A Potential Peptide Therapeutic Derived from the Juxtamembrane Domain of the Epidermal Growth Factor Receptor

The epidermal growth factor receptor (EGFR) is involved in many cancers and EGFR has been heavily pursued as a drug target. Drugs targeting EGFR have shown promising clinical results for several cancer types. However, resistance to EGFR inhibitors often occurs, such as with KRAS mutant cancers, therefore new methods of targeting EGFR are needed. The juxtamembrane (JXM) domain of EGFR is critical for receptor activation and targeting this region could potentially be a new method of inhibiting EGFR. We hypothesized that the structural role of the JXM region could be mimicked by peptides encoding a JXM amino acid sequence, which could interfere with EGFR signaling and consequently could have anti-cancer activity. A peptide encoding EGFR 645–662 conjugated to the Tat sequence (TE-64562) displayed anti-cancer activity in multiple human cancer cell types with diminished activity in non-EGFR expressing cells and non-cancerous cells. In nude mice, TE-64562 delayed MDA-MB-231 tumor growth and prolonged survival, without inducing toxicity. TE-64562 induced non-apoptotic cell death after several hours and caspase-3-mediated apoptotic cell death with longer treatment. Mechanistically, TE-64562 bound to EGFR, inhibited its dimerization and caused its down-regulation. TE-64562 reduced phosphorylated and total EGFR levels but did not inhibit kinase activity and instead prolonged it. Our analysis of patient data from The Cancer Genome Atlas supported the hypothesis that down-regulation of EGFR is a potential therapeutic strategy, since phospho- and total-EGFR levels were strongly correlated in a large majority of patient tumor samples, indicating that lower EGFR levels are associated with lower phospho-EGFR levels and presumably less proliferative signals in breast cancer. Akt and Erk were inhibited by TE-64562 and this inhibition was observed in vivo in tumor tissue upon treatment with TE-64562. These results are the first to indicate that the JXM domain of EGFR is a viable drug target for several cancer types.     

Human Epidermal Growth Factor Receptor 2 (HER2) Impedes MLK3 Kinase Activity to Support Breast Cancer Cell Survival

Human epidermal growth factor receptor 2 (HER2) is amplified in ∼15–20% of human breast cancer and is important for tumor etiology and therapeutic options of breast cancer. Up-regulation of HER2 oncogene initiates cascades of events cumulating to the stimulation of transforming PI3K/AKT signaling, which also plays a dominant role in supporting cell survival and efficacy of HER2-directed therapies. Although investigating the underlying mechanisms by which HER2 promotes cell survival, we noticed a profound reduction in the kinase activity of a pro-apoptotic mixed lineage kinase 3 (MLK3) in HER2-positive (HER2+) but not in HER2-negative (HER2−) breast cancer tissues, whereas both HER2+ and HER2− tumors expressed a comparable level of MLK3 protein. Furthermore, the kinase activity of MLK3 was inversely correlated with HER2+ tumor grades. Moreover, HER2-directed drugs such as trastuzumab and lapatinib as well as depletion of HER2 or HER3 stimulated MLK3 kinase activity in HER2+ breast cancer cell lines. In addition, the noted inhibitory effect of HER2 on MLK3 kinase activity was mediated via its phosphorylation on Ser⁶⁷⁴ by AKT and that pharmacological inhibitors of PI3K/AKT prevented trastuzumab- and lapatinib-induced stimulation of MLK3 activity. Consistent with the pro-apoptotic function of MLK3, stable knockdown of MLK3 in the HER2+ cell line blunted the pro-apoptotic effects of trastuzumab and lapatinib. These findings suggest that HER2 activation inhibits the pro-apoptotic function of MLK3, which plays a mechanistic role in mediating anti-tumor activities of HER2-directed therapies. In brief, MLK3 represents a newly recognized integral component of HER2 biology in HER2+ breast tumors.     

Plakophilin-2 Promotes Activation of Epidermal Growth Factor Receptor

While growth factor-driven dimerization of receptor tyrosine kinases (RTKs) is a simple and intuitive mechanism of activating RTKs, K.-I. Arimoto et al. (Mol. Cell. Biol. 34:3843–3854, 2014, doi:10.1128/MCB.00758-14) describe a novel means of promoting the activity of RTKs. Namely, plakophilin-2 (PKP2) associates with the epidermal growth factor receptor (EGFR) and enhances its ligand-dependent and ligand-independent activity. This discovery suggests that antagonizing PKP2 may be a new therapeutic opportunity to combat tumors in which activation of EGFR contributes to pathogenesis.     

Conformational Analysis of the Phosphorylated Epidermal Growth Factor Receptor

Phosphorylation-induced conformational changes have been well documented with different receptor tyrosine kinases. However, the susceptible epitopes and the tyrosine residue(s) involved in particular structural alteration mostly remain to be determined. Using a conformation-specific anti-peptide antibody, we have not only identified one such domain in the C-terminal tail of the EGF receptor but also identified the phosphate acceptor sites that are involved in the conformational change.     

Identification and Characterization of a Novel Evolutionarily Conserved Lysine-specific Methyltransferase Targeting Eukaryotic Translation Elongation Factor 2 (eEF2)

The components of the cellular protein translation machinery, such as ribosomal proteins and translation factors, are subject to numerous post-translational modifications. In particular, this group of proteins is frequently methylated. However, for the majority of these methylations, the responsible methyltransferases (MTases) remain unknown. The human FAM86A (family with sequence similarity 86) protein belongs to a recently identified family of protein MTases, and we here show that FAM86A catalyzes the trimethylation of eukaryotic elongation factor 2 (eEF2) on Lys-525. Moreover, we demonstrate that the Saccharomyces cerevisiae MTase Yjr129c, which displays sequence homology to FAM86A, is a functional FAM86A orthologue, modifying the corresponding residue (Lys-509) in yeast eEF2, both in vitro and in vivo. Finally, Yjr129c-deficient yeast cells displayed phenotypes related to eEF2 function (i.e. increased frameshifting during protein translation and hypersensitivity toward the eEF2-specific drug sordarin). In summary, the present study establishes the function of the previously uncharacterized MTases FAM86A and Yjr129c, demonstrating that these enzymes introduce a functionally important lysine methylation in eEF2. Based on the previous naming of similar enzymes, we have redubbed FAM86A and Yjr129c as eEF2-KMT and Efm3, respectively.     

靶向表皮生长因子受体的全新小分子配体筛选

肿瘤靶向分子的筛选一直是肿瘤治疗和早期诊断的研究热点 . 表皮生长因子受体 (EGFR) 在很多肿瘤细胞表面过量表达,是一个理想的药物输送靶点 . 选择了 EGFR 表面不同于表皮生长因子 (EGF) 结合位点的一个凹陷部位作为计算机模拟筛选的结合位点,然后使用 DOCK 软件包对 DTP-Plated 有机小分子数据库进行了两遍筛选,最后选择了 7 个有机小分子作为可能的靶向分子 . BIAcore 体外结合实验对所选择的小分子样品进行了进一步的验证,结果表明,小分子 NSC51186 能特异地与 EGFR 结合 . 小分子 NSC51186 和 EGFR 之间的动力学常数也得到进一步的测定 . 新的靶向分子和药物、纳米粒子或者基因载体相连,将有可能用于靶向于 EGFR 的肿瘤治疗和诊断 .     

表皮生长因子及受体在甲状腺肿瘤中的表达

目的:研究表皮生长因子(Epidermal Growth Factor,EGF)及受体(Epidermal Growth Factor Receptor,EGFR)及在甲状腺肿瘤中的表达。方法:应用免疫组织化学法检测91例甲状腺病变组织中EGFR和EGF的表达情况。结果:结节性甲状腺肿、甲状腺腺瘤、分化型甲状腺癌标本中EGFR表达的阳性率分别为15%、25%、68.62%,EGF表达的阳性率分别为10%、15%、68.62%,其中EGFR、EGF在分化型甲状腺癌与其余两组间差异均有统计学意义(P<0.05)。EGFR和EGF在甲状腺乳头状癌中的表达与性别、年龄、肿瘤大小、淋巴结转移、临床分期等临床因素无明显相关。结论:EGF和EGFR的表达可作为鉴别甲状腺肿瘤良恶性的一个指标。     

表皮生长因子受体(EGFR)的细胞内信号传导

表皮生长因子受体（EGFR）是一种存在于细胞表面的多功能跨膜蛋白分子,具有酪氨酸蛋白激酶活性,EGFR与配体结合后启动细胞内信号传导通路,不同的通路之间存在交叉对话（Cross-talks）共同完成细胞生理功能.对EGFR的深入研究,不仅可阐明细胞生长和发育等重要的生命过程,而且在医药和工业上也将有广泛的应用.     

人EGFR显性负性突变体负调控内源性EGFR功能的机制分析

通过定向克隆法构建真核表达载体pEGFPN1-DNEGFR,脂质体介导下转染体外培养的SGC-7901细胞,应用Western blotting检测DNEGFR-EGFP蛋白的表达,激光共聚焦显微镜对DNEGFR-EGFP亚细胞结构定位检测;并经RT-PCR、Western blotting检测DNEGFR-EGFP对内源性EGFRmRNA水平、蛋白及磷酸化水平的影响.成功检测到DNEGFR-EGFP蛋白的表达,DNEGFR-EGFP蛋白主要定位于细胞膜,DNEGFR-EGFP能降低内源性EGFR蛋白磷酸化水平,而对内源性EGFRmRNA水平及蛋白水平无影响.研究证明DNEGFR通过降低内源性EGFR蛋白磷酸化水平负调控EGFR功能,为靶向EGFR显性负性策略在肿瘤生物治疗中的进一步研究打下基础.     

Growth Differentiation Factor (GDF)-15 Blocks Norepinephrine-induced Myocardial Hypertrophy via a Novel Pathway Involving Inhibition of Epidermal Growth Factor Receptor Transactivation

Accumulating evidence suggests that growth differentiation factor 15 (GDF-15) is associated with the severity and prognosis of various cardiovascular diseases. However, the effect of GDF-15 on the regulation of cardiac remodeling is still poorly understood. In this present study, we demonstrate that GDF-15 blocks norepinephrine (NE)-induced myocardial hypertrophy through a novel pathway involving inhibition of EGFR transactivation. Both in vivo and in vitro assay indicate that NE was able to stimulate the synthesis of GDF-15. The up-regulation of GDF-15 feedback inhibits NE-induced myocardial hypertrophy, including quantitation of [³H]leucine incorporation, protein/DNA ratio, cell surface area, and ANP mRNA level. Further research shows that GDF-15 could inhibit the phosphorylation of EGF receptor and downstream kinases (AKT and ERK1/2) induced by NE. Clinical research also shows that serum GDF-15 levels in hypertensive patients were significant higher than in healthy volunteers and were positively correlated with the thickness of the posterior wall of the left ventricle, interventricular septum, and left ventricular mass, as well as the serum level of norepinephrine. In conclusion, NE induces myocardial hypertrophy and up-regulates GDF-15, and this up-regulation of GDF-15 negatively regulates NE-induced myocardial hypertrophy by inhibiting EGF receptor transactivation following NE stimulation.     

Intracellular Transactivation of the Insulin-like Growth Factor I Receptor by an Epidermal Growth Factor Receptor

Growth factor receptors may be transactivated not only by homologous receptors, but also by heterologous receptors. We have investigated this possibility, using for this purpose R⁻/EGFR cells, which are mouse embryo cells devoid of IGF-I receptors, but overexpressing the EGF receptor. At variance with mouse embryo cells with a wild-type number of IGF-I receptors and overexpressing the EGF receptor, R⁻/EGFR cells cannot grow in EGF only, nor can they form colonies in soft agar. However, if a wild type human IGF-I receptor is stably transfected into R⁻/EGFR cells, growth in EGF and colony formation in soft agar are restored. To determine a possible interaction between the two receptors, we transfected into R⁻/EGFR cells a number of IGF-I receptor mutants with different impaired functions. The only IGF-I receptor that cannot reverse the growth phenotype of R⁻/EGFR cells is a receptor with a point mutation at the ATP-binding site. All other mutant receptors, even when incapable of responding to IGF-I with a mitogenic signal, made R⁻/EGFR cells fully capable of responding with growth to EGF stimulation. IGF-I receptor mutants that are mitogenic but not transforming made R⁻/EGFR cells grow in EGF only, but were incapable of inducing the transformed phenotype. The mutant IGF-I receptors are activated (tyrosyl phosphorylation of IRS-1) in response to EGF. These experiments indicate that certain IGF-I receptor mutants with loss of function can be reactivated intracellularly by an overexpressed EGF receptor and confirm that the C-terminus of the IGF-IR is required for its transforming activity.     

The Constitutive Activity of Epidermal Growth Factor Receptor vIII Leads to Activation and Differential Trafficking of Wild-type Epidermal Growth Factor Receptor and erbB2

A constitutively active epidermal growth factor receptor (EGFR) mutant, EGFR variant III (EGFRvIII), has been detected at high frequencies in certain human cancers. This study evaluated transactivation and trafficking of erbB family members as a result of constitutive EGFR activity in a cancer cell line. Expression of EGFRvIII modulated erbB family members through different mechanisms; the erbB3 mRNA level was reduced, whereas wild-type EGFR (wtEGFR) and erbB2 protein levels were diminished, with no change in their mRNA levels, and there was no change in the erbB4 expression level. Both EGFR and erbB2 were internalized as a result of EGFRvIII''s activity and redistributed to the cell surface upon addition of AG1478, an inhibitor of wtEGFR/EGFRvIII catalytic activity. Acute activation of EGFRvIII by removing AG1478 from cells increased phosphorylation of both wtEGFR and erbB2 and caused differential trafficking of EGFRvIII''s activation partners; wtEGFR was directed primarily to lysosomal compartments and partially to recycling compartments, whereas erbB2 was directed primarily to recycling compartments and partially to lysosomal compartments. Our data demonstrate that the constitutive activity of EGFRvIII is sufficient to trigger endocytosis and trafficking of wtEGFR and erbB2, which may play a role in activating signaling pathways that are triggered during receptor endocytosis. (J Histochem Cytochem 58:529–541, 2010)     

The F-BAR Protein PACSIN2 Regulates Epidermal Growth Factor Receptor Internalization

Signaling via growth factor receptors, including the epidermal growth factor (EGF) receptor, is key to various cellular processes, such as proliferation, cell survival, and cell migration. In a variety of human diseases such as cancer, aberrant expression and activation of growth factor receptors can lead to disturbed signaling. Intracellular trafficking is crucial for proper signaling of growth factor receptors. As a result, the level of cell surface expression of growth factor receptors is an important determinant for the outcome of downstream signaling. BAR domain-containing proteins represent an important family of proteins that regulate membrane dynamics. In this study, we identify a novel role for the F-BAR protein PACSIN2 in the regulation of EGF receptor signaling. We show that internalized EGF as well as the (activated) EGF receptor translocated to PACSIN2-positive endosomes. Furthermore, loss of PACSIN2 increased plasma membrane expression of the EGF receptor in resting cells and increased EGF-induced phosphorylation of the EGF receptor. As a consequence, EGF-induced activation of Erk and Akt as well as cell proliferation were enhanced in PACSIN2-depleted cells. In conclusion, this study identifies a novel role for the F-BAR-domain protein PACSIN2 in regulating EGF receptor surface levels and EGF-induced downstream signaling.     

Systems Biological Analysis of Epidermal Growth Factor Receptor Internalization Dynamics for Altered Receptor Levels

Epidermal growth factor (EGF) receptor (EGFR) overexpression is a hallmark of many cancers. EGFR endocytosis is a critical step in signal attenuation, raising the question of how receptor expression levels affect the internalization process. Here we combined quantitative experimental and mathematical modeling approaches to investigate the role of the EGFR expression level on the rate of receptor internalization. Using tetramethylrhodamine-labeled EGF, we established assays for quantifying EGF-triggered EGFR internalization by both high resolution confocal microscopy and flow cytometry. We determined that the flow cytometry approach was more sensitive for examining large populations of cells. Mathematical modeling was used to investigate the relationship between EGF internalization kinetics, EGFR expression, and internalization machinery. We predicted that the standard parameter used to assess internalization kinetics, the temporal evolution r(t) of the ratio of internalized versus surface-located ligand·receptor complexes, does not describe a straight line, as proposed previously. Instead, a convex or concave curve occurs depending on whether initial receptor numbers or internalization adaptors are limiting the uptake reaction, respectively. To test model predictions, we measured EGF-EGFR binding and internalization in cells expressing different levels of green fluorescent protein-EGFR. As expected, surface binding of rhodamine-labeled EGF increased with green fluorescent protein-EGFR expression level. Unexpectedly, internalization of ligand· receptor complexes increased linearly with increasing receptor expression level, suggesting that receptors and not internalization adaptors were limiting the uptake in our experimental model. Finally, determining the ratio of internalized versus surface-located ligand·receptor complexes for this cell line confirmed that it follows a convex curve, supporting our model predictions.The epidermal growth factor receptor (EGFR)³ belongs to the family of transmembrane receptor tyrosine kinases and mediates diverse actions, including proliferation, differentiation, and apoptosis (1, 2). Overexpression and/or mutations of the EGFR occur in ∼40% of neoblastomas (3) and correlate with poor prognosis (4–6). Unstimulated EGFR is located at the plasma membrane as a monomer and pre-formed dimer (7). Upon ligand binding, EGFR forms a dimer, and trans-phosphorylation occurs at specific residues of the cytoplasmic domain (8). Phosphorylated EGFR recruits adaptor proteins from which different conserved signaling pathways are activated, namely the MAPK (9), phosphatidylinositol 3-kinase, and protein kinase C pathways (10).Furthermore, activated EGFR recruits various adaptor proteins that mediate receptor internalization by endocytosis (2). Endocytosis occurs via the recruitment of adaptor proteins to phosphorylated tyrosine residues of the receptor and formation of membrane invaginations, which eventually pinch off to form internalized early endosomes (2, 11) (see Fig. 1). Both constitutive endocytosis and ligand-induced EGFR endocytosis are critical events in EGF signal regulation (2, 12). Endosomal EGFR can be transited back to the plasma membrane or to the late endosome/lysosome for degradation (2). As the majority of internalized receptors are targeted for lysosomal degradation upon EGF stimulation (13), endocytic entry of active EGFR is a crucial step for signal attenuation, which is also highlighted by the findings that impaired or delayed internalization is highly oncogenic (14, 15).Open in a separate windowFIGURE 1.Scheme of ligand-induced internalization. EGF binds membrane-located EGFR to give rise to surface-bound EGF·EGFR complex RE_s. Via diffusion events, the activated receptor binds internalization adaptors IC, which leads to internalized receptors R_i.In light of the role of endocytosis in EGFR signal attenuation and the oncogenicity of EGFR overexpression, it is important to elucidate the relationship between high receptor expression levels relative to internalization pathway capacity and their effect on internalization dynamics.Mathematical modeling is an important tool in elucidating EGFR signaling, at the level of EGFR internalization (16–19) and, more recently, at the level of the integration of input signals into signaling events downstream of the EGFR, such as the MAPK cascade (20, 21). In earlier models, pioneering concepts such as the nonlinearity of the uptake reaction, because of the existence of alternative pathways that are entered with different affinities, were developed (16, 19). Also, the notion of saturability of the EGFR endocytosis system, in contrast to internalization of the transferrin receptor, for example, was introduced (18).Importantly, in mathematical formulations of EGFR endocytosis, the standard parameter used to estimate the rate of the internalization step (16) and to assess the effect of certain perturbations on internalization (22–24) is the temporal evolution of the ratio of internalized versus surface-located ligand·receptor complexes r(t). In Refs. 16, 17, it was mathematically determined that, under certain assumptions, this ratio describes a straight line with the slope corresponding to the rate of the internalization step. These assumptions were as follows: (i) that the number of surface-bound ligand·receptor complexes (RE_s) remains approximately constant during the measurements, and (ii) that the internalization step is a first-order process, i.e. it is directly proportional to RE_s and independent of a potentially limiting availability of internalization adaptors.The presence of multiple endocytotic routes (23, 25) and different EGFR affinities for EGF (26) argue against first-order kinetics. Moreover, the possible limited capacity of internalization adaptors may restrict EGFR internalization in cells expressing abnormally high numbers of EGFR (18). In this work we investigated the potential of EGFR internalization to occur as a nonlinear process by combining mathematical modeling with novel quantitative, live cell measurements of EGF internalization.We extended the previous derivation of the ratio of internalized versus surface-located ligand·receptor complexes r(t) (16, 17, 19) by eliminating above assumptions i and ii, which allowed us to investigate in silico different scenarios for the shape of r(t) as a function of the relative concentrations of EGFR and internalization adaptors. We predicted that r(t) is not a straight line as derived previously but is a convex or concave curve depending on whether receptors or internalization components are limiting the reaction, respectively.In earlier studies, quantitative measurements of parameters of EGFR endocytosis have been performed using classical biochemical techniques to detect cellular ligand uptake using radioactively labeled EGF (16, 24, 27) or biotin-labeled EGF (28). Importantly, both methods do not reach single cell precision and instead yield an integrated signal over a population of cells. To test our mathematical predictions we combined the following: (i) quantitative laser scanning confocal microscopy, and (ii) multiple parametric flow cytometry, using a custom Beckman Coulter FC500 equipped with a 488 and 561 nm laser excitation, to quantitatively measure the temporal and spatial dynamics of EGFR endocytosis using tetramethylrhodamine-tagged EGF (Rh-EGF) and GFP-EGFR. We show that both quantitative imaging and flow cytometry measurements were highly sensitive, allowing for live cell investigations and confirmation of the mathematical predictions.     

A Novel Glycoengineered Bispecific Antibody Format for Targeted Inhibition of Epidermal Growth Factor Receptor (EGFR) and Insulin-like Growth Factor Receptor Type I (IGF-1R) Demonstrating Unique Molecular Properties

In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the “knob-into-hole” technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats.     

Selected Reaction Monitoring (SRM) Analysis of Epidermal Growth Factor Receptor (EGFR) in Formalin Fixed Tumor Tissue

Background

Analysis of key therapeutic targets such as epidermal growth factor receptor (EGFR) in clinical tissue samples is typically done by immunohistochemistry (IHC) and is only subjectively quantitative through a narrow dynamic range. The development of a standardized, highly-sensitive, linear, and quantitative assay for EGFR for use in patient tumor tissue carries high potential for identifying those patients most likely to benefit from EGFR-targeted therapies.

Methods

A mass spectrometry-based Selected Reaction Monitoring (SRM) assay for the EGFR protein (EGFR-SRM) was developed utilizing the Liquid Tissue®-SRM technology platform. Tissue culture cells (n = 4) were analyzed by enzyme-linked immunosorbent assay (ELISA) to establish quantitative EGFR levels. Matching formalin fixed cultures were analyzed by the EGFR-SRM assay and benchmarked against immunoassay of the non-fixed cultured cells. Xenograft human tumor tissue (n = 10) of non-small cell lung cancer (NSCLC) origin and NSCLC patient tumor tissue samples (n = 23) were microdissected and the EGFR-SRM assay performed on Liquid Tissue lysates prepared from microdissected tissue. Quantitative curves and linear regression curves for correlation between immunoassay and SRM methodology were developed in Excel.

Results

The assay was developed for quantitation of a single EGFR tryptic peptide for use in FFPE patient tissue with absolute specificity to uniquely distinguish EGFR from all other proteins including the receptor tyrosine kinases, IGF-1R, cMet, Her2, Her3, and Her4. The assay was analytically validated against a collection of tissue culture cell lines where SRM analysis of the formalin fixed cells accurately reflects EGFR protein levels in matching non-formalin fixed cultures as established by ELISA sandwich immunoassay (R² = 0.9991). The SRM assay was applied to a collection of FFPE NSCLC xenograft tumors where SRM data range from 305amol/μg to 12,860amol/μg and are consistent with EGFR protein levels in these tumors as previously-reported by western blot and SRM analysis of the matched frozen tissue. In addition, the SRM assay was applied to a collection of histologically-characterized FFPE NSCLC patient tumor tissue where EGFR levels were quantitated from not detected (ND) to 670amol/μg.

Conclusions

This report describes and evaluates the performance of a robust and reproducible SRM assay designed for measuring EGFR directly in FFPE patient tumor tissue with accuracy at extremely low (attomolar) levels. This assay can be used as part of a complementary or companion diagnostic strategy to support novel therapies currently under development and demonstrates the potential to identify candidates for EGFR-inhibitor therapy, predict treatment outcome, and reveal mechanisms of therapeutic resistance.     

Acute Regulation of the Epidermal Growth Factor Receptor in Response to Nerve Growth Factor

PC12 cells possess specific receptors for both nerve growth factor and epidermal growth factor, and by an unknown mechanism, nerve growth factor is able to attenuate the propagation of a mitogenic response to epidermal growth factor. The differentiation response of PC12 cells to nerve growth factor, therefore, predominates over the proliferative response to epidermal growth factor. We have observed that the addition of nerve growth factor to PC12 cells rapidly produces a decrease in surface 125I-epidermal growth factor binding capacity. Unlike previously described nerve growth factor effects on 125I-epidermal growth factor binding capacity, which required several days of nerve growth factor exposure, the decreases we report occur within minutes of nerve growth factor addition: A 50% decrease in 125I-epidermal growth factor binding capacity is evident at 10 min. This rapid nerve growth factor response is concentration dependent; inhibition of 125I-epidermal growth factor binding is detectable at nerve growth factor levels as low as 0.2 ng/ml and is maximal at approximately 50 ng/ml, consistent with known ranges of biological activity. No demonstrable differences in the rate of epidermal growth factor receptor synthesis or degradation were observed in cells acutely exposed to nerve growth factor. Scatchard analysis revealed that acute nerve growth factor treatment decreased the number of both high- and low-affinity 125I-epidermal growth factor binding sites, while the receptor affinity remained unchanged. We have also investigated the involvement of various potential intracellular mediators of nerve growth factor action and of known intracellular modulatory systems of the epidermal growth factor receptor for their capacity to participate in this nerve growth factor activity.     

Cardiac-Specific Over-Expression of Epidermal Growth Factor Receptor 2 (ErbB2) Induces Pro-Survival Pathways and Hypertrophic Cardiomyopathy in Mice

Background

Emerging evidence shows that ErbB2 signaling has a critical role in cardiomyocyte physiology, based mainly on findings that blocking ErbB2 for cancer therapy is toxic to cardiac cells. However, consequences of high levels of ErbB2 activity in the heart have not been previously explored.

Methodology/Principal Findings

We investigated consequences of cardiac-restricted over-expression of ErbB2 in two novel lines of transgenic mice. Both lines develop striking concentric cardiac hypertrophy, without heart failure or decreased life span. ErbB2 transgenic mice display electrocardiographic characteristics similar to those found in patients with Hypertrophic Cardiomyopathy, with susceptibility to adrenergic-induced arrhythmias. The hypertrophic hearts, which are 2–3 times larger than those of control littermates, express increased atrial natriuretic peptide and β-myosin heavy chain mRNA, consistent with a hypertrophic phenotype. Cardiomyocytes in these hearts are significantly larger than wild type cardiomyocytes, with enlarged nuclei and distinctive myocardial disarray. Interestingly, the over-expression of ErbB2 induces a concurrent up-regulation of multiple proteins associated with this signaling pathway, including EGFR, ErbB3, ErbB4, PI3K subunits p110 and p85, bcl-2 and multiple protective heat shock proteins. Additionally, ErbB2 up-regulation leads to an anti-apoptotic shift in the ratio of bcl-xS/xL in the heart. Finally, ErbB2 over-expression results in increased activation of the translation machinery involving S6, 4E-BP1 and eIF4E. The dependence of this hypertrophic phenotype on ErbB family signaling is confirmed by reduction in heart mass and cardiomyocyte size, and inactivation of pro-hypertrophic signaling in transgenic animals treated with the ErbB1/2 inhibitor, lapatinib.

Conclusions/Significance

These studies are the first to demonstrate that increased ErbB2 over-expression in the heart can activate protective signaling pathways and induce a phenotype consistent with Hypertrophic Cardiomyopathy. Furthermore, our work suggests that in the situation where ErbB2 signaling contributes to cardiac hypertrophy, inhibition of this pathway may reverse this process.