20 research outputs found
Oncogenic Transformation by Inhibitor-Sensitive and -Resistant EGFR Mutants
BACKGROUND: Somatic mutations in the kinase domain of the epidermal growth factor receptor tyrosine kinase gene EGFR are common in lung adenocarcinoma. The presence of mutations correlates with tumor sensitivity to the EGFR inhibitors erlotinib and gefitinib, but the transforming potential of specific mutations and their relationship to drug sensitivity have not been described. METHODS AND FINDINGS: Here, we demonstrate that EGFR active site mutants are oncogenic. Mutant EGFR can transform both fibroblasts and lung epithelial cells in the absence of exogenous epidermal growth factor, as evidenced by anchorage-independent growth, focus formation, and tumor formation in immunocompromised mice. Transformation is associated with constitutive autophosphorylation of EGFR, Shc phosphorylation, and STAT pathway activation. Whereas transformation by most EGFR mutants confers on cells sensitivity to erlotinib and gefitinib, transformation by an exon 20 insertion makes cells resistant to these inhibitors but more sensitive to the irreversible inhibitor CL-387,785. CONCLUSION: Oncogenic transformation of cells by different EGFR mutants causes differential sensitivity to gefitinib and erlotinib. Treatment of lung cancers harboring EGFR exon 20 insertions may therefore require the development of alternative kinase inhibition strategies
Transformation of Human and Murine Fibroblasts without Viral Oncoproteins
Murine embryo fibroblasts are readily transformed by the introduction of specific combinations of oncogenes; however, the expression of those same oncogenes in human cells fails to convert such cells to tumorigenicity. Using normal human and murine embryonic fibroblasts, we show that the transformation of human cells requires several additional alterations beyond those required to transform comparable murine cells. The introduction of the c-Myc and H-RAS oncogenes in the setting of loss of p53 function efficiently transforms murine embryo fibroblasts but fails to transform human cells constitutively expressing hTERT, the catalytic subunit of telomerase. In contrast, transformation of multiple strains of human fibroblasts requires the constitutive expression of c-Myc, H-RAS, and hTERT, together with loss of function of the p53, RB, and PTEN tumor suppressor genes. These manipulations permit the development of transformed human fibroblasts with genetic alterations similar to those found associated with human cancers and define specific differences in the susceptibility of human and murine fibroblasts to experimental transformation
Sensitivity of Cell Transformation Induced by Expression of Mutant <i>EGFR</i> Characterized by Missense Mutation or Exon 19 Deletion, but not Exon 20 Insertion, to Gefitinib and Erlotinib
<div><p>(A) Anchorage-independent growth of clonal NIH-3T3 cells transformed with mutant <i>EGFR</i> or EGF-stimulated wild-type <i>EGFR</i> treated with the indicated concentrations of erlotinib immediately prior to suspension in soft agar. Transformation induced by expression of L858R, G719S, and L747_E749del A750P <i>EGFR,</i> but not EGF-stimulated wild-type <i>EGFR</i> or D770_N771insNPG <i>EGFR,</i> was inhibited by 0.1 μM erlotinib. Representative photographs are shown.</p>
<p>(B) Number of colonies formed in soft agar by clonal NIH-3T3 cells expressing L858R <i>EGFR</i> and D770_N771insNPG <i>EGFR</i> treated with the indicated concentrations of gefitinib or erlotinib immediately prior to suspension in soft agar. Transformation by cells expressing the L858R <i>EGFR</i> was inhibited by 0.1 μM gefitinib or erlotinib, whereas transformation by cells expressing the insertion mutant was resistant to low concentrations of these inhibitors. Colonies were quantitated by counting ten fields each of triplicate wells photographed with a 10× objective; mean ± standard deviation is shown. Ins, D770_N771insNPG <i>EGFR</i>.</p>
<p>(C) Transformation induced by expression of D770_N771insNPG <i>EGFR</i> is inhibited 10-fold more efficiently by the irreversible EGFR inhibitor CL-387,785 [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0020313#pmed-0020313-b35" target="_blank">35</a>]. Clonal NIH-3T3 cells expressing the insertion mutant were treated with the indicated concentrations of gefitinib, erlotinib, or CL-387,785 immediately prior to suspension in soft agar. This assay was not done in triplicate, but the results are representative of two independent experiments. The number of colonies was normalized to maximum colony formation for each treatment, and sigmoidal dose response curves were fitted to the data using Prism Graphpad software to determine IC50s.</p></div
Ligand-Independent Activation of the Mutant EGFR
<div><p>(A) Cells expressing the wild-type or mutant <i>EGFR</i> were lysed and immunoblotted with antibodies to total EGFR or antibodies that recognize specific phosphorylation sites in the EGFR C-terminal tail as labeled. All four mutant EGFR proteins, representative of the four classes of <i>EGFR</i> mutations observed in lung adenocarcinoma tumor DNA, exhibited constitutive phosphorylation on the indicated C-terminal autophosphorylation sites. Note that the nomenclature for the anti-phospho-EGFR antibodies reflects elimination of the 24-amino acid signal peptide. Due to difficulties in isolating clonal cell lines with the same levels of mutant EGFR expression, G719S is expressed at higher levels and D770_N771ins NPG at lower levels than the other mutant EGFR. del, L747_E749del A750P; ins, D770_N771insNPG; pBp, pBabe-Puro vector control; wt, wild-type EGFR.</p>
<p>(B) Cells expressing the wild-type or L858R <i>EGFR</i> were placed in media containing 0.5% CS for 24 h. A combination of three neutralizing antibodies (anti-EGF, anti-TGFα, and anti-EGFR) was added 3 h prior to EGF stimulation and lysis. Upper row of blots show the anti-phospho-EGFR Y1068 immunoblots. The lower row shows anti-EGFR immunoblots. No inhibition of L858R EGFR autophosphorylation was observed upon treatment with a combination of three neutralizing antibodies (“neutr Ab”) sufficient to prevent EGF stimulation of autophosphorylation of the wild-type EGFR.</p></div
Sensitivity of Mutant EGFR Autophosphorylation to EGFR Inhibitors Reflects Inhibition of Anchorage-Independent Growth
<p>Cells expressing wild-type, L858R, or D770_N771insNPG <i>EGFR</i> were treated for 2 h with the indicated concentrations of gefitinib or CL-387,785. Cells expressing the wild-type EGFR were then stimulated for 10 min with 7 ng/ml EGF, and all plates were lysed. Whole-cell lysates were immunoblotted for phospho-EGFR Y1068 (upper row of blots), total EGFR (middle row), and actin as a loading control (lower row). Although compound concentrations necessary for inhibition of autophosphorylation do not exactly correspond to inhibition of anchorage-independent growth, the relative sensitivity of autophosphorylation of the wild-type and mutant EGFR to gefitinib or CL-387,785 mirrors the relative sensitivity of colony formation to these inhibitors.</p
Mammalian Cells Expressing the Lung Cancer-Derived Mutant EGFR Grow in an Anchorage-Independent Manner
<div><p>(A) NIH-3T3 cells infected with retroviruses encoding the indicated wild-type or mutant <i>EGFR</i> were selected in the presence of 2.5 μg /ml puromycin for 4 d. In the top photomicrographs, 1 × 10<sup>5</sup> cells were suspended in soft agar for a colony formation assay and photographed after 3 wk incubation at 37 °C. Expression of lung cancer-derived missense <i>EGFR</i> mutants, but not wild-type or kinase-inactive D837A <i>EGFR,</i> induces colony formation in soft agar. In the bottom photomicrographs, samples were identical, but 20 ng/ml EGF was added to the top agar. Representative photomicrographs are shown.</p>
<p>(B) Anti-EGFR immunoblot analysis of pooled stable NIH-3T3 cells infected as described in (A). All <i>EGFR</i> constructs are expressed at similar levels. pBp, pBabe-Puro vector; wt, wild-type EGFR.</p>
<p>(C) Lysates from 4 × 10<sup>4</sup> cells from the human lung adenocarcinoma cell line H3255, harboring the L858R mutation in <i>EGFR,</i> or the wild-type or L858R <i>EGFR</i>-overexpressing NIH-3T3 cells, were immunoblotted for total <i>EGFR</i> levels. Although total protein levels per cell are lower for the H3255 than the NIH-3T3 cells, EGFR expression levels are slightly higher in the H3255s.</p>
<p>(D) NIH-3T3 cells infected with retroviruses encoding the mutant <i>EGFR</i> were selected in the presence of 2 μg/ml puromycin for 9 d. Selected cells (1 × 10<sup>5</sup>) were suspended in soft agar for a colony formation assay and photographed after 3 wk incubation at 37 °C. Expression of the deletion and insertion <i>EGFR</i> mutants induced formation of colonies in soft agar with higher efficiency than expression of L858R. Representative photos are shown. Polyoma mT, NIH-3T3 cells infected with positive control pBabe-Puro retrovirus encoding the polyoma middle T antigen.</p>
<p>(E) hTBE cells expressing the SV40 early region and hTERT were infected with control virus pBabe-Puro (pBp) or with viruses encoding the indicated <i>EGFR</i> alleles. Cells were plated in 0.4% Noble agar, and colonies were counted with an automated imager at 6 wk. Mean ± standard deviation is shown for three independent determinations. Control cells (pBp) formed many microscopic colonies, but colonies formed by cells expressing <i>EGFR</i> mutants were more numerous and larger. del, L747_E749del A750P mutated EGFR; ins, D770_N771insNPG mutated EGFR; pBp, pBabe-Puro vector; RasV12, V12 H-Ras; wt, wild-type EGFR.</p>
<p>(F) Anti-EGFR immunoblot analysis of hTBE cells infected as described in (E). All <i>EGFR</i> constructs were expressed at similar levels. pBp, pBabe-Puro vector; wt, wild-type EGFR.</p></div
Population-specific variations of the genetic architecture of sex determination in wild European sea bass Dicentrarchus labrax L.
Polygenic sex determination (PSD) may show variations in terms of genetic and environmental components between populations of fish species exposed/adapted to different environments. The European sea bass (Dicentrarchus labrax) is an interesting model, combining both a PSD system and a genetic subdivision into an Atlantic and a Mediterranean lineage, with genetic substructures within the Mediterranean Sea. Here, we produced experimental progeny crosses (N = 927) from broodstock sampled in four wild populations (North Atlantic, NAT; Western Mediterranean, WEM; North-Eastern Mediterranean, NEM; South-Eastern Mediterranean, SEM). We found less females than males in the progeny, both in the global dataset (32.5%) and within each paternal group (from 25.1% for NEM to 39.0% for WEM), with significant variation among populations, dams, and sires. Sex, body weight (BW), and body length (BL) showed moderate heritability (0.52 ± 0.17, 0.46 ± 0.17, 0.34 ± 0.15, respectively) and sex was genetically correlated with BW and BL, with rAsex/BW = 0.69 ± 0.12 and rA sex/BL = 0.66 ± 0.13. A weighted GWAS performed both on the global dataset and within each paternal group revealed a different genetic architecture of sex determination between Atlantic and Mediterranean populations (9 QTLs found in NAT, 7 in WEM, 5 in NEM, and 4 in SEM, with a cumulated variance explained of 27.04%, 21.87%, 15.89%, and 12.10%, respectively) and a more similar genetic architecture among geographically close populations compared to geographically distant populations, consistent with the hypothesis of a population-specific evolution of polygenic sex determination systems in different environments