Subfamily Of Receptor Tyrosine Kinases Research Articles

Nephric duct insertion requires EphA4/EphA7 signaling from the pericloacal mesenchyme.

The vesico-ureteric junction (VUJ) forms through a complex developmental program that connects the primordium of the upper urinary tract [the nephric duct (ND)] with that of the lower urinary tract (the cloaca). The signals that orchestrate the various tissue interactions in this program are poorly understood. Here, we show that two members of the EphA subfamily of receptor tyrosine kinases, EphA4 and EphA7, are specifically expressed in the mesenchyme surrounding the caudal ND and the cloaca, and that Epha4(-/-);Epha7(+/-) and Epha4(-/-);Epha7(-/-) (DKO) mice display distal ureter malformations including ureterocele, blind and ectopically ending ureters with associated hydroureter, megaureter and hydronephrosis. We trace these defects to a late or absent fusion of the ND with the cloaca. In DKO embryos, the ND extends normally and approaches the cloaca but the tip subsequently looses its integrity. Expression of Gata3 and Lhx1 and their downstream target Ret is severely reduced in the caudal ND. Conditional deletion of ephrin B2 from the ND largely phenocopies these changes, suggesting that EphA4/EphA7 from the pericloacal mesenchyme signal via ephrin B2 to mediate ND insertion. Disturbed activity of this signaling module may entail defects of the VUJ, which are frequent in the spectrum of congenital anomalies of the kidney and the urinary tract (CAKUT) in human newborns.

Function of the Ryk intracellular domain in C. elegans vulval development.

Ryk is a subfamily of receptor tyrosine kinases, which along with Frizzled and Ror, function as Wnt receptors. Vertebrate Ryk intracellular domain (ICD) is released from the cell membrane by a proteolytic cleavage in the transmembrane region and localizes to the nucleus. In C. elegans, Ryk is encoded by the lin-18 gene and regulates the polarity of the P7.p vulval cell. Based on Western blots, we were unable to detect the presence of the cleaved LIN-18 ICD fragment. Functional assays found that LIN-18 intracellular domain is not absolutely required for LIN-18 function, consistent with previous results. However, overexpression of the LIN-18 intracellular domain fragment (LIN-18ICD) weakly enhanced the phenotype of lin-18 loss-of-function mutants. Furthermore, this activity was specific to the serine-rich juxtamembrane region. We also found that the nuclear localization of LIN-18ICD fragment can be regulated by Wnt pathway components including CAM-1/Ror, and by PAR-5/14-3-3. Release of LIN-18ICD by cleavage at the membrane is not the main mechanism of LIN-18 signaling in vulval cells. However, our results suggest that LIN-18 intracellular domain interacts with Wnt pathway components and a 14-3-3 protein and likely plays a minor role in LIN-18 signaling.

Expression of Eph A4, Eph B2 and Eph B4 receptors in AML.

Eph receptors represent the largest subfamily of receptor tyrosine kinases (RTKs). The up- regulation of Eph receptors has been documented in various solid tumors, where it often correlates with poor prognosis. Their significance in hematologic malignancies is still unclear. This study aimed to investigate the expression of Eph A4, Eph B2, and Eph B4 mRNA in non - M3 AML patients and determine their prognostic significance. Bone marrow samples from 101 newly diagnosed non - M3 AML patients and 26 healthy controls for comparison were quantified by real time reverse transcriptase polymerase chain reaction (RT-PCR), and the comparative cycle threshold (Ct) method was used to determine their relative expression levels to GUS control gene. The results showed that expression of all selected Eph receptors was significantly lower in AML patients comparing to controls. It also differed according to FAB subtypes. The decreased expression levels of Eph A4 were associated with higher leukocytes (p = 0.022) and blast cell counts (p = 0.001), and unfavorable FLT3-ITD mutation. Our study revealed significant correlation between lower EphB2 expression levels, and higher complete remission rate (p = 0.009724) and longer overall survival. Additionally, we found that patients with shorter RFS had decreased EphB4 expression (p = 0.00). In conclusion, the results suggest the prognostic impact of decreased expression levels of some Eph receptors in AML patients.

Structural Mechanisms Determining Inhibition of the Collagen Receptor DDR1 by Selective and Multi-Targeted Type II Kinase Inhibitors

The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1. Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC50 of 9nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and αD helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a β-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket. Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer.

Poster #M135 A GENOME-WIDE ANALYSIS ON ANTIPSYCHOTIC-INDUCED WEIGHT GAIN USING REFINED CRITERIA IN THE CATIE SAMPLE

The discoidin domain receptors, DDR1 and DDR2, are nonintegrin collagen receptors that are members of the receptor tyrosine kinase family. Both DDRs bind a number of different collagen types and play important roles in embryo development. Dysregulated DDR function is associated with progression of various human diseases, including fibrosis, arthritis, and cancer. By interacting with key components of the extracellular matrix and displaying distinct activation kinetics, the DDRs form a unique subfamily of receptor tyrosine kinases. DDR-facilitated cellular functions include cell migration, cell survival, proliferation, and differentiation, as well as remodeling of extracellular matrices. This review summarizes the current knowledge of DDR–ligand interactions, DDR-initiated signal pathways and the molecular mechanisms that regulate receptor function. Also discussed are the roles of DDRs in development and disease progression.

OP1-05 INTEGRATIVE DNA METHYLATION AND GENE EXPRESSION ANALYSES IDENTIFIES DISCOIDIN DOMAIN RECEPTOR 1 (DDR1) ASSOCIATION WITH IDIOPATHIC NONOBSTRUCTIVE AZOOSPERMIA

You have accessJournal of UrologyOutstanding Posters: Research1 Apr 2014OP1-05 INTEGRATIVE DNA METHYLATION AND GENE EXPRESSION ANALYSES IDENTIFIES DISCOIDIN DOMAIN RECEPTOR 1 (DDR1) ASSOCIATION WITH IDIOPATHIC NONOBSTRUCTIVE AZOOSPERMIA Ranjith Ramasamy, Alex Ridgeway, Josephine Addai, Larry Lipshultz, and Dolores Lamb Ranjith RamasamyRanjith Ramasamy More articles by this author , Alex RidgewayAlex Ridgeway More articles by this author , Josephine AddaiJosephine Addai More articles by this author , Larry LipshultzLarry Lipshultz More articles by this author , and Dolores LambDolores Lamb More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.613AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Spermatogenesis is a complex process that involves proliferation, differentiation, and cell adhesion. Spermatogenic failure or non−obstructive azoospermia (NOA) results from mechanisms involved are incompletely understood. DDR1 is a member of a small subfamily of receptor tyrosine kinases that is involved in adhesion, migration, proliferation, apoptosis, cell morphogenesis and differentiation. Since, DDR1 is expressed in human post−meiotic germ cells of testis, we hypothesized that abnormal DDR1 expression could be a possible mechanism that can compromise spermatogenesis in a subset of men with idiopathic NOA. METHODS We used the high resolution Infinium 450K methylation array and compared fibrobalsts cultured from testicular biopsies of 19 NOA men and 4 fertile controls. Microarray data was analyzed using Minfi (R software package) utilizing subset−quantile within array normalization. We investigated the functional role of abnormal promoter DNA methylation for selected genes using mRNA expression by quantitative RT−real time PCR. Immunohistochemistry was used to confirm testicular expression and potential importance in spermatogenesis RESULTS Differentially methylated CpG sites (∼20K) were identified using an F−Test (p<0.05) in the NOA samples. We identified 24 genes with the >30% difference in methylation within promoter region of men with NOA and fertile controls. Of the aberrantly methylated CpGs, 13 were hypomethylated and 11 were hypermethylated groups. From the top 11 hypermethylated genes, six genes (MRI1, DCAF12L1, TMEM95, CECR2, DDR1, NPHS2) were selected for validation since they were shown to be expressed in testis. Of the 6 genes validated with qPCR, DDR1 showed aberrant gene expression pattern. Four (21%) patients out of the 19 NOA men had lower expression levels (1.8x) of DDR1, whereas two (10.5%) men had higher expression levels (2.5x) of DDR1compared to fertile men (p<0.05). Immunohistochemical analysis suggests presence of DDR1 within cytoplasm of germ cells in fertile men and men with maturation arrest histology. DDR1 protein is absent in men with Sertoli−cell only or germ cell aplasia. CONCLUSIONS Aberrant expression of DDR1 is associated with NOA. The functional relevance of abnormal methylation of DDR1 to NOA warrants further investigation © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e163-e164 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Ranjith Ramasamy More articles by this author Alex Ridgeway More articles by this author Josephine Addai More articles by this author Larry Lipshultz More articles by this author Dolores Lamb More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MerTK inhibition is a novel therapeutic approach for glioblastoma multiforme

Glioblastoma is an aggressive tumor that occurs in both adult and pediatric patients and is known for its invasive quality and high rate of recurrence. Current therapies for glioblastoma result in high morbidity and dismal outcomes. The TAM subfamily of receptor tyrosine kinases includes Tyro3, Axl, and MerTK. Axl and MerTK exhibit little to no expression in normal brain but are highly expressed in glioblastoma and contribute to the critical malignant phenotypes of survival, chemosensitivity and migration. We have found that Foretinib, a RTK inhibitor currently in clinical trial, inhibited phosphorylation of TAM receptors, with highest efficacy against MerTK, and blocked downstream activation of Akt and Erk in adult and pediatric glioblastoma cell lines, findings that are previously unreported. Survival, proliferation, migration, and collagen invasion were hindered in vitro. Foretinib treatment in vivo abolished MerTK phosphorylation and reduced tumor growth 3-4 fold in a subcutaneous mouse model. MerTK targeted shRNA completely prevented intracranial and subcutaneous glioma growth further delineating the impact of MerTK inhibition on glioblastoma. Our findings provide additional target validation for MerTK inhibition in glioblastoma and demonstrate that robust MerTK inhibition can be achieved with the multi-kinase inhibitor Foretinib as an innovative and translational therapeutic approach to glioblastoma.

Chapter Two - Discoidin Domain Receptor Functions in Physiological and Pathological Conditions

The discoidin domain receptors, DDR1 and DDR2, are nonintegrin collagen receptors that are members of the receptor tyrosine kinase family. Both DDRs bind a number of different collagen types and play important roles in embryo development. Dysregulated DDR function is associated with progression of various human diseases, including fibrosis, arthritis, and cancer. By interacting with key components of the extracellular matrix and displaying distinct activation kinetics, the DDRs form a unique subfamily of receptor tyrosine kinases. DDR-facilitated cellular functions include cell migration, cell survival, proliferation, and differentiation, as well as remodeling of extracellular matrices. This review summarizes the current knowledge of DDR-ligand interactions, DDR-initiated signal pathways and the molecular mechanisms that regulate receptor function. Also discussed are the roles of DDRs in development and disease progression.

TNF-α/NF-κB Signaling in the CNS: Possible Connection to EPHB2

Tumor necrosis factor-alpha, TNF-α, is a cytokine that is a well-known factor in multiple disease conditions and is recognized for its major role in central nervous system signaling. TNF-α signaling is most commonly associated with neurotoxicity, but in some conditions it has been found to be neuroprotective. TNF-α has long been known to induce nuclear factor-kappa B, NF-κB, signaling by, in most cases, translocating the p65 (RelA) DNA binding factor to the nucleus. p65 is a key member of NF-κB, which is well established as a family of transcription factors that regulates many signaling events, including growth and process development, in neuronal cell populations. NF-κB has been shown to affect both the receiving aspect of neuronal signaling events in dendritic development as well as the sending of neuronal signals in axonal development. In both cases, NK-κB functions as a promoter and/or inhibitor of growth, depending on the environmental conditions and signaling cascade. In addition, NF-κB is involved in memory formation or neurogenesis, depending on the region of the brain in which the signaling occurs. The ephrin (Eph) receptor family represents a subfamily of receptor tyrosine kinases, RTKs, which received much attention due to its potential involvement in neuronal cell health and function. There are two subsets of ephrin receptors, Eph A and Eph B, each with distinct functions in cardiovascular and skeletal development and axon guidance and synaptic plasticity. The presence of multiple binding sites for NF-κB within the regulatory region of EphB2 gene and its potential regulation by NF-κB pathway suggests that TNF-α may modulate EphB2 via NF-κB and that this may contribute to the neuroprotective activity of TNF-α.

EphA2 Signaling Following Endocytosis: Role of Tiam1

Eph receptors and their membrane‐bound ligands, the ephrins, represent a complex subfamily of receptor tyrosine kinases (RTKs). Eph/ephrin binding can lead to various and opposite cellular behaviors such as adhesion versus repulsion, or cell migration versus cell‐adhesion. Recently, Eph endocytosis has been identified as one of the critical steps responsible for such diversity. Eph receptors, as many RTKs, are rapidly endocytosed following ligand‐mediated activation and traffic through endocytic compartments prior to degradation. However, it is becoming obvious that endocytosis controls signaling in many different manners. Here we showed that activated EphA2 are degraded in the lysosomes and that about 35% of internalized receptors are recycled back to the plasma membrane. Our study is also the first to demonstrate that EphA2 retains the capacity to signal in endosomes. In particular, activated EphA2 interacted with the Rho family GEF Tiam1 in endosomes. This association led to Tiam1 activation, which in turn increased Rac1 activity and facilitated Eph/ephrin endocytosis. Disrupting Tiam1 function with RNA interference impaired both ephrinA1‐dependent Rac1 activation and ephrinA1‐induced EphA2 endocytosis. In summary, our findings shed new light on the regulation of EphA2 endocytosis, intracellular trafficking and signal termination and establish Tiam1 as an important modulator of EphA2 signaling.

EphA2-Induced Angiogenesis in Ewing Sarcoma Cells Works through bFGF Production and Is Dependent on Caveolin-1

Angiogenesis is the result of the combined activity of the tumor microenvironment and signaling molecules. The angiogenic switch is represented as an imbalance between pro- and anti-angiogenic factors and is a rate-limiting step in the development of tumors. Eph receptor tyrosine kinases and their membrane-anchored ligands, known as ephrins, constitute the largest receptor tyrosine kinase (RTK) subfamily and are considered a major family of pro-angiogenic RTKs. Ewing sarcoma (EWS) is a highly aggressive bone and soft tissue tumor affecting children and young adults. As other solid tumors, EWS are reliant on a functional vascular network for the delivery of nutrients and oxygen and for the removal of waste. Based on the biological roles of EphA2 in promoting angiogenesis, we explored the functional role of this receptor and its relationship with caveolin-1 (CAV1) in EWS angiogenesis. We demonstrated that lack of CAV1 results in a significant reduction in micro vascular density (MVD) on 3 different in vivo models. In vitro, this phenomenon correlated with inactivation of EphA2 receptor, lack of AKT response and downregulation of bFGF. We also demonstrated that secreted bFGF from EWS cells acted as chemoattractant for endothelial cells. Furthermore, interaction between EphA2 and CAV1 was necessary for the right localization and signaling of the receptor to produce bFGF through AKT and promote migration of endothelial cells. Finally, introduction of a dominant-negative form of EphA2 into EWS cells mostly reproduced the effects occurred by CAV1 silencing, strongly suggesting that the axis EphA2-CAV1 participates in the promotion of endothelial cell migration toward the tumors favoring EWS angiogenesis.

Prolonged Apoptotic Cell Accumulation in Germinal Centers of Mer-Deficient Mice Causes Elevated B Cell and CD4+ Th Cell Responses Leading to Autoantibody Production

Mer receptor tyrosine kinase is a member of the Tyro-3/Axl/Mer (TAM) subfamily of receptor tyrosine kinases, and its expression on phagocytes facilitates their clearance of apoptotic cells (ACs). Mer expression in germinal centers (GCs) occurs predominantly on tingible body macrophages. B and T cells do not express Mer. In this study, we show that Mer deficiency ((Mer(-/-)) resulted in the long-term accumulation of ACs primarily in GCs and not in the T cell zone, marginal zone, or red pulp areas of the spleen. AC accumulation in GCs led to augmented Ab-forming cell, GC, and IgG2 Ab responses in Mer(-/-) mice, which were sustained for at least 80 d. Enhanced responses in Mer(-/-) mice were due to increased activation and proliferation of B cells and CD4(+) Th cells, including follicular helper T cells, which resulted in high titers of anti-nuclear Abs in Mer(-/-) mice compared with wild-type controls. Secondary IgG-producing Ab-forming cell, total IgG, and IgG2 Ab responses were also increased in Mer(-/-) mice. Finally, compared with wild-type controls, Mer(-/-) mice had increased percentage of IFN-γ-producing CD4(+) Th cells and elevated levels of Th1 (i.e., IL-2 and IFN-γ) and proinflammatory (i.e., TNF and IL-6) cytokines, consistent with elevated levels of Th1-biased IgG2 Abs in Mer(-/-) mice. Together, our results demonstrate that Mer deficiency induces prolonged accumulation of ACs in GCs, resulting in dysregulation of GC B cell and CD4(+) Th cell responses and Th1 cytokine production, leading to alteration of B cell tolerance and the development of autoantibodies.

CYT-1 isoform of ErbB4 is an independent prognostic factor in serous ovarian cancer and selectively promotes ovarian cancer cell growth in vitro

ObjectiveErbB4 is a member of the ErbB subfamily of receptor tyrosine kinases with a poorly understood biological role in ovarian cancer. Here, we have addressed the expression, subcellular localization, and prognostic relevance of ErbB4 and its alternatively spliced isoforms in serous ovarian adenocarcinoma. MethodsA tissue microarray including 482 samples was analyzed by immunohistochemistry, and a series of 198 samples by isoform-specific real-time RT-PCR. The data were statistically analyzed for associations with clinicopathological markers and survival. The functional effect of expressing the relevant ErbB4 isoforms in ovarian cancer cells was addressed by measuring colony formation in soft agar. ResultsWhile ErbB4 immunoreactivity was present in 90% of the samples, total ErbB4 protein expression was not significantly associated with prognostic markers. However, real-time RT-PCR analysis of serous ovarian cancer samples indicated the presence of two alternatively spliced cytoplasmic isoforms of ERBB4, CYT-1 and CYT-2, previously demonstrated to mediate significantly different cellular activities. Expression of CYT-1, but not of CYT-2, was significantly associated with tumor grade (P=0.014) and poor overall survival (P=0.0028). CYT-1 expression was also an independent prognostic factor (P=0.021) in multivariate analysis of survival. Consistent with a biological effect specific for the one isoform, overexpression of ErbB4 CYT-1, but not of ErbB4 CYT-2, increased anchorage-independent growth of ovarian adenocarcinoma cells in vitro. ConclusionsThese results suggest that expression of a specific ErbB4 isoform, CYT-1, is associated with poor survival and enhanced growth in serous ovarian cancer.

Abstract 9929: Novel Mutations Identified by Whole Exome Sequencing in Families With Congenital Heart Disease

Congenital heart disease (CHD) is a complex disorder with multifactorial etiology and high heritability. We used whole exome sequencing to identify novel variants associated with familial CHD. Methods: CHD patients were enrolled in a prospective biobank registry (n=2800). Whole exome sequencing was performed in 12 affected members with LV outflow tract defects and 7 non-affected members from 5 families using Illumina Hi-Seq 2000, and Agilent SureSelect Human All Exon v.2 Kit for sequence capture. Results: Sequence alignment to the reference human genome (GRCh37/hg19) identified over 48K single nucleotide variants (SNVs) and indels (average 820 novel coding variants per individual). SNVs were called using SOAP tools and SIFT, Polyphen and Mutation Taster algorithms were used to predict pathogenicity. Affected and unaffected members were compared using the dominant model to identify novel CHD-associated variants. We identified 216 novel SNVs that segregated within affected members (not seen in unaffected members). These included 82 potentially pathogenic variants on 69 genes expressed in the heart and/or vasculature. Of these, 51 were inherited and 31 were de novo. Novel inherited variants in two genes on the Eph (ephrin) family receptor interacting proteins segregated within 4 affected members from 2 families. Eph receptors form the largest subfamily of receptor tyrosine kinases that are components of cell adhesion, migration, and signalling pathways and are involved in organ development. Additional inherited and de novo variants are being further characterized to determine if they are related to cardiac developmental pathways. Conclusions: Next-generation sequencing can detect novel variants on susceptibility genes that identify new pathways involved in CHD causation. Replication in additional CHD families and candidate gene resequencing will help validate the association of observed variants with CHD.

Gas6 and TAM Receptors in Mouse Platelets

Abstract 5199 Objective:Growth Arrest Specific-6 (Gas6) is a new vitamin K-dependent protein and is a ligand for TAM receptors, three receptors (Tyro, Axl and Mer) belonging to the receptor tyrosine kinase subfamily. The number of publications concerning the physiological importance of the new vitamin-K dependent proteins, which are not members of the coagulation cascade, is growing. Gas6 has a structural homology to natural anticoagulant of protein S, but has no anticoagulant activity. Some research groups have reported contradictory results concerning the presence of Gas6 and its receptors in mouse platelets. The purpose of this study is to investigate the presence of Gas6 and its receptors in mouse platelets as mRNA and protein levels. Material and Methods:25 specific pathogen-free, 8–10 week old, 25–30 g in weight, race of Balb-C male/female mice were anesthetized under light ether anesthesia and blood samples were taken from their hearts. Total RNAs were isolated from platelets, then mRNAs encoding Gas6 and TAM receptors were detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Protein concentrations of Gas6 and TAM receptors in platelets were measured by ELISA. We were unable to measure the Mer protein level because of the absence of any commercial ELISA kit for mouse Mer. Results:RT-PCR results indicated the presence of mRNAs encoding to Gas6 and Mer in mouse platelets. However, although RT-PCR reactions were performed at various temperatures and cycles, we could not detect the presence of mRNAs encoding Axl and Tyro. Receptor levels of Axl and Tyro were below the detection limits of the ELISA method. Plasma Gas6 concentration was found to be 26 ng/ml. Conclusion:We found the presence of mRNAs encoding Gas6 and only receptor Mer in mouse platelets but not Axl and Tyro. Protein Gas6, Axl and Tyro levels were below the detection limits of the ELISA methods. The presence of mRNA is not evidence of protein expression in platelets because they have no DNA. Further studies are required to clarify the presence of Gas6 and TAM receptors in platelets using Real Time PCR, and more sensitive immunological methods, and then, as studies of their effects on mechanisms. Original results from these further studies will provide reliable information as to whether Gas6 and its TAM receptors may be a pharmacological target in thrombosis, hemostasis, restenosis and atherosclerosis. Disclosures:No relevant conflicts of interest to declare.

Advances of EphrinBs/EphBs signaling pathway in the nociceptive process

Background Ephs is the largest subfamily of receptor tyrosine kinases (RTKs) in the human genome,which binds to the ligand,Ephrins,to regulate many development processes,including tissue-border formation,re-angiogenesis,axon guidance,and synaptic plasticity.Recent studies indicate that EphrinBs/EphBs signaling regulates the nociceptive process.Objective Here we retrospectively summarized the mechanism of EphrinBs/EphBs signaling pathway in regulating the process of pain. Content A great deal of studies have shown that EphrinBs/EphBs signaling pathway modulates the development of nociceptive process through upregulating the excitability of nociceptive dorsal root ganglia and wide dynamic range neurons in spinal dorsal horn,producing central sensitization,and activating the downstream mitogen-activated protein kinase (MAPKs) pathway.Trend Clarifying the downstream mechanism of EphrinBs/EphBs signaling pathway in the nociceptive process will be helpful helpful to find a new potential target for the treatment of pain. Key words: EphrinBs/EphBs; Nociceptive process; Central sensitization; Mitogen-activated protein kinase

Inhibitors of the TAM subfamily of tyrosine kinases: Synthesis and biological evaluation

The TAM subfamily of Receptor Tyrosine Kinases (RTKs) contains three human proteins of therapeutical interest, Axl, Mer, and Tyro3. Our goal was to design a type II inhibitor specific for this family, i.e. able to interact with the allosteric pocket and with the hinge region of the kinase. We report the synthesis of several series of purine analogues of BMS-777607. The structural diversity of the designed inhibitors was expected to modify the interactions formed in the binding site and consequently to modulate their selectivity profiles. The most potent inhibitor 6g exhibits Kds of 39, 42, 65 and 200 nM against Axl, Mer, Met and Tyro3 respectively. Analysis of the affinity of 6g for active and inactive forms of Abl1, an RTK protein that does not belong to the TAM subfamily, together with the binding modes of 6g predicted by docking studies, indicates that 6g displays some selectivity for the TAM family and may act as a type II inhibitor.

Abstract 3434: EphB3 mediates kinase-dependent suppression of non-small cell lung cancer metastasis

Abstract Eph family is one of the largest subfamily of receptor tyrosine kinases. Their important but controversial roles in cancer development have drawn growing attentions in recent years. We have recently reported that EphB3 acts as a tumor promoter in human non-small-cell lung cancer (NSCLC) independent of its kinase activity (Cancer Research. 71(3):1156, 2011). In the present study, we find that despite overexpression of EphB3 in NSCLC, the expression of its cognate ligands, ephrin-B1 and ephrin-B2, is significantly down-regulated, leading to reduced tyrosine phosphorylation of EphB3. Activation of EphB3 kinase with either ligand stimulation or genomic engineering (Y608/614E) in EphB3-overexpressing NSCLC cells inhibits cell migratory capability in vitro as well as in vivo metastatic seeding. Furthermore, we have identified a novel EphB3-binding protein, the receptor for activated C-kinase 1 (RACK1), which mediates the assembly of a ternary signal complex comprising protein phosphatase 2A (PP2A), Akt and itself in response to EphB3 activation, leading to reduced Akt phosphorylation and subsequent inhibition of cell migration. Our study reveals a novel tumor-suppressing signaling pathway associated with kinase-activated EphB3 in NSCLC, and provides a potential therapeutic strategy by activating EphB3 signaling, thus inhibiting tumor metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3434. doi:1538-7445.AM2012-3434

Abstract 1197: EphA3 kinase ablation induces glioblastoma differentiation and prevents tumor progression

Abstract Purpose: Eph receptors constitute the largest sub-family of receptor tyrosine kinases and interact with membrane-bound ligands termed ephrins. Eph and ephrins have many vital functions including cell adhesion, migration and axon guidance. Eph and ephrins have been found to be aberrantly expressed in many malignancies including brain tumor. The purpose of this study was to investigate EphA3 receptor function in the most common and aggressive form of brain tumor, Glioblastoma (GBM). Methods: Gene expression was investigated by Q-PCR, IHC and flow cytometry in high grade glioma (HGG) surgical specimens and primary derived serum free cell cultures. Targeted reduction of Eph expression was performed using both a constitutive and inducible shRNA system. Murine in-vivo studies were performed using both subcutaneous and orthotopic ‘intracranial’ xenografts in immuno-compromised animals. Signaling pathways were assessed by western blotting. Results: To establish whether the receptor tyrosine kinase EphA3 was over expressed in HGG we assessed 12 normal human brain specimens, 56 HGG specimens and 26 HGG primary cultures. EphA3 mRNA expression was negligible in normal brain while 30% of clinical specimens and 46% of primary cultured tumor cells expressed EphA3. EphA3 protein was also detected in HGG clinical specimens using IHC. To further investigate EphA3 function the receptor was down regulated using shRNA in an EphA3+ GBM neurosphere cell line. Constitutive and inducible down regulation of the EphA3 receptor resulted in initiation of neuronal and glial cell differentiation following activation of the ERK/MAPK pathway. A reduction in stem/progenitor cell proliferation was also observed following EphA3 knockdown by shRNA (46%) or by alternately inhibiting EphA3 function using soluble ephrin A5-Fc (33%). CFSE division tracking identified slower cell division in populations in which EphA3 signaling was attenuated. In-vivo studies were performed using a NOD/SCID mouse subcutaneous and intracranial xenograft model. Results highlighted a marked reduction in tumor formation in the EphA3 knockdown as opposed to control tumors. Subcutaneous control tumors formed with a median survival of 66 days while EphA3 knockdown animals survived beyond 100 days (p&amp;lt;0.05). Similar to the subcutaneous xenograft model a marked lack of intracranial tumor formation was observed when EphA3 was neutralized. Control mice formed large well vascularized invasive tumors with a median survival of 62 days. All EphA3 knockdown animals were free of tumor following autopsy at 145 days when the experiment was terminated (p&amp;lt;0.05). Importantly a mutant EphA3 rescue of the knockdown culture returned the tumorigenic potential of these cells. Conclusions: We propose EphA3, in part, regulates cancer stem cell self renewal and cell division rate in GBM and could prove a potential therapeutic target and marker of brain tumor initiating stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1197. doi:10.1158/1538-7445.AM2011-1197

Abstract SY23-02: RTK endocytosis and other feedback mechanisms in cancer

Abstract The view of signal transduction pathways as a set of parallel pipelines connecting transmembrane receptor tyrosine kinases (RTKs) and the genome has given way, over the last decade, to the notion of layered networks, highly rich in internal connections. This paradigm is well exemplified by the RTK subfamily called ERBB, namely receptors for growth factors of the EGF (epidermal growth factor) and neuregulin groups: whereas ERBB-1 (also called EGFR) binds seven different growth factors and ERBB-4 binds several isoforms of neuregulins, ERBB-2/HER2 binds no known growth factor and the kinase activity of ErbB-3/HER3 is almost dead. Because both EGFR and HER2 frequently display aberrant expression and mutant forms in human tumors, their mode of action and regulation are of interest. Like other autonomous bi-stable systems, the ERBB network is regulated by feedback loops, both positive and negative. For example, one outcome of activation by growth factors is the transcriptional induction of EGF-like molecules (positive feedback). On the other hand, once activated the receptors are rapidly endocytosed and degraded in lysosomes (negative feedback). According to recent studies, negative feedback loops are often defective in tumors, whereas positive loops are frequently enhanced. In general, negative feedback regulation may be divided into two temporal phases: the immediate phase (approximately 20 minutes from stimulation) comprises posttranslational protein modifications, such as protein phosphorylation and ubiquitylation, as well as intracellular translocations of enzymes and adaptor proteins, including receptor endocytosis. The late phase of feedback regulation harnesses the transcriptional machinery, and it comprises newly synthesized proteins (e.g., phosphatases of the mitogen-activated protein kinases, MKPs), which restrain signaling. Interestingly, microRNAs complement the roles played by mRNA synthesis: multiple microRNAs undergo rapid turnover within minutes after growth factor stimulation, while another set is newly synthesized. In addition to the immediately down-regulated microRNAs (ID-miRs) and E3 ubiquitin ligases like CBL and Nedd4 family members, the first phase of negative RTK regulation entails tyrosine phosphatases like DEP-1, which dephosphorylates EGFR, and de-ubiquitinating enzymes (DUBs). E3 ligases, in concert with ubiquitin-binding proteins, which nucleate four different endosomal sorting complexes called ESCRTs, coordinate receptor sorting for either lysosomal degradation or recycling to the plasma membrane. In line with their negative regulatory functions, certain components of ESCRTs have been implicated in tumor suppression, and, on the other hand, inactive forms of the RTK-specific E3 ligase, namely CBL, are oncogenic. In analogy with regulators of the endocytic pathway, the group of immediate early genes (IEGs; e.g., FOS and JUN), which is induced by growth factors within the first 30 minutes, is endowed with an oncogenic potential. Likewise, tumors of epithelial and brain origins deviate from the physiological tight regulation of the IEGs: both ID-miRs and several RNA- and DNA-binding proteins, collectively termed delayed early genes (DEGs; e.g., the RNA-binding protein ZFP-36) shape the pulse like profile of IEG induction, but transformed cells escape this type of regulation. In summary, the emerging landscape of feedback regulation in malignant cells proposes multi-site manipulation of systems control: RTKs are often derailed to a recycling route by means of hyperactive RAB-coupling proteins (e.g., RAB11FIP1) and other aberrations, while both the ID-miRs and the DEGs are frequently down-regulated in tumors. Collectively, these aberrations enhance cytoplasmic signaling and augment expression of oncogenic transcription factors, which result in accelerated cell proliferation and higher invasiveness. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY23-02. doi:10.1158/1538-7445.AM2011-SY23-02