Aggressive human brain tumours (gliomas) often express a truncated and oncogenic form of the epidermal growth factor receptor, known as EGFRvIII. Within each tumour only a small percentage of glioma cells may actually express EGFRvIII; however, most of the cells exhibit a transformed phenotype. Here we show that EGFRvIII can be 'shared' between glioma cells by intercellular transfer of membrane-derived microvesicles ('oncosomes'). EGFRvIII expression in indolent glioma cells stimulates formation of lipid-raft related microvesicles containing EGFRvIII. Microvesicles containing this receptor are then released to cellular surroundings and blood of tumour-bearing mice, and can merge with the plasma membranes of cancer cells lacking EGFRvIII. This event leads to the transfer of oncogenic activity, including activation of transforming signalling pathways (MAPK and Akt), changes in expression of EGFRvIII-regulated genes (VEGF, Bcl-x(L), p27), morphological transformation and increase in anchorage-independent growth capacity. Thus, membrane microvesicles of cancer cells can contribute to a horizontal propagation of oncogenes and their associated transforming phenotype among subsets of cancer cells.

The EPH-related transmembrane tyrosine kinases constitute the largest known family of receptor-like tyrosine kinases, with many members displaying specific patterns of expression in the developing and adult nervous system. A family of cell surface-bound ligands exhibiting distinct, but overlapping, specificities for these EPH-related kinases was identified. These ligands were unable to act as conventional soluble factors. However, they did function when presented in membrane-bound form, suggesting that they require direct cell-to-cell contact to activate their receptors. Membrane attachment may serve to facilitate ligand dimerization or aggregation, because antibody-mediated clustering activated previously inactive soluble forms of these ligands.

Tissue factor (TF) is the primary cellular initiator of blood coagulation and a modulator of angiogenesis and metastasis in cancer. Indeed, systemic hypercoagulability in patients with cancer and TF overexpression by cancer cells are both closely associated with tumor progression, but their causes have been elusive. We now report that in human colorectal cancer cells, TF expression is under control of 2 major transforming events driving disease progression (activation of K-ras oncogene and inactivation of the p53 tumor suppressor), in a manner dependent on MEK/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K). Furthermore, the levels of cell-associated as well as circulating (microvesicle-associated) TF activity are linked to the genetic status of cancer cells. Finally, RNA interference experiments suggest that TF expression is an important effector of the K-ras-dependent tumorigenic and angiogenic phenotype in vivo. Thus, this study establishes a causal link between cancer coagulopathy, angiogenesis, and genetic tumor progression. ( IntroductionCancer is believed to arise and progress toward increasing malignancy as a result of cumulative genetic "hits" sustained by the tumor cell genome. Paradigmatic in this regard is the development of colorectal carcinoma (CRC), where sequential transition through clinical stages of the disease is paralleled by a series of well-characterized alterations in proto-oncogenes and tumor suppressor genes. 1 In this tumor type, activation of mutant K-ras and subsequent inactivation/loss of p53 are key changes, which drive many interrelated aspects of the malignant phenotype including aberrant mitogenesis and survival. 2 Moreover, both of these genetic alterations are thought to contribute to proangiogenic properties of affected cancer cells, 3,4 and thereby enable them to exploit the host vascular system to advance malignant growth and metastasize in vivo. 5 The involvement of the vascular system in malignancy encompasses not only angiogenesis but also systemic hypercoagulability. Blood clotting abnormalities are detected in up to 90% of patients with metastatic disease, and thrombosis represents the second most frequent cause of cancer-related mortality. 6 Cancer coagulopathy is often linked to up-regulation of tissue factor (TF), the primary cellular initiator of the blood coagulation cascade. 7,8 Interaction of coagulation factor VIIa with TF on the cell surface leads to activation of factor X and generation of thrombin, with subsequent involvement of platelets and formation of a fibrin clot. 9 Remarkably, as a member of the class II cytokine receptor family, TF is also capable of transducing intracellular signals and regulating gene expression. 10,11 Interestingly, elements of the coagulation/fibrinolytic system in general, 12 and TF in particular, have been implicated in regulation of angiogenesis, 13,14 as well as tumor growth 15 and metastasis 16 in various experimental settings. This is consistent with the observed up-regulation of TF in huma...

The elk gene encodes a novel receptorlike protein-tyrosine kinase, which belongs to the eph subfamily. We have previously identified a partial cDNA encompassing the elk catalytic domain (K. Letwin, S.-P. Yee, and T. Pawson, Oncogene 3:621-678, 1988). Using this cDNA as a probe, we have isolated cDNAs spanning the entire rat elk coding sequence. The predicted Elk protein contains all the hallmarks of a receptor tyrosine kinase, including an N-terminal signal sequence, a cysteine-rich extraceliular domain, a membrane-spanning segment, a cytoplasmic tyrosine kinase domain, and a C-terminal tail. In both amino acid sequence and overall structure, Elk is most similar to the Eph and Eck protein-tyrosine kinases, suggesting that the eph, elk, and eck genes encode members of a new subfamily of receptorlike tyrosine kinases. Among rat tissues, elk expression appears restricted to brain and testes, with the brain having higher levels of both elk RNA and protein. Elk protein immunoprecipitated from a rat brain lysate becomes phosphorylated on tyrosine in an in vitro kinase reaction, consistent with the prediction that the mammalian elk gene encodes a tyrosine kinase capable of autophosphorylation. The characteristics of the Elk tyrosine kinase suggest that it may be involved in cell-cell interactions in the nervous system. A frequently used mechanism by which the cells of metazoan organisms communicate involves the binding of growth factors to transmembrane receptors with proteintyrosine kinase activity. In vertebrates, receptor tyrosine kinases and their ligands participate in embryonic development, differentiation of specific cell lineages, biological activities of mature cells, cellular proliferation, and regulation of cell metabolism (17). All receptorlike tyrosine kinases have a similar structural organization, with an extracellular ligand-binding domain, a membrane-spanning segment, and a cytoplasmic enzymatic domain. However, a more detailed comparison allows the division of receptor tyrosine kinases into subfamilies whose members are particularly closely related to each other. Prototypes for these subfamilies include the epidermal growth factor receptor, the a-type platelet-derived growth factor receptor (P-PDGFR), and the insulin receptor (reviewed in references 22 and 27). It seems likely that the members of each subfamily have evolved by a process of gene duplication to accommodate the complex cell-cell interactions of higher organisms. Consistent with this notion, the genes for ,B-PDGFR and the macrophage colony-stimulating factor receptor are located in a tandem array on human chromosome 5 (19), while the a-PDGFR and c-kit genes are similarly linked on chromosome 4 (1).The eph gene encodes a transmembrane protein-tyrosine kinase that differs from previously identified receptors primarily in the structure of its extracellular domain, which contains a single cysteine-rich region (9). The eph enzymatic domain also appears distinct from that of other subclasses of transmembrane tyrosine kinases. The suggestion tha...

Eph, Elk, and Eck are prototypes of a large family of transmembrane protein-tyrosine kinases, which are characterized by a highly conserved cysteine-rich domain and two fibronectin tpe m repeats in their extracellular regions. Despite the extent of the Eph family, no extracellular ligands for any family member have been identified, and hence, little is known about the biological and biochemical properties of these receptor-like tyrosine kinases. In the absence of a physiological ligand for the Elk receptor, we constructed chimeric receptor molecules, in which the extracellular region of the Elk receptor is replaced by the extraceliular, ligand-binding domain of the epidermal growth factor (EGF) receptor. These chimeric receptors were expressed in NIH 3T3 cells that lack endogenous EGF receptors to analyze their signaling properties. The chimeric EGF-Elk receptors became glycosylated, were correctly localized to the plasma membrane, and bound EGF with high affinity. The chimeric receptors underwent autophosphorylation and induced the yrosine phosphorylation of a specific set of cellular proteins in response to EGF. EGF stimulation also induced DNA synthesis in fibroblasts stably expressing the EGF-Elk receptors. In contrast, EGF stimulation of these cells did not lead to visible changes in cellular morphology, nor did it induce loss of contact inhibition in confluent monolayers or growth in semisolid media. The Elk cytoplasmic domain is therefore able to induce tyrosine phosphorylation and DNA synthesis in response to an extracellular ligand, suggesting that Elk and related polypeptides function as ligand-dependent receptor tyrosine kinases.A variety of growth factors, cytokines, and hormones, involved in regulation of diverse aspects of cell behavior such as cellular proliferation, differentiation, survival, and metabolism, bind to receptors with protein-tyrosine kinase activity. In addition, a considerable number of transmembrane, receptor-like tyrosine kinases (RTKs) whose ligands and biological activities remain to be identified (orphan receptors) have been isolated (27,34 primarily expressed in the nervous system (18,25). In addition, their temporal pattern of expression often coincides with the time of maximal proliferation and differentiation of neuronal precursors (8,13,23,25,29), suggesting involvement of these kinases in the development of neural structures. Despite the large number of RTKs in the Eph family, no ligand for any of these receptors has been identified, and this has hindered the experimental analysis of their biological and biochemical properties.A strategy that has been used successfully for the study of receptors without known extracellular ligands involves the construction of chimeric receptor molecules, in which the extracellular domain of an orphan RTK is replaced by the ligand-binding extracellular region of another, well-characterized RTK whose ligand is available. Studies of such hybrid molecules, consisting of heterologous extracellular and cytoplasmic domains, have demonstrated th...

No abstract

Eph, Elk, and Eck are prototypes of a large family of transmembrane protein-tyrosine kinases, which are characterized by a highly conserved cysteine-rich domain and two fibronectin type III repeats in their extracellular regions. Despite the extent of the Eph family, no extracellular ligands for any family member have been identified, and hence, little is known about the biological and biochemical properties of these receptor-like tyrosine kinases. In the absence of a physiological ligand for the Elk receptor, we constructed chimeric receptor molecules, in which the extracellular region of the Elk receptor is replaced by the extracellular, ligand-binding domain of the epidermal growth factor (EGF) receptor. These chimeric receptors were expressed in NIH 3T3 cells that lack endogenous EGF receptors to analyze their signaling properties. The chimeric EGF-Elk receptors became glycosylated, were correctly localized to the plasma membrane, and bound EGF with high affinity. The chimeric receptors underwent autophosphorylation and induced the tyrosine phosphorylation of a specific set of cellular proteins in response to EGF. EGF stimulation also induced DNA synthesis in fibroblasts stably expressing the EGF-Elk receptors. In contrast, EGF stimulation of these cells did not lead to visible changes in cellular morphology, nor did it induce loss of contact inhibition in confluent monolayers or growth in semisolid media. The Elk cytoplasmic domain is therefore able to induce tyrosine phosphorylation and DNA synthesis in response to an extracellular ligand, suggesting that Elk and related polypeptides function as ligand-dependent receptor tyrosine kinases.

The elk gene encodes a novel receptorlike protein-tyrosine kinase, which belongs to the eph subfamily. We have previously identified a partial cDNA encompassing the elk catalytic domain (K. Letwin, S.-P. Yee, and T. Pawson, Oncogene 3:621-678, 1988). Using this cDNA as a probe, we have isolated cDNAs spanning the entire rat elk coding sequence. The predicted Elk protein contains all the hallmarks of a receptor tyrosine kinase, including an N-terminal signal sequence, a cysteine-rich extracellular domain, a membrane-spanning segment, a cytoplasmic tyrosine kinase domain, and a C-terminal tail. In both amino acid sequence and overall structure, Elk is most similar to the Eph and Eck protein-tyrosine kinases, suggesting that the eph, elk, and eck genes encode members of a new subfamily of receptorlike tyrosine kinases. Among rat tissues, elk expression appears restricted to brain and testes, with the brain having higher levels of both elk RNA and protein. Elk protein immunoprecipitated from a rat brain lysate becomes phosphorylated on tyrosine in an in vitro kinase reaction, consistent with the prediction that the mammalian elk gene encodes a tyrosine kinase capable of autophosphorylation. The characteristics of the Elk tyrosine kinase suggest that it may be involved in cell-cell interactions in the nervous system.