addition, our assay may be used to determine the response of resistant EGFR mutants to novel second-generation TKIs.</p

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

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-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-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