Autophosphorylation promotes complex formation of recombinant hepatocyte growth factor receptor with cytoplasmic effectors containing SH2 domains.

Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a polypeptide which induces motility and/or mitogenesis in epithelial cells. The receptor for HGF/SF, p190MET, is a two-chain transmembrane tyrosine kinase encoded by the MET proto-oncogene. To identify the cytoplasmic effectors involved in signal transduction we have produced the human HGF/SF receptor in insect cells (Sf9) by means of a recombinant baculovirus. Two 170-kDa forms of the receptor were synthesized in Sf9 cells: the uncleaved single-chain precursor (which is by far the more abundant) and the proteolytically processed two-chain molecule. Both receptor species are phosphorylated on tyrosine in vivo and are active kinases in vitro. The recombinant receptor binds and phosphorylates in vitro four known cytoplasmic transducers containing src homology region 2 (SH2) domains: the 85-kDa subunit of phosphatidylinositol 3-kinase (Pl 3-kinase), rasGAP, phospholipase-C gamma (PLC-gamma), and p59Fyn, a tyrosine kinase of the src family. In all cases the association is strictly dependent on tyrosine phosphorylation of the receptor, indicating that it occurs via specific interaction with the SH2 domains. These results show that the HGF/SF receptor has the sequence requirements for binding a spectrum of cytoplasmic transducers whose different combinations in target cells may result in the observed pleiotropic biological response.     

Impaired ligand‐dependent MET activation caused by an extracellular SEMA domain missense mutation in lung cancer

Aberrant activation of the MET/hepatocyte growth factor (HGF) receptor participates in the malignant behavior of cancer cells, such as invasion‐metastasis and resistance to molecular targeted drugs. Many mutations in the MET extracellular region have been reported, but their significance is largely unknown. Here, we report the dysregulation of mutant MET originally found in a lung cancer patient with Val370 to Asp370 (V370D) replacement located in the extracellular SEMA domain. MET‐knockout cells were prepared and reconstituted with WT‐MET or V370D‐MET. HGF stimulation induced MET dimerization and biological responses in cells reconstituted with WT‐MET, but HGF did not induce MET dimerization and failed to induce biological responses in V370D‐MET cells. The V370D mutation abrogated HGF‐dependent drug resistance of lung cancer cells to epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKI). Compared with WT‐MET cells, V370D‐MET cells showed different activation patterns in receptor tyrosine kinases upon exposure to survival/growth‐stressed conditions. Surface plasmon resonance analysis indicated that affinity between the extracellular region of V370D‐MET and HGF was reduced compared with that for WT‐MET. Further analysis of the association between V370D‐MET and the separate domains of HGF indicated that the SP domain of HGF was unchanged, but its association with the NK4 domain of HGF was mostly lost in V370D‐MET. These results indicate that the V370D mutation in the MET receptor impairs the functional association with HGF and is therefore a loss‐of‐function mutation. This mutation may change the dependence of cancer cell growth/survival on signaling molecules, which may promote cancer cell characteristics under certain conditions.     

Inactivation of von Hippel-Lindau gene induces constitutive phosphorylation of MET protein in clear cell renal carcinoma

It is well known that inactivation of von Hippel-Lindau (VHL) gene predisposes for human clear cell renal carcinoma (CCRC). However, details about critical roles of VHL inactivation during tumorigenesis are still unknown. MET protein is a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), which regulates cell growth, cell morphology, and cell motility. We showed that MET protein overexpressed in CCRC cells was phosphorylated without HGF/SF. This constitutive phosphorylation of MET protein in CCRC cells was inhibited by the rescue of exogenous wild-type VHL gene without a decrease in expression level of MET protein. Interestingly, wild-type VHL gene suppressed the phosphorylation of MET protein only under high cell density conditions. Additionally, MET protein activated by the inactivation of VHL gene modified cell adherence, including N-cadherin and beta-catenin. When activation of MET protein in CCRC cells was inhibited by the MET inhibitor K252a, the growth of CCRC cells in vitro and the tumorigenesis induced by CCRC cells in nude mice were suppressed. From these results, we concluded that inactivation of VHL gene induced constitutive phosphorylation of MET protein and modified intercellular adherence structure to trigger the cell growth released from contact inhibition, finally resulting in tumorigenesis. This is one of the mechanisms of CCRC oncogenesis, and MET protein has potential as a molecular target for novel CCRC therapies.     

Role of hepatocyte growth factor activator (HGF activator) in invasive growth of human glioblastoma cells in vivo

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor that is involved in invasive growth of tumor cells via its receptor MET, a protein product of c-met proto-oncogene. HGF activator (HGFA) is a serine proteinase responsible for the activation of proform of HGF/SF (proHGF/SF). In our study, we examined the effects of engineered expression of HGFA on 2 human glioblastoma cell lines (YKG-1 and U251). Both cells expressed MET, while only YKG-1 expressed endogenous proHGF/SF. Enhanced MET phosphorylation and increased migratory activity were induced by the expression of HGFA in YKG-1 cells in vitro in the presence of thrombin, which is a known activator of proHGFA. In contrast, MET phosphorylation was consistently observed in U251 that lacked endogenous HGF/SF, suggesting ligand-independent activation of MET in this cell line. Consequently, the expression of HGFA in U251 did not enhance the MET phosphorylation and following cellular response even with the thrombin treatment. However, addition of exogenous proHGF/SF resulted in enhanced migratory activity of HGFA-expressing U251 cells in the presence of thrombin in vitro. The engineered HGFA expression resulted in significantly enhanced tumor growth with increased vascular density in vivo when YKG-1 cells were implanted in nude mouse brain. This effect was not observed in U251 lacking endogenous proHGF/SF. These results indicate the possible existence of multiple mechanisms of MET activation in glioblastomas and that the activation system of proHGF/SF is important in progression of glioblastomas that express endogenous proHGF/SF and require ligand-dependent MET activation.     

Identification of Grb10 as a direct substrate for members of the Src tyrosine kinase family

Treatment of cells with insulin and protein tyrosine phosphatase inhibitors such as vanadate and pervanadate resulted in the tyrosine phosphorylation of Grb10, a Src homology 2 (SH2) and pleckstrin homology domain-containing adaptor protein which binds to a number of receptor tyrosine kinases including the insulin receptor (IR). Although Grb10 binds directly to the kinase domain of the IR, our data show that Grb10 is not a direct substrate for the IR tyrosine kinase. Consistent with this finding, Grb10 tyrosine phosphorylation in cells was inhibited by herbimycin A, a relatively specific inhibitor for members of the Src tyrosine kinase family, and by the expression of dominant negative Src or Fyn. In addition, Grb10 tyrosine phosphorylation was stimulated by expression of constitutively active Src or Fyn in cells and by incubation with purified Src or Fyn in vitro. The insulin stimulated or Src/Fyn-mediated tyrosine phosphorylation in vivo was significantly reduced when Grb10 tyrosine 67 was changed to glycine. This mutant form of Grb10 bound with higher affinity to the IR in cells than that of the wild-type protein, suggesting that tyrosine phosphorylation of Grb10 may normally negatively regulate its binding to the IR. Our data show that Grb10 is a new substrate for members of the Src tyrosine kinase family and that the tyrosine phosphorylation of the protein may play a potential role in cell signaling processes mediated by these kinases. Oncogene (2000).     

Targeting MET in cancer: rationale and progress

Uncontrolled cell survival, growth, angiogenesis and metastasis are essential hallmarks of cancer. Genetic and biochemical data have demonstrated that the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, the tyrosine kinase MET, have a causal role in all of these processes, thus providing a strong rationale for targeting these molecules in cancer. Parallel progress in understanding the structure and function of HGF/SF, MET and associated signalling components has led to the successful development of blocking antibodies and a large number of small-molecule MET kinase inhibitors. In this Review, we discuss these advances, as well as results from recent clinical studies that demonstrate that inhibiting MET signalling in several types of solid human tumours has major therapeutic value.     

The MET axis as a therapeutic target

The MET receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) have been implicated in transformation of a variety of malignancies. Chronic or dysregulated activation of the MET/HGF pathway may lead to increased cell growth, invasion, angiogenesis, and metastasis, reduced apoptosis, altered cytoskeletal functions and other biological changes. It has been suggested that ligand activated MET stimulation can be sufficient for a transforming phenotype. In addition, amplification and activation mutations (germline and/or somatic) within the tyrosine kinase domain, juxtamembrane domain, or semaphorin domain have been identified for MET. MET gain-of-function mutations lead to either deregulated or prolonged tyrosine kinase activity, which are instrumental to its transforming activity. A number of therapeutic strategies targeting ligand-dependent activation or the kinase domain have been employed to inhibit MET. The different structural requirements for activation of signaling events and biological functions regulated by MET will be summarized. Therapeutic targets and current pre-clinical and clinical approaches will be described. Targeting the HGF/MET pathway, alone or in combination with standard therapies, is likely to improve present therapies in MET-dependent malignancies.     

Expression and localization of scatter factor/hepatocyte growth factor in human astrocytomas

Scatter factor/hepatocyte growth factor (SF/HGF) is a pleiotropic cytokine that has been implicated in glioma invasion and angiogenesis. The SF/HGF receptor, MET, has been found to be expressed in neoplastic astrocytes as well as in endothelial cells of the tumor vasculature. Both SF/HGF and MET expression have also been described to correlate with the malignancy grade of human gliomas. However, most glioblastoma cell lines lack SF/HGF expression, raising the question of the cellular origin of SF/HGF in vivo. Using in situ hybridization, we analyzed glioblastomas, anaplastic astrocytomas, diffuse astrocytomas, pilocytic astrocytomas, and normal brain for the expression of SF/HGF mRNA. We detected strong SF/HGF expression by the majority of the tumor cells and by vascular endothelial cells in all glioblastoma specimens analyzed. Combined use of in situ hybridization with fluorescence immunohistochemistry confirmed the astrocytic origin of the SF/HGF-expressiong cells. In contrast, CD68-immunoreactive microglia/macrophages, as well as vascular smooth muscle cells reactive to alpha-smooth muscle actin, lacked SF/HGF expression. In anaplastic, diffuse, and pilocytic astrocytomas, SF/HGF expression was confined to a subset of tumor cells, and signals were less intense than in glioblastomas. In addition, we detected SF/HGF mRNA in cortical neurons. SF/HGF expression was not up regulated around necroses or at tumor margins. MET immunoreactivity was observed in GFAP-expressing astrocytic tumor cells and endothelial cells as well as in a subset of microglia/macrophages. We conclude that in vivo, both autocrine and paracrine stimulation of tumor cells and endothelium through the SF/HGF-MET system are likely to contribute to tumor invasion and angiogenesis. Lack of SF/HGF expression by most cultured glioblastoma cells is not representative of the in vivo situation and most likely represents a culture artifact.     

Multiple Grb2-protein complexes in human cancer cells

Grb2 is an SH2/SH3 domain-containing adaptor protein that links receptor tyrosine kinases to the ras signaling pathway. The Grb2-SH2 domain binds phosphotyrosine sequences on activated tyrosine kinases, and one target of the SH3 domains is the ras-nucleotide-exchange factor Sos1. We have examined Grb2-protein interactions in human cancer cells that over-express the receptor tyrosine kinase erbB2. Our results show that the 2 Grb2-SH3 domains complex with Sos1, dynamin and at least 4 other proteins (p228, p140, p55, p28) in these cells. The 2 Grb2-SH3 domains bind these proteins differently, with the N-terminal SH3 domain interacting preferentially with p228, Sos1, p140 and dynamin. The C-terminal SH3 domain has higher affinity toward p28. The Grb2-SH3 domain interactions appear to be similar in erbB2 over-expressing breast, ovarian and lung cancer cells. Also, the major tyrosine-phosphorylated proteins that associate with Grb2 in erbB2 over-expressing cancer cells appear to be erbB2 and Shc. The multiple Grb2-SH3 domain interactions in these cells may mediate novel cellular functions. Int. J. Cancer, 70:208–213, 1977. © 1997 Wiley-Liss Inc.     

Discovery of Fully Human Anti-MET Monoclonal Antibodies with Antitumor Activity against Colon Cancer Tumor Models In Vivo

The receptor tyrosine kinase MET is a major component controlling the invasive growth program in embryonic development and in invasive malignancies. The discovery of therapeutic antibodies against MET has been difficult, and antibodies that compete with hepatocyte growth factor (HGF) act as agonists. By applying phage technology and cell-based panning strategies, we discovered two fully human antibodies against MET (R13 and R28), which synergistically inhibit HGF binding to MET and elicit antibody-dependent cellular cytotoxicity. Cell-based phosphorylation assays demonstrate that R13 and R28 abrogate HGF-induced activation of MET, AKT1, ERK1/2, and HGF-induced migration and proliferation. FACS experiments suggest that the inhibitory effect is mediated by “locking” MET receptor in a state with R13, which then increases avidity of R28 for the extracellular domain of MET, thus blocking HGF binding without activating the receptor. In vivo studies demonstrate that the combination of R13/28 significantly inhibited tumor growth in various colon tumor xenograft models. Inhibition of tumor growth was associated with induction of hypoxia. Global gene expression analysis shows that inhibition of HGF/MET pathway significantly upregulated the tumor suppressors KLF6, CEACAM1, and BMP2, the negative regulator of phosphatidylinositol-3-OH-kinase PIK3IP1, and significantly suppressed SCF and SERPINE2, both enhancers of proliferation and invasiveness. Moreover, in an experimental metastasis model, R13/28 increased survival by preventing the recurrence of otherwise lethal lung metastases. Taken together, these results underscore the utility of a dual-antibody approach for targeting MET and possibly other receptor tyrosine kinases. Our approach could be expanded to drug discovery efforts against other cell surface proteins.     

The adapter protein, Grb10, is a positive regulator of vascular endothelial growth factor signaling

Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis and angiogenesis. Activation of VEGF receptors leads to the recruitment of SH2 containing proteins which link the receptors to the activation of signaling pathways. Here we report that Grb10, an adapter protein of which the biological role remains unknown, is tyrosine phosphorylated in response to VEGF in endothelial cells (HUVEC) and in 293 cells expressing the VEGF receptor KDR. An intact SH2 domain is required for Grb10 tyrosine phosphorylation in response to VEGF, and this phosphorylation is mediated in part through the activation of Src. In HUVEC, VEGF increases Grb10 mRNA level. Expression of Grb10 in HUVEC or in KDR expressing 293 cells results in an increase in the amount and in the tyrosine phosphorylation of KDR. In 293 cells, this is correlated with the activation of signaling molecules, such as MAP kinase. By expressing mutants of Grb10, we found that the positive action of Grb10 is independent of its SH2 domain. Moreover, these Grb10 effects on KDR seem to be specific since Grb10 has no effect on the insulin receptor, and Grb2, another adapter protein, does not mimic the effect of Grb10 on KDR. In conclusion, we propose that VEGF up-regulates Grb10 level, which in turn increases KDR molecules, suggesting that Grb10 could be involved in a positive feedback loop in VEGF signaling.     

Met and hepatocyte growth factor/scatter factor signal transduction in normal melanocytes and melanoma cells.

The proto-oncogene c-MET encodes a transmembrane tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF stimulates the proliferation and motility of various cell types. Because HGF/SF is also a melanocyte mitogen, we investigated the biological role of HGF/SF, including c-Met expression, activation and signal transduction, in normal and malignant human melanocytes. We show that HGF/SF is mitogenic in the presence of synergistic factors, such as basic fibroblast growth factor (bFGF) and mast cell growth factor (MGF) and that, by itself, it stimulates the motility of normal human melanocytes. The ligand also maintained high levels of tyrosinase activity and melanin content in theses cells. Signal transduction by HGF/SF included phosphorylation of tyrosyl residues on c-Met, a cascade of tyrosine phosphorylations on several other proteins and activation of microtubule-associated protein kinase/extracellular signal-regulated kinase. Met expression and activity are normal in human melanomas, and constitutive activity of HGF/SF in retrovirally infected autonomously proliferative mouse melanocytes is insufficient to confer the malignant phenotype. Our findings suggest that activation of Met in response to HGF/SF may contribute to malignant progression synergistically with the aberrant expression of bFGF in malignant melanocytes and that, in addition, the peptide may promote dispersion of factor-dependent melanocytes from early stages of primary melanomas to ectopic sites.     

Nk4, a new HGF/SF variant, is an antagonist to the influence of HGF/SF on the motility and invasion of colon cancer cells

Hepatocyte Growth Factor/Scatter Factor (HGF/SF) is a heterodimeric molecule that plays a key role in the regulation of migration, invasion and angiogenesis in cancer, via activation of its receptor, c-met. HGF/SF is composed of an alpha-chain, containing the N-terminal hairpin domain and 4 kringle domains, plus the serine protease-like beta-chain. We have examined here the properties of NK4, an HGF variant containing the N-terminal hairpin plus the 4 kringle domains, on tumour cell proliferation, dissociation and invasion using human colorectal cancer cells (HT115). The expression of HGF/SF and its receptor was also examined by RT-PCR and Western blotting. Analysis revealed NK4 to be an HGF/SF antagonist that, at a wide range of concentrations, did not exert any biological effects of its own. HT115 cells were shown to express the HGF/SF receptor mRNA and protein. HGF/SF-induced receptor tyrosine phosphorylation was suppressed, in a dose-dependent manner, upon addition of NK4, whereas the addition of NK4 alone caused no phosphorylation. Tumour cell motility was induced by HGF and inhibited by NK4. Furthermore, HGF/SF induced the invasion of cells through Matrigel basement membrane components, and again this induced invasion was suppressed by NK4. Our results show that the ability of HGF/SF to stimulate tumour cell motility and invasion, properties required for metastatic spread, can be inhibited by NK4. Thus, NK4 may have an important role in the control of cancer metastasis.     

Gab1 phosphorylation: a novel mechanism for negative regulation of HGF receptor signaling

Signal transduction by HGF receptor, the tyrosine kinase encoded by the MET oncogene, switches on a genetic program called 'invasive growth' inducing epithelial cell dissociation, migration, growth, and ultimately leading to differentiation into branched tubular structures. Sustained tyrosine phosphorylation of the downstream adaptor protein Gab1 is required for the HGF response. Here we show that serine/threonine phosphorylation of Gab1 provides a control mechanism for negative regulation. Treatment with okadaic acid, a potent inhibitor of the serine/threonine protein phosphatases PP1 and PP2A, was followed by activation of a number of serine/threonine kinases, hyper-phosphorylation in serine and threonine of Gab1 and severe inhibition of the HGF-induced biological responses. Under these conditions, Gab1 was found to be concomitantly hypo-phosphorylated in tyrosine, and thus endowed with reduced ability to recruit SH2 containing signal transducers such as PI3 kinase. Among the serine-threonine kinases activated by PP1 and PP2A inhibition, we found that PKC-alpha and PKC-beta1 are required for negative regulation of Gab1. These data provide a novel negative mechanism for the HGF receptor signaling pathways and highlight a potentially useful target for inhibitors of invasive growth.     

The scatter factor/hepatocyte growth factor: c-met pathway in human embryonal central nervous system tumor malignancy

Embryonal central nervous system (CNS) tumors, which comprise medulloblastoma, are the most common malignant brain tumors in children. The role of the growth factor scatter factor/hepatocyte growth factor (SF/HGF) and its tyrosine kinase receptor c-Met in these tumors has been until now completely unknown. In the present study, we show that human embryonal CNS tumor cell lines and surgical tumor specimens express SF/HGF and c-Met. Furthermore, c-Met mRNA expression levels statistically significantly correlate with poor clinical outcome. Treatment of medulloblastoma cells with SF/HGF activates c-Met and downstream signal transduction as evidenced by c-Met, mitogen-activated protein kinase, and Akt phosphorylation. SF/HGF induces tumor cell proliferation, anchorage-independent growth, and cell cycle progression beyond the G1-S checkpoint. Using dominant-negative Cdk2 and a degradation stable p27 mutant, we show that cell cycle progression induced by SF/HGF requires Cdk2 function and p27 inhibition. SF/HGF also protects medulloblastoma cells against apoptosis induced by chemotherapy. This cytoprotective effect is associated with reduction of proapoptotic cleaved poly(ADP-ribose) polymerase and cleaved caspase-3 proteins and requires phosphoinositide 3-kinase activity. SF/HGF gene transfer to medulloblastoma cells strongly enhances the in vivo growth of s.c. and intracranial tumor xenografts. SF/HGF-overexpressing medulloblastoma xenografts exhibit increased invasion and morphologic changes that resemble human large cell anaplastic medulloblastoma. This first characterization establishes SF/HGF:c-Met as a new pathway of malignancy with multifunctional effects in human embryonal CNS tumors.     

HGF/SF and its receptor c-MET play a minor role in the dissemination of human B-lymphoma cells in SCID mice.

The MET protooncogene, c-MET, encodes a cell surface tyrosine kinase receptor. The ligand for c-MET is hepatocyte growth factor (HGF), also known as scatter factor (SF), which is known to affect proliferation and motility of primarily epithelial cells. Recently, HGF/SF was also shown to affect haemopoiesis. Studies with epithelial and transfected NIH3T3 cells indicated that the HGF/SF-c-MET interaction promotes invasion in vitro and in vivo. We previously demonstrated that HGF/SF induces adhesion of c-MET-positive B-lymphoma cells to extracellular matrix molecules, and promoted migration and invasion in in vitro assays. Here, the effect of HGF/SF on tumorigenicity of c-MET-positive and c-MET-negative human B-lymphoma cell lines was studied in C.B-17 scid/scid (severe combined immune deficient) mice. Intravenously (i.v.) injected c-MET-positive (BJAB) as well as c-MET-negative (Daudi and Ramos cells) B-lymphoma cells formed tumours in SCID mice. The B-lymphoma cells invaded different organs, such as liver, kidney, lymph nodes, lung, gonads and the central nervous system. We assessed the effect of human HGF/SF on the dissemination of the B-lymphoma cells and found that administration of 5 microg HGF/SF to mice, injected (i.v.) with c-MET-positive lymphoma cells, significantly (P = 0.018) increased the number of metastases in lung, liver and lymph nodes. In addition, HGF/SF did not significantly influence dissemination of c-MET-negative lymphoma cells (P = 0.350 with Daudi cells and P= 0.353 with Ramos cells). Thus the effect of administration of HGF/SF on invasion of lymphoma cells is not an indirect one, e.g. via an effect on endothelial cells. Finally, we investigated the effect of HGF/SF on dissemination of c-MET-transduced Ramos cells. In response to HGF/SF, c-MET-transduced Ramos cells showed an increased migration through Matrigel in Boyden chambers compared to wild-type and control-transduced Ramos cells. The dissemination pattern of c-MET-transduced cells did not differ from control cells in in vivo experiments using SCID mice. Also no effect of HGF/SF administration could be documented, in contrast to the in vitro experiments. From our experiments can be concluded that the HGF/SF-c-MET interaction only plays a minor role in the dissemination of human B-lymphoma cells.     

Hepatocyte growth factor induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin and enhances cell-matrix interactions

This study examined the effects of hepatocyte growth factor/scatter factor (HGF/SF) on the adhesion of HT115 (human colon) and MDA MB 231 (human breast) tumour cells to an extracellular matrix, Matrigel, together with the tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. Treatment of cells with HGF increased cell adhesion to matrix. Following adhesion to Matrigel, FAK and paxillin were quickly phosphorylated and located to the focal adhesion area. HGF/SF increased both tyrosine phosphorylation of FAK and paxillin and also increased formation of focal adhesion. HGF also induced an increased spreading on matrix. It is concluded therefore that HGF/SF stimulated FAK and paxillin phosphorylation resulting in an increased tumour-matrix adhesion.