Epidermal Growth Factor Receptor Stimulation Research Articles

DUSP1/MKP-1 represents another piece in the P2X7R intracellular signaling puzzle in cerebellar cells: our last journey with Mª Teresa along the purinergic pathways of Eden

The P2X7 receptor (P2X7R) stands out within the purinergic family as it has exclusive pharmacological and regulatory features, and it fulfills distinct roles depending on the type of stimulation and cellular environment. Tonic activation of P2X7R promotes cell proliferation, whereas sustained activation is associated with cell death. Yet strikingly, prolonged P2X7R activation in rat cerebellar granule neurons and astrocytes does not affect cell survival. The intracellular pathways activated by P2X7Rs involve proteins like MAPKs, ERK1/2 and p38, and interactions with growth factor receptors could explain their behavior in populations of rat cerebellar cells. In this study, we set out to characterize the intracellular mechanisms through which P2X7Rs and Trk receptors, EGFR (epidermal growth factor receptor) and BDNFR (brain-derived neurotrophic factor receptor), regulate the dual-specificity phosphatase DUSP1. In cerebellar astrocytes, the regulation of DUSP1 expression by P2X7R depends on ERK and p38 activation. EGFR stimulation can also induce DUSP1 expression, albeit less strongly than P2X7R. Conversely, EGF was virtually ineffective in regulating DUSP1 in granule neurons, a cell type in which BDNF is the main regulator of DUSP1 expression and P2X7R only induces a mild response. Indeed, the regulation of DUSP1 elicited by BDNF reflects the balance between both transcriptional and post-transcriptional mechanisms. Importantly, when the regulation of DUSP1 expression is compromised, the viability of both astrocytes and neurons is impaired, suggesting this phosphatase is essential to maintain proper cell cytoarchitecture and functioning.

Major histocompatibility complex class I molecule expression by pancreatic cancer cells is regulated by activation and inhibition of the epidermal growth factor receptor.

Pancreatic cancer is one of the deadliest neoplasms, with a dismal 5-year survival rate of only 10%. The ability of pancreatic cancer cells to evade the immune system hinders an anti-tumor response and contributes to the poor survival rate. Downregulation of major histocompatibility complex (MHC) class I cell-surface expression can aid in immune evasion by preventing endogenous tumor antigens from being presented to cytotoxic T cells. Earlier studies suggested that epidermal growth factor receptor (EGFR) signaling can decrease MHC class I expression on certain cancer cell types. However, even though erlotinib (a tyrosine kinase inhibitor that targets EGFR) is an approved drug for advanced pancreatic cancer treatment, the impact of EGFR inhibition or stimulation on pancreatic cancer cell MHC class I surface expression has not previously been analyzed. In this current study, we discovered that EGFR affects MHC class I mRNA and protein expression by human pancreatic cancer cell lines. We demonstrated that cell-surface MHC class I expression is downregulated upon EGFR activation, and the MHC class I level at the surface is elevated following EGFR inhibition. Furthermore, we found that EGFR associates with MHC class I molecules. By defining a role in pancreatic cancer cells for activated EGFR in reducing MHC class I expression and by revealing that EGFR inhibitors can boost MHC class I expression, our work supports further investigation of combined usage of EGFR inhibitors with immunotherapies against pancreatic cancer.

Abstract 5197: A systems approach to evaluating the effects of two EGFR-targeted therapies on cellular models of human skin and lung cancer using an integrated imaging and analysis platform and the rapid quantification of multiple key biomarkers

Abstract The physiological function of the epidermal growth factor receptor (EGFR) is to regulate epithelial tissue development and homeostasis, but its overexpression and activation are strongly linked to oncogenesis and tumor progression in a variety of human cancers. Many EGFR-targeted therapies have been developed which alone have proven to be effective, but resistance often develops, indicating a need for more studies probing the efficacy of combinatorial drug treatments. These studies will further require the discovery and characterization of biomarkers that can predict therapeutic efficacy and resistance. The purpose of this study was to examine the effects of individual and combined treatment with two classical EGFR-targeted therapies with different modes of action on cellular models of human skin cancer and non-small cell lung carcinoma (NSCLC). We characterized the effects of a two-day treatment with varying concentrations of EGF on cellular health and proliferation and on the expression of several key biomarker proteins in A431 and A549 cell lines. We then examined the individual and combined effects of treatment with the tyrosine kinase inhibitor (TKI) gefitinib and the humanized monoclonal antibody cetuximab. Cellular growth, proliferation and health were assessed with ATPlite luminescence assays and well-imaging and cytometry measures using a high-throughput multimodal imager and plate reader. Biomarkers measured included EGFR, immune checkpoint proteins (PD-L1 and CD276/B7-H3), chemokines (IL-8/CXCL8 and GRO-α/CXCL1), a protease inhibitor (TIMP-2), and growth factor (VEGF). All these biomarker proteins were rapidly assessed using AlphaLISA no-wash, mix-and-read assays on 5 μL samples collected from the same wells of the culture plate. We observed that EGF treatment resulted in decreased EGFR expression and upregulation of PD-L1 protein in a dose-dependent manner in both culture models. CD276, VEGF and TIMP-2 expression levels, however, were upregulated with EGF treatment only in the A431 cells. Complex drug treatment effects on biomarker expression are presented. In addition, treatment effects on EGFR phosphorylation and downstream signaling pathways (including MEK/ERK and AKT) were probed using AlphaLISA SureFire Ultra phosphorylation assays and demonstrate the different pathways effected by either drug or a combination of both. These data illustrate the effects of EGFR stimulation on the expression of an array of biomarkers and signaling pathways and in the cellular tolerance for individual and combined treatment with two EGFR-targeted therapies with different mechanisms of action between two different cellular models of human cancer. Citation Format: Jeanine M. Hinterneder, Lauren Berstler, Adam Carlson, Jen Carlstrom. A systems approach to evaluating the effects of two EGFR-targeted therapies on cellular models of human skin and lung cancer using an integrated imaging and analysis platform and the rapid quantification of multiple key biomarkers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5197.

Analysis of Virus and Host Proteomes During Productive HSV-1 and VZV Infection in Human Epithelial Cells.

Herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) are two closely related human alphaherpesviruses that persistently infect most adults worldwide and cause a variety of clinically important diseases. Herpesviruses are extremely well adapted to their hosts and interact broadly with cellular proteins to regulate virus replication and spread. However, it is incompletely understood how HSV-1 and VZV interact with the host proteome during productive infection. This study determined the temporal changes in virus and host protein expression during productive HSV-1 and VZV infection in the same cell type. Results demonstrated the temporally coordinated expression of HSV-1 and VZV proteins in infected cells. Analysis of the host proteomes showed that both viruses affected extracellular matrix composition, transcription, RNA processing and cell division. Moreover, the prominent role of epidermal growth factor receptor (EGFR) signaling during productive HSV-1 and VZV infection was identified. Stimulation and inhibition of EGFR leads to increased and decreased virus replication, respectively. Collectively, the comparative temporal analysis of viral and host proteomes in productively HSV-1 and VZV-infected cells provides a valuable resource for future studies aimed to identify target(s) for antiviral therapy development.

Proteomic and Phosphoproteomic Analysis Reveals that Neurokinin-1 Receptor (NK1R) Blockade with Aprepitant in Human Keratinocytes Activates a Distinct Subdomain of EGFR Signaling: Implications for the Anti-Pruritic Activity of NK1R Antagonists.

Background: Epidermal growth factor receptor (EGFR) inhibitors can cause serious cutaneous toxicities, including pruritus and papulopustular acneiform skin eruptions. Increasingly, the neurokinin-1 receptor (NK1R) antagonist aprepitant is being utilized as an anti-pruritic agent in the treatment of EGFR-inhibitor induced pruritus. Aprepitant is believed to reduce itching by blocking NK1R on the surface of dermal mast cells. However, the effects of aprepitant on human keratinocytes remains unexplored. Methods: Herein, we examine the effects of aprepitant on EGFR stimulation in HaCaT cells using a phosphoproteomic approach including reverse phase protein arrays and Ingenuity Pathway Analysis. Changes in EGFR phosphorylation were visualized using Western blotting and the effect of EGF and aprepitant on the growth of HaCaT cells was determined using the WST-1 Cell Proliferation Assay System. Results: We found that aprepitant increased the phosphorylation of EGFR, as well as 10 of the 23 intracellular proteins phosphorylated by EGF. Analysis of phosphoproteomic data using Ingenuity Pathway Analysis software revealed that 5 of the top 10 pathways activated by EGF and aprepitant are shared. Conclusions: We propose that aprepitant produces its antipruritic effects by partially activating EGFR. Activation of EGFR by aprepitant was also seen in primary human keratinocytes. In addition to itch reduction through partial activation of shared EGFR pathways, aprepitant exerts a dose-dependent cytotoxicity to epithelial cells, which may contribute to its antitumor effects.

Abstract 2644: Histone deacetylase 6 plays a critical role in sulfiredoxin-mediated activation of the epidermal growth factor receptor signaling in human colorectal cancer

Abstract Mammalian cells develop various transmembrane receptors including the family of receptor tyrosine kinases (RTKs) to respond to extracellular stimuli. Epidermal growth factor receptor (EGFR) is the first identified member of the RTKs and it plays a pivotal role in colorectal cancer by stimulating cell proliferation and survival. Binding of extracellular ligands, mainly EGF, leads to the dimerization of EGFR that triggers the activation of downstream mitogen activated protein kinases. In addition to the amount of ligand binding, the strength and duration of EGFR signaling are also affected by the posttranslational modifications of the receptor itself, such as phosphorylation, ubiquitination, acetylation, etc. In the previous study, we demonstrated that Sulfiredoxin (Srx) enhances the EGFR signaling in colorectal cancer cells through the inhibition of receptor acetylation at lysine residue 1061. However, the molecular mechanism by which Srx regulates the level of EGFR acetylation is still unclear. In this study, we investigated (1) the mechanism of EGFR acetylation and its regulation by Srx; (2) the functional consequence of EGFR acetylation on EGF-induced receptor dimerization, recycling, trafficking and degradation. Human colon cancer cell lines were cultured and maintained in standard conditions. Western blotting, immunoprecipitation and Mass spectrometry were used to identify cysteine oxidation of HDAC6 and characterize enzymes that are involved in the acetylation of EGFR. Stable cells that overexpress or loss of target genes, such as Srx, HDAC6, EGFR and/or mutants, were established using the 3rd generation lentiviral ShRNA infection or the CRISPR-Cas9 system. The activity of histone deacetylases was determined using commercial kit in the presence/absence of pan- or isoform-specific chemical inhibitors of HDACs. Upon EGF stimulation, the dimerization, recycling, trafficking and degradation of EGFR were demonstrated through a combination of immunoblotting, flow-cytometry and immunofluorescent imaging. We demonstrated that HDAC6 plays a central role in the regulation of EGFR acetylation through its interaction with EGFR. Depletion of Srx in human colon cancer cells increases the levels of intracellular reactive oxygen species, leading to inhibition of HDAC6 activity through regulating the redox state of cysteines 407/417. Depletion of HDAC6 not only leads to increased level of EGFR acetylation but also suppresses the binding between EGFR and its adaptor proteins. EGF-induced receptor dimerization and recycling are altered in HDAC6 knockout cells, result in inhibition of EGFR downstream signaling. Taken together, this study indicates that Srx enhances the strength and duration of EGF signaling through the ROS-HDAC6-EGFR axis in human colon cancer cells. Citation Format: Qi Ying, Hong Jiang, B. Mark Evers, Qiou Wei. Histone deacetylase 6 plays a critical role in sulfiredoxin-mediated activation of the epidermal growth factor receptor signaling in human colorectal 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 2644.

A protective Langerhans cell-keratinocyte axis that is dysfunctional in photosensitivity.

Photosensitivity, or skin sensitivity to ultraviolet radiation (UVR), is a feature of lupus erythematosus and other autoimmune and dermatologic conditions, but the mechanistic underpinnings are poorly understood. We identify a Langerhans cell (LC)-keratinocyte axis that limits UVR-induced keratinocyte apoptosis and skin injury via keratinocyte epidermal growth factor receptor (EGFR) stimulation. We show that the absence of LCs in Langerin-diphtheria toxin subunit A (DTA) mice leads to photosensitivity and that, in vitro, mouse and human LCs can directly protect keratinocytes from UVR-induced apoptosis. LCs express EGFR ligands and a disintegrin and metalloprotease 17 (ADAM17), the metalloprotease that activates EGFR ligands. Deletion of ADAM17 from LCs leads to photosensitivity, and UVR induces LC ADAM17 activation and generation of soluble active EGFR ligands, suggesting that LCs protect by providing activated EGFR ligands to keratinocytes. Photosensitive systemic lupus erythematosus (SLE) models and human SLE skin show reduced epidermal EGFR phosphorylation and LC defects, and a topical EGFR ligand reduces photosensitivity. Together, our data establish a direct tissue-protective function for LCs, reveal a mechanistic basis for photosensitivity, and suggest EGFR stimulation as a treatment for photosensitivity in lupus erythematosus and potentially other autoimmune and dermatologic conditions.

Abstract 5443: Methylation-dependent suppression of Tissue Factor is a key contributor to the less aggressive phenotype in IDH1 mutant versus IDH1 wild-type gliomas

Abstract Gliomas with mutations in isocitrate dehydrogenase 1 (IDH1mut) have global genomic hypermethylation, are less aggressive than IDH1 wild-type (IDH1wt) gliomas, and generally grow poorly in vitro and in vivo. Yet little data exist that connect specific hypermethylation events to this unique phenotype. We previously reported that the gene encoding Tissue Factor (TF), F3, is among the most hypermethylated and downregulated genes in IDH1mut gliomas relative to IDH1wt gliomas. TF was originally described in blood coagulation, but also can enhance tumor malignancy via protease-activated receptor 2 (PAR2) signaling, though its role in gliomas is not as well understood. In this study, we further explored the significance of TF on malignancy in IDH1wt and IDH1mut gliomas, and showed that receptor tyrosine kinases (RTKs) and β-catenin are downstream effectors of TF-PAR2 signaling. Studies were performed using 6 patient derived glioma cell lines, 3 IDH1wt (GBM6, GBM12, GBM43) and 3 IDH1mut (TB09, BT142, GBM164). DNA methylation was assessed by Illumina Human 850K. TF procoagulant activity (PCA) was determined by FXa generation, using conditioned medium. Markers of malignancy included BrdU incorporation for cell proliferation, transwell invasion, and soft agar colony formation. Nude mice (N≥6/group) were transplanted with glioma cells and monitored for growth using bioluminescence imaging. The TF gene, F3, was significantly hypermethylated in IDH1mut cells compared to IDH1wt cells, with corresponding lower TF protein in IDH1mut cells. Treatment of IDH1mut glioma cells with a demethylating agent, decitabine, increased F3 transcription 5-fold and PCA 19-fold in IDH1mut cells. TF increased the in vivo "take rate" of IDH1mut GBM164 from 0% to 100% (P=0.0001, Fisher's exact test). Conversely, TF knockdown in IDH1wt cells greatly reduced cell proliferation, invasion, colony formation, and extended median survival of engrafted mice by 86% (P=0.001). In both IDH1wt and IDH1mut cells, TF-PAR2 activated β-catenin, ERK1/2, and Akt, and was associated with multiple pathways that increase high-grade behavior. Disrupting TF-PAR2 signaling had the greatest impact on tumor growth in IDH1wt gliomas driven by epidermal growth factor receptor (EGFR), a major receptor tyrosine kinase (RTK) involved in glioma malignancy. TF-PAR2 transactivated EGFR through a Src-dependent intracellular pathway, even when extracellular stimulation of EGFR was blocked, and further enhanced phosphorylation of the constitutively activated EGFRvIII. In IDH1mut cells, TF-PAR2 signaled through another RTK, PDGFR. In contrast, TF-PAR2 stimulated invasion in all glioma cell lines through an RTK-independent, β-catenin-dependent mechanism. These results demonstrate the importance of TF-PAR2 in gliomas, and show that its suppression is a critical component of the IDH1mut phenotype. Citation Format: Dusten J. Unruh, Snezana Mirkov, Seamus Caragher, Jann Sarkaria, Atique Ahmed, C. David James, Craig Horbinski. Methylation-dependent suppression of Tissue Factor is a key contributor to the less aggressive phenotype in IDH1 mutant versus IDH1 wild-type gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5443.

Effect of EGFR on Calcium Mobilization and Epithelial Repair in Gastric Organoids

BackgroundIn vivo, addition of calcium (Ca2+) accelerates epithelial repair of damage; intracellular Ca2+ mobilization is known to be an essential factor for gastric epithelial repair. We are investigating upstream effectors that play a role in Ca2+ mobilization and repair. Epidermal growth factor receptor (EGFR) is implicated in repair as stimulation of EGFR and subsequent activation of its downstream MAP kinases (e.g. ERK) has been shown to occur in the healing gut mucosa. Additionally, ERK activation has been shown to affect intracellular Ca2+ signaling in several cell lines. However the connection between EGFR stimulation and Ca2+ during the gastric repair process has yet to be elucidated in native tissue, in part because of the limitations of working in vivo. In the present study, we utilize the 3D primary culture of gastric epithelial cells known as gastric organoids (gastroids), which mimic in vivo gastric epithelium, to test the role of EGFR in Ca2+‐driven restitution.MethodsGastroids were generated from isolated corpus tissue of yellow cameleon Nano15 (YC‐Nano) transgenic mice, and cultured 4–5 days prior to experiments. YC‐Nano is a fluorescent calcium reporter which utilizes Förster resonance energy transfer (FRET) from CFP to YFP to indicate changes in intracellular Ca2+ mobilization. Damage was induced in gastroids by 1–2s exposure of single epithelial cells to high intensity two‐photon 840 nm light (photodamage, PD), resulting in damage to the targeted cell, subsequent cell death, and repair of the epithelium over 10–15 min. Damage and repair progress was evaluated using confocal/2‐photon microscopy. YC‐Nano was excited by low intensity two‐photon 840 nm light and CFP and YFP (FRET) fluorescence imaged simultaneously. Ca2+ levels reported as YFP/CFP ratio (FRET ratio) were measured in the migrating cells that neighbor the damage area as well as intact cells 2–3 cells away from the site of damage.ResultsSingle epithelial cell PD injury in gastroids resulted in the paired events of dead cell exfoliation and migration of undamaged neighboring cells to restore a continuous epithelium in the damaged site. Additionally, PD injury resulted in intracellular Ca2+ increase within 1–3 min after PD, selectively in migrating cells adjacent to the damage site. The observed peak FRET ratio corresponds to the time of maximum damage area. In contrast, PD did not affect intracellular Ca2+ level in 2–3 cells away from the site of damage. FRET ratio increased more in the lateral side of the cell adjacent to the damaged area versus whole cell FRET ratio values, suggesting localized Ca2+ mobilization at the site of cell migration. Gastroids supplemented with 200 nM AG1478 (EGFR inhibitor) resulted in delayed repair. AG1478 significantly decreased the observed FRET ratio peak within gastroids, suggesting the presence of EGFR within the gastroid model. In preliminary data, addition of 10 μM FR180204 (ERK1/2 inhibitor) also prevented prompt repair and affected the intracellular Ca2+ peak.ConclusionResults demonstrate that EGFR is required for efficient epithelial repair in gastroids. The parallel effects of AG1478 to decrease intracellular Ca2+ levels and delay repair suggests EGFR acts upstream in the pathway of calcium‐driven repair.Support or Funding InformationFunded by R01 DK102551‐01 and F31 DK115126‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ADAM17 is required for EGF-R-induced intestinal tumors via IL-6 trans-signaling.

Colorectal cancer is treated with antibodies blocking epidermal growth factor receptor (EGF-R), but therapeutic success is limited. EGF-R is stimulated by soluble ligands, which are derived from transmembrane precursors by ADAM17-mediated proteolytic cleavage. In mouse intestinal cancer models in the absence of ADAM17, tumorigenesis was almost completely inhibited, and the few remaining tumors were of low-grade dysplasia. RNA sequencing analysis demonstrated down-regulation of STAT3 and Wnt pathway components. Because EGF-R on myeloid cells, but not on intestinal epithelial cells, is required for intestinal cancer and because IL-6 is induced via EGF-R stimulation, we analyzed the role of IL-6 signaling. Tumor formation was equally impaired in IL-6-/- mice and sgp130Fc transgenic mice, in which only trans-signaling via soluble IL-6R is abrogated. ADAM17 is needed for EGF-R-mediated induction of IL-6 synthesis, which via IL-6 trans-signaling induces β-catenin-dependent tumorigenesis. Our data reveal the possibility of a novel strategy for treatment of colorectal cancer that could circumvent intrinsic and acquired resistance to EGF-R blockade.

Heterologous phosphorylation–induced formation of a stability lock permits regulation of inactive receptors by β-arrestins

β-Arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptors and β-arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether β-arrestins are able to bind second messenger kinase-phosphorylated, but inactive receptors as well. Because heterologous phosphorylation is a common phenomenon among GPCRs, this mode of β-arrestin activation may represent a novel mechanism of signal transduction and receptor cross-talk. Here we demonstrate that activation of protein kinase C (PKC) by phorbol myristate acetate, Gq/11-coupled GPCR, or epidermal growth factor receptor stimulation promotes β-arrestin2 recruitment to unliganded AT1 angiotensin receptor (AT1R). We found that this interaction depends on the stability lock, a structure responsible for the sustained binding between GPCRs and β-arrestins, formed by phosphorylated serine-threonine clusters in the receptor's C terminus and two conserved phosphate-binding lysines in the β-arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters β-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor-β-arrestin interaction, but also governs the structural rearrangements within β-arrestins. Furthermore, we found that β-arrestin2 binds to PKC-phosphorylated AT1R in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results provide new insights into the activation of β-arrestins and reveal their novel role in receptor cross-talk.

The EGF receptor inhibits the signaling of dopamine D3 receptor through the phosphorylation of GRK2 on tyrosine residues

Receptor transactivation or crosstalk are terms referring to instances in which the signaling of a given receptor is regulated by a different class of receptor. The epidermal growth factor (EGF) and the dopaminergic systems in the brain are closely related to schizophrenia with respect to both etiology and treatment. Thus, we investigated the functional interactions between the EGF receptor (EGFR), which belongs to the receptor tyrosine kinase family, and the dopamine D2-like receptors (D2R, D3R, and D4R), which are members of the G protein-coupled receptor (GPCR) family. Among D2-like receptors, the signaling of D3R was selectively inhibited by EGFR stimulation. Moreover loss-of-function assays showed that tyrosine-phosphorylated GRK2 mediates this inhibition by acting on the second intracellular loop of D3R. Considering that both EGFR and D3R are closely related to schizophrenia, this study could provide new molecular insight into the etiology of the disorder.

Autocrine expression of the epidermal growth factor receptor ligand heparin-binding EGF-like growth factor in cervical cancer.

In cervical cancer, the epidermal growth factor receptor (EGFR) is overexpressed in 70–90% of the cases and has been associated with poor prognosis. EGFR-based therapy is currently being explored in cervical cancer. We investigated which EGFR ligand is primarily expressed in cervical cancer and which cell type functions as the major source of this ligand. We hypothesized that macrophages are the main source of EGFR ligands and that a paracrine loop between tumor cells and macrophages is responsible for ligand expression. mRNA expression analysis was performed on 32 cervical cancer cases to determine the expression of the EGFR ligands amphiregulin, β-cellulin, epidermal growth factor (EGF), epiregulin, heparinbinding EGF-like growth factor (HB-EGF) and transforming growth factor α (TGFα). Subsequently, protein expression was determined immunohistochemically on 36 additional cases. To assess whether macrophages are the major source of EGFR ligands, immunohistochemical double staining was performed on four representative tissue slides. Expression of the chemokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and C-C motif ligand 2 (CCL2) was determined by mRNA in situ hybridization. Of the known EGFR ligands, HB-EGF had the highest mRNA expression and HB-EGF and EGFR protein expression were highly correlated. Tumor specimens with high EGFR expression showed higher numbers of macrophages, and higher expression of GM-CSF and CCL2, but only a small subset (9%) of macrophages was found to be HB-EGF-positive. Strikingly, 78% of cervical cancer specimens were found to express HB-EGF. Standardized assessment of staining intensity, using spectral imaging analysis, showed that HB-EGF expression was higher in the tumor compartment than in the stromal compartment. These results suggest that HB-EGF is an important EGFR ligand in cervical cancer and that cervical cancer cells are the predominant source of HB-EGF. Therefore, we propose an autocrine EGFR stimulation model in cervical carcinomas.

EGFR-mediated apoptosis via STAT3

The Epidermal Growth Factor Receptor (EGFR) is a cell surface receptor with primary implications in cell growth in both normal and malignant tissue. Paradoxically, cell lines that hyperexpress the EGFR have been documented to undergo receptor-mediated apoptosis. The underlying mechanism by which EGF-induced apoptosis occurs however remains inexplicit. In an attempt to identify this mechanism, we assessed downstream effectors of EGFR in MDA-MB-468 cells during conditions of EGF-induced apoptosis. The effector assessment revealed STAT3 as a potential mediator of EGF-induced apoptosis. Alternative strategies for activating STAT3, independent of EGFR stimulation, resulted in the induction of the apoptotic pathways. A reduction in STAT3 expression via RNAi resulted in a significant attenuation of EGF-induced PARP cleavage. Our findings support STAT3 as a positive mediator of EGF-induced apoptosis in MDA-MB-468 cells.

Epithelial Cell-Derived a Disintegrin and Metalloproteinase-17 Confers Resistance to Colonic Inflammation Through EGFR Activation

Epithelial regeneration is a key process for the recovery from ulcerative colitis (UC). Here we demonstrate that a disintegrin and metalloproteinase-17 (ADAM17), a main sheddase for tumor necrosis factor (TNF)-α, is essential for defensive epithelial properties against UC by promoting epithelial cell growth and goblet cell differentiation in mouse and human. Mice with systemic deletion of Adam17 developed severe dextran sulfate sodium-induced colitis when compared to mice with myeloid cell Adam17 deletion or control littermates. ADAM17 was predominantly expressed by regenerating epithelia in control mice, and its loss or inhibition attenuated epidermal growth factor receptor (EGFR) activation, epithelial proliferation, mucus production and barrier functions. Conversely, ectopic EGFR stimulation promoted epithelial regeneration thereby partially rescuing the severe colitis caused by ADAM17 deficiency. In UC patients, epithelial ADAM17 expression positively correlated with both cell proliferation and goblet cell number. These findings suggest that maintaining ADAM17–EGFR epithelial signaling is necessary for the recovery from UC and would be beneficial to therapeutic strategies targeting ADAM17-mediated TNF-α shedding.

STAT1 Activation Is Enhanced by Cisplatin and Variably Affected by EGFR Inhibition in HNSCC Cells.

Cisplatin is a cytotoxic chemotherapeutic drug frequently used to treat many solid tumors, including head and neck squamous cell carcinoma (HNSCC). EGF receptor (EGFR) inhibitors have also shown efficacy as alternatives to cisplatin in some situations. However, large clinical trials have shown no added survival benefit from the use of these two drugs in combination. Possible explanations for this include overlapping downstream signaling cascades. Using in vitro studies, we tested the hypothesis that cisplatin and EGFR inhibitors rely on the activation of the tumor suppressor STAT1, characterized by its phosphorylation at serine (S727) or tyrosine (Y701) residues. Cisplatin consistently increased the levels of p-S727-STAT1, and STAT1 siRNA knockdown attenuated cisplatin-induced cell death. EGFR stimulation also activated p-S727-STAT1 and p-Y701-STAT1 in a subset of cell lines, whereas EGFR inhibitors alone decreased levels of p-S727-STAT1 and p-Y701-STAT1 in these cells. Contrary to our hypothesis, EGFR inhibitors added to cisplatin treatment caused variable effects among cell lines, with attenuation of p-S727-STAT1 and enhancement of cisplatin-induced cell death in some cells and minimal effect in other cells. Using HNSCC tumor specimens from a clinical trial of adjuvant cisplatin plus the anti-EGFR antibody panitumumab, higher intratumoral p-S727-STAT1 appeared to correlate with worse survival. Together, these results suggest that cisplatin-induced cell death is associated with STAT1 phosphorylation, and the addition of anti-EGFR therapy to cisplatin has variable effects on STAT1 and cell death in HNSCC.

Adenovirus E4-ORF1 Dysregulates Epidermal Growth Factor and Insulin/Insulin-Like Growth Factor Receptors To Mediate Constitutive Myc Expression.

The E4-ORF1 protein encoded by human adenovirus stimulates viral replication in human epithelial cells by binding and activating cellular phosphatidylinositol 3-kinase (PI3K) at the plasma membrane and cellular Myc in the nucleus. In this study, we showed that E4-ORF1 hijacks the tyrosine kinase activities of cellular epidermal growth factor receptor (EGFR) and insulin receptor (InsR)/insulin-like growth factor receptor 1 (IGF1R), as well as the lipid kinase activity of PI3K, to mediate constitutive Myc protein expression. We additionally demonstrated that EGFR contributes to constitutive Myc expression through the capacity of E4-ORF1 to induce ligand-independent EGFR activation and stimulation of the Ras/Mek/Erk pathway, the latter activity of which was conserved by human adenoviruses. Results further suggested that EGFR normally forms a complex with the cellular PDZ protein Discs Large 1 (Dlg1), a component of the Dlg1:E4-ORF1:PI3K ternary complex that mediates E4-ORF1-induced PI3K activation, and that E4-ORF1 binds the Dlg1:EGFR complex and promotes the association of EGFR with InsR and IGF1R. In addition to its role in constitutive Myc expression, InsR/IGF1R also negatively regulates EGFR autophosphorylation and EGFR-mediated Ras/Mek/Erk signaling, and data suggested that E4-ORF1 binding to Dlg1 antagonizes these activities. Collectively, our findings suggest that in human epithelial cells, E4-ORF1 targets EGFR, InsR/IGF1R, and PI3K at the plasma membrane to activate cytosolic signaling pathways that sustain Myc protein levels in the nucleus. We postulate that E4-ORF1-induced constitutive Myc expression functions to ensure the formation of nuclear E4-ORF1:Myc complexes, which have been shown to activate Myc and to enhance adenovirus replication. While human adenoviruses primarily produce self-limited acute infections in humans, these agents are associated with life-threatening diseases in immunocompromised patients and in otherwise healthy individuals infected with certain virulent serotypes. The adenovirus E4-ORF1 protein enhances viral replication by activating the cellular lipid kinase PI3K and the cellular transcription factor Myc. Here we report that E4-ORF1 usurps the functions of the cellular tyrosine kinase receptors EGFR and InsR /: IGF1R, as well as PI3K, to sustain Myc protein expression in cells. Furthermore, sustained Myc expression depended on E4-ORF1-induced ligand-independent EGFR activation that stimulated Ras/Mek/Erk signaling, a function found to be conserved by human adenoviruses. Given the established roles of PI3K, the Ras/Mek/Erk pathway, and Myc in the adenovirus life cycle, our findings may aid in the development of safer, more effective therapeutic strategies to treat severe adenovirus infections as well as improved adenovirus vectors for use in vaccination and gene and cancer therapy.

Significant cytostatic effect of everolimus on a gefitinib-resistant anaplastic thyroid cancer cell line harboring PI3KCA gene mutation.

We previously demonstrated the efficacy of gefitinib, a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR), on an anaplastic thyroid cancer (ATC) cell line. We also observed that gefitinib was not effective in regulating cell growth in a different ATC cell line that exhibited an altered EGFR-initiated signal transduction pathway. In the present study, we attempted to regulate the downstream effector of EGFR-Akt-mammalian target of rapamycin (mTOR) pathway by an mTOR inhibitor, everolimus. A total of 8 ATC cell lines were employed, 7 of which were established in our institute. OCUT-2 was known to carry a mutation in the phosphoinositide-3-kinase, catalytic, α polypeptide gene (PI3KCA) and to be gefitinib-resistant, whereas ACT-1 exhibited a remarkable growth arrest by gefitinib. All the cell lines were tested for the cytotoxic effect of everolimus. The mechanisms of cellular toxicity were investigated by EGFR stimulation, cell cycle and concurrent exposure to paclitaxel. In OCUT-2, but not in any of the other cell lines, everolimus achieved a significant growth inhibition (inhibition of 30 and 50% was achieved by concentrations of 0.8 and 5 nM, respectively). The growth in OCUT-2 was inhibited by everolimus, even with concordant EGFR stimulation. This effect was demonstrated by a G2M cell cycle arrest. An additive effect of everolimus onto the cytotoxic effect of paclitaxel was demonstrated at a dose of 1-2 nM. A significant growth inhibitory effect of everolimus on the gefitinib-resistant ATC cell line was demonstrated, suggesting a possible correlation between the efficacy of everolimus and PI3KCA gene mutation and the significance of molecular-targeted therapy in the management of ATC.

Zn2+-stimulation of sperm capacitation and of the acrosome reaction is mediated by EGFR activation

Extracellular zinc regulates cell proliferation via the MAP1 kinase pathway in several cell types, and has been shown to act as a signaling molecule. The testis contains a relatively high concentration of Zn2+, required in both the early and late stages of spermatogenesis. Despite the clinical significance of this ion, its role in mature sperm cells is poorly understood. In this study, we characterized the role of Zn2+ in sperm capacitation and in the acrosome reaction. Western blot analysis revealed the presence of ZnR of the GPR39 type in sperm cells. We previously demonstrated the presence of active epidermal growth factor receptor (EGFR) in sperm, its possible transactivation by direct activation of G-protein coupled receptor (GPCR), and its involvement in sperm capacitation and in the acrosome reaction (AR). We show here that Zn2+ activates the EGFR during sperm capacitation, which is mediated by activation of trans-membrane adenylyl cyclase (tmAC), protein kinase A (PKA), and the tyrosine kinase, Src. Moreover, the addition of Zn2+ to capacitated sperm caused further stimulation of EGFR and phosphatydil-inositol-3-kinase (PI3K) phosphorylation, leading to the AR. The stimulation of the AR by Zn2+ also occurred in the absence of Ca2+ in the incubation medium, and required the tmAC, indicating that Zn2+ activates a GPCR. The AR stimulated by Zn2+ is mediated by GPR39 receptor, PKA, Src and the EGFR, as well as the EGFR down-stream effectors PI3K, phospholipase C (PLC) and protein kinase C (PKC). These data support a role for extracellular zinc, acting through the ZnR, in regulating multiple signaling pathways in sperm capacitation and the acrosome reaction.

Abstract 5255: EGFR is a conspiring kinase in gene fusion positive lung cancer

Abstract The use of kinase inhibitors directed at dominant oncogenes has proven a successful treatment strategy for non-small cell lung cancer (NSCLC) harboring ALK gene fusions, but intrinsic or acquired drug resistance limits clinical benefit. We recently identified a novel class of oncogenes generated by translocations of the NTRK1 gene, which encodes the TRKA receptor tyrosine kinase. ROS1 and RET gene fusions represent additional oncogene targets in lung cancer and gene fusion positive tumors currently comprise ∼10% of lung adenocarcinomas. Previous results from our laboratory have demonstrated that EGFR signaling can mediate both intrinsic and acquired resistance in a ROS1+ NSCLC cell line. We asked whether EGFR might play a common role in other NSCLC cell lines harboring gene fusions with the aim of developing combination therapies that may delay drug resistance to oncogene-targeted kinase inhibitors in these tumors. We have observed high protein expression of the epidermal growth factor receptor (EGFR) in NSCLC cell lines positive for ALK (H3122, H2228, and STE-1), ROS1 (HCC78 and CUTO-2), RET (LC-2/Ad), and NTRK1 gene fusions (CUTO-3), and in a NTRK1 patient-derived xenograft (PDX) model. We observed increased inhibition of downstream signaling and in vitro cellular proliferation with the addition of EGFR tyrosine kinase inhibitors in these gene fusion positive cell lines. Furthermore, stimulation of EGFR with EGF rescues targeted inhibition with oncogene-specific kinase inhibitors in the critical downstream signaling pathways such as PI3K/AKT and MEK/ERK. Inhibition of cellular proliferation of these cell lines using oncogene-specific kinase inhibitors was also rescued by stimulation of EGFR. Interestingly, we observed transactivation of the TRKA and RET kinase domains by EGFR stimulation in the CUTO-3 and LC-2/Ad cell lines, respectively, despite treatment with a TRKA or RET kinase inhibitor. Additionally, co-immunoprecipitation experiments show that EGFR and the chimeric protein RIP-TRKA (encoded by the fusion gene MPRIP-NTRK1) associate in the CUTO-3 cells or when ectopically expressed in 293T cells. Collectively, these data demonstrate a critical role for EGFR in gene fusion positive lung cancer and suggest that signaling via this receptor may provide a survival mechanism for cancer cells treated with an oncogene-specific kinase inhibitor. Further investigation of EGFR inhibition in addition to oncogene-specific kinase inhibitors may be warranted. Citation Format: Aria Vaishnavi, Anh T. Le, Stephen B. Keysar, Christine M. Lovly, Severine Kako, Marileila Varella-Garcia, Antonio Jimeno, Robert C. Doebele. EGFR is a conspiring kinase in gene fusion positive lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5255. doi:10.1158/1538-7445.AM2014-5255