Extracellular domain mutations of the EGF receptor differentially modulate high-affinity and low-affinity responses to EGF receptor ligands.

The EGF receptor is mutated in a number of cancers. In most cases, the mutations occur in the intracellular tyrosine kinase domain. However, in glioblastomas, many of the mutations are in the extracellular ligand binding domain. To determine what changes in receptor function are induced by such extracellular domain mutations, we analyzed the binding and biological response to the seven different EGF receptor ligands in three common glioblastoma mutants-R84K, A265V, and G574V. Our data indicate that all three mutations significantly increase the binding affinity of all seven ligands. In addition, the mutations increase the potency of all ligands for stimulating receptor autophosphorylation, phospholipase Cγ, Akt, and MAP kinase activity. In all mutants, the rank order of ligand potency seen at the wild-type receptor was retained, suggesting that the receptors still discriminate among the different ligands. However, the low-affinity ligands, EPR and EPG, did show larger than average enhancements of potency for stimulating Akt and MAPK but not receptor autophosphorylation and phospholipase Cγ activation. Relative to the wild-type receptor, these changes lead to an increase in the responsiveness of these mutants to physiological concentrations of ligands and an alteration in the ratio of activation of the different pathways. This may contribute to their oncogenic potential. In the context of recent findings, our data also suggest that so-called "high"-affinity biological responses arise from activation by isolated receptor dimers, whereas "low"-affinity biological responses require clustering of receptors which occurs at higher concentrations of ligand.

Follicular cells protect Xenopus oocyte from abnormal maturation via integrin signaling downregulation and O-GlcNAcylation control.

Xenopus oocytes are encompassed by a layer of follicular cells that contribute to oocyte growth and meiosis in relation to oocyte maturation. However, the effects of the interaction between follicular cells and the oocyte surface on meiotic processes are unclear. Here, we investigated Xenopus follicular cell function using oocyte signaling and heterologous-expressing capabilities. We found that oocytes deprotected from their surrounding layer of follicular cells and expressing the epidermal growth factor (EGF) receptor (EGFR) and the Grb7 adaptor undergo accelerated prophase I to metaphase II meiosis progression upon stimulation by EGF. This unusual maturation unravels atypical spindle formation but is rescued by inhibiting integrin ß1 or Grb7 binding to the EGFR. In addition, we determined that oocytes surrounded by their follicular cells expressing EGFR-Grb7 exhibit normal meiotic resumption. These oocytes are protected from abnormal meiotic spindle formation through the recruitment of O-GlcNAcylated Grb7, and OGT (O-GlcNAc transferase), the enzyme responsible for O-GlcNAcylation processes, in the integrin ß1-EGFR complex. Folliculated oocytes can be forced to adopt an abnormal phenotype and exclusive Grb7 Y338 and Y188 phosphorylation instead of O-GlcNAcylation under integrin activation. Furthermore, an O-GlcNAcylation increase (by inhibition of O-GlcNAcase), the glycosidase that removes O-GlcNAc moieties, or decrease (by inhibition of OGT) amplifies oocyte spindle defects when follicular cells are absent highlighting a control of the meiotic spindle by the OGT-O-GlcNAcase duo. In summary, our study provides further insight into the role of the follicular cell layer in oocyte meiosis progression.

Crosstalk between KDEL receptor and EGF receptor mediates cell proliferation and migration via STAT3 signaling.

Hostile microenvironment of cancer cells provoke a stressful condition for endoplasmic reticulum (ER) and stimulate the expression and secretion of ER chaperones, leading to tumorigenic effects. However, the molecular mechanism underlying these effects is largely unknown. In this study, we reveal that the last four residues of ER chaperones, which are recognized by KDEL receptor (KDELR), is required for cell proliferation and migration induced by secreted chaperones. By combining proximity-based mass spectrometry analysis, split venus imaging and membrane yeast two hybrid assay, we present that EGF receptor (EGFR) may be a co-receptor for KDELR on the surface. Prior to ligand addition, KDELR spontaneously oligomerizes and constantly undergoes recycling near the plasma membrane. Upon KDEL ligand binding, the interactions of KDELR with itself and with EGFR increase rapidly, leading to augmented internalization of KDELR and tyrosine phosphorylation in the C-terminus of EGFR. STAT3, which binds the phosphorylated tyrosine motif on EGFR, is subsequently activated by EGFR and mediates cell growth and migration. Taken together, our results suggest that KDELR serves as a bona fide cell surface receptor for secreted ER chaperones and transactivates EGFR-STAT3 signaling pathway.

Synthesis and biological evaluation of EGFR binding peptides for near-infrared photoimmunotherapy.

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that involves photoimmunotherapy drug injection and NIR light exposure. In NIR-PIT, antibodies are commonly used as target-directed molecules carrying IRDye700DX (IR700). However, antibodies have disadvantages, such as high cost, complex development strategies, and poor tumor penetration. In contrast, peptides have lower production costs, can be easy to chemically synthesize and modify, and can also be used for tumor-targeting like antibodies. In this study, we developed a novel PIT drug using a peptide as the target-directed molecule. Epidermal growth factor receptor (EGFR) was selected as the target, and monovalent and bivalent EGFR-binding peptides were synthesized. The bivalent peptide showed sufficient binding to EGFR-positive cells, and a bivalent peptide-IR700 conjugate with a long linker induced morphological changes in EGFR-positive cells. Additionally, the drug significantly reduced cell viability in vitro in an NIR light-dose- and drug-concentration-dependent manner. These results indicate the feasibility of NIR-PIT in treating cancer using peptide-based drugs.

Oocyte Competence of Prepubertal Sheep and Goat Oocytes: An Assessment of Large-Scale Chromatin Configuration and Epidermal Growth Factor Receptor Expression in Oocytes and Cumulus Cells.

The oocyte competence of prepubertal females is lower compared to that of adults, mainly because they originate from small follicles. In adult females, the germinal vesicle (GV) and epidermal growth factor receptor (EGFR) have been associated with oocyte competence. This study aimed to analyze GV chromatin configuration and EGFR expression in prepubertal goat and sheep oocytes obtained from small (<3 mm) and large (≥3 mm) follicles and compare them with those from adults. GV chromatin was classified from diffuse to condensed as GV1, GVn, and GVc for goats and NSN, SN, and SNE for sheep. EGFR was quantified in cumulus cells (CCs) by Western blotting and in oocytes by immunofluorescence. Oocytes from prepubertal large follicles and adults exhibited highly condensed chromatin in goats (71% and 69% in GVc, respectively) and sheep (59% and 75% in SNE, respectively). In both species, EGFR expression in CCs and oocytes was higher in prepubertal large follicles than in small ones. In adult females, EGFR expression in oocytes was higher than in prepubertal large follicles. In conclusion, GV configuration and EGFR expression in CCs and oocytes were higher in the large than small follicles of prepubertal females.

Quantitative modeling of EGF receptor ligand discrimination via internalization proofreading.

The epidermal growth factor receptor (EGFR) is a central regulator of cell physiology that is stimulated by multiple distinct ligands. Although ligands bind to EGFR while the receptor is exposed on the plasma membrane, EGFR incorporation into endosomes following receptor internalization is an important aspect of EGFR signaling, with EGFR internalization behavior dependent upon the type of ligand bound. We develop quantitative modeling for EGFR recruitment to and internalization from clathrin domains, focusing on how internalization competes with ligand unbinding from EGFR. We develop two model versions: a kinetic model with EGFR behavior described as transitions between discrete states and a spatial model with EGFR diffusion to circular clathrin domains. We find that a combination of spatial and kinetic proofreading leads to enhanced EGFR internalization ratios in comparison to unbinding differences between ligand types. Various stages of the EGFR internalization process, including recruitment to and internalization from clathrin domains, modulate the internalization differences between receptors bound to different ligands. Our results indicate that following ligand binding, EGFR may encounter multiple clathrin domains before successful recruitment and internalization. The quantitative modeling we have developed describes competition between EGFR internalization and ligand unbinding and the resulting proofreading.

Current treatment strategies for EGFR-mutated non-small cell lung cancer: from first line to beyond osimertinib resistance.

Osimertinib, a third-generation EGFR TKI, is the standard therapy for previously untreated EGFR-mutated non-small cell lung cancer patients following the landmark FLAURA study. However, resistance inevitably hinders patient prognosis, increasing the need for new therapeutic strategies beyond osimertinib. Frontline osimertinib-based combination strategies (platinum-based chemotherapy and angiogenesis inhibitors) are currently being tested primarily to prevent initial resistance. In the later-line setting after osimertinib, many next-line therapeutic candidates have been actively examined in clinical trials. Notably, several drugs with novel mechanisms of action, such as antibody-drug conjugates and EGFR -MET bispecific antibodies, have shown promising efficacy despite the resistance mechanisms and are close to clinical application. In addition, genotype-based target strategies have been investigated for a better understanding of osimertinib resistance mechanisms based on molecular profiling tests at relapse. The C797S mutation and MET gene alterations are commonly identified following osimertinib resistance, for which targeting strategies are actively tested. This review describes current pharmacotherapeutic strategies for EGFR-mutated non-small cell lung cancer based on the results of clinical trials and the latest published data, broadly grouped into two sections: 1) EGFR TKIs-based combination therapy in the front-line setting and 2) novel therapeutic strategies after osimertinib resistance.

Identification of EGF Receptor and Thrombospondin-1 as Endogenous Targets of ER-Associated Degradation Enhancer EDEM1 in HeLa Cells.

Secretory and membrane proteins are vital for cell activities, including intra- and intercellular communication. Therefore, protein quality control in the endoplasmic reticulum (ER) is an essential and crucial process for eukaryotic cells. Endoplasmic reticulum-associated degradation (ERAD) targets misfolded proteins during the protein maturation process in the ER and leads to their disposal. This process maintains the ER productive function and prevents misfolded protein stress (i.e., ER stress). The ERAD-stimulating factor ER degradation-enhancing α mannosidase-like 1 protein (EDEM1) acts on misfolded proteins to accelerate ERAD, thereby maintaining the productivity of the ER. However, the detail mechanism underlying the function of EDEM1 in ERAD is not completely understood due to a lack of established physiological substrate proteins. In this study, we attempted to identify substrate proteins for EDEM1 using siRNA. The matrix component thrombospondin-1 (TSP1) and epidermal growth factor receptor (EGFR) were identified as candidate targets of EDEM1. Their protein maturation status and cellular localization were markedly affected by knockdown of EDEM1. We also showed that EDEM1 physically associates with EGFR and enhances EGFR degradation via ERAD. Our data highlight the physiological role of EDEM1 in maintaining specific target proteins and provide a potential approach to the regulation of expression of clinically important proteins.

STS-1 and STS-2, Multi-Enzyme Proteins Equipped to Mediate Protein-Protein Interactions.

STS-1 and STS-2 form a small family of proteins that are involved in the regulation of signal transduction by protein-tyrosine kinases. Both proteins are composed of a UBA domain, an esterase domain, an SH3 domain, and a PGM domain. They use their UBA and SH3 domains to modify or rearrange protein-protein interactions and their PGM domain to catalyze protein-tyrosine dephosphorylation. In this manuscript, we discuss the various proteins that have been found to interact with STS-1 or STS-2 and describe the experiments used to uncover their interactions.

Overexpression of N-acetylglucosaminyltransferase V promotes human parotid gland acinar cell immortalization via the epidermal receptor activation.

The purpose of this study is to maintain the proliferation capability of human parotid gland acinar cells (ACs) in vitro to extend passage number and to study the mechanism that regulates AC stemness. N-acetylglucosaminyltransferase V (GnT-V) is the Golgi enzyme, and it has been reported that the ß1,6GlcNAc-branched N-linked glycans are associated with various cell behaviors. Therefore, we modify the gene expression of ACs by transfection of the GnT-V-overexpression plasmid, and we found that upregulation of GnT-V extensively increased ACs proliferation and stemness properties in ACs/GnT-V compared to ACs transfected with Mock plasmid. More importantly, we observed that high levels of GnT-V positively correlated with ALDH1A3 expression via increasing phosphorylation of cell surface receptors and activating the downstream signaling transduction. Hence, the current study suggested that GnT-V is a significant factor for cell immortalization in the ACs model by activating the EGFR/ERK/ALDH1A3 signaling pathway.

GAREM1 is involved in controlling body mass in mice and humans.

The adaptor protein GAREM has two subtypes. Each is involved in Erk activation signaling downstream of the cell growth factor receptor in cultured cells. Regarding their role in individual animals, we have previously reported that mice deficient in GAREM2, which is highly expressed in the brain, exhibit emotional changes. In this paper, we report an amino acid substitution mutation (K291R) in GAREM1, in a patient with idiopathic short stature, which indicates that the mutant exhibits dominant-negative properties. The GAREM K291R mutant did not promote Erk activation in EGF-stimulated cultured cells. Similar features were also observed in cells in which GAREM1 expression was suppressed by genome editing; along with Erk, phosphorylation of S6 kinase and 4EBP1, whose activation is necessary for cell proliferation and biological growth, were inhibited Furthermore, we generated mice deficient in GAREM1 and showed that the mutant mice are lighter in weight. Overall, the results of this paper suggest that GAREM1 is required for normal growth and for maintaing average body size in humans and mice.

The Hippo pathway effectors YAP and TAZ interact with EGF-like signaling to regulate expansion-related events in bovine cumulus cells in vitro.

PURPOSE: To determine if the inhibition of the interaction between the Hippo effector YAP or its transcriptional co-activator TAZ with the TEAD family of transcription factors is critical for the cumulus expansion-related events induced by the EGF network in cumulus-oocyte complexes (COCs). METHODS: We performed a series of experiments using immature bovine COCs subjected to an IVM protocol for up 24 h in which cumulus expansion was stimulated with EGF recombinant protein or FSH. RESULTS: The main results indicated that EGFR activity stimulation in bovine cumulus cells (CC) increases mRNA levels encoding the classic YAP/TAZ-TEAD target gene CTGF. To determine if important genes for cumulus expansion are transcriptional targets of YAP/TAZ-TEAD interaction in CC, COCs were then subjected to IVM in the presence of FSH with or without distinct concentrations of Verteporfin (VP; a small molecule inhibitor that interferes with YAP/TAZ binding to TEADs). COCs were then collected at 6, 12, 18, and 24 h for total RNA extraction and RT-qPCR analyses. This experiment indicated that VP inhibits in a time- and concentration-dependent manner distinct cumulus expansion and oocyte maturation-related genes, by regulating EGFR and CTGF expression in CC. CONCLUSIONS: Taken together, the results presented herein represent considerable insight into the functional relevance of a completely novel signaling pathway underlying cumulus expansion and oocyte maturation in monovulatory species. YAP/TAZ or CTGF may represent potential targets to improve the efficiency of IVM systems, not only for monovulatory species of agricultural importance as the cow, but for human embryo production.

Soluble SORLA Enhances Neurite Outgrowth and Regeneration through Activation of the EGF Receptor/ERK Signaling Axis.

SORLA is a transmembrane trafficking protein associated with Alzheimer's disease risk. Although SORLA is abundantly expressed in neurons, physiological roles for SORLA remain unclear. Here, we show that cultured transgenic neurons overexpressing SORLA feature longer neurites, and accelerated neurite regeneration with wounding. Enhanced release of a soluble form of SORLA (sSORLA) is observed in transgenic mouse neurons overexpressing human SORLA, while purified sSORLA promotes neurite extension and regeneration. Phosphoproteomic analyses demonstrate enrichment of phosphoproteins related to the epidermal growth factor (EGFR)/ERK pathway in SORLA transgenic mouse hippocampus from both genders. sSORLA coprecipitates with EGFR in vitro, and sSORLA treatment increases EGFR Y1173 phosphorylation, which is involved in ERK activation in cultured neurons. Furthermore, sSORLA triggers ERK activation, whereas pharmacological EGFR or ERK inhibition reverses sSORLA-dependent enhancement of neurite outgrowth. In search for downstream ERK effectors activated by sSORLA, we identified upregulation of Fos expression in hippocampus from male mice overexpressing SORLA by RNAseq analysis. We also found that Fos is upregulated and translocates to the nucleus in an ERK-dependent manner in neurons treated with sSORLA. Together, these results demonstrate that sSORLA is an EGFR-interacting protein that activates EGFR/ERK/Fos signaling to enhance neurite outgrowth and regeneration.SIGNIFICANCE STATEMENT SORLA is a transmembrane trafficking protein previously known to reduce the levels of amyloid-ß, which is critical in the pathogenesis of Alzheimer's disease. In addition, SORLA mutations are a risk factor for Alzheimer's disease. Interestingly, the SORLA ectodomain is cleaved into a soluble form, sSORLA, which has been shown to regulate cytoskeletal signaling pathways and cell motility in cells outside the nervous system. We show here that sSORLA binds and activates the EGF receptor to induce downstream signaling through the ERK serine/threonine kinase and the Fos transcription factor, thereby enhancing neurite outgrowth. These findings reveal a novel role for sSORLA in promoting neurite regeneration through the EGF receptor/ERK/Fos pathway, thereby demonstrating a potential neuroprotective mechanism involving SORLA.

Prostaglandin E2 promotes pathological retinal neovascularisation via EP4R-EGFR-Gab1-AKT signaling pathway.

Proliferative retinopathies, such as proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP) are major causes of visual impairment and blindness in industrialized countries. Prostaglandin E2 (PGE2) is implicated in cellular proliferation and migration via E-prostanoid receptor (EP4R). The aim of this study was to investigate the role of PGE2/EP4R signaling in the promotion of retinal neovascularisation. In a streptozotocin (STZ)-induced diabetic model and an oxygen-induced retinopathy (OIR) model, rats received an intravitreal injection of PGE2, cay10598 (an EP4R agonist) or AH23848 (an EP4R antagonist). Optical coherence tomography, retinal histology and biochemical markers were assessed. Treatment with PGE2 or cay10598 accelerated pathological retinal angiogenesis in STZ and OIR-induced rat retina, which was ameliorated in rats pretreated with AH23848. Serum VEGF-A was upregulated in the PGE2-treated diabetic rats vs non-treated diabetic rats and significantly downregulated in AH23848-treated diabetic rats. PGE2 or cay10598 treatment also significantly accelerated endothelial tip-cell formation in new-born rat retina. In addition, AH23848 treatment attenuated PGE2-or cay10598-induced proliferation and migration by repressing the EGF receptor (EGFR)/Growth factor receptor bound protein 2-associated binder protein 1 (Gab1)/Akt/NF-κB/VEGF-A signaling network in human retinal microvascular endothelial cells (hRMECs). PGE2/EP4R signaling network is thus a potential therapeutic target for pathological intraocular angiogenesis.

Effect of rebiopsy methods and clinical features on T790M mutation after first-line EGFR-TKI treatment failure.

Aim: This study investigated the association between clinical data and T790M mutation in rebiopsy after EGFR tyrosine kinase inhibitors (EGFR-TKIs) failure, and explored the prognosis of T790M-positive patients. Methods: Patients with non-small-cell lung cancer undergoing rebiopsy after first-generation TKI failure were reviewed. Results & conclusion: Patients with brain metastases, negative TP53, initial 19del and longer initial PFS had higher positive rate of T790M. The median progression-free survival (PFS) of T790M-positive patients with cytology and tissue rebiopsy were longer than patients with liquid rebiopsy. The median PFS of T790M-positive patients rebiopsied by ordinary bronchoscope and endobronchial ultrasound-guided transbronchial lung biopsy with a guided sheath (EBUS-GS-TBLB) were longer than that of the patients rebiopsied by EBUS transbronchial needle aspiration (TBNA).

Lay abstract A specific genetic mutation, T790M, is the main cause of drug resistance in patients with lung cancer receiving a type of drug called EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib. We explored the factors that influence the prognosis of T790M-positive patients and how this mutation causes resistance. We found a number of biomarkers that are linked to higher expression of T790M, including brain metastases and longer initial treatment period of EGFR-TKI. The median length of time before tumors started progressing in T790M-positive patients who underwent cytology and tissue rebiopsy was longer than patients who received a blood biopsy. The length of time before progression in patients undergoing different rebiopsy methods (such as ordinary bronchoscope and endobronchial ultrasound bronchoscope) was different.

Effect of the glycine-rich domain in GAREM2 on its unique subcellular localization upon EGF stimulation.

BACKGROUND: In mammals, there are two subtypes of Grb2-associated regulator of Erk/MAPK (GAREM), an adaptor protein that functions downstream of the cell growth factor receptor. GAREM1 is ubiquitously expressed, whereas GAREM2 is mainly expressed in the brain. However, the precise mechanism of the translocation of each GAREM subtype in growth factor-stimulated cells is still unclear. METHODS: In this study, immunofluorescence staining with specific antibodies against each GAREM subtype and time-lapse analysis using GFP fusion proteins were used to analyze the subcellular localization of each GAREM subtype in a cell growth stimulus-dependent manner. We also biochemically analyzed the correlation between its subcellular localization and tyrosine phosphorylation of GAREM2. RESULTS: We found that endogenously and exogenously expressed GAREM2 specifically aggregated and formed granules in NGF-stimulated PC-12 cells and in EGF-stimulated COS-7 cells. Based on the observed subcellular localizations of chimeric GAREM1 and GAREM2 proteins, a glycine-rich region, which is present only in GAREM2, is required for the observed granule formation. This region also regulates the degree of EGF-stimulation-dependent tyrosine phosphorylation of GAREM2. CONCLUSIONS: Our results, showing that aggregation of GAREM2 in response to EGF stimulation is dependent on a glycine-rich region, suggest that GAREM2 aggregation may be involved in neurodegenerative diseases.

Design, Synthesis and Biological Evaluation of Novel Pyrazolo[1,2,4]triazolopyrimidine Derivatives as Potential Anticancer Agents.

Three novel pyrazolo-[4,3-e][1,2,4]triazolopyrimidine derivatives (1, 2, and 3) were designed, synthesized, and evaluated for their in vitro biological activity. All three compounds exhibited different levels of cytotoxicity against cervical and breast cancer cell lines. However, compound 1 showed the best antiproliferative activity against all tested tumor cell lines, including HCC1937 and HeLa cells, which express high levels of wild-type epidermal growth factor receptor (EGFR). Western blot analyses demonstrated that compound 1 inhibited the activation of EGFR, protein kinase B (Akt), and extracellular signal-regulated kinase (Erk)1/2 in breast and cervical cancer cells at concentrations of 7 and 11 µM, respectively. The results from docking experiments with EGFR suggested the binding of compound 1 at the ATP binding site of EGFR. Furthermore, the crystal structure of compound 3 (7-(4-bromophenyl)-9-(pyridin-4-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine) was determined by single crystal X-ray analysis. Our work represents a promising starting point for the development of a new series of compounds targeting EGFR.

ErbB1-dependent signalling and vesicular trafficking in primary afferent nociceptors associated with hypersensitivity in neuropathic pain.

Effective analgesic treatment for neuropathic pain remains an unmet need, so previous evidence that epidermal growth factor receptor inhibitors (EGFRIs) provide unexpected rapid pain relief in a clinical setting points to a novel therapeutic opportunity. The present study utilises rodent models to address the cellular and molecular basis for the findings, focusing on primary sensory neurons because clinical pain relief is provided not only by small molecule EGFRIs, but also by the anti-EGFR antibodies cetuximab and panitumumab, which are unlikely to access the central nervous system in therapeutic concentrations. We report robust, rapid and dose-dependent analgesic effects of EGFRIs in two neuropathic pain models, matched by evidence with highly selective antibodies that expression of the EGFR (ErbB1 protein) is limited to small nociceptive afferent neurons. As other ErbB family members can heterodimerise with ErbB1, we investigated their distribution, showing consistent co-expression of ErbB2 but not ErbB3 or ErbB4, with ErbB1 in cell bodies of nociceptors, as well as providing evidence for direct molecular interaction of ErbB1 with ErbB2 in situ. Co-administration of selective ErbB1 and ErbB2 inhibitors produced clear evidence of greater-than-additive, synergistic analgesia; highlighting the prospect of a unique new combination therapy in which enhanced efficacy could be accompanied by minimisation of side-effects. Peripheral (intraplantar) administration of EGF elicited hypersensitivity only following nerve injury and this was reversed by local co-administration of selective inhibitors of either ErbB1 or ErbB2. Investigating how ErbB1 is activated in neuropathic pain, we found evidence for a role of Src tyrosine kinase, which can be activated by signals from inflammatory mediators, chemokines and cytokines during neuroinflammation. Considering downstream consequences of ErbB1 activation in neuropathic pain, we found direct recruitment to ErbB1 of an adapter for PI 3-kinase and Akt signalling together with clear Akt activation and robust analgesia from selective Akt inhibitors. The known Akt target and regulator of vesicular trafficking, AS160 was strongly phosphorylated at a perinuclear location during neuropathic pain in an ErbB1-, ErbB2- and Akt-dependent manner, corresponding to clustering and translocation of an AS160-partner, the vesicular chaperone, LRP1. Exploring whether neuronal ion channels that could contribute to hyperexcitability might be transported by this vesicular trafficking pathway we were able to identify Nav1.9, (Nav1.8) and Cav1.2 moving towards the plasma membrane or into proximal axonal locations - a process prevented by ErbB1 or Akt inhibitors. Overall these findings newly reveal both upstream and downstream signals to explain how ErbB1 can act as a signalling hub in neuropathic pain models and identify the trafficking of key ion channels to neuronal subcellular locations likely to contribute to hyperexcitability. The new concept of combined treatment with ErbB1 plus ErbB2 blockers is mechanistically validated as a promising strategy for the relief of neuropathic pain.

Epidermal growth factor alleviates cerebral ischemia-induced brain injury by regulating expression of neutrophil gelatinase-associated lipocalin.

Neutrophil gelatinase-associated lipocalin (NGAL) may be a biomarker candidate for brain injury and a novel therapeutic target in ischemic stroke. Epidermal growth factor (EGF) has protective effects on ischemic injury via activating EGF receptor (EGFR). Whether the protection mechanism of activating EGF-EGFR axis against brain injury is involved in regulating NGAL is still unknown. In the present study, we attempted to explore the expression of NGAL in ischemic brain and the effects of EGF on the NGAL expression in a mouse model of middle cerebral artery occlusion (MCAO). Results suggested that the NGAL expression in ischemic brain was markedly increased after cerebral ischemic damage, and specific NGAL-siRNA can attenuate ischemia-triggered infarct volume and neurological deficit. Then, we found that intracerebroventricular EGF treatment may reduce the level of NGAL in ischemic brain, accompanied by functional improvements. Meanwhile, specific JAK2/STAT3 inhibitor AG490 can reverse EGF-induced reduction of NGAL level. Therefore, the elevated NGAL level in ischemic brain may be an important participant in ischemic brain injury. EGF/EGFR activation ameliorated infarct volume of brain tissues and neurological deficit, and the underlying mechanism is involved in regulating the expression of NGAL via the activation of JAK2/STAT3 pathway.

The Role of BEHAB/Brevican in the Tumor Microenvironment: Mediating Glioma Cell Invasion and Motility.

Malignant gliomas are the most common tumors in the central nervous system (CNS) and, unfortunately, are also the most deadly. The lethal nature of malignant gliomas is due in large part to their unique and distinctive ability to invade the surrounding neural tissue. The invasive and dispersive nature of these tumors makes them particularly challenging to treat, and currently there are no effective therapies for malignant gliomas. The brain tumor microenvironment plays a particularly important role in mediating the invasiveness of gliomas, and, therefore, understanding its function is key to developing novel therapies to treat these deadly tumors. A defining aspect of the tumor microenvironment of gliomas is the unique composition of the extracellular matrix that enables tumors to overcome the typically inhibitory environment found in the CNS. One conspicuous component of the glioma tumor microenvironment is the neural-specific ECM molecule, brain-enriched hyaluronan binding (BEHAB)/brevican (B/b). B/b is highly overexpressed in gliomas, and its expression in these tumors contributes importantly to the tumor invasiveness and aggressiveness. However, B/b is a complicated protein with multiple splice variants, cleavage products, and glycoforms that contribute to its complex functions in these tumors and provide unique targets for tumor therapy. Here we review the role of B/b in glioma tumor microenvironment and explore targeting of this protein for glioma therapy.