Constitutive Epidermal Growth Factor Receptor Research Articles

A Constitutive EGFR Kinase Dimer to Study Inhibitor Pharmacology.

Lung cancer is commonly caused by activating mutations in the epidermal growth factor receptor (EGFR). Allosteric kinase inhibitors are unaffected by common ATP-site resistance mutations and represent a promising therapeutic strategy for targeting drug-resistant EGFR variants. However, allosteric inhibitors are antagonized by kinase dimerization, and understanding this phenomenon has been limited to cellular experiments. To facilitate the study of allosteric inhibitor pharmacology, we designed and purified a constitutive EGFR kinase dimer harboring the clinically relevant L858R/T790M mutations. Kinetic characterization revealed that the EGFR kinase dimer is more active than monomeric EGFR(L858R/T790M) kinase and has the same Km,ATP Biochemical profiling of a large panel of ATP-competitive and allosteric EGFR inhibitors showed that allosteric inhibitor potency decreased by >500-fold in the kinase dimer compared with monomer, yielding IC50 values that correlate well with Ba/F3 cellular potencies. Thus, this readily purifiable constitutive asymmetric EGFR kinase dimer represents an attractive tool for biochemical evaluation of EGFR inhibitor pharmacology, in particular for allosteric inhibitors. SIGNIFICANCE STATEMENT: Drugs targeting epidermal growth factor receptor (EGFR) kinase are commonly used to treat lung cancers but are affected by receptor dimerization. Here, we describe a locked kinase dimer that can be used to study EGFR inhibitor pharmacology.

Identification of Activating Mutations in the Transmembrane and Extracellular Domains of EGFR.

The epidermal growth factor receptor (EGFR) is frequently mutated in human cancer, most notably non-small-cell lung cancer and glioblastoma. While many frequently occurring EGFR mutations are known to confer constitutive EGFR activation, the situation is less clear for rarely detected variants. In fact, more than 1000 distinct EGFR mutations are listed in the Catalogue of Somatic Mutations in Cancer (COSMIC), but for most of them, the functional consequence is unknown. To identify additional, previously unknown activating mutations in EGFR, we screened a randomly mutated EGFR library for constitutive EGFR phosphorylation using a recently developed high-throughput approach termed PhosphoFlowSeq. Enrichment of the well-known activating mutations S768I, T790M, and L858R validated the experimental approach. Importantly, we also identified the activating mutations S442I and L658Q located in the extracellular and transmembrane domains of EGFR, respectively. To the best of our knowledge, neither S442I nor L658Q has been associated with an activating phenotype before. However, both have been detected in cancer samples. Interestingly, molecular dynamics (MD) simulations suggest that the L658Q mutation located in the hydrophobic transmembrane region forms intermolecular hydrogen bonds, thereby promoting EGFR dimerization and activation. Based on these findings, we screened the COSMIC database for additional hydrophilic mutations in the EGFR transmembrane region and indeed detected moderate constitutive activation of EGFR-G652R. Together, this study demonstrates that unbiased screening for activating mutations in EGFR not only yields well-established substitutions located in the kinase domain but also activating mutations in other regions of EGFR, including the extracellular and transmembrane domains.

EGFR ligand shifts the role of EGFR from oncogene to tumour suppressor in EGFR-amplified glioblastoma by suppressing invasion through BIN3 upregulation.

The epidermal growth factor receptor (EGFR) is a prime oncogene that is frequently amplified in glioblastomas. Here we demonstrate a new tumour-suppressive function of EGFR in EGFR-amplified glioblastomas regulated by EGFR ligands. Constitutive EGFR signalling promotes invasion via activation of a TAB1-TAK1-NF-κB-EMP1 pathway, resulting in large tumours and decreased survival in orthotopic models. Ligand-activated EGFR promotes proliferation and surprisingly suppresses invasion by upregulating BIN3, which inhibits a DOCK7-regulated Rho GTPase pathway, resulting in small hyperproliferating non-invasive tumours and improved survival. Data from The Cancer Genome Atlas reveal that in EGFR-amplified glioblastomas, a low level of EGFR ligands confers a worse prognosis, whereas a high level of EGFR ligands confers an improved prognosis. Thus, increased EGFR ligand levels shift the role of EGFR from oncogene to tumour suppressor in EGFR-amplified glioblastomas by suppressing invasion. The tumour-suppressive function of EGFR can be activated therapeutically using tofacitinib, which suppresses invasion by increasing EGFR ligand levels and upregulating BIN3.

Abstract 2092: Metabolic approach to EGFR-targeted therapy in non-small cell lung cancer

Abstract Mutation of the epidermal growth factor receptor (EGFR) is a major genetic driver of non-small cell lung cancer (NSCLC). While first-line tyrosine-kinase inhibitors (TKIs) have improved patient survival, many patients eventually develop resistance to these drugs. Combination therapies are now being researched to overcome this resistance. Pathways that may synergize with EGFR signaling are of interest since a liability created with one compound may then be exploited by a second. It is well known that glycolysis is controlled by EGFR signaling in cancer cells. However, the interplay of cell metabolism and EGFR inhibitors is not fully understood. Using PC9 cells which have known constitutive EGFR activation, we examined the relative poise of ATP production from mitochondria and glycolysis. In response to any of four different TKIs: afatinib, CO-1686, dacomitinib and erlotinib, a rapid decrease in glycolytic activity was induced. However, the total ATP production rates were not changed significantly as a result of increased mitochondrial ATP production. We next examined the mitochondrial fuel sources which support this increase by selectively blocking individual fuel pathways. In the presence of either a mitochondrial pyruvate carrier or glutaminase inhibitor, total ATP production rate decreased, and cell viability followed. Taken together, these data suggest that a combinational application of EGFR-targeted therapy and mitochondrial-targeted therapy may provide metabolic stress to cancer cells. Further study is required to understand the interplay of the tumor microenvironment and metabolic niches that may be found therein. Citation Format: Yoonseok Kam, Pamela Swain, Natalia Romero, Brian P. Dranka. Metabolic approach to EGFR-targeted therapy in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2092.

Cardiomyocyte‐Specific EGFR Deletion Differentially Alters Cardiac Contractility and Remodeling

Epidermal Growth Factor Receptor (EGFR), a member of the Erbb receptor tyrosine kinase family, is essential to the development of multiple tissues and organs. Due to the embryonic lethality of global EGFR deletion, the impact of EGFR signaling in the adult heart, normally or in response to pathological stimuli, has not been well‐explored. Using mice with floxed EGFR alleles crossed with αMHC‐Cre mice, we have generated a cardiomyocyte‐specific constitutive EGFR knockout mouse model (CM‐EGFR‐KO) to address its role in the heart. Compared to their wild‐type (WT) littermate controls, CM‐EGFR‐KO mice displayed age‐related development of cardiac contractile dysfunction, marked by impaired diastolic relaxation and Ca2+ handling at the individual myocyte level, as monitored via echocardiographic, hemodynamic, myocyte contractility and Ca2+ transient analyses. RNASeq analysis of CM‐EGFR‐KO versus WT littermate left ventricular samples revealed temporally‐dependent alterations in the expression of a number of myofilament, Ca2+ handling and fetal genes. The contractile defect initially occurs between 7 and 9 weeks of age and is maintained over time without overt cardiac hypertrophic or fibrotic remodeling until 10 months of age, by which time the mice ultimately succumb to severe heart failure and reduced lifespan. Chronic administration of the β‐adrenergic receptor (βAR) agonist isoproterenol effectively compensated for the contractile dysfunction in CM‐EGFR‐KO mice, an effect that was reversible upon cessation of infusion. Notably, βAR‐mediated cardiomyocyte hypertrophy was attenuated in CM‐EGFR‐KO mice compared to WT littermate controls. Treatment of 16 week‐old CM‐EGFR‐KO mice with AAV9‐cTnT‐EGFR permanently rescued cardiomyocyte‐specific EGFR expression and contractile function, suggesting that the contractile dysfunction phenotype observed in these mice is not secondary to aberrant cardiac development, but is specific to EGFR‐dependent regulation of contractile signaling.Support or Funding InformationNIH grant HL105414 to DGTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 423: Cardiomyocyte-Specific Epidermal Growth Factor Receptor Deletion Differentially Alters Cardiac Contractility and Remodeling During Physiologic versus Pathologic Development

Epidermal Growth Factor Receptor (EGFR), a member of the Erbb receptor tyrosine kinase family, is essential to the development of multiple tissue and organs. Due to the embryonic lethality of global EGFR deletion, the impact of EGFR signaling in the adult heart, normally or in response to pathological stimuli, has not been well-explored. Using recently attained mice with floxed EGFR alleles crossed with αMHC-Cre mice, we have generated a cardiomyocyte-specific constitutive EGFR knockout mouse model (CM-EGFR KO) to address its role in the heart. Compared to their wild-type (WT) littermate controls, CM-EGFR-KO mice displayed age-related development of cardiac dysfunction and remodeling, occurring between 7 and 9 weeks of age, as monitored via echocardiography and immunohistochemistry analyses. Although contractile responsiveness to β-adrenergic receptor stimulation was unaffected by EGFR deletion, RNASeq analysis of CM-EGFR-KO hearts within this timeframe revealed alterations in myofilament protein expression profiles. Additionally, there was enhanced cardiomyocyte death in the 7-9 week window of development, which persisted to 10 months of age, at which time the CM-EGFR KO mice had progressively worsened cardiac function, dilation and cardiomyocyte loss. To investigate the impact of cardiomyocyte-specific EGFR deletion on the response to pathologic stress, we challenged 12-14 week old WT and CM-EGFR KO mice with chronic isoproterenol infusion. Notably, while chronic isoproterenol treatment diminished cardiac function and induced hypertrophic remodeling in WT mouse hearts, CM-EGFR-KO mouse hearts displayed progressively improved contractility and ablated hypertrophy over time, suggesting that EGFR differentially regulates cardiac function and remodeling processes during physiological versus pathological development.

Detection of EGFR Gene Mutation by Mutation-oriented LAMP Method

Epidermal growth factor receptor (EGFR) is a target of molecular therapeutics for non-small cell lung cancer. EGFR gene mutations at codons 746-753 promote constitutive EGFR activation and result in worst prognosis. However, these mutations augment the therapeutic effect of EGFR-tyrosine kinase inhibitor. Therefore, the detection of EGFR gene mutations is important for determining treatment planning. The aim of the study was to establish a method to detect EGFR gene mutations at codons 746-753. EGFR gene mutation at codons 746-753 in six cancer cell lines were investigated. A loop-mediated isothermal amplification (LAMP)-based procedure was developed, that employed peptide nucleic acid to suppress amplification of the wild-type allele. This mutation-oriented LAMP can amplify the DNA fragment of the EGFR gene with codons 746-753 mutations within 30 min. Moreover, boiled cells can work as template resources. Mutation oriented-LAMP assay for EGFR gene mutation is sensitive on extracted DNA. This procedure would be capable of detecting EGFR gene mutation in sputum, pleural effusion, broncho-alveolar lavage fluid or trans-bronchial lung biopsy by chair side.

OP.5A.08] HCARG/COMMD5 IMPROVES KIDNEY REPAIR BY REGULATING EGF RECEPTOR TRAFFICKING

Objective: Epidermal Growth Factor Receptor (EGFR) is crucially involved in nephrogenesis. Although, constitutive EGFR signaling is needed for normal kidney development and can enhance recovery of renal function and structure following acute kidney injury, its overexpression may leads to excess renal tissue fibrosis and functional deterioration in diabetic and hypertensive nephropathy. Consequently, targeting EGFR in renal diseases is complex. Our group has identified HCaRG (Hypertension-related, Calcium-Regulated Gene), currently named COMMD5, whose overexpression in an in vivo model of renal carcinoma inhibited tumor growth and angiogenesis by inhibiting EGFR activation and downstream signaling. Overexpression of COMMD5 in renal proximal tubules of transgenic mice increased mice survival and recovery of renal tissue integrity and function after acute kidney injury. Our goal is to demonstrate that COMMD5 accelerated kidney repair by controlling EGFR activity, endocytosis and trafficking. Design and method: COMMD5 was silenced by siRNA and EGFR expression and activation were evaluated in renal cell lines. Intracellular localization of COMMD5, EGFR and endosome associated protein, such as Rab, were analyzed by confocal microscopy and live cell imaging. Co-immunoprecipitations were used to investigate interactions between COMMD5 and proteins known to be involved in vesicle trafficking. Results: We demonstrated that COMMD5 is an adaptor protein forming a bridge between endosomes and cytoskeleton by binding Rab protein and microtubules/actin. This tethering is essential because silencing of COMMD5 delayed the intracellular transport dynamic and consequently cargo protein such as EGFR. Defect in intracellular trafficking led to loss of cell polarity and oriented migration which are crucial for tissue repair. Conclusions: Our results demonstrated that COMMD5 plays a crucial role in controlling EGFR signaling/trafficking and suggest that COMMD5 could be used as a novel target for EGFR-targeted therapy in kidney diseases.

Analysis of Constitutive EGFR Signaling Regulating IRF3 Transcriptional Activity in Cancer Cells.

Epidermal growth factor receptor (EGFR) plays an important role in various types of human cancers. Overexpression of EGFR leads to a constitutive tyrosine phosphorylation of multiple tyrosine residues in the EGFR. Recently, we have demonstrated that overexpressed EGFR oscillates between two distinct and mutually exclusive modes of signaling depending on the presence or absence of ligand. EGFR constitutively activates transcription factor IRF3, which results in transcription of its target genes. Addition of EGF causes a loss of IRF3 transcriptional activity and activation of canonical signaling pathways such as ERK. The mechanistic basis of this bimodal signaling appears to be the association of a distinct set of signaling proteins with EGFR in the absence or presence of ligand. In this chapter, we describe a detailed protocol for analyses of constitutive EGFR signaling with a focus on IRF3 target genes.

Abstract 1949: Oncogenic signaling in amphiregulin- and EGFR-expressing PTEN-null human breast cancer

Abstract A subset of triple negative breast cancer (TNBC) is characterized by overexpression of the epidermal growth factor receptor (EGFR) and loss of PTEN, and patients with these factors have a poor prognosis. In addition, approximately half of all basal breast cancers show autocrine activation of EGFR by amphiregulin (AREG), resulting in altered receptor trafficking and downstream signaling. However, despite the strong rationale for targeting EGFR in the treatment of TNBC, EGFR inhibitors have performed poorly in clinical trials. To further analyze the signaling processes active in this subset of TNBC, we performed reverse-phase protein array (RPPA) analysis on SUM149 cells, a highly tumorigenic and metastatic PTEN-null cell line that expresses high levels of AREG and EGFR, and MCF10A cells, a non-transformed mammary epithelial cell line cultured in the presence of either EGF or AREG. This analysis identified a number of signaling components mediated specifically by amphiregulin, including marked changes in integrin expression and increased levels of fibronectin, which is known to be a mediator of invasive capacity via interaction with integrin β1. RPPA analysis also revealed elevated AKT activity in SUM-149 cells, which was not surprising given the lack of PTEN expression. Interestingly, this AKT activity is not abrogated by treatment with the EGFR inhibitor gefitinib, nor by treatment with PI3K inhibitors. These findings and the results of preliminary experiments in SUM149 cells re-expressing PTEN suggest that AKT activity in these cells is, at least partly, independent of EGFR and PI3K activity. In addition, expression profiling demonstrated that AREG-activated EGFR regulates gene expression differently than EGF-activated EGFR, and functional analysis via genome-scale shRNA screening identified a set of genes, including PLK1 and BIRC5, that are regulated by EGFR in MCF10A cells but uncoupled from EGFR signaling in SUM149 cells, although they are essential for the survival of SUM-149 cells. Thus, our results demonstrate that in cells with constitutive EGFR activation and PTEN loss, critical survival genes are uncoupled from regulation by EGFR, a mechanism that is likely to mediate resistance to EGFR inhibitors. Citation Format: Christiana S. Kappler, Stephen T. Guest, Ericka L. Smith, Jonathan C. Irish, Elizabeth Garrett-Mayer, Zachary Kratche, Stephen Ethier. Oncogenic signaling in amphiregulin- and EGFR-expressing PTEN-null human breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1949. doi:10.1158/1538-7445.AM2015-1949

Abstract B14: Cisplatin induced activation of the epidermal growth factor receptor as a mechanism for epigenetic silencing in ovarian cancer cell platinum resistance

Abstract The 5-year survival rate for women diagnosed with ovarian cancer remains dismally unchanged at 44%. Resistance to platinum compounds in ovarian cancer imparts a major obstacle to increasing patient survival. The epidermal growth factor receptor (EGFR) plays a role in the regulation of growth and differentiation in normal cells, and proliferation in malignant cells. It is known that disregulation of the EGFR pathway, including prolonged receptor activation, is associated with platinum resistance in cellular models and our lab revealed a novel mechanism by which constitutive EGFR activation regulates DNA methyltransferase (DNMT) activity and DNA methylation. Platinum resistance is associated with epigenetic downregulation (via DNA methylation) of genes involved in platinum drug response, particularly genes important for drug uptake. Given the established link between platinum resistance and alterations to DNA methylation, our studies investigated the effects of acute cisplatin treatment on EGFR activation and DNMT activity. We hypothesized that acquired chemoresistance resulting from cisplatin treatment is initially triggered by cisplatin-induced EGFR activation and subsequent alterations to DNMT activity. We show activation of EGFR upon treatment of OVCA433 cells with cisplatin. Functional EGFR activation following cisplatin treatment was also observed by evaluating activation of downstream targets, Jak2 and Akt. Hence, cisplatin facilitates activation of EGFR and its downstream signaling pathways. Moreover, there was also a significant increase in DNMT activity in cisplatin treated cells when compared to untreated passage controls. These observations unmask a potential mechanism by which initial exposure to cisplatin can activate EGFR signaling and cause epigenetic alterations that may contribute to the development of platinum resistance. By understanding the underlying mechanism for development of platinum resistance and discovering targets of methylation, we may be able to enhance sensitivity to platinum based chemotherapeutics. Citation Format: Michaela Lee Granados, Laurie G. Hudson, Sabrina Leigh Samudio-Ruiz. Cisplatin induced activation of the epidermal growth factor receptor as a mechanism for epigenetic silencing in ovarian cancer cell platinum resistance. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B14.

Constitutive and ligand-induced EGFR signalling triggers distinct and mutually exclusive downstream signalling networks

EGFR overexpression plays an important oncogenic role in cancer. Regular EGFR protein levels are increased in cancer cells and the receptor then becomes constitutively active. However, downstream signals generated by constitutively activated EGFR are unknown. Here we report that the overexpressed EGFR oscillates between two distinct and mutually exclusive modes of signaling. Constitutive or non-canonical EGFR signaling activates the transcription factor IRF3 leading to expression of IFI27, IFIT1, and TRAIL. Ligand-mediated activation of EGFR switches off IRF3 dependent transcription, activates canonical ERK and Akt signals, and confers sensitivity to chemotherapy and virus-induced cell death. Mechanistically, the distinct downstream signals result from a switch of EGFR associated proteins. EGFR constitutively complexes with IRF3 and TBK1 leading to TBK1 and IRF3 phosphorylation. Addition of EGF dissociates TBK1, IRF3, and EGFR leading to a loss of IRF3 activity, Shc-EGFR association and ERK activation. Finally, we provide evidence for non-canonical EGFR signaling in glioblastoma.

Oncogenic signaling in amphiregulin and EGFR-expressing PTEN-null human breast cancer

A subset of triple negative breast cancer (TNBC) is characterized by overexpression of the epidermal growth factor receptor (EGFR) and loss of PTEN, and patients with these determinants have a poor prognosis. We used cell line models of EGFR-positive/PTEN null TNBC to elucidate the signaling networks that drive the malignant features of these cells and cause resistance to EGFR inhibitors. In these cells, amphiregulin (AREG)-mediated activation of EGFR results in up-regulation of fibronectin (FN1), which is known to be a mediator of invasive capacity via interaction with integrin β1. EGFR activity in this PTEN null background also results in Wnt/beta-catenin signaling and activation of NF-κB. In addition, AKT is constitutively phosphorylated in these cells and is resistant to gefitinib. Expression profiling demonstrated that AREG-activated EGFR regulates gene expression differently than EGF-activated EGFR, and functional analysis via genome-scale shRNA screening identified a set of genes, including PLK1 and BIRC5, that are essential for survival of SUM-149 cells, but are uncoupled from EGFR signaling. Thus, our results demonstrate that in cells with constitutive EGFR activation and PTEN loss, critical survival genes are uncoupled from regulation by EGFR, which likely mediates resistance to EGFR inhibitors.

Persistent N-cadherin Expression Induced by Extended Epidermal Growth Factor Exposure Regulates Multicellular Aggregate Compaction and Sensitivity to Cisplatin

Objective: In ovarian cancer, activation of the epidermal growth factor receptor (EGFR) is associated with poor prognosis. The presence of EGFR activators in patient ascites fluid may cause constitutive EGFR activation thereby contributing to metastasis and/or resistance to therapy. Our goal was to identify alterations resulting from constitutive EGFR activation that influence cell behavior and drug sensitivity. Methods: We used an in vitro model (OVCA 433 cells) to evaluate changes in mesenchymal marker levels and multicellular aggregate (MCA) formation following long term epidermal growth factor (EGF) treatment. We determined sensitivity to cisplatin following EGF treatment and evaluated the role of the mesenchymal marker, N-cadherin, in aggregate formation and sensitivity using siRNA. Results: We found that EGFR activation led to phenotypic and functional changes in ovarian tumor cells that were retained after ligand was withdrawn (removal). Expression levels of the mesenchymal markers N-cadherin and vimentin were elevated in EGF treated and removal cells. This persistent increase in mesenchymal markers was associated with a significant increase in (MCA) compaction and cell spreading when MCAs were plated on collagen. N-cadherin silencing decreased MCA compaction and spreading in cells following extended exposure to EGF or in cells with high endogenous levels of N-cadherin. Furthermore, the compact MCA structure in EGF treated cells with increased N-cadherin expression conferred resistance to cisplatin and N-cadherin silencing largely restored cisplatin sensitivity. Conclusion: Our results indicate that prolonged EGFR activation causes a persistent change in mesenchymal marker expression, which regulates compaction and drug sensitivity. The findings implicate N-cadherin as a key regulator of the EGFR-dependent functional changes in MCAs such as compaction, spreading and sensitivity to platinum-based chemotherapeutics. These findings suggest a mechanism by which persistent EGFR activation in the microenvironment may drive changes in ovarian cancer cells that contribute to the poor prognosis associated with EGFR activation.

Quantitative Analysis of [11C]-Erlotinib PET Demonstrates Specific Binding for Activating Mutations of the EGFR Kinase Domain

Activating mutations of the epidermal growth factor receptor (EGFR) occur in multiple tumor types, including non-small cell lung cancer (NSCLC) and malignant glioma, and have become targets for therapeutic intervention. The determination of EGFR mutation status using a noninvasive, molecular imaging approach has the potential for clinical utility. In this study, we investigated [11C]-erlotinib positron emission tomography (PET) imaging as a tool to identify activating mutations of EGFR in both glioma and NSCLC xenografts. Radiotracer specific binding was determined for high and low specific activity (SA) [11C]-erlotinib PET scans in mice bearing synchronous human cancer xenografts with different EGFR expression profiles (PC9, HCC827, U87, U87 ΔEGFR, and SW620). Although xenograft immunohistochemistry demonstrated constitutive EGFR phosphorylation, PET scan analysis using the Simplified Reference Tissue Model showed that only kinase domain mutant NSCLC (HCC827 and PC9) had significantly greater binding potentials in high versus low SA scans. Xenografts with undetectable EGFR expression (SW620), possessing wild-type EGFR (U87), and expressing an activating extracellular domain mutation (U87 ΔEGFR) were indistinguishable under both high and low SA scan conditions. The results suggest that [11C]-erlotinib is a promising radiotracer that could provide a novel clinical methodology for assessing EGFR and erlotinib interactions in patients with tumors that harbor EGFR-activating kinase domain mutations.

Malignant Peripheral Nerve Sheath Tumor Invasion Requires Aberrantly Expressed EGF Receptors and Is Variably Enhanced by Multiple EGF Family Ligands

Aberrant epidermal growth factor receptor (EGFR) expression promotes the pathogenesis of malignant peripheral nerve sheath tumors (MPNSTs), the most common malignancy associated with neurofibromatosis type 1, but the mechanisms by which EGFR expression promotes MPNST pathogenesis are poorly understood. We hypothesized that inappropriately expressed EGFRs promote MPNST invasion and found that these kinases are concentrated in MPNST invadopodia in vitro. Epidermal growth factor receptor knockdown inhibited the migration of unstimulated MPNST cells in vitro, and exogenous EGF further enhanced MPNST migration in a substrate-specific manner, promoting migration on laminin and, to a lesser extent, collagen. In this setting, EGF acts as a chemotactic factor. We also found that the 7 known EGFR ligands (EGF, betacellulin, epiregulin, heparin-binding EGF, transforming growth factor-α [TGF-α], amphiregulin, and epigen) variably enhanced MPNST migration in a concentration-dependent manner, with TGF-α being particularly potent. With the exception of epigen, these factors similarly promoted the migration of nonneoplastic Schwann cells. Although transcripts encoding all 7 EGFR ligands were detected in human MPNST cells and tumor tissues, only TGF-α was consistently overexpressed and was found to colocalize with EGFR in situ. These data indicate that constitutive EGFR activation, potentially driven by autocrine or paracrine TGF-α signaling, promotes the aggressive invasive behavior characteristic of MPNSTs.

Epidermal Growth Factor Receptor: Is It a Feasible Target for the Treatment of Osteosarcoma?

PurposeFeatures of epidermal growth factor receptor (EGFR) expression in osteosarcoma and in vitro efficacies of EGFR inhibitors against osteosarcoma cells were evaluated.Materials and MethodsThirty biopsy samples of osteosarcoma patients were retrospectively analyzed for EGFR protein expression by immunohistochemistry. Relationships between EGFR expression and clinicopathologic characteristics and treatment outcomes were evaluated. Four osteosarcoma cell lines were analyzed for EGFR and p-EGFR expression by western blotting. Efficacies of gefitinib and BIBW2992 on osteosarcoma cells were evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Tyrosine kinase domains in exons 18 to 21 were sequenced and gene expression analyses of EGFR and PTEN were performed in four osteosarcoma cell lines.ResultsEGFR protein was expressed in 27 (90%) samples (6 low, 12 intermediate, 9 high) and in three cell lines. Intermediate or high staining for EGFR was related to a tumor volume<150 mL (p<0.001) and histologic subtype other than osteoblastic type (p=0.03). However, EGFR expression was not associated with histologic response to preoperative chemotherapy or survival. Gefitinib and BIBW 2992 did not have any significant inhibitory effect on cell viabilities. DNA sequencing analysis revealed three osteosarcoma cell lines have single base changes at codon 2361 of exon 20 (G to A), without affecting translation results. Furthermore, no mutation was found to be associated with constitutive EGFR activation.ConclusionIn the present study, gefitinib and BIBW2992 were not effective against osteosarcoma cells. However, as osteosarcoma cells express EGFR, further studies are necessary to explore the potential of other therapeutic agents targeting EGFR.

Abstract 1891: NF-κB signaling plays a certain role in apoptosis resistance in 3D-cultured EGFR-mutant lung adenocarcinoma cells

Abstract It is generally thought that suspended tumor cells show reduced proliferative activity and are more resistant to conventional chemotherapy compared with adherent tumor cells. Two epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cell lines, HCC827 (E746-A750 deletion) and H1975 (L858R + T790M), cultured in suspension, however, undergo apoptosis much more easily than these two cell lines cultured in conventional monolayer dishes, i.e. anchorage dependent conditions, when the kinase activity of EGFR is completely inhibited by treatment with EGFR tyrosine kinase inhibitors (TKIs) regardless of suspension or monolayer culture conditions. These findings indicate that attached EGFR-mutant lung adenocarcinoma cells appear less dependent on constitutive EGFR signaling for survival than detached cells, probably because integrin ligation enhances cell survival in adherent cells. However, the microenvironment of cells in monolayer culture conditions is considerably different from that of cells in vivo. We thus tested whether EGFR-mutated lung adenocarcinoma cells in three dimensional (3D) conditions using Matrigel resist EGFR TKIs-induced apoptosis compared with suspended cells. We uncovered three major findings in this study as follows: 1) both HCC827 cells and H1975 cells cultured in 3D conditions were significantly less sensitive to EGFR TKIs-induced apoptosis than suspended cells. 2) Marked degradation of IαBβ, the inhibitor of NF-κB pathway, was observed only in 3D-cultured EGFR-mutated lung adenocarcinoma cells, resulting in NF-κB signaling activation. 3) TPCA-1, a potent IKKα inhibitor, can enhance the levels of apoptosis in 3D-cultured EGFR-mutant cells treated with EGFR TKIs. These results suggest that combined EGFR and NF-κB inhibition is required to induce apoptosis effectively in EGFR-mutated lung adenocarcinoma cells in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1891. doi:1538-7445.AM2012-1891

Abstract 4083: Increased DNA methyltransferase activity and DNA methylation following epidermal growth factor stimulation in ovarian cancer cells

Abstract Ovarian cancer progression is correlated with accumulation of aberrant CpG island methylation. In patients with ovarian cancer, ascites fluid builds up and contains abundant levels of Epidermal-Growth-Factor-Receptor (EGFR) activators. This fosters a tumor microenvironment of constant EGFR activation. Signaling pathways downstream of EGFR, such as RAS, regulate DNA methylation and expression of DNA methyltransferases. We hypothesized that mimicking this microenvironment of chronic EGFR activation in culture could alter DNA methylation or expression of DNA methyltransferases in ovarian cancer cells. We found evidence that EGFR activation increased DNA methyltransferase activity by ∼3 fold acutely and by ∼7 fold following long term EGFR activation. Furthermore, this increase in activity was dependent on continued EGFR activity and resulted in a 50% increase in global DNA methylation. We also found that constitutive EGFR activation can decrease expression of DNMT3A. However, this decrease does not explain the observed increases in DNMT activity or methylation, thus future experiments will focus on a potential mechanism that mediates these EGF induced alterations to DNMT activity and DNA methylation. The data support a role for EGFR in the process of DNA methylation and accumulation during ovarian cancer progression; thus, suggesting that epigenetic therapy, used in combination with conventional chemotherapy, may be beneficial at preventing cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4083. doi:1538-7445.AM2012-4083

Blocking Epidermal Growth Factor Receptor Activation by 3,3′-Diindolylmethane Suppresses Ovarian Tumor Growth In Vitro and In Vivo

Genetic alterations, including the overexpression of epidermal growth factor receptor (EGFR) (in approximately 70% of ovarian tumors), play a crucial role in the signal transduction pathways that regulate key cellular functions, such as cell survival and proliferation, and are responsible for compromising traditional chemotherapy. 3,3'-Diindolylmethane (DIM) is an indole compound present in Brassica vegetables. In our previous studies, we demonstrated that BR-DIM, a formulated version of DIM, suppressed the growth of ovarian cancer cells by causing cell cycle arrest and apoptosis. In the present study, we delineated the mechanism by which DIM suppressed the growth of SKOV-3, OVCAR-3, and TOV-21G human ovarian cancer cells. DIM treatment caused significant down-regulation of the constitutive EGFR protein level as well as phosphorylation of EGFR at Tyr1068, Tyr992, Tyr845, and Tyr1173 in various ovarian cancer cells. To determine whether DIM suppressed the activation of EGFR by activating phosphorylation, cells were treated with epidermal growth factor. Epidermal growth factor treatment significantly blocked the DIM-mediated inhibition of EGFR activation and apoptosis in both SKOV-3 and OVCAR-3 cells. In addition, DIM treatment drastically reduced the phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK), which are downstream to EGFR, without affecting their protein levels. DIM treatment also inhibited the kinase activity of ERK, as observed by the down-regulation of phospho-E twenty-six like transcription factor 1 (p-ELK1) in all three ovarian cancer cell lines. DIM significantly suppressed the growth of ovarian tumors in vivo. Tumor growth suppressive effects of DIM in SKOV-3 tumor xenografts were associated with reduced phosphorylation of EGFR, MEK, and ERK. These results indicate that DIM induces apoptosis in ovarian cancer cells by inhibiting the EGFR-ERK pathway in vitro and in vivo.