Activation of HGF/c-Met pathway contributes to the reactive oxygen species generation and motility of small cell lung cancer cells

Small cell lung cancer (SCLC) is a difficult disease to treat and sometimes has overexpression or mutation of c-Met receptor tyrosine kinase. The effects of c-Met/hepatocyte growth factor (c-Met/HGF, ligand for c-Met) on activation of reactive oxygen species (ROS) was determined. HGF stimulation of c-Met-overexpressing H69 SCLC cells (40 ng/ml, 15 min) resulted in an increase of ROS, measured with fluorescent probe 2'-7'-dichlorofluorescein diacetate (DCFH-DA) or dihydroethidine (DHE) but not in c-Met-null H446 cells. ROS was increased in juxtamembrane (JM)-mutated variants (R988C and T1010I) of c-Met compared with wild-type c-Met-expressing cells. ROS was significantly inhibited by preincubation of SCLC cells with pyrrolidine dithiocarbamate (PDTC, 100 microM) and/or SU11274 (small molecule c-Met tyrosine kinase inhibitor, 2 microM) for 3 h. PDTC and SU11274 also abrogated the HGF proliferative signal and cell motility in a cooperative fashion. H(2)O(2) treatment of SCLC cells (over 15 min) led to phosphorylation of c-Met receptor tyrosine kinase and further upregulated downstream phosphorylation of phospho-AKT, ERK1/2, and paxillin in a dose-dependent manner (125 microM to 500 microM). c-Met is an important target in lung cancer, and the pathways responsible for ROS generation together may provide novel therapeutic intervention.     

Mutations in the met oncogene unveil a "dual switch" mechanism controlling tyrosine kinase activity

The met oncogene, encoding the high affinity hepatocyte growth factor receptor, is the only known gene inherited in human cancer that is invariably associated with somatic duplication of the mutant locus. Intriguingly, mutated Met requires ligand stimulation in order to unleash its transforming potential. Furthermore, individuals bearing a germ line met mutation develop cancer only late in life and with incomplete penetrance. To date, there is no molecular explanation for this unique behavior, which is unusual for a dominant oncogene. Here we investigate the molecular mechanisms underlying met oncogenic conversion by generating antibodies specific for the differently phosphorylated forms of the Met protein. Using these antibodies, we show that activation of wild-type Met is achieved through sequential phosphorylation of Tyr1235 and Tyr1234 in the activation loop and that mutagenesis of either tyrosine dramatically impairs kinase function. Surprisingly, oncogenic Met mutants never become phosphorylated on Tyr1234 despite their high enzymatic activity, and mutagenesis of Tyr1234 does not affect their biochemical or biological function. By analyzing the enzymatic properties of the mutant proteins in different conditions, we demonstrate that oncogenic mutations do not elicit constitutive kinase activation but simply overcome the requirement for the second phosphorylation step, thus reducing the threshold for activation. In the presence of activating signals, these mutations result therefore in a dynamic imbalance toward the active conformation of the kinase. This explains why mutant met provides an oncogenic predisposition but needs a second activating "hit," provided by sustained ligand stimulation or receptor overexpression, to achieve a fully transformed phenotype.     

Crosstalk of the EphA2 receptor with a serine/threonine phosphatase suppresses the Akt-mTORC1 pathway in cancer cells

Receptor tyrosine kinases of the Eph family play multiple roles in the physiological regulation of tissue homeostasis and in the pathogenesis of various diseases, including cancer. The EphA2 receptor is highly expressed in most cancer cell types, where it has disparate activities that are not well understood. It has been reported that interplay of EphA2 with oncogenic signaling pathways promotes cancer cell malignancy independently of ephrin ligand binding and receptor kinase activity. In contrast, stimulation of EphA2 signaling with ephrin-A ligands can suppress malignancy by inhibiting the Ras-MAP kinase pathway, integrin-mediated adhesion, and epithelial to mesenchymal transition. Here we show that ephrin-A1 ligand-dependent activation of EphA2 decreases the growth of PC3 prostate cancer cells and profoundly inhibits the Akt-mTORC1 pathway, which is hyperactivated due to loss of the PTEN tumor suppressor. Our results do not implicate changes in the activity of Akt upstream regulators (such as Ras family GTPases, PI3 kinase, integrins, or the Ship2 lipid phosphatase) in the observed loss of Akt T308 and S473 phosphorylation downstream of EphA2. Indeed, EphA2 can inhibit Akt phosphorylation induced by oncogenic mutations of not only PTEN but also PI3 kinase. Furthermore, it can decrease the hyperphosphorylation induced by constitutive membrane-targeting of Akt. Our data suggest a novel signaling mechanism whereby EphA2 inactivates the Akt-mTORC1 oncogenic pathway through Akt dephosphorylation mediated by a serine/threonine phosphatase. Ephrin-A1-induced Akt dephosphorylation was observed not only in PC3 prostate cancer cells but also in other cancer cell types. Thus, activation of EphA2 signaling represents a possible new avenue for anti-cancer therapies that exploit the remarkable ability of this receptor to counteract multiple oncogenic signaling pathways.     

Neuregulin-1alpha and beta isoform expression in cardiac microvascular endothelial cells and function in cardiac myocytes in vitro

Neuregulins (NRGs) are a family of alternatively spliced growth factors that act through receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family in diverse tissues. The NRG-erbB signaling axis is a critical mediator of cardiac development, and growing evidence supports a role for this system in the intricate cross-talk between the microvascular endothelium and myocytes in the adult heart. The purpose of this study was first to examine the expression of splice variants of the NRG1 gene in adult rat cardiac microvascular endothelial cells and second to compare the function of these variants in cardiac myocytes. We demonstrate that cardiac microvascular endothelial cells in rat culture express multiple Type I NRG1 gene products, including both alpha and beta variants. Comparison of the activity of recombinant NRG1alpha and NRG1beta EGF-like domain proteins in cardiac myocytes shows that the beta ligand is a more potent activator of receptor phosphorylation and intracellular signaling than the alpha ligand, and only the beta ligand stimulated glucose uptake and protein synthesis in these culture conditions. Thus, cardiac microvascular endothelial cells express multiple NRG1 isotypes, but only beta-variants are biologically active on cardiac myocytes.     

Tyrosine kinases. New target of anticancer therapy

Recently, clinical studies of new drugs development to target specific forms of cancer were reported. Herceptin, a monoclonal antibody against the Her2/neu receptor tyrosine kinase, prolonged the survival of women with Her2/neu positive metastatic breast cancer. STI571, a small molecule inhibitor of the BCR/ABL, c-Kit and platelet derived growth factor receptor tyrosine kinase, produced pronounced clinical responses in patients with BCR/ABL positive chronic myeloid leukemia and c-Kit positive gastrointestial stromal tumors. In order to consider the use of the inhibitor of tyrosine kinases activity as anticancer drug, their mechanisms of the oncogenic activation and their impact on tumor transformation should be studied. The treatment with tyrosine kinase inhibitors such as STI571 or herceptin was a spectacular clinical success which stimulated research on the structure and function of both kinases and their inhibitors.     

Discovery of novel furanylbenzamide inhibitors that target oncogenic tyrosine phosphatase SHP2 in leukemia cells

Disturbance of the dynamic balance between tyrosine phosphorylation and dephosphorylation of signaling molecules, controlled by protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is known to lead to the development of cancer. While most approved targeted cancer therapies are tyrosine kinase inhibitors, PTPs have long been stigmatized as undruggable and have only recently gained renewed attention in drug discovery. One PTP target is the Src-homology 2 domain–containing phosphatase 2 (SHP2). SHP2 is implicated in tumor initiation, progression, metastasis, and treatment resistance, primarily because of its role as a signaling nexus of the extracellular signal–regulated kinase pathway, acting upstream of the small GTPase Ras. Efforts to develop small molecules that target SHP2 are ongoing, and several SHP2 allosteric inhibitors are currently in clinical trials for the treatment of solid tumors. However, while the reported allosteric inhibitors are highly effective against cells expressing WT SHP2, none have significant activity against the most frequent oncogenic SHP2 variants that drive leukemogenesis in several juvenile and acute leukemias. Here, we report the discovery of novel furanylbenzamide molecules as inhibitors of both WT and oncogenic SHP2. Importantly, these inhibitors readily cross cell membranes, bind and inhibit SHP2 under physiological conditions, and effectively decrease the growth of cancer cells, including triple-negative breast cancer cells, acute myeloid leukemia cells expressing either WT or oncogenic SHP2, and patient-derived acute myeloid leukemia cells. These novel compounds are effective chemical probes of active SHP2 and may serve as starting points for therapeutics targeting WT or mutant SHP2 in cancer.     

Survey of tyrosine kinase signaling reveals ROS kinase fusions in human cholangiocarcinoma

Cholangiocarcinoma, also known as bile duct cancer, is the second most common primary hepatic carcinoma with a median survival of less than 2 years. The molecular mechanisms underlying the development of this disease are not clear. To survey activated tyrosine kinases signaling in cholangiocarcinoma, we employed immunoaffinity profiling coupled to mass spectrometry and identified DDR1, EPHA2, EGFR, and ROS tyrosine kinases, along with over 1,000 tyrosine phosphorylation sites from about 750 different proteins in primary cholangiocarcinoma patients. Furthermore, we confirmed the presence of ROS kinase fusions in 8.7% (2 out of 23) of cholangiocarcinoma patients. Expression of the ROS fusions in 3T3 cells confers transforming ability both in vitro and in vivo, and is responsive to its kinase inhibitor. Our data demonstrate that ROS kinase is a promising candidate for a therapeutic target and for a diagnostic molecular marker in cholangiocarcinoma. The identification of ROS tyrosine kinase fusions in cholangiocarcinoma, along with the presence of other ROS kinase fusions in lung cancer and glioblastoma, suggests that a more broadly based screen for activated ROS kinase in cancer is warranted.     

An oncogenic point mutation confers high affinity ligand binding to the neu receptor. Implications for the generation of site heterogeneity.

The neu protooncogene encodes a receptor tyrosine kinase homologous to the receptor for the epidermal growth factor. The oncogenic potential of neu is released upon chemical carcinogenesis, which replaces a glutamic acid for a valine residue, within the single transmembrane domain. This results in constitutive receptor dimerization and activation of the intrinsic catalytic function. To study the implications of the oncogenic mutation and the consequent receptor dimerization on the interaction with the yet incompletely characterized ligand of p185neu, we constructed chimeric proteins between the ligand binding domain of the epidermal growth factor receptor and the transmembrane and cytoplasmic domains of the normal or the transforming Neu proteins. The chimeric receptors displayed cellular and biochemical differences characteristic of the normal and the transforming Neu proteins and therefore may reliably represent the ligand binding functions of the two receptor forms. Analyses of ligand binding revealed qualitative and quantitative differences that were a result of the single mutation; whereas the normal chimera (valine version) displayed two populations of binding sites with approximately 90% of the receptors in the low affinity state, the transforming receptor (glutamic acid version) showed a single population of binding sites with relatively high affinity. Kinetics measurements indicated that the difference in affinities was because of slower rates of both ligand association and ligand dissociation from the constitutively dimerized mutant receptor. It therefore appears that the oncogenic mutation, by permanently dimerizing the receptor, establishes a high affinity ligand binding state which is functionally equivalent to the ligand-occupied normal receptor. Our conclusion is further supported by the rates of endocytosis of the wild-type and the mutant receptor. Hence, these results provide the first experimental evidence from living cells which supports a model that attributes the heterogeneity of ligand binding sites to the state of oligomerization of receptor tyrosine kinases.     

Exploitation of phage display for the development of anti-cancer agents targeting fibroblast growth factor signaling pathways: New strategies to tackle an old challenge

A tumor is defined as a group of cancer cells and ‘surrounding’ stromal bio-entities. Alongside the extracellular matrix (ECM) in the tumor microenvironment (TME), the stromal cells play key roles in cancer affliction and progression. Carcinoma-associated fibroblasts (CAFs) in the area of the tumor, whether activated or not, dictate the future of tumor cells. The CAFs and corresponding secreted growth factors (GFs), which mediate the crosstalk within the TME, can be targeted in therapies directed at the stroma. The impact of the fibroblast growth factor-fibroblast growth factor receptor (FGF-FGFR) signaling pathway in different kinds of tumors has been explored. Several tyrosine kinase inhibitors (TKIs), monoclonal antibodies (mAbs), and ligand traps targeting the formation of FGF-FGFR complex are in preclinical or early development phases. Moreover, there are numerous studies in the literature reporting the application of phage display technology for the development of peptides and proteins capable of functioning as FGF mimetics or traps, which are able to modulate FGF-related signaling pathways. In this review, prominent research in relation to phage display-assisted ligand identification for the FGF/FGFR system is discussed.     

Activation of Rho is required for ligand-independent oncogenic signaling by a mutant epidermal growth factor receptor

Mutations in the epidermal growth factor receptor have been identified in several human tumor types, including gliomas. These receptor mutants have deletions in their extracellular ligand-binding domains and are, therefore, no longer regulated by ligand, resulting in constitutive activation of the receptor kinase. These mutants have been proposed to transduce oncogenic signals via ligand-independent signaling pathways. Avian viral homologues of these oncogenic epidermal growth factor receptors exhibit structurally homologous deletions and form tumors in chickens. One such mutant, S3v-ErbB, transforms fibroblasts in vitro, and transformation has been correlated with the formation of a novel tyrosine phosphoprotein complex. V-ErbB-mediated complex formation and transformation have been shown to occur independently of Ras activation. The major aims of this study are to further characterize this ligand-independent v-ErbB oncogenic signaling pathway. Here we show that both v-ErbB-mediated phosphoprotein complex formation and transformation are inhibited by a dominant negative mutant of Rho. This inhibition is specific for dominant negative Rho; dominant negative mutants of Rac and Cdc42 have no effect on transformation or on tyrosine phosphorylation of the phosphoprotein complex. Based on these observations, we propose that S3v-ErbB stimulates a Rho-dependent tyrosine kinase, resulting in complex formation and ultimately oncogenic transformation.     

EPH receptors in cancer

EPH receptors and their ephrin ligands constitute the largest sub-family of receptor tyrosine kinases (RTKs) and are components of cell signaling pathways involved in animal development. The ability of the EPH/ephrin guidance system to position cells and modulate cell morphology underlies their various roles in development. In addition, EPH signaling plays an important role in oncogenic processes observed in several organs. These receptors are involved in a wide range of processes directly related with tumorigenesis and metastasis, including cell attachment and shape, migration, and angiogenesis. Accordingly, deregulation of EPH expression and signaling activity could be crucial for the tumorigenic process. This review focuses on EPH receptors' roles in oncogenic transformation and tumor progression.     

FES kinase participates in KIT-ligand induced chemotaxis

FES is a cytoplasmic tyrosine kinase activated by several membrane receptors, originally identified as a viral oncogene product. We have recently identified FES as a crucial effector of oncogenic KIT mutant receptor. However, FES implication in wild-type KIT receptor function was not addressed. We report here that FES interacts with KIT and is phosphorylated following activation by its ligand SCF. Unlike in the context of oncogenic KIT mutant, FES is not involved in wild-type KIT proliferation signal, or in cell adhesion. Instead, FES is required for SCF-induced chemotaxis. In conclusion, FES kinase is a mediator of wild-type KIT signalling implicated in cell migration.     

Degradation of the Met tyrosine kinase receptor by the ubiquitin-proteasome pathway.

The Met tyrosine kinase receptor is a widely expressed molecule which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). In this communication we demonstrate that significant Met degradation is induced by HGF/SF and that this degradation can be blocked by lactacystin, an inhibitor of proteasome activity. We also show that Met is rapidly polyubiquitinated in response to ligand and that polyubiquitinated Met molecules, which are normally unstable, are stabilized by lactacystin. Both HGF/SF-induced degradation and polyubiquitination of Met were shown to be dependent on the receptor possessing intact tyrosine kinase activity. Finally, we found that a normally highly labile 55-kDa fragment of the Met receptor is stabilized by lactacystin and demonstrate that it represents a cell-associated remnant that is generated following the ligand-independent proteolytic cleavage of the Met receptor in its extracellular domain. This truncated Met molecule encompasses the kinase domain of the receptor and is itself tyrosine phosphorylated. We conclude that the ubiquitin-proteasome pathway plays a significant role in the degradation of the Met tyrosine kinase receptor as directed by ligand-dependent and -independent signals. We propose that this proteolytic pathway may be important for averting cellular transformation by desensitizing Met signaling following ligand stimulation and by eliminating potentially oncogenic fragments generated via extracellular cleavage of the Met receptor.     

Regulation of EGFR endocytic trafficking by rab proteins

The Epidermal Growth Factor Receptor (EGFR) is a member of the receptor tyrosine kinase family and has important roles in development and cancer. Through ligand stimulation, the EGFR initiates a number of biochemical pathways that integrate to form specific physiological responses. In addition to these signaling pathways, the ligand stimulation also causes the EGFR to internalize and be transported through the endocytic pathway. The endocytic pathway regulates the rate of EGFR degradation and recycling, as well as the signaling mediated by the EGFR. In this review, the role of rabs, a family of small molecular weight guanine nucleotide binding proteins, is examined in how they regulate endocytic trafficking.     

Specificity of HCPTP variants toward EphA2 tyrosines by quantitative selected reaction monitoring

EphA2 receptor tyrosine kinase and the human cytoplasmic protein tyrosine phosphatase (HCPTP) are overexpressed in a number of epithelial cancers. Overexpressed EphA2 in these cancers shows a significant decrease in phosphotyrosine content which results in suppression of receptor signaling and endocytosis and an increase in metastatic potential. The decreased phosphotyrosine content of EphA2 has been associated with decreased contact with its ligand, ephrin A1 and dephosphorylation by HCPTP. Potential specificity of the two HCPTP variants for tyrosines on EphA2 has not been investigated. We have used a mass spectrometry assay to measure relative rates of dephosphorylation for the two HCPTP variants at phosphotyrosine sites associated with control of the EphA2 kinase activity or interaction with downstream targets. Our results suggest that although both variants dephosphorylate the EphA2 receptor, the rate and specificity of dephosphorylation for specific tyrosines are different for HCPTP-A and HCPTP-B. The SAM domain tyrosine Y960 which has been implicated in downstream PI3K signaling is dephosphorylated exclusively by HCPTP-B. The activation loop tyrosine (Y772) which directly controls kinase activity is dephosphorylated about six times faster by HCPTP-A. In contrast, the juxtamembrane tyrosines (Y575, Y588 and Y594) which are implicated in both control of kinase activity and downstream signaling are dephosphorylated by both variants with similar rates. This difference in preference for dephosphorylation sites on EphA2 not only illuminates the different roles of the two variants of the phosphatase in EphA2 signaling, but also explains why both HCPTP variants are highly conserved in most mammals.     

Regulated coupling of the Neu receptor to phosphatidylinositol 3'-kinase and its release by oncogenic activation.

The neu protooncogene encodes a tyrosine kinase receptor that is involved in the regulation of normal growth and malignant transformation. To circumvent the use of the incompletely characterized ligand of Neu, we constructed a chimeric protein composed of the ligand-binding domain of the epidermal growth factor receptor and the transmembrane and cytoplasmic portions of Neu. By expressing this Neu-epidermal growth factor receptor chimera (termed NEC), we found that following stimulation by the heterologous ligand, the tyrosine kinase of Neu became associated with a phosphatidylinositol (PI) kinase activity. The association was dependent on the concentration of the ligand and was almost maximal within 30 s after ligand binding. The lipid kinase was identified as a type I PI 3'-kinase on the basis of its inhibition by Nonidet P-40 and high pressure liquid chromatography of the phosphorylated product. To confirm the identification of PI 3'-kinase as an effector of Neu, we raised antibodies to the alpha-isoform of the regulatory subunit of PI 3'-kinase (p85). Using these antibodies, it was possible to directly demonstrate ligand-dependent formation of a tyrosine-phosphorylated complex of NEC and PI 3'-kinase. Apparently, both PI 3'-kinase and phospholipase C gamma, another substrate of the Neu kinase, simultaneously associated with the same activated NEC molecule. Nevertheless, immunofluorescence localization of PI 3'-kinase revealed no significant cellular redistribution of the enzyme after activation of the Neu kinase. Interestingly, PI 3'-kinase was localized primarily to the cell nucleus and to confined regions of the plasma membrane. Analysis of mutants of the Neu protein indicated that the oncogenic point-mutated Neu (Glu664) was permanently coupled to PI 3'-kinase; but two nontransforming versions of the oncoprotein, a kinase-defective protein and a carboxyl-terminally deleted Neu, were devoid of the constitutive association with PI 3'-kinase. Hence, we concluded that phosphatidylinositol 3'-kinase is a physiological substrate of the Neu receptor, but the regulation of this coupling is released upon oncogenic activation.     

Developmental control by the Drosophila EGF receptor homolog DER.

The identification of receptor tyrosine kinases in Drosophila has provided an opportunity to study the requirement for these proteins during the development of a multicellular organism. Genetic analysis of the function of the Drosophila epidermal growth factor (EGF) receptor homolog (DER) has revealed an extremely diverse set of roles for this protein throughout the life cycle of the organism, for example in eye development and in the establishment of dorsoventral polarity in the oocyte. We discuss the possible basis for the pleiotropic activity of DER, and the similarities and differences in the function of the homologous proteins in other invertebrates and vertebrates.     

Multimerization of polyomavirus middle-T antigen.

The oncogenic protein of polyomavirus, middle-T antigen, associated with cell membranes and interacts with a variety of cellular proteins involved in mitogenic signalling. Middle-T antigen may therefore mimic the function of cellular tyrosine kinase growth factor receptors, like the platelet-derived growth factor or epidermal growth factor receptor. Growth factor receptor signalling is initiated upon the binding of a ligand to the extracellular domain of the receptor. This results in activation of the intracellular tyrosine kinase domain of the receptor, followed by receptor phosphorylation, presumably as a consequence of dimerization of two receptor molecules. Similar to middle-T antigen, phosphorylation of growth factor receptors leads to recruitment of cellular signalling molecules downstream in the signalling cascade. In this study, we investigated whether middle-T antigen, similar to tyrosine kinase growth factor receptors, is able to form dimeric signalling complexes. We found that association with cellular membranes was a prerequisite for multimerization, most likely dimer formation. A chimeric middle-T antigen carrying the membrane-targeting sequence of the vesicular stomatitis virus G protein instead of the authentic polyomavirus sequence still dimerized. However, mutants of middle-T antigen unable to associate with 14-3-3 proteins, like d18 and S257A, did not form dimers but were still oncogenic. This indicates that both membrane association and binding of 14-3-3 are necessary for dimer formation of middle-T antigen but that only the former is essential for cell transformation.