No abstract

RON is a member of the c-MET receptor tyrosine kinase family. Like c-MET, RON is expressed by a variety of epithelial-derived tumors and cancer cell lines and it is thought to play a functional role in tumorigenesis. To date, antagonists of RON activity have not been tested in vivo to validate RON as a potential cancer target. In this report, we used an antibody phage display library to generate IMC-41A10, a human immunoglobulin G1 (IgG1) antibody that binds with high affinity (ED 50 = 0.15 nmol/L) to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein (MSP; IC 50 = 2 nmol/L). We found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migration, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, mitogen-activated protein kinase (MAPK), and AKT in several cancer cell lines. In HT-29 colon, NCI-H292 lung, and BXPC-3 pancreatic cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent, and in BXPC-3 xenografts, it led to tumor regressions when combined with Erbitux. Western blot analyses of HT-29 and NCI-H292 xenograft tumors treated with IMC-41A10 revealed a decrease in MAPK phosphorylation compared with control IgG-treated tumors, suggesting that inhibition of MAPK activity may be required for the antitumor activity of IMC-41A10. To our knowledge, this is the first demonstration that a RON antagonist and specifically an inhibitory antibody of RON negatively affects tumorigenesis. Another major contribution of this report is an extensive analysis of RON expression in f100 cancer cell lines and f300 patient tumor samples representing 10 major cancer types. Taken together, our results highlight the potential therapeutic usefulness of RON activity inhibition in human cancers. (Cancer Res 2006; 66(18): 9162-70)

Sphingosine-1-phosphate (S1P) is a potent biomediator that can act as either an intracellular or an intercellular messenger. In the nervous system it exerts a wide range of actions, and specific membrane receptors for it have been identified in various regions. However, the physiological origin of extracellular S1P in the nervous system is largely unknown. We investigated cerebellar granule cells at different stages of differentiation and astrocytes in primary cultures as possible origins of extracellular S1P. Although these cells show marked differences in S1P metabolism, we found that they can all release S1P and express mRNAs for S1P specific receptors. Extracellular S1P derives from the export of newly synthesized intracellular S1P, and not from the action of a released sphingosine kinase. S1P release is rapid, efficient, and can be regulated by exogenous stimuli. Phorbol ester treatment resulted in an increase in sphingosine kinase 1 activity in the membranes, accompanied by a significant increase in extracellular S1P. S1P release in cells from the cerebellum emerges as a regulated mechanism, possibly related to a specific pool of newly synthesized S1P. To our knowledge, this is the first evidence of the extracellular release of S1P by primary cells from the CNS, which supports a role of S1P as autocrine/ paracrine physiological messenger in the cerebellum.

Current studies indicate that ceramide is involved in the regulation of important cell functions, namely cell growth, differentiation, and apoptosis. In the present study, the possible role of ceramide in the differentiation of neuroblastoma Neuro2a cells was investigated.The following results were obtained. (a) Ceramide content of Neuro2a cells, induced to differentiate by retinoic acid (RA) treatment rapidly increased after addition of RA, was maintained at high levels in RAdifferentiated cells and returned to the starting levels with removal of RA and reversal of differentiation; under the same conditions, the sphingosine content remained unchanged. (b) After a short pulse with Increasing evidence indicates important roles for molecules of sphingoid nature in the modulation of cell response to different extracellular signals. These molecules include sphingosine, ceramide, and some derivatives of them, N-methylated forms of sphingosine, sphingosine-1-phosphate, and ceramide-1-phosphate (2, 3). Ceramide (N-acyl-erythro-sphingosine) has been shown to possess bioeffector properties and to act as a key molecule in a new signal transduction pathway, the sphingomyelin pathway or cycle (4 -7). In fact, in several cell lines, especially of the immune system, the activation of certain growth factor receptors by vitamin D3 and cytokines (tumor necrosis factor ␣, interleukin-1␤, and ␥-interferon) induces sphingomyelin hydrolysis by activation of sphingomyelinase, resulting in the elevation of the intracellular levels of ceramide. This, in turn, acts as mediator of the elicited physiological effects, presumably by controlling the activity of specific protein kinases and protein phosphatases. In particular, ceramide has emerged as a candidate for regulatory roles in biological processes that are intimately connected to each other, including cell proliferation, oncogenesis, differentiation, and apoptosis (reviewed in Refs. 4 -10).A process that is based on the regulation of proliferation/ differentiation and differentiation/apoptosis is neural development. Several cell systems (neurons, glial cells, neurotumoral cells) that undergo morphological and functional differentiation in culture are available to study this process in vitro. Some studies suggest that sphingolipids and sphingoid molecules may be involved in the regulation of neural development. In fact, exogenously added glycosphingolipids are capable to affect differentiation of neurons in primary culture and to induce differentiation of neuroblastoma cells in vitro (for a review, see Ref. 11). Moreover, in cultured hippocampal neurons, sphingolipid biosynthesis is necessary for axonal outgrowth (12), and inhibition of sphingolipid biosynthesis and degradation causes opposite effects on axonal branching (13). Furthermore, induced expression of G D3 and/or b-series gangliosides is followed by differentiation of Neuro2a cells (14). Finally, in T9 glioma cells, addition of a cell-permeable ceramide analog (C 2 -ceramide) causes growth inhibition and formation of process...

The vascular endothelial growth factor (VEGF) receptor fetal liver kinase 1 (flk1; VEGFR-2, KDR) is an endothelial cell–specific receptor tyrosine kinase that mediates physiological and pathological angiogenesis. We hypothesized that an active immunotherapy approach targeting flk1 may inhibit tumor angiogenesis and metastasis. To test this hypothesis, we first evaluated whether immune responses to flk1 could be elicited in mice by immunization with dendritic cells pulsed with a soluble flk1 protein (DC-flk1). This immunization generated flk1-specific neutralizing antibody and CD8+ cytotoxic T cell responses, breaking tolerance to self-flk1 antigen. Tumor-induced angiogenesis was suppressed in immunized mice as measured in an alginate bead assay. Development of pulmonary metastases was strongly inhibited in DC-flk1–immunized mice challenged with B16 melanoma or Lewis lung carcinoma cells. DC-flk1 immunization also significantly prolonged the survival of mice challenged with Lewis lung tumors. Thus, an active immunization strategy that targets an angiogenesis-related antigen on endothelium can inhibit angiogenesis and may be a useful approach for treating angiogenesis-related diseases.