Epidermal Growth Factor Receptor Cascade Research Articles

Epidermal growth factor receptor cascade prioritizes the maximization of signal transduction

Many studies have been performed to quantify cell signaling. Cell signaling molecules are phosphorylated in response to extracellular stimuli, with the phosphorylation sequence forming a signal cascade. The information gain during a signal event is given by the logarithm of the phosphorylation molecule ratio. The average information gain can be regarded as the signal transduction quantity (ST), which is identical to the Kullback–Leibler divergence (KLD), a relative entropy. We previously reported that if the total ST value in a given signal cascade is maximized, the ST rate (STR) of each signaling molecule per signal duration (min) approaches a constant value. To experimentally verify this theoretical conclusion, we measured the STR of the epidermal growth factor (EGF)-related cascade in A431 skin cancer cells following stimulation with EGF using antibody microarrays against phosphorylated signal molecules. The results were consistent with those from the theoretical analysis. Thus, signaling transduction systems may adopt a strategy that prioritizes the maximization of ST. Furthermore, signal molecules with similar STRs may form a signal cascade. In conclusion, ST and STR are promising properties for quantitative analysis of signal transduction.

Mechanistic insights into differential requirement of receptor dimerization for oncogenic activation of mutant EGFR and its clinical perspective

The epidermal growth factor receptor (EGFR), a member of the ErbB family (EGFR, ErbB2, ErbB3 and ErbB4), plays a crucial role in regulating various cellular responses such as proliferation, differentiation, and survival. As a result, aberrant activation of EGFR, mostly mediated through different classes of genomic alterations occurring within EGFR, is closely associated with the pathogenesis of numerous human cancers including lung adenocarcinoma, glioblastoma, and colorectal cancer. Thus, specific suppression of oncogenic activity of mutant EGFR with its targeted drugs has been routinely used in the clinic as a very effective anti-cancer strategy in treating a subset of tumors driven by such oncogenic EGFR mutants. However, the clinical efficacy of EGFR-targeted therapy does not last long due to several resistance mechanisms that emerge in the patients following the drug treatment. Thus, there is an urgent need for the development of novel therapeutic tactics specifically targeting mutant EGFR with the focus on the unique biological features of various mutant EGFR. Regarding this point, our review specifically emphasizes the recent findings about distinct requirements of receptor dimerization and autophosphorylation, which are critical steps for enzymatic activation of EGFR and signaling cascades, respectively, among wildtype and mutant EGFR and further discuss their clinical significance. In addition, the molecular mechanisms regulating EGFR dimerization and enzymatic activity by a key negative feedback inhibitor Mig6 as well as the clinical use for developing potential novel drugs targeting it are described in this review.

Epidermal Growth Factor Receptor and Transforming Growth Factor β Signaling Pathways Cooperate To Mediate Chlamydia Pathogenesis.

Human genital Chlamydia infection is a major public health concern due to the serious reproductive system complications. Chlamydia binds several receptor tyrosine kinases (RTKs) on host cells, including the epidermal growth factor receptor (EGFR), and activates cellular signaling cascades for host invasion, cytoskeletal remodeling, optimal inclusion development, and induction of pathogenic epithelial-mesenchyme transition (EMT). Chlamydia also upregulates transforming growth factor beta (TGF-β) expression, whose signaling pathway synergizes with the EGFR cascade, but its role in infectivity, inclusions, and EMT induction is unknown. We hypothesized that the EGFR and TGF-β signaling pathways cooperate during chlamydial infection for optimal inclusion development and stable EMT induction. The results revealed that Chlamydia upregulated TGF-β expression as early as 6 h postinfection of epithelial cells and stimulated both the EGFR and TGF-β signaling pathways. Inhibition of either the EGFR or TGF-βR1 signaling substantially reduced inclusion development; however, the combined inhibition of both EGFR and TGF-βR1 signaling reduced inclusions by over 90% and prevented EMT induction. Importantly, EGFR inhibition suppressed TGF-β expression, and an inhibitory thrombospondin-1 (Tsp1)-based peptide inhibited chlamydia-induced EMT, revealing a major source of active TGF-β during infection. Finally, TGF-βR signaling inhibition suppressed the expression of transforming acidic coiled-coil protein-3 (TACC3), which stabilizes EGFR signaling, suggesting reciprocal regulation between TGF-β and EGFR signaling during chlamydial infection. Thus, RTK-mediated host invasion by chlamydia upregulated TGF-β expression and signaling, which cooperated with other cellular signaling cascades and cytoskeletal remodeling to support optimal inclusion development and EMT induction. This finding may provide new targets for chlamydial disease biomarkers and prevention.

EGFL7: Master regulator of cancer pathogenesis, angiogenesis and an emerging mediator of bone homeostasis.

Epidermal growth factor-like domain-containing protein 7 (EGFL7), a member of the epidermal growth factor (EGF)-like protein family, is a potent angiogenic factor expressed in many different cell types. EGFL7 plays a vital role in controlling vascular angiogenesis during embryogenesis, organogenesis, and maintaining skeletal homeostasis. It regulates cellular functions by mediating the main signaling pathways (Notch, integrin) and EGF receptor cascades. Accumulating evidence suggests that Egfl7 plays a crucial role in cancer biology by modulating tumor angiogenesis, metastasis, and invasion. Dysregulation of Egfl7 has been frequently found in several types of cancers, such as malignant glioma, colorectal carcinoma, oral and oesophageal cancers, gastric cancer, hepatocellular carcinoma, pancreatic cancer, breast cancer, lung cancer, osteosarcoma, and acute myeloid leukemia. In addition, altered expression of miR-126, a microRNA associated with Egfl7, was found to play an important role in oncogenesis. More recently, our study has shown that EGFL7 is expressed in both the osteoclast and osteoblast lineages and promotes endothelial cell activities via extracellular signal-regulated kinase (ERK), signal transducer and activator of transcription 3 (STAT3), and integrin signaling cascades, indicative of its angiogenic regulation in the bone microenvironment. Thus, understanding the role of EGFL7 may provide novel insights into the development of improved diagnostics and therapeutic treatment for cancers and skeletal pathological disorders, such as ischemic osteonecrosis and bone fracture healing.

Inactivation of EGFR/AKT signaling enhances TSA-induced ovarian cancer cell differentiation.

Ovarian tumor is one of the most lethal gynecologic cancers, but differentiation therapy for this cancer is poorly characterized. Here, we show that thrichostatin A (TSA), the well known inhibitor of histone deacetylases (HDACs), can induce cell differentiation in HO8910 ovarian cancer cells. TSA-induced cell differentiation is characterized by typical morphological change, increased expression of the differentiation marker FOXA2, decreased expression of the pluripotency markers SOX2 and OCT4, suppressing cell proliferation, and cell cycle arrest in the G1 phase. TSA also induces an elevated expression of cell cycle inhibitory protein p21Cip1 along with a decrease in cell cycle regulatory protein cyclinD1. Significantly, blockage of epidermal growth factor receptor(EGFR) signaling pathway with specific inhibitors of this signaling cascade promotes the TSA-induced differentiation of HO8910 cells. These results imply that the EGFR cascade inhibitors in combination with TSA may represent a promising differentiation therapy strategy for ovarian cancer.

Deep Phosphotyrosine Proteomics by Optimization of Phosphotyrosine Enrichment and MS/MS Parameters.

Phosphorylation is a major post-translational modification that regulates protein function, with phosphotyrosine (pY) modifications being implicated in oncogenesis. However, global profiling of pY statuses without treatment with a tyrosine phosphatase inhibitor such as pervanadate is still challenging due to the low occupancy of pY sites. In this study, we greatly improved the identification of pY sites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) by optimization of both the pY-immunoprecipitation (pY-IP) protocol and the LC-MS/MS parameters. Our highly sensitive method reproducibly identified more than 1000 pY sites from 8 mg of protein lysate without the need for tyrosine phosphatase inhibitor treatment. Furthermore, >30% of the identified pY sites were not assigned in the PhosphositePlus database. We further applied our method to the comparison of pY status between PC3 cells with and without treatment using the epidermal growth factor receptor (EGFR) inhibitor Erlotinib. Under Erlotinib treatment, we observed not only a decrease in well-known modes of EGFR downstream signaling but also modulations of kinases that are not relevant to the EGFR cascade, such as PTK6 and MAPK13. Our newly developed method for pY proteomics has the potential to reveal unknown pY signaling modes and to identify novel kinase anticancer targets.

Oncolytic Activity of a Recombinant Measles Virus, Blind to Signaling Lymphocyte Activation Molecule, Against Colorectal Cancer Cells.

Oncolytic virotherapy is a distinctive antitumor therapy based on the cancer-cell-specific infectivity and killing activity of viruses, which exert a considerable antitumor effect with only a few treatments. Because colorectal cancer cells often acquire resistance to the molecular-targeted therapies and alternative treatments are called for, in this study, we evaluated the oncolytic activity against colorectal cancer cells of a recombinant measles virus (rMV-SLAMblind), which is blind to signaling lymphocytic activation molecule (SLAM) and infects target cells via nectin-4/poliovirus receptor-related 4 protein. We examined 10 cell lines including 8 cell lines that were resistant to epidermal-growth-factor-receptor (EGFR) targeted therapy. rMV-SLAMblind infected and lysed the nectin-4-positive cell lines dependently on nectin-4 expression, in spite of mutation in EGFR cascade. Tumour progression in xenograft models was also abrogated by the virus, and the infection of cancer cells in vivo by the virus was demonstrated with both flow cytometry and a histological analysis. Therefore, rMV-SLAMblind is considered a novel therapeutic agent for colorectal cancers, including those resistant to molecular-targeted therapies.

Resveratrol Alleviates Cadmium-Induced Damage and Overexpression of Epidermal Growth Factor Receptor and its Downstream Signaling Proteins in the Reproductive System of Male Swiss Albino Mice.

Nowadays, exposure to heavy metals and their detrimental effects in humans are grave health concerns. In this study, we investigated the protective effect of resveratrol (RES) against CdCl2 (cadmium chloride)-induced impairment of spermatogenesis, histopathological alterations, and the up-regulation of epidermal growth factor receptor (EGFR) signaling cascade in Swiss albino mice. Two different doses of CdCl2 were injected intraperitoneally into two groups of mice, and in the third group RES was administered orally before injecting CdCl2 (3 times/wk) for 14 days. Sperm motility, count, vitality, and morphology were analyzed. Hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and western blot analyses were performed on testis tissue. In CdCl2-administered animals, significant perturbations of spermatogenesis and histoarchitecture of seminiferous tubules were observed. p-EGFR, p-AKT, AKT1/2/3, NF-κβ (p50), and COX-2 of the EGFR cascade were up-regulated. Although there was significant negative correlation between percentage of motile cells and protein expression, we found positive correlation between morphologically abnormal cells and overexpression of proteins in CdCl2-only treated groups. Marked improvement of sperm parameters and histopathological damages as well as down-regulation of the EGFR signaling cascade were observed in the RES-pretreated mice. Hence, the present study elucidates that RES protects against CdCl2-induced perturbation of spermatogenesis and overexpression of EGFR and its downstream signaling proteins.

Bronchial epithelial cell lines and primary nasal epithelial cells from cystic fibrosis respond differently to cigarette smoke exposure

The effects of cigarette smoke extract (CSE) on airway epithelial cells (AECs) from cystic fibrosis (CF) and non-cystic fibrosis (non-CF) individuals are not fully understood. It has been suggested that CSE modulates inflammatory cytokine release from AECs by modulating the epidermal growth factor receptor (EGFR) pathway; these pathways could reveal novel therapeutic targets. We compared the effect of CSE pre-incubation on IL-8 release from CF and non-CF bronchial epithelial cell lines, and separately, with primary nasal epithelial cells (NECs) retrieved from CF and non-CF individuals. We also determined if the EGFR pathway regulates IL-8 release by LPS or cytomix in non-CF and CF AECs at baseline and following CSE exposure.CF and non-CF cell lines, NECs derived from both CF patients (R117H heterozygous and F508del homozygous), and from healthy subjects, were cultured in the presence or absence of CSE, and subsequently exposed to inflammatory stimuli. In cell lines CSE significantly reduced IL-8 release following inflammatory challenge. Conversely, CSE pre-treatment was pro-inflammatory in primary NECs. In NECs from control subjects, CSE increased cytomix and LPS induced IL-8 release, and for the R117H heterozygous NEC cultures, CSE enhanced basal IL-8 release. Cytomix and LPS induced IL-8 release from F508del homozygous NEC cultures was further heightened following CSE pre-treatment.EGFR inhibition mitigated IL-8 release from immortalised and primary non-CF and CF AECs, suggesting that constitutive and CSE elicited IL-8 release from AECs is partly regulated via the EGFR pathway.This study demonstrates the importance of the EGFR cascade in the regulation of constitutive and CSE induced inflammatory mediator release from immortalised and primary AECs. Moreover, it clearly highlights the significance of using primary cells to confirm results obtained from immortalised cell studies, as these model systems may respond very differently to the stimuli under investigation.

Cannabis smoke can be a major risk factor for early-age laryngeal cancer--a molecular signaling-based approach.

Epidermal growth factor receptor (EGFR) and its downstream elements are overexpressed in most cases of the head and neck squamous cell carcinoma. This study investigated the expression pattern of key proteins linked to the EGFR pathway in laryngeal carcinoma patients with a history of cannabis smoking. We selected 83 male glottic cancer patients, aged between 45 to 75 years with three distinct populations-nonsmoker, cigarette smoker, and cannabis smoker. Immunohistochemical staining was performed for EGFR, protein kinase B (PKB or Akt), nuclear factor kappa B p50 (NF-КB), and cyclooxygenase-2 (COX-2) followed by boolean scoring for statistical analysis. Experimental data showed upregulation of the selected EGFR cascade in tumor cells, stromal expression of EGFR, and nuclear localization of COX-2 in metaplastic gland cells of laryngeal cancer tissue sample. Statistical analyses indicated that overexpression of the EGFR cascade is significantly correlated to cannabis smoking. Cannabis smokers had higher expression (p < 0.01) of these onco-proteins with respect to both nonsmokers as well as cigarette smokers. Risk factor analysis showed high risk of these proteins expression in age <60 years (odds ratio (OR) > 1.5) as the lower age group had relatively higher number of cannabis smokers. This study provides evidence for a direct association between cannabis smoking and increased risk of laryngeal cancer. Higher expression of the EGFR cascade in cannabis smokers revealed that cannabis smoking may be a major cause for the early onset of aggressive laryngeal cancer.

Associations of positive epidermal growth factor receptor expression and K-RAS gene mutations with various clinicopathological parameters and survival of colorectal carcinoma patients

Epidermal growth factor receptor (EGFR) is a member of the transmembrane receptor tyrosine kinase family. In normal and malignant cells, activation of the EGFR cascade is involved in the regulation of various cellular activities. The objective of this study was to identify and assess associations of positive EGFR expression and K-RAS mutations with various clinicopathological parameters and survival of colorectal carcinoma patients. EGFR of colorectal cancer (CRC) tissue specimens was subjected to immunohistochemical analysis, polymerase chain reaction, and DNA sequencing. Immunohistochemical staining was performed using monoclonal antibodies against EGFR antigens and examination of mutations was performed to detect mutations in codons 12 and 13 of the K-RAS. Statistical analysis was performed using SPSS version 16.0. The results of this study showed that of the 40 study participants, 62.5% (25/40) showed positive EGFR overexpression. Of the patients showing positive EGFR expressions, 52% had mutations in the K-RAS. Mutations were spread in codon 12 (64.3%) and codon 13 (35.7%) and there was one sample with mutations in codons 12 and 13 at the same time. A statistically significant association was found between the presence of metastasis and EGFR overexpression and survival of CRC patients. In addition, a significant association was found between K-RAS mutations and metastasis and survival of CRC patients. In conclusion, EGFR overexpression and K-RAS mutations were found in CRC patients. Both factors are known to be associated with poor prognosis of cancer patients in terms of patient survival. Early detection of K-RAS mutations in CRC patients is a crucial component in the determination of the type of therapy and treatment for the patient.

Normal CFTR Inhibits Epidermal Growth Factor Receptor-Dependent Pro-Inflammatory Chemokine Production in Human Airway Epithelial Cells

Mutations in cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis, a disease characterized by exaggerated airway epithelial production of the neutrophil chemokine interleukin (IL)-8, which results in exuberant neutrophilic inflammation. Because activation of an epidermal growth factor receptor (EGFR) signaling cascade induces airway epithelial IL-8 production, we hypothesized that normal CFTR suppresses EGFR-dependent IL-8 production and that loss of CFTR at the surface exaggerates IL-8 production via activation of a pro-inflammatory EGFR cascade. We examined this hypothesis in human airway epithelial (NCI-H292) cells and in normal human bronchial epithelial (NHBE) cells containing normal CFTR treated with a CFTR-selective inhibitor (CFTR-172), and in human airway epithelial (IB3) cells containing mutant CFTR versus isogenic (C38) cells containing wild-type CFTR. In NCI-H292 cells, CFTR-172 induced IL-8 production EGFR-dependently. Pretreatment with an EGFR neutralizing antibody or the metalloprotease TACE inhibitor TAPI-1, or TACE siRNA knockdown prevented CFTR-172-induced EGFR phosphorylation (EGFR-P) and IL-8 production, implicating TACE-dependent EGFR pro-ligand cleavage in these responses. Pretreatment with neutralizing antibodies to IL-1R or to IL-1alpha, but not to IL-1beta, markedly suppressed CFTR-172-induced EGFR-P and IL-8 production, suggesting that binding of IL-1alpha to IL-1R stimulates a TACE-EGFR-IL-8 cascade. Similarly, in NHBE cells, CFTR-172 increased IL-8 production EGFR-, TACE-, and IL-1alpha/IL-1R-dependently. In IB3 cells, constitutive IL-8 production was markedly increased compared to C38 cells. EGFR-P was increased in IB3 cells compared to C38 cells, and exaggerated IL-8 production in the IB3 cells was EGFR-dependent. Activation of TACE and binding of IL-1alpha to IL-1R contributed to EGFR-P and IL-8 production in IB3 cells but not in C38 cells. Thus, we conclude that normal CFTR suppresses airway epithelial IL-8 production that occurs via a stimulatory EGFR cascade, and that loss of normal CFTR activity exaggerates IL-8 production via activation of a pro-inflammatory EGFR cascade.

Protein Phosphorylation Profiling Using an In Situ Proximity Ligation Assay: Phosphorylation of AURKA-Elicited EGFR-Thr654 and EGFR-Ser1046 in Lung Cancer Cells

The epidermal growth factor receptor (EGFR), which is up-regulated in lung cancer, involves the activation of mitogenic signals and triggers multiple signaling cascades. To dissect these EGFR cascades, we used 14 different phospho-EGFR antibodies to quantify protein phosphorylation using an in situ proximity ligation assay (in situ PLA). Phosphorylation at EGFR-Thr654 and -Ser1046 was EGF-dependent in the wild-type (WT) receptor but EGF-independent in a cell line carrying the EGFR-L858R mutation. Using a ProtoAarray™ containing ∼5000 recombinant proteins on the protein chip, we found that AURKA interacted with the EGFR-L861Q mutant. Moreover, overexpression of EGFR could form a complex with AURKA, and the inhibitors of AURKA and EGFR decreased EGFR-Thr654 and -Ser1046 phosphorylation. Immunohistochemical staining of stage I lung adenocarcinoma tissues demonstrated a positive correlation between AURKA expression and phosphorylation of EGFR at Thr654 and Ser1046 in EGFR-mutant specimens, but not in EGFR-WT specimens. The interplay between EGFR and AURKA provides an explanation for the difference in EGF dependency between EGFR-WT and EGFR-mutant cells and may provide a new therapeutic strategy for lung cancer patients carrying EGFR mutations.

Drosophila Zpr1 (Zinc Finger Protein 1) Is Required Downstream of Both EGFR And FGFR Signaling in Tracheal Subcellular Lumen Formation

The cellular and molecular cues involved in creating branched tubular networks that transport liquids or gases throughout an organism are not well understood. To identify factors required in branching and lumen formation of Drosophila tracheal terminal cells, a model for branched tubular networks, we performed a forward genetic-mosaic screen to isolate mutations affecting these processes. From this screen, we have identified the first Drosophila mutation in the gene Zpr1 (Zinc finger protein 1) by the inability of Zpr1-mutant terminal cells to form functional, gas-filled lumens. We show that Zpr1 defective cells initiate lumen formation, but are blocked from completing the maturation required for gas filling. Zpr1 is an evolutionarily conserved protein first identified in mammalian cells as a factor that binds the intracellular domain of the unactivated epidermal growth factor receptor (EGFR). We show that down-regulation of EGFR in terminal cells phenocopies Zpr1 mutations and that Zpr1 is epistatic to ectopic lumen formation driven by EGFR overexpression. However, while Zpr1 mutants are fully penetrant, defects observed when reducing EGFR activity are only partially penetrant. These results suggest that a distinct pathway operating in parallel to the EGFR pathway contributes to lumen formation, and this pathway is also dependent on Zpr1. We provide evidence that this alternative pathway may involve fibroblast growth factor receptor (FGFR) signaling. We suggest a model in which Zpr1 mediates both EGFR and FGFR signal transduction cascades required for lumen formation in terminal cells. To our knowledge, this is the first genetic evidence placing Zpr1 downstream of EGFR signaling, and the first time Zpr1 has been implicated in FGFR signaling. Finally, we show that down-regulation of Smn, a protein known to interact with Zpr1 in mammalian cells, shows defects similar to Zpr1 mutants.

Multidrug-resistant cells overexpressing P-glycoprotein are susceptible to DNA crosslinking agents due to attenuated Src/nuclear EGFR cascade-activated DNA repair activity

We synthesized several novel bifunctional alkylating derivatives of 3a-aza-cyclopenta[a]indene (BO-1012, BO-1005, BO-1099 and BO-1101) that are potent DNA interstrand crosslinking agents. In in vitro cytotoxicity assay, these compounds were more cytotoxic to multidrug-resistant (MDR) cells, such as KBvin10, KBtax50 and CEM/VBL, than their parental cells. Using a xenograft model, BO-1012, at a dose of 5 mg/kg, partially suppressed the growth of parental KB cells but completely suppressed the growth of KBvin10 cells in nude mice. In exploring the possible mechanism, we found that DNA double-strand break (DSB) repair activity in MDR cells, KBvin10 and CEM/VBL, was significantly reduced compared with their parental cells, KB and CEM. Reduced DSB repair activity in KBvin10 cells was likely due to a defect in nuclear translocation of DNA-dependent protein kinase (DNA-PK), a component of the non-homologous end-joining repair machinery. Furthermore, BO-1012-induced DNA-PK translocation from the cytosol into the nucleus in KB cells is associated with the activation of the Src/nuclear epidermal growth factor receptor (EGFR) cascade, which is defective in MDR cells. As knockdown of P-glycoprotein (P-gp) by siRNA reactivated the Src/nuclear EGFR cascade, DNA-PK translocation and DNA repair activity in MDR cells, overexpression of P-gp attenuates the activity of DNA DSB repair through suppression of Src/nuclear EGFR cascade. Therefore, DNA interstrand crosslinking agents may have potential therapeutic use against P-gp-overexpressing MDR cells.

Complex Oncogenic Signaling Networks Regulate Brain Tumor‐Initiating Cells and Their Progenies: Pivotal Roles of Wild‐Type EGFR, EGFRvIII Mutant and Hedgehog Cascades and Novel Multitargeted Therapies

Complex signaling cross-talks between different growth factor cascades orchestrate the primary brain cancer development. Among the frequent deregulated oncogenic pathways, the ligand-activated wild-type epidermal growth factor receptor (EGFR), constitutively activated EGFRvIII mutant and sonic hedgehog pathways have attracted much attention because of their pivotal roles in pediatric medulloblastomas and adult glioblastoma multiformes (GBM) brain tumors. The enhanced expression levels and activation of EGFR, EGFRvIII mutant and hedgehog signaling elements can provide key roles for the sustained growth, migration and local invasion of brain tumor-initiating cells (BTICs) and their progenies, resistance to current therapies and disease relapse. These tumorigenic cascades also can cooperate with Wnt/β-catenin, Notch, platelet-derived growth factor (PDGF)/PDGF receptors (PDGFRs), hepatocyte growth factor (HGF)/c-Met receptor and vascular endothelial growth factor (VEGF)/VEGF receptors (VEGFRs) for the acquisition of a more malignant behavior and survival advantages by brain tumor cells during disease progression. Therefore, the simultaneous targeting of these oncogenic signaling components including wild-type EGFR, EGFRvIII mutant and hedgehog pathways may constitute a potential therapeutic approach of great clinical interest to eradicate BTICs and improve the efficacy of current clinical treatments by radiation and/or chemotherapy against aggressive and recurrent medulloblastomas and GBMs.

Mouse Cumulus-Oocyte Complexes from In Vitro-Cultured Preantral Follicles Suggest an Anti-Luteinizing Role for the EGF Cascade in the Cumulus Cells1

The mammalian ovulatory process is a fairly complex succession of events that leads to the release of a competent oocyte. The luteinizing hormone (LH) triggers the cascade of events, which starts with the production of secondary messengers in the follicular wall and ends with the release of a fertilizable oocyte. Most of these events can be reproduced using in vitro models, which offer a wide range of possibilities for study strategies. Although it is accepted that epidermal growth factor receptor (EGFR) activation is required for transmission of the LH-initiated signal, we hypothesized that LH receptor activation might also play a role in oocyte meiotic resumption and cumulus cell response, because the current mouse preantral follicle in vitro model expresses functional LH receptor. To separate the LH-mediated response and the epidermal growth factor (EGF)-mediated response (following LH stimulus), in vitro-grown mouse ovarian follicles were stimulated for ovulation with a combination of human chorionic gonadotropin (hCG) plus galardin (inhibitor for the release of endogenous EGF-like factors) or hCG plus galardin plus EGF. Results suggest that the stimulation provided by LH (hCG) is insufficient to induce a maximum oocyte meiotic resumption and that EGFR activation is also required. Analysis of transcript levels of Egfr, Ereg, Cyp19a1, Hsd3b1, Adamts1, and Has2 in cumulus cells further indicate that the triggers for the EGFR cascade preserve the expression profile of the studied transcripts. Therefore, it is proposed that within this in vitro mouse model, EGF signaling during ovulation might protect the cumulus cells from the potential luteinizing effects of LH.

Cytotoxic Effects Induced by Docetaxel, Gefitinib, and Cyclopamine on Side Population and Nonside Population Cell Fractions from Human Invasive Prostate Cancer Cells

The present study has been undertaken to establish the therapeutic benefit of cotargeting epidermal growth factor receptor (EGFR) and sonic hedgehog pathways by using gefitinib and cyclopamine, respectively, for improving the efficacy of the current chemotherapeutic drug docetaxel to counteract the prostate cancer progression from locally invasive to metastatic and recurrent disease stages. The data from immuofluorescence analyses revealed that EGFR/Tyr(1173)-pEGFR, sonic hedgehog ligand, smoothened coreceptor, and GLI-1 were colocalized with the CD133(+) stem cell-like marker in a small subpopulation of prostate cancer cells. These signaling molecules were also present in the bulk tumor mass of CD133(-) prostate cancer cells with a luminal phenotype detected in patient's adenocarcinoma tissues. Importantly, the results revealed that the CD133(+)/CD44(high)/AR(-/low) side population (SP) cell fraction endowed with a high self-renewal potential isolated from tumorigenic and invasive WPE1-NB26 cells by the Hoechst dye technique was insensitive to the current chemotherapeutic drug, docetaxel. In contrast, the docetaxel treatment induced significant antiproliferative and apoptotic effects on the CD133(-)/CD44(low)/AR(+) non-SP cell fraction isolated from the WPE1-NB26 cell line. Of therapeutic interest, the results have also indicated that combined docetaxel, gefitinib, and cyclopamine induced greater antiproliferative and apoptotic effects on SP and non-SP cell fractions isolated from WPE1-NB26 cells than individual drugs or two-drug combinations. Altogether, these observations suggest that EGFR and sonic hedgehog cascades may represent the potential therapeutic targets of great clinical interest to eradicate the total prostate cancer cell mass and improve the current docetaxel-based therapies against locally advanced and invasive prostate cancers, and thereby prevent metastases and disease relapse.

Epidermal growth factor receptor-mediated innate immune responses and their roles in airway diseases

Inhaled air is contaminated with pathogens and particulates that may deposit in the airways and damage the host. In response to these invaders, the airway epithelium has developed innate immune responses that provide a defence against the invaders and protect the airway structure and function. Thus, the epithelium of conducting airways becomes the "battleground" between the invaders and the host. Recent evidence suggests that airway epithelial surface signalling through the epidermal growth factor receptor (EGFR) is a convergent pathway producing innate immune responses to a variety of infectious and noninfectious noxious stimuli. In the present review, the EGFR signalling pathways leading to airway mucin production, neutrophil recruitment (via interleukin-8 production) and airway epithelial repair were examined. The importance of these findings in human airway diseases was also investigated. The current authors suggest that the exaggerated innate immune responses found in chronic inflammatory airway diseases (e.g. chronic obstructive pulmonary disease, cystic fibrosis and severe asthma) contribute to the pathogenesis or the aggravation of these diseases. Potential therapies include inhibition of the various elements of the described epidermal growth factor receptor cascade. In considering each therapeutic intervention, the potential benefits must be considered in relation to potential deleterious effects.

Role of epidermal growth factor receptor in maintaining airway goblet cell hyperplasia in rats sensitized to allergen

Stimulation of epidermal growth factor receptor (EGFR) induces airway goblet cell hyperplasia, but the role of this molecule in the maintenance of this pathologic change remains uncertain. To determine the mechanisms by which goblet cell hyperplasia is maintained in airway epithelium, we investigated EGFR-induced signalling pathways that lead to both mucin production and antiapoptosis in vitro. We also tested whether the inhibition of EGFR tyrosine kinase speeds reversal of established goblet cell hyperplasia to normal epithelial phenotype in vivo. MUC5AC production was measured by immunoassay, and antiapoptotic responses were determined by Bcl-2 expression and terminal deoxynucleotidyl transferase-mediated dUTP-biotin Nick End Labelling staining using NCI-H292 cells. The effect of an inhibitor of EGFR tyrosine kinase (AG1478) on goblet cell hyperplasia was also determined in rats sensitized with ovalbumin (OVA). MUC5AC was constitutively expressed and few apoptotic cells were observed in NCI-H292 cells under non-stimulated condition. TGF-alpha increased MUC5AC and Bcl-2 expression, an effect that was prevented by inhibitors of EGFR tyrosine kinase (AG1478), MEK (PD98059), and NF-kappaB (CAPE). After the addition of TGF-alpha, AG1478 and an inhibitor of phosphatidylinositol 3 kinase/Akt (LY294002), but not PD98059, induced a marked apoptotic response, which was prevented by the caspase inhibitor Z-VAD fmk. Goblet cell hyperplasia and EGFR expression in airway epithelium were noted in the OVA-sensitized rats. Intratracheal instillation of AG1478 induced apoptosis of goblet cells, reverting the airway epithelium to normal epithelial phenotype. These findings indicate that EGFR plays an important role in the maintenance of goblet cell hyperplasia. We speculate that inhibitors of the EGFR cascade might be an effective therapy of airway remodelling.