Potent blockade of hepatocyte growth factor-stimulated cell motility, matrix invasion and branching morphogenesis by antagonists of Grb2 Src homology 2 domain interactions

Hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of cellular targets during development, homeostasis and tissue regeneration. Inappropriate HGF signaling occurs in several human cancers, and the ability of HGF to initiate a program of protease production, cell dissociation, and motility has been shown to promote cellular invasion and is strongly linked to tumor metastasis. Upon HGF binding, several tyrosines within the intracellular domain of its receptor, c-Met, become phosphorylated and mediate the binding of effector proteins, such as Grb2. Grb2 binding through its SH2 domain is thought to link c-Met with downstream mediators of cell proliferation, shape change, and motility. We analyzed the effects of Grb2 SH2 domain antagonists on HGF signaling and observed potent blockade of cell motility, matrix invasion, and branching morphogenesis, with ED(50) values of 30 nm or less, but only modest inhibition of mitogenesis. These compounds are 1000-10,000-fold more potent anti-motility agents than any previously characterized Grb2 SH2 domain antagonists. Our results suggest that SH2 domain-mediated c-Met-Grb2 interaction contributes primarily to the motogenic and morphogenic responses to HGF, and that these compounds may have therapeutic application as anti-metastatic agents for tumors where the HGF signaling pathway is active.     

The Met receptor tyrosine kinase transduces motility, proliferation, and morphogenic signals of scatter factor/hepatocyte growth factor in epithelial cells

Depending on the target cells and culture conditions, scatter factor/hepatocyte growth factor (SF/HGF) mediates several distinct activities, i.e., cell motility, proliferation, invasiveness, tubular morphogenesis, angiogenesis, or cytotoxicity. A small isoform of SF/HGF encoded by a natural splice variant, which consists of the NH2-terminal hairpin structure and the first two kringle domains but not the protease homology region, induces cell motility but not mitogenesis. Two types of SF/HGF receptors have recently been discovered in epithelial cells, the high affinity c-Met receptor tyrosine kinase, and low affinity/high capacity binding sites, which are probably located on heparan sulfate proteoglycans. In the present study, we have addressed the question whether the various biological activities of SF/HGF are transduced into cells by a single type of receptor. We have here examined MDCK epithelial cells transfected with a hybrid cDNA encoding the ligand binding domain of the nerve growth factor (NGF) receptor and the membrane-spanning and tyrosine kinase domains of the Met receptor. We demonstrate that all biological effects of SF/HGF upon epithelial cells such as the induction of cell motility, proliferation, invasiveness, and tubular morphogenesis can now be triggered by the addition of NGF. Thus, it is likely that all known biological signals of SF/HGF are transduced through the receptor tyrosine kinase encoded by the c-Met protooncogene.     

Differential mitogenic effects of single chain hepatocyte growth factor (HGF)/scatter factor and HGF/NK1 following cleavage by factor Xa

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that is involved in many normal as well as pathological conditions. HGF/NK1, a splice variant of HGF/SF, has been reported to have either antagonistic or agonistic effects with regard to c-Met signaling depending on the cell type. In these experiments, we have determined that HGF/NK1 is a potent mitogen for rat hepatocytes in culture. Furthermore, we have found that coagulation factor Xa (fXa) is capable of cleaving HGF/NK1 and single chain HGF/SF (scHGF/SF). The products resulting from cleavage of HGF/NK1 or scHGF/SF by fXa appear as single bands under non-reducing conditions. The reaction products from the digestion of HGF/NK1 by fXa were separated under reducing conditions, and the cleavage site, as determined by N-terminal sequencing, was located C-terminal to arginine 134. Previous work established that the heparin-binding domain for HGF/SF is located in the N domain of HGF/SF. Additionally, the dimerization of the HGF/SF receptor (c-Met) by the ligand HGF/NK1 is facilitated by heparin and related sulfonated sugars on the cell surface, whereas heparin is not required for HGF/SF-mediated dimerization. Cleavage of single chain HGF/SF or HGF/NK1 by factor Xa does not alter the affinity of the respective molecules for heparin, but it did variably affect the associated mitogenic activity of these factors. The associated mitogenic activity of HGF/NK1 was reduced by more than 90%, whereas the mitogenic activity of scHGF/SF was unaffected. This suggests mandatory maintenance of a steric interaction of the N domain and the first kringle domain for HGF/NK1 to act as an agonist for rat hepatocyte growth but is not required by full-length HGF/SF.     

Heparin induces dimerization and confers proliferative activity onto the hepatocyte growth factor antagonists NK1 and NK2

Hepatocyte growth factor (HGF) is a potent epithelial mitogen whose actions are mediated through its receptor, the proto-oncogene c-Met. Two truncated variants of HGF known as NK1 and NK2 have been reported to be competitive inhibitors of HGF binding to c-Met, and to function as HGF antagonists (Lokker, N.A., and P.J. Godowski. 1993. J. Biol. Chem. 268: 17145-17150; Chan, A.M., J.S. Rubin, D.P. Bottaro, D.W. Hirschfield, M. Chedid, and S.A. Aaronson. 1991. Science (Wash. DC). 254:1382-1387). We show here, however, that NK1 acts as a partial agonist in mink lung cells. Interestingly, NK1, which is an HGF antagonist in hepatocytes in normal conditions, was converted to a partial agonist by adding heparin to the culture medium. The interaction of NK1 and heparin was further studied in BaF3 cells, which express little or no cell surface heparan sulfate proteoglycans. In BaF3 cells transfected with a plasmid encoding human c-Met, heparin and NK1 synergized to stimulate DNA synthesis and cell proliferation. There was no effect of heparin on the IL-3 sensitivity of BaF3-hMet cells, and no effect of NK1 plus heparin in control BaF3 cells, indicating that the response was specific and mediated through c-Met. The naturally occurring HGF splice variant NK2 also stimulated DNA synthesis in mink lung cells and exerted a heparin-dependent effect on BaF3-hMet cells, but not on BaF3-neo cells. The activating effect of heparin was mimicked by a variety of sulfated glycosaminoglycans. Mechanistic studies revealed that heparin increased the binding of NK1 to BaF3-hMet cells, stabilized NK1, and induced dimerization of NK1. Based on these studies, we propose that the normal agonist activity of NK1 and NK2 in mink lung cells is due to an activating interaction with an endogenous glycosaminoglycan. Consistent with that model, a large portion of the NK1 binding to mink lung cells could be blocked by heparin. Moreover, a preparation of glycosaminoglycans from the surface of mink lung cells induced dimerization of NK1. These data show that the activity of NK1 and NK2 can be modulated by heparin and other related glycosaminoglycans to induce proliferation in cells expressing c-Met.     

Src homology 2-containing inositol 5-phosphatase 1 binds to the multifunctional docking site of c-Met and potentiates hepatocyte growth factor-induced branching tubulogenesis

Hepatocyte growth factor (HGF)/scatter factor is a multifunctional cytokine that induces mitogenesis, motility, and morphogenesis in epithelial, endothelial, and neuronal cells. The receptor for HGF/scatter factor was identified as c-Met tyrosine kinase, and activation of the receptor induces multiple signaling cascades. To gain further insight into c-Met-mediated multiple events at a molecular level, we isolated several signaling molecules including a novel binding partner of c-Met, SH2 domain-containing inositol 5-phosphatase 1 (SHIP-1). Western blot analysis revealed that SHIP-1 is expressed in the epithelial cell line, Madin-Darby canine kidney (MDCK) cells. SHIP-1 binds at phosphotyrosine 1356 at the multifunctional docking site. Because a number of signaling molecules such as Grb2, phosphatidylinositol 3-kinase, and Gab1 bind to the multifunctional docking site, we further performed an in vitro competition study using glutathione S-transferase- or His-tagged signaling molecules with c-Met tyrosine kinase. Our binding study revealed that SHIP-1, Grb2, and Gab1 bound preferentially over phosphatidylinositol 3-kinase. Surprisingly, MDCK cells that overexpress SHIP-1 demonstrated branching tubulogenesis within 2 days after HGF treatment, whereas wild-type MDCK cells showed tubulogenesis only after 6 days following treatment without altering cell scattering or cell growth potency. Furthermore, overexpression of a mutant SHIP-1 lacking catalytic activity impaired HGF-mediated branching tubulogenesis.     

Identification and dynamics of a heparin-binding site in hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a heparin-binding, multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. Heparin or closely related heparan sulfate has profound effects on HGF signaling. A heparin-binding site in the N-terminal (N) domain of HGF was proposed on the basis of the clustering of surface positive charges [Zhou, H., Mazzulla, M. J., Kaufman, J. D., Stahl, S. J., Wingfield, P. T., Rubin, J. S., Bottaro, D. P., and Byrd, R. A. (1998) Structure 6, 109-116]. In the present study, we confirmed this binding site in a heparin titration experiment monitored by nuclear magnetic resonance spectroscopy, and we estimated the apparent dissociation constant (K(d)) of the heparin-protein complex by NMR and fluorescence techniques. The primary heparin-binding site is composed of Lys60, Lys62, and Arg73, with additional contributions from the adjacent Arg76, Lys78, and N-terminal basic residues. The K(d) of binding is in the micromolar range. A heparin disaccharide analogue, sucrose octasulfate, binds with similar affinity to the N domain and to a naturally occurring HGF isoform, NK1, at nearly the same region as in heparin binding. (15)N relaxation data indicate structural flexibility on a microsecond-to-millisecond time scale around the primary binding site in the N domain. This flexibility appears to be dramatically reduced by ligand binding. On the basis of the NK1 crystal structure, we propose a model in which heparin binds to the two primary binding sites and the N-terminal regions of the N domains and stabilizes an NK1 dimer.     

Hepatocyte growth factor-induced differential activation of phospholipase cgamma 1 and phosphatidylinositol 3-kinase is regulated by tyrosine phosphatase SHP-1 in astrocytes

Hepatocyte growth factor (HGF) elicits pleiotropic effects on various types of cells through the c-Met receptor tyrosine kinase. However, the mechanisms underlying the diverse responses of cells remain unknown. We show here that HGF promoted chemokinesis of rat primary astrocytes through the activation of phosphatidylinositol 3 (PI3)-kinase without any influence on mitogenesis of the cells. Under the same condition, phospholipase Cgamma1 (PLCgamma1), which is another signal mediator of c-Met, was not tyrosine-phosphorylated during HGF stimulation. However, treatment of the cells with orthovanadate, a tyrosine phosphatase inhibitor, restored the HGF-induced tyrosine phosphorylation of PLCgamma1. A tyrosine phosphatase, SHP-1, was associated with both PI3-kinase and PLCgamma1 before HGF stimulation, but it was dissociated only from PI3-kinase after the stimulation. Furthermore, transfectants of catalytically inactive mutant of SHP-1 showed tyrosine phosphorylation of PLCgamma1 and mitogenic responses to HGF, and the mitogenic response was blocked with, an inhibitor of phosphatidylinositol-specific PLC, and calphostin C, an inhibitor of protein kinase C downstream of the PLCgamma1. These results indicate that PLCgamma1 is selectively prevented from being a signal mediator by constitutive association of SHP-1, and that this selective inhibition of PLCgamma1 may determine the cellular response of astrocytes to HGF.     

Hepatocyte growth factor is a pleiotropic factor protecting epithelial cells from apoptosis

Hepatocyte Growth Factor (HGF) is a pleiotropic factor, capable of evoking complex biological responses such as mitogenesis, motogenesis and morphogenesis in a variety of epithelial and endothelial cells. Nonetheless, the meaning of the acronym is consistent with the key role of the factor in liver regeneration, in vivo and in liver development during embryogenesis. The receptor for HGF is the tyrosine kinase encoded by the c-MET proto-oncogene. Upon ligand binding, the receptor kinase is activated by tyrosine autophosphorylation and recruits cytoplasmic transducers involved in HGF-triggered signal transduction. We investigated the role of HGF as a survival factor in protecting cells from apoptosis and we show that HGF is able to counteract staurosporin-induced apoptosis of epithelial cells.     

Activated c-Met signals through PI3K with dramatic effects on cytoskeletal functions in small cell lung cancer

Small cell lung cancer (SCLC) is an aggressive illness with early metastases. There are several receptor tyrosine kinases (RTKs) overexpressed in SCLC, including c-Met. c-Met contains an external semaphorin-like domain, a cytoplasmic juxtamembrane domain, tyrosine kinase domain and multiple tyrosines that bind to adapter molecules. We have previously reported that c-Met is abundantly expressed in the NCI-H69 SCLC cell line and now have determined the downstream effects of stimulating c-Met via its ligand hepatocyte growth factor (HGF). Utilizing unique phospho-specific antibodies generated against various tyrosines of c-Met, we show that Y1003 (binding site for c-Cb1 and a negative regulatory site), Y1313 (binding site for PI3K), Y1230/Y1234/Y1235 (autophosphorylation site), Y1349 (binding site for Grb2), Y1365 (important in cell morphogenesis) are phosphorylated in response to HGF (40 ng/ml, 7.5 min) in H69 cells. Since multiple biological and biochemical effects are transduced through the PI3K pathway, we determine the role of PI3K in the c-Met/HGF stimulation pathway. We initially determined that by inhibiting PI3K with LY294002 (50μM over 72 hours), there was at least a 55% decrease in viability of H69 cells. Since H69 SCLC cells form clusters in cell culture, we determined the effects of HGF and LY294002 on cell motility of the clusters by time-lapse video microscopy. In response to HGF, SCLC moved much faster and formed more clusters, and this was inhibited by LY294002. Finally, we determined the downstream signal transduction of HGF stimulation of c-Met with and without inhibition of c-Met (with geldanamycin, an anisamycin antibiotic that inhibits c-Met in SCLC) or PI3K (with LY294002). We show that association of c-Met with PI3K and GAB2 is diminished by inhibiting c-Met. In summary, activation of the c-Met pathway targets the PI3K pathway in SCLC and this may be an important therapeutic target.     

Insights into the structure/function of hepatocyte growth factor/scatter factor from studies with individual domains

Hepatocyte growth factor/scatter factor (HGF/SF), the ligand for the receptor tyrosine kinase encoded by the c-Met proto-oncogene, is a multidomain protein structurally related to the pro-enzyme plasminogen and with major roles in development, tissue regeneration and cancer. We have expressed the N-terminal (N) domain, the four kringle domains (K1 to K4) and the serine proteinase homology domain (SP) of HGF/SF individually in yeast or mammalian cells and studied their ability to: (i) bind the Met receptor as well as heparan sulphate and dermatan sulphate co-receptors, (ii) activate Met in target cells and, (iii) map their binding sites onto the beta-propeller domain of Met. The N, K1 and SP domains bound Met directly with comparable affinities (K(d)=2.4, 3.3 and 1.4 microM). The same domains also bound heparin with decreasing affinities (N>K1>SP) but only the N domain bound dermatan sulphate. Three kringle domains (K1, K2 and K4) displayed agonistic activity on target cells. In contrast, the N and SP domains, although capable of Met binding, displayed no or little activity. Further, cross-linking experiments demonstrated that both the N domain and kringles 1-2 bind the beta-chain moiety (amino acid residues 308-514) of the Met beta-propeller. In summary, the K1, K2 and K4 domains of HGF/SF are sufficient for Met activation, whereas the N and SP domains are not, although the latter domains contribute additional binding sites necessary for receptor activation by full length HGF/SF. The results provide new insights into the structure/function of HGF/SF and a basis for engineering the N and K1 domains as receptor antagonists for cancer therapy.     

Biochemical and pharmacological characterization of human c-Met neutralizing monoclonal antibody CE-355621

The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase that is stimulated by its ligand, hepatocyte growth factor (HGF), to induce cell growth, motility and morphogenesis. Dysregulation of c-Met function, through mutational activation or overexpression, has been observed in many types of cancer and is thought to contribute to tumor growth and metastasis by affecting mitogenesis, invasion, and angiogenesis. We identified human monoclonal antibodies that bind to the extracellular domain of c-Met and inhibit tumor growth by interfering with ligand-dependent c-Met activation. We identified antibodies representing four independent epitope classes that inhibited both ligand binding and ligand-dependent activation of c-Met in A549 cells. In cells, the antibodies antagonized c-Met function by blocking receptor activation and by subsequently inducing downregulation of the receptor, translating to phenotypic effects in soft agar growth and tubular morphogenesis assays. Further characterization of the antibodies in vivo revealed significant inhibition of c-Met activity (≥ 80% lasting for 72–96 h) in excised tumors corresponded to tumor growth inhibition in multiple xenograft tumor models. Several of the antibodies identified inhibited the growth of tumors engineered to overexpress human HGF and human c-Met (S114 NIH 3T3) when grown subcutaneously in athymic mice. Furthermore, lead candidate antibody CE-355621 inhibited the growth of U87MG human glioblastoma and GTL-16 gastric xenografts by up to 98%. The findings support published pre-clinical and clinical data indicating that targeting c-Met with human monoclonal antibodies is a promising therapeutic approach for the treatment of cancer.     

Adaptor molecule Crk is required for sustained phosphorylation of Grb2-associated binder 1 and hepatocyte growth factor-induced cell motility of human synovial sarcoma cell lines

Activation of the c-Met receptor tyrosine kinase through its ligand, hepatocyte growth factor (HGF), promotes mitogenic, motogenic, and morphogenic cellular responses. Aberrant HGF/c-Met signaling has been strongly implicated in tumor cell invasion and metastasis. Both HGF and its receptor c-Met have been shown to be overexpressed in human synovial sarcoma, which often metastasizes to the lung; however, little is known about HGF-mediated biological effects in this sarcoma. Here, we provide evidence that Crk adaptor protein is required for the sustained phosphorylation of c-Met-docking protein Grb2-associated binder 1 (Gab1) in response to HGF, leading to the enhanced cell motility of human synovial sarcoma cell lines SYO-1, HS-SY-II, and Fuji. HGF stimulation induced the sustained phosphorylation on Y307 of Gab1 where Crk was recruited. Crk knockdown by RNA interference disturbed this HGF-induced tyrosine phosphorylation of Gab1. By mutational analysis, we identified that Src homology 2 domain of Crk is indispensable for the induction of the phosphorylation on multiple Tyr-X-X-Pro motifs containing Y307 in Gab1. HGF remarkably stimulated cell motility and scattering of synovial sarcoma cell lines, consistent with the prominent activation of Rac1, extreme filopodia formation, and membrane ruffling. Importantly, the elimination of Crk in these cells induced the disorganization of actin cytoskeleton and complete abolishment of HGF-mediated Rac1 activation and cell motility. Time-lapse microscopic analysis revealed the significant attenuation in scattering of Crk knockdown cells following HGF treatment. Furthermore, the depletion of Crk remarkably inhibited the tumor formation and its invasive growth in vivo. These results suggest that the sustained phosphorylation of Gab1 through Crk in response to HGF contributes to the prominent activation of Rac1 leading to enhanced cell motility, scattering, and cell invasion, which may support the crucial role of Crk in the aggressiveness of human synovial sarcoma.     

Biochemical and pharmacological characterization of human c-Met neutralizing monoclonal antibody CE-355621

The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase that is stimulated by its ligand, hepatocyte growth factor (HGF), to induce cell growth, motility and morphogenesis. Dysregulation of c-Met function, through mutational activation or overexpression, has been observed in many types of cancer and is thought to contribute to tumor growth and metastasis by affecting mitogenesis, invasion, and angiogenesis. We identified human monoclonal antibodies that bind to the extracellular domain of c-Met and inhibit tumor growth by interfering with ligand-dependent c-Met activation. We identified antibodies representing four independent epitope classes that inhibited both ligand binding and ligand-dependent activation of c-Met in A549 cells. In cells, the antibodies antagonized c-Met function by blocking receptor activation and by subsequently inducing downregulation of the receptor, translating to phenotypic effects in soft agar growth and tubular morphogenesis assays. Further characterization of the antibodies in vivo revealed significant inhibition of c-Met activity (≥ 80% lasting for 72–96 h) in excised tumors corresponded to tumor growth inhibition in multiple xenograft tumor models. Several of the antibodies identified inhibited the growth of tumors engineered to overexpress human HGF and human c-Met (S114 NIH 3T3) when grown subcutaneously in athymic mice. Furthermore, lead candidate antibody CE-355621 inhibited the growth of U87MG human glioblastoma and GTL-16 gastric xenografts by up to 98%. The findings support published pre-clinical and clinical data indicating that targeting c-Met with human monoclonal antibodies is a promising therapeutic approach for the treatment of cancer.     

Mechanisms and significance of bifunctional NK4 in cancer treatment

Based on the background that hepatocyte growth factor (HGF) and c-Met/HGF receptor tyrosine kinase play a definite role in tumor invasion and metastasis, NK4, four-kringles containing intramolecular fragment of HGF, was isolated as a competitive antagonist for the HGF-c-Met system. Independent of its HGF-antagonist action, NK4 inhibited angiogenesis induced by vascular endothelial cell growth factor and basic fibroblast growth factor, as well as HGF, indicating that NK4 is a bifunctional molecule that acts as an HGF-antagonist and angiogenesis inhibitor. Interestingly, kringle domains in distinct types of proteins, e.g., plasminogen, prothrombin, plasminogen activators, apolipoprotein(a), and HGF, share angioinhibitory actions. In experimental models of distinct types of cancers, NK4 protein administration or NK4 gene therapy inhibited tumor invasion, metastasis, and angiogenesis-dependent tumor growth. Cancer treatment with NK4 may prove to suppress malignant tumors to be 'static' in both tumor growth and spreading, as based on biological characteristics of malignant tumors.     

Matriptase is required for the active form of hepatocyte growth factor induced Met,focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility

Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.     

Engineering the NK1 fragment of hepatocyte growth factor/scatter factor as a MET receptor antagonist

The growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor MET, the tyrosine kinase encoded by the c-MET proto-oncogene, exert major roles in cancer invasion and metastasis and are key targets for therapy. NK1 is an alternative spliced variant of HGF/SF that consists of the N-terminal (N) and first kringle (K1) domains and has partial agonistic activity. NK1 crystallises as a head-to-tail dimer with an extensive inter-protomeric interface resulting from contacts between the two short interdomain linkers and reciprocal contacts between the N and K1 domains. Here we show that a subset of mutants at the NK1 dimer interface, such as the linker mutants Y124A or N127A or the kringle mutant V140A:I142A, bind the MET receptor with affinities comparable to wild-type NK1 but fail to assemble a dimeric, signalling competent NK1-MET complex. These NK1 variants have no detectable agonistic activity on, behave as bona fide receptor antagonists by blocking cell migration and DNA synthesis in target cells and have strong prospects as therapeutics for human cancer.     

A novel recognition motif for phosphatidylinositol 3-kinase binding mediates its association with the hepatocyte growth factor/scatter factor receptor.

The pleiotropic effects (mitogenesis, motogenesis, and morphogenesis) elicited by hepatocyte growth factor/scatter factor (HGF/SF) are mediated by the activation of the tyrosine kinase receptor encoded by the MET proto-oncogene. Following autophosphorylation, the receptor associates with the p85/110 phosphatidylinositol (PI) 3-kinase complex in vivo and in vitro. By a combination of two complementary approaches, competition with synthetic phosphopeptides and association with Tyr-Phe receptor mutants, we have identified Y-1349 and Y-1356 in the HGF/SF receptor as the binding sites for PI 3-kinase. Y-1349VHV and Y-1356VNV do not conform to the canonical consensus sequence YXXM for PI 3-kinase binding and thus define YVXV as a novel recognition motif. Y-1349 and Y-1356 are located within the C-terminal portion of the HGF/SF receptor and are phosphorylation sites. The affinity of the N- and C-terminal src homology region 2 (SH2) domains of p85 for the phosphopeptides including Y-1349 and Y-1356 is 2 orders of magnitude lower than that measured for Y-751 in the platelet-derived growth factor receptor binding site. However, the closely spaced duplication of the novel recognition motif in the native HGF/SF receptor may allow binding with both SH2 domains of p85, thus generating an efficient docking site for PI 3-kinase. In agreement with this model, we have observed that a phosphopeptide including both Y-1349 and Y-1356 activates PI 3-kinase in vitro.     

Human Monoclonal Antibody Targeting the Heparan Sulfate Chains of Glypican-3 Inhibits HGF-Mediated Migration and Motility of Hepatocellular Carcinoma Cells

Heparan sulfate proteoglycans (HSPGs) participate in many processes related to tumor development, including tumorigenesis and metastasis. HSPGs contain one or more heparan sulfate (HS) chains that are covalently linked to a core protein. Glypican-3 (GPC3) is a cell surface-associated HSPG that is highly expressed in hepatocellular carcinoma (HCC). GPC3 is involved in Wnt3a-dependent HCC cell proliferation. Our previous study reported that HS20, a human monoclonal antibody targeting the HS chains on GPC3, inhibited Wnt3a/β-catenin activation. In the current study, we showed that the HS chains of GPC3 could mediate HCC cells’ migration and motility. Knocking down GPC3 or targeting the HS chains by HS20 inhibited HCC cell migration and motility. However, HS20 had no effect on GPC3 knockdown cells or GPC3 negative cells. In addition, an antibody that recognizes the core protein of GPC3 did not change the rate of cell motility. HCC cell migration and motility did not respond to either canonical or non-canonical Wnt induction, but did increase under hepatocyte growth factor (HGF) treatment. HS20-treated HCC cells exhibited less ability for HGF-mediated migration and motility. Furthermore, HS20 inhibited in vitro HCC spheroid formation and liver tumor growth in mice. GPC3 interacted with HGF; however, a mutant GPC3 lacking the HS chain showed less interaction with HGF. Blocking the HS chains on GPC3 with HS20 reduced c-Met activation in HGF-treated HCC cells and 3D-cultured spheroids. Taken together, our study suggests that GPC3 is involved in HCC cell migration and motility through HS chain-mediated cooperation with the HGF/Met pathway, showing how HS targeting has potential therapeutic implications for liver cancer.     

肝细胞生长因子及受体传递的调节与内吞作用

肝细胞生长因子(HGF)是一种具有多重功能的细胞调控因子。HGF与其受体Met酪氨酸激酶(c-Met)的结合可激发多种生物学反应,从而调节细胞的增殖、分化、形态发生和侵袭运动等。有多种因素参与了HGF/c-Met信号传导的调控,从而防止信号的过度放大,其中Cbl1、Rab、泛素化激酶和HGF/c-Met的内吞等发挥了重要的作用。因此,对HGF/c-Met内吞过程的研究,了解内吞对于HGF/c-Met的信号传导及其调控的影响,探讨HGF/c-Met信号传导通路的调控机理和相互作用模式,可进一步阐明HGF/c-Met信号传导的调控机制,从而验证肝细胞中内吞作用直接调节HGF/c-Met信号通路的作用机制。