Isoquercitrin,ingredients in Tetrastigma hemsleyanum Diels et Gilg,inhibits hepatocyte growth factor/scatter factor-induced tumor cell migration and invasion

ABSTRACT

Aberrant activation of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, is involved in the development and progression of many human cancers. In the screening assay of extracts from the root tuber of Tetrastigma hemsleyanum Diels et Gilg, isoquercitrin inhibited HGF/SF-Met signaling as indicated by its inhibitory activity on HGF/SF-induced cell scattering. Further analysis revealed that isoquercitrin specifically inhibited HGF/SF-induced tyrosine phosphorylation of Met. We also found that isoquercitrin decreased HGF-induced migration and invasion by parental or HGF/SF-transfected bladder carcinoma cell line NBT-II cells. Furthermore, isoquercitrin inhibited HGF/SF-induced epithelial mesenchymal transition in vitro and the invasion/metastasis of HGF/SF-transfected NBT-II cells in vivo. Our data suggest the possible use of isoquercitrin in human cancers associated with dysregulated HGF/SF-Met signaling.     

Epithelio-mesenchymal transition in a neoplastic ovarian epithelial hybrid cell line

A hybrid cell line, IOSE-Ov29, was created through fusion of cells from the human ovarian adenocarcinoma line OVCAR3 and the non-tumorigenic SV40 Tag-transfected human ovarian surface epithelial line IOSE-29. OVCAR3 cells exhibit a differentiated epithelial phenotype, whereas line IOSE-29 expresses mesenchymal characteristics that were acquired in culture by epithelio-mesenchymal transition. Microsatellite analysis, comparative genomic hybridization (CGH), and MFISH showed the genotype of the IOSE-Ov29 cells to contain components of both parent cell lines, but to be predominantly OVCAR3 derived. IOSE-Ov29 resembled OVCAR3 and differed from IOSE-29 as shown by its unlimited life span, tumorigenicity, epithelial morphology, keratin, occludin, E-cadherin and CA125 expression, increased expression of kinases of the PI3K pathway, and loss of cGMP-dependent protein kinase expression. IOSE-29-derived properties included SV40 Tag expression, growth inhibition by activin, collagen type III secretion, increased adhesion and spreading on tissue culture plastic, and increased growth rate. Proliferation of all three lines was stimulated by FSH and ATP and inhibited by GnRH I and GnRH II. Interestingly, IOSE-Ov29 was more anchorage independent than either parent line and was the only line that invaded Matrigel in Boyden chambers and formed invasive branches in collagen gels. The results indicate that IOSE-Ov29 is an IOSE-29/OVCAR3 hybrid, which differs from both parent lines genetically and phenotypically. Unexpectedly, fusion with the non-tumorigenic IOSE-29 cells enhanced malignancy-associated characteristics of OVCAR3, presumably as a result of the expression of IOSE-29-derived mesenchymal properties that are usually acquired by carcinoma cells through epithelio-mesenchymal transition during metastatic progression.     

HGF/MET signaling in ovarian cancer

Ovarian cancer is the leading cause of death from gynecological cancers in North America and Europe. Despite its clinical significance, the factors that regulate the development and progression of ovarian cancer are among the least understood of all major human malignancies. A growth factor with pleiotropic effects, which has attracted increasing attention in recent years, is the hepatocyte growth factor (HGF) and its receptor MET. While deregulated HGF/MET signaling is observed in many tumors, the consequences of MET activation are complex and context dependent. Recent observations have demonstrated a cross-talk of other signaling pathways with MET signaling. This review summarizes the key findings and recent advances in our understanding of HGF and MET in the transformation and progression of ovarian cancer. We will begin with a brief discussion on the role of HGF and MET in the physiology of normal ovarian surface epithelium (OSE) and ovarian cancer development. In particular, the coexpression of HGF and MET in OSE of women with hereditary ovarian cancer syndromes emphasizes their importance in neoplastic transformation of OSE. The involvement of HGF in other aspects of tumor progression, such as invasion and metastasis, and novel downstream target genes activated by HGF is summarized next. The therapeutic potential of HGF to treat ovarian cancer and to improve response to conventional chemotherapy is also described. Finally, the most recent progress in drug development and future areas of research in terms of their potential clinical implications are discussed.     

Osteopontin contributes to hepatocyte growth factor-induced tumor growth and metastasis formation

The cytokine hepatocyte growth factor (HGF)/scatter factor-1 and its cognate receptor, Met, are involved in the etiology and progression of many types of cancer. Despite recent advances in understanding the signal transduction pathways activated by HGF, the mechanism by which HGF exerts its tumorigenic effect is not well understood. To identify proteins that may be involved in mediating HGF-induced cell motility, invasiveness, and tumorigenesis, we used two separate differential display screening methods to identify changes in gene expression that are initiated by HGF in an epithelial cell culture system. Among several known and unknown genes whose expression was modified, osteopontin (OPN), a protein previously associated with tumorigenesis, was found to be upregulated within 6 h following HGF stimulation. OPN expression was dependent on activation of the PI-3 kinase pathway. Autocrine secretion of HGF resulted in sustained expression of OPN. Downregulation of opn expression by stable antisense transfection attenuated OPN expression and repressed HGF-induced invasiveness in vitro and decreased HGF-mediated tumor growth and metastasis formation in vivo. Constitutive expression of OPN in itself exerted partial invasiveness in vitro, but its expression itself was not sufficient to initiate tumor growth or metastasis formation in vivo. Thus, together with other molecules, OPN activity contributes to HGF-induced tumor growth and invasiveness.     

Sphingosine kinase activation regulates hepatocyte growth factor induced migration of endothelial cells

Hepatocyte growth factor (HGF)-induced migration of endothelial cells is critical for angiogenesis. Sphingosine kinase (SPK) is a key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through both intracellular and extracellular mechanisms. The aim of this study was to investigate whether activation of SPK is involved in the migration of endothelial cells induced by HGF. The biological functions of HGF are mediated through the activation of its high-affinity tyrosine kinase receptor, c-met protooncogene. In the present study, Treatment of ECV304 endothelial cells with HGF resulted in tyrosine phosphorylation of c-Met and activation of SPK in a concentration-dependent manner. Either Ly294002 or PD98059, specific inhibitor of the PI3K and ERK/MAPK pathways, respectively, blocked the HGF-induced activation of SPK. HGF stimulation significantly increased intracellular S1P level, but no detectable secretion of S1P into the cell culture medium was observed. Treatment of ECV304 cells with pertussis toxin (PTX) has no effect on the HGF-induced migration, indicating extracellular S1P is dispensable for this process. Overexpression of wild-type SPK gene in ECV 304 cells increased the intracellular S1P and enhanced the HGF-induced migration, whereas inhibition of cellular SPK activity by N,N-dimethylsphingosine (DMS), a potent inhibitor of SPK, or by expression of a dominant-negative SPK (DN-SK) blocked the HGF-induced migration of ECV 304 cells. It is suggested that PI3K and ERK/MAPK mediated the activation of SPK and would be involved in the HGF-induced migration of endothelial cells. These results elucidate a novel mechanism by which intracellularly generated S1P mediates signaling from HGF/c-Met to the endothelial cell migration.     

Met degradation by SAIT301, a Met monoclonal antibody,reduces the invasion and migration of nasopharyngeal cancer cells via inhibition of EGR-1 expression

Nasopharyngeal carcinoma (NPC) is a common malignant tumor with high invasive and metastatic potential. The hepatocyte growth factor (HGF)-Met signaling pathway has a critical role in mediating the invasive growth of many different types of cancer, including head and neck squamous cell carcinoma. HGF also stimulates NPC cell growth and invasion in the cell line model. In this study, we determined the inhibitory effect of Met, using a Met-targeting monoclonal antibody (SAIT301), on the invasive and growth potential of NPC cell lines. Met inhibition by SAIT301 resulted in highly significant inhibition of cell migration and invasion in both the HONE1 and HNE1 cell lines. In addition, we also found that co-treatment of SAIT301 and HGF decreased the anchorage-independent growth induced by HGF in HNE1 cell lines. After SAIT301 treatment, Met, together with its downstream signaling proteins, showed downregulation of p-Met and p-ERK, but not p-AKT, in both HONE1 and HNE1 cell lines. Interestingly, we found that HGF treatment of NPC cell lines induced early growth response protein (EGR-1) expression, which is involved in cell migration and invasion. In addition, co-treatment with SAIT301 and HGF inhibited the HGF-induced expression of EGR-1. Next, knockdown of EGR-1 using small-interfering RNA inhibited HGF-induced cell invasion in NPC cell lines, suggesting that the expression level of EGR-1 is important in HGF-induced cell invasion of NPC cells. Therefore, the results support that SAIT301 inhibited Met activation as well as the downstream EGR-1 expression and could have therapeutic potential in NPC. Taken together, we suggest that Met is an anticancer therapeutic target for NPC that warrants further investigation and clinical trials and SAIT301 may be a promising tool for NPC therapy.     

Hepatocyte growth factor triggers signaling cascades mediating vascular smooth muscle cell migration

A key event in neointima formation and atherogenesis is the migration of vascular smooth muscle cells (VSMCs) into the intima. This is controlled by cytokines and extracellular matix (ECM) components within the microenvironment of the diseased vessel wall. At present, these signals have only been partially identified. In this study, we demonstrate that Met, the receptor tyrosine kinase for hepatocyte growth factor (HGF), is expressed on VSMCs isolated from the intima of atherosclerotic plaques of carotid arteries. Stimulation with HGF led to activation of Met as well as to activation of PI3-K, PKB/Akt, MEK, and the MAP kinases Erk1 and -2. Moreover, HGF induced lamellipodia formation, a characteristic feature of motile cells, and promoted VSMC migration across fibronectin-coated filters. The HGF-induced cell migration was mediated by beta1 integrins and required PI3-K activation. Our results suggest a role for the HGF-Met signaling pathway in the pathogenesis of atherosclerosis and restenosis.     

Hepatocyte growth factor upregulates α2β1 integrin in Madin-Darby canine kidney cells: Implications in tubulogenesis

It has been well established that hepatocyte growth factor (HGF) induces branching tubule formation of Madin-Darby canine kidney (MDCK) cells cultured in collagen gel. Tubulogenesis per se requires the involvement of cell proliferation, migration, focalization proteolysis, cell-cell interaction and differentiation. However, signaling pathways and proteins involved in HGF-induced tubulogenesis by MDCK cells have not been thoroughly studied. Because cell-matrix interactions play important roles in tubulogenesis, we analyzed whether HGF altered the expression of extracellular matrix receptor (alpha2, alpha3, beta1 and alphavbeta3 integrin). We found that among those proteins examined, alpha2beta1 integrin levels were enhanced by HGF. HGF-induced upregulation of alpha2beta1 integrin was mediated via upregulation of alpha2 integrin mRNA abundance. Cycloheximide blocked the HGF-induced increase in alpha2 integrin mRNA expression. To understand the signaling pathways leading to an HGF-induced increase in alpha2beta1 integrin levels, PD98059 (MEK1 inhibitor), LY294002 (PI3-kinase inhibitor), and GF109203X (PKC inhibitor) were used. We found that PD98059 blocked the HGF-induced increase in alpha2beta1 integrin expression. Furthermore, 5E8 (specific anti-alpha2beta1 integrin antibody) was employed to elucidate the potential role of HGF-induced upregulation of alpha2beta1 integrin in branching morphogenesis. 5E8 did not alter HGF-induced scattering effects but disrupted HGF-induced branching tubulogenesis in collagen gel via inhibition of cell-cell interactions and growth. Taken together, HGF upregulates alpha2beta1 integrin expression via an indirect pathway, the results of which contribute to the regulation of cell-cell interactions and cell growth during branching morphogenesis in collagen gel.     

Hepatocyte growth factor and Met in tumor biology and therapeutic approach with NK4

Hepatocyte growth factor (HGF) and Met/HGF receptor tyrosine kinase play a role in the progression to invasive and metastatic cancers. A variety of cancer cells secrete molecules that enhance HGF expression in stromal fibroblasts, while fibroblast-derived HGF, in turn, is a potent stimulator of the invasion of cancer cells. In addition to the ligand-dependent activation, Met receptor activation is negatively regulated by cell-cell contact and Ser985 phosphorylation in the juxtamembrane of Met. The loss of intercellular junctions may facilitate an escape from the cell-cell contact-dependent suppression of Met-signaling. Significance of juxtamembrane mutations found in human cancers is assumed to be a loss-of-function in the negative regulation of Met. In attempts to block the malignant behavior of cancers, NK4 was isolated as a competitive antagonist against HGF-Met signaling. Independently on its HGF-antagonist action, NK4 inhibited angiogenesis induced by vascular endothelial cell growth factor and basic fibroblast growth factor, as well as HGF. In experimental models of distinct types of cancers, NK4 inhibited Met activation and this was associated with inhibition of tumor invasion and metastasis. NK4 inhibited tumor angiogenesis, thereby suppressing angiogenesis-dependent tumor growth. Cancer treatment with NK4 suppresses malignant tumors to be "static" in both tumor growth and spreading.     

Low Dose Geldanamycin Inhibits Hepatocyte Growth Factor- and Hypoxia-Stimulated Invasion of Cancer Cells

Hepatocyte growth factor (HGF) receptor Met and hypoxia-inducible factor-1 (HIF-1) signaling pathways are commonly activated in aggressive tumors and promote progression. Since both Met and HIF-1α proteins are heat shock protein (Hsp) 90 clients, Hsp90 inhibitors might be expected to positively impact tumor progression. Here, we systematically evaluated the inhibitory effects of the prototypical Hsp90 inhibitor geldanamycin (GA) on cellular processes involved in invasion and angiogenesis in T24 bladder cancer cells stimulated with HGF and chemical hypoxia. First, we demonstrated the positive feedback loop between Met and HIF-1 pathways, which serves to sustain and amplifies their signaling in T24 cells. GA down-regulated Met by inhibiting new protein maturation, thereby dampening HGF signaling. HGF and chemical hypoxia with CoCl2 cooperatively promoted in vitro invasion and vascular endothelial growth factor (VEGF) secretion, while CoCl2 but not HGF activated urokinase-type plasminogen activator and matrix metalloproteinase 2, both of which promote invasion and angiogenesis. Low dose GA (100 nmol/L) inhibited these processes by suppressing both HGF and HIF-1 pathways. Notably, brief GA pretreatment inhibited in vitro invasion and VEGF secretion induced by HGF as effectively as did continuous treatment. Moreover, we found that GA inhibited activation of focal adhesion kinase, focal adhesion assembly, and actin reorganization induced by HGF and integrin engagement by extracellular matrix. Thus, GA widely suppresses extrinsic stimuli-induced signaling that contribute to tumor invasion and angiogenesis in this bladder carcinoma model, suggesting the utility of Hsp90 inhibitors in preventing tumor progression and metastasis.     

The tetraspanin CD151 is required for Met-dependent signaling and tumor cell growth

CD151, a transmembrane protein of the tetraspanin family, is implicated in the regulation of cell-substrate adhesion and cell migration through physical and functional interactions with integrin receptors. In contrast, little is known about the potential role of CD151 in controlling cell proliferation and survival. We have previously shown that β4 integrin, a major CD151 partner, not only acts as an adhesive receptor for laminins but also as an intracellular signaling platform promoting cell proliferation and invasive growth upon interaction with Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF). Here we show that RNAi-mediated silencing of CD151 expression in cancer cells impairs HGF-driven proliferation, anchorage-independent growth, protection from anoikis, and tumor progression in xenograft models in vivo. Mechanistically, we found that CD151 is crucially implicated in the formation of signaling complexes between Met and β4 integrin, a known amplifier of HGF-induced tumor cell growth and survival. CD151 depletion hampered HGF-induced phosphorylation of β4 integrin and the ensuing Grb2-Gab1 association, a signaling pathway leading to MAPK stimulation and cell growth. Accordingly, CD151 knockdown reduced HGF-triggered activation of MAPK but not AKT signaling cascade. These results indicate that CD151 controls Met-dependent neoplastic growth by enhancing receptor signaling through β4 integrin-mediated pathways, independent of cell-substrate adhesion.     

BCL-2-induced glioma cell invasiveness depends on furin-like proteases

Migration and invasion are prerequisites for the neoplastic phenotype of malignant glioma. Ectopic expression of BCL-2 enhances migration and invasion of glioma cells and promotes their synthesis of transforming growth factor-beta2 (TGF-beta2). We here report that BCL-2-expressing cells show enhanced expression and activity of the proprotein convertase, furin, which processes metalloproteinases (MMP) and TGF-beta. Consistent with a biological role for a BCL-2-dependent increase in furin-like protease (FLP) activity, BCL-2-expressing cells exhibit enhanced MMP activity. Both a pseudosubstrate furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk), or alpha 1-anti-trypsin Portland (PDX), a recombinant furin-inhibitory protein, suppress constitutive and BCL-2-mediated MMP activity and invasion. This inhibition is not overcome by TGF-beta or hepatocyte growth factor (HGF). A neutralizing TGF-beta antibody attenuates, but not abrogates, the invasive properties conferred by exogenous expression of BCL-2, whereas the MMP inhibitor o-phenantroline (o-PA) abolishes the pro-invasive action of BCL-2. Exogenous HGF results in enhanced, and expression of dominant-negative ezrin in reduced, FLP activity, and dec-RVKR-cmk blunts the HGF-induced expression of mature TGF-beta2. Consequently, HGF and BCL-2 family proteins use a furin-dependent pathway to promote invasion via TGF-beta and MMP in human malignant glioma cells and the pro-invasive properties of TGF-beta require furin- dependent MMP activity.     

Green tea (-)-epigallocatechin-3-gallate inhibits HGF-induced progression in oral cavity cancer through suppression of HGF/c-Met

Hepatocyte growth factor (HGF) and c-Met have recently attracted a great deal of attention as prognostic indicators of patient outcome, and they are important in the control of tumor growth and invasion. Epigallocatechin-3-gallate (EGCG) has been shown to modulate multiple signal pathways in a manner that controls the unwanted proliferation and invasion of cells, thereby imparting cancer chemopreventive and therapeutic effects. In this study, we investigated the effects of EGCG in inhibiting HGF-induced tumor growth and invasion of oral cancer in vitro and in vivo. We examined the effects of EGCG on HGF-induced cell proliferation, migration, invasion, induction of apoptosis and modulation of HGF/c-Met signaling pathway in the KB oral cancer cell line. We investigated the antitumor effect and inhibition of c-Met expression by EGCG in a syngeneic mouse model (C3H/HeJ mice, SCC VII/SF cell line). HGF promoted cell proliferation, migration, invasion and induction of MMP (matrix metalloproteinase)-2 and MMP-9 in KB cells. EGCG significantly inhibited HGF-induced phosphorylation of Met and cell growth, invasion and expression of MMP-2 and MMP-9. EGCG blocked HGF-induced phosphorylation of c-Met and that of the downstream kinases AKT and ERK, and inhibition of p-AKT and p-ERK by EGCG was associated with marked increases in the phosphorylation of p38, JNK, cleaved caspase-3 and poly-ADP-ribose polymerase. In C3H/HeJ syngeneic mice, as an in vivo model, tumor growth was suppressed and apoptosis was increased by EGCG. Our results suggest that EGCG may be a potential therapeutic agent to inhibit HGF-induced tumor growth and invasion in oral cancer.     

Cabozantinib inhibits AXL- and MET-dependent cancer cell migration induced by growth-arrest-specific 6 and hepatocyte growth factor

Cabozantinib is known as an inhibitor of receptor tyrosine kinases mainly targeting AXL receptor tyrosine kinase (AXL), MET proto-oncogene-encoded receptor tyrosine kinase (MET), and vascular endothelial growth factor receptor 2. Growth arrest-specific 6 (GAS6) and hepatocyte growth factor (HGF), the natural ligands of AXL and MET, respectively, are associated with the induction of cancer cell proliferation or metastasis. Currently, it is still unclear how cabozantinib regulates cancer cell migration and invasion by inhibiting AXL and MET. This study was conducted to investigate the mechanism underlying the anti-cancer effects of cabozantinib through regulation of AXL and MET signaling.The results of Boyden chamber assays showed that cancer cell migration was induced by GAS6 and HGF in SKOV3 cells in serum-free medium. Combinatorial treatment with GAS6 and HGF exerted an additive effect on cell migration. Furthermore, we examined the role of AXL and MET signaling in cell migration. Short interfering RNA targeting AXL and MET inhibited GAS6- and HGF-induced migration, respectively. Double knockdown of AXL and MET completely suppressed cell migration induced by combination treatment with GAS6 and HGF compared to AXL or MET inhibition alone. Finally, we investigated the effects of cabozantinib on cell migration and invasion. Cabozantinib inhibited AXL and MET phosphorylation and downregulated the downstream mediators, phosphorylated SRC in the presence of both GAS6 and HGF in SKOV3 cells. The cell migration and invasion induced by combined GAS6 and HGF treatment were suppressed by cabozantinib, but not by capmatinib, a selective MET inhibitor.Our data indicate that the GAS6-AXL and HGF-MET signal pathways markedly contribute to cancer cell migration and invasion in an independent manner, suggesting that simultaneous inhibition of these two pathways contributes to the anti-cancer effects of cabozantinib.     

Role of HGF/c‐Met in the treatment of colorectal cancer with liver metastasis

The system of hepatocyte growth factor (HGF) and its receptor c‐Met plays a critical role in tumor invasive growth and metastasis. The mortality rate of colorectal cancer (CRC), one of the most commonly diagnosed malignancies, is increased by it gradual development into metastasis, most frequently in the liver. Overexpression of c‐Met, the protein tyrosine kinase receptor for the HCF/scatter factor, has been implicated in the progression and metastasis of human colorectal carcinoma. In this study, we aimed to investigate the role of c‐Met in CRC liver metastasis and illustrate the clinical impact of regulating HGF/c‐Met signaling in patients with CRC liver metastasis. We found that (I) higher levels of c‐Met expression (mRNA and Protein) in CRC liver metastasis than primary CRC by assessing the patient tissue samples; (II) a positive correlation of c‐Met expression with tumor stages of CRC liver metastasis, as well as c‐Met expression in CRC, live metastasis concurred with regional lymph node metastasis; (III) the clinical impact of downregulation of HGF/c‐Met signaling on the reduction of proliferation and invasion in CRC liver metastasis. Therefore, we demonstrate that the regulation of HGF/c‐Met pathways may be a promising strategy in the treatment of patients with CRC liver metastasis.     

Interleukin 4 inhibits hepatocyte growth factor-induced invasion and migration of colon carcinomas

Hepatocyte growth factor (HGF) is known to have a number of biological properties including promoting tumor progression of human carcinomas. Metastasis involves a number of events that are attributed to induction by paracrine factors such as HGF. Identification of natural inhibitors of these events would allow better control of tumor progression. Recently we demonstrated that interleukin 4 (IL-4) can regulate proliferation of various human carcinoma cell lines. In the present study, we used established human colon carcinoma cell lines and primary colon carcinoma cell cultures to determine if IL-4 could regulate HGF-induced cell proliferation and other events of tumor progression such as MMP (matrix metalloproteinases)-1, -2, and -9 production, cell migration and cell-matrix invasive activity. All colon carcinoma cell lines expressed HGF and IL-4 receptors. IL-4 significantly inhibited HGF-induced proliferation of one cell line. Cell-matrix invasion was significantly enhanced by HGF (0.1–10 ng/ml); IL-4 (1–10 U/ml) significantly inhibited HGF-induced invasion in a dose-dependent manner. IL-4 also inhibited HGF-induced cell-matrix invasion of metastatic colon carcinoma cells and HGF-induced cell migration. HGF enhanced MMP-1, -2, and -9 production by cell lines. This effect could be inhibited by IL-4. These findings indicate that IL-4 is a potent inhibitor of HGF-induced invasion and metastasis-related functions of human colon carcinoma cells. © 1996 Wiley-Liss, Inc.     

c-Cbl is involved in Met signaling in B cells and mediates hepatocyte growth factor-induced receptor ubiquitination

Hepatocyte growth factor/scatter factor (HGF) and its receptor tyrosine kinase Met are key regulators of epithelial motility and morphogenesis. Recent studies indicate that the HGF/Met pathway also plays a role in B cell differentiation, whereas uncontrolled Met signaling may lead to B cell neoplasia. These observations prompted us to explore HGF/Met signaling in B cells. In this study, we demonstrate that HGF induces strong tyrosine phosphorylation of the proto-oncogene product c-Cbl in B cells and increases Cbl association with the Src family tyrosine kinases Fyn and Lyn, as well as with phosphatidylinositol-3 kinase and CrkL. In addition, we demonstrate that c-Cbl mediates HGF-induced ubiquitination of Met. This requires the juxtamembrane tyrosine Y1001 (Y2) of Met, but not the multifunctional docking site (Y14/15) or any additional C-terminal tyrosine residues (Y13-16). In contrast to wild-type c-Cbl, the transforming mutants v-Cbl and 70Z/3 Cbl, which lack the ubiquitin ligase RING finger domain, suppress Met ubiquitination. Our findings identify c-Cbl as a negative regulator of HGF/Met signaling in B cells, mediating ubiquitination and, consequently, proteosomal degradation of Met, and suggest a role for Cbl in Met-mediated tumorigenesis.