Despite the improvement in clinical outcomes derived by the introduction of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) in the treatment of patients with advanced non-small cell lung cancer (NSCLC) whose tumours harbour EGFR-activating mutations, prognosis remains unfavourable because of the occurrence of either intrinsic or acquired resistance. We reviewed the published literature and abstracts of oral and poster presentations from international conferences addressing EGFR-TKIs resistance mechanisms discovered in preclinical models and in patients with NSCLC. The molecular heterogeneity of lung cancer has several implications in terms of possible mechanisms of either intrinsic or acquired resistance to EGFR-targeted inhibitors. Several mechanisms of resistance have been described to EGFR-TKIs, such as the occurrence of secondary mutation (T790M, C797S), the activation of alternative signalling (Met, HGF, AXL, Hh, IGF-1R), the aberrance of the downstream pathways (AKT mutations, loss of PTEN), the impairment of the EGFR-TKIs-mediated apoptosis pathway (BCL2-like 11/BIM deletion polymorphism) and histological transformation. Although some of the mechanisms of resistance have been identified, much additional information is needed to understand and overcome resistance to EGFR-TKI agents. The majority of resistance mechanisms described are the result of a selection of pre-existing clones; thus, studies on the mechanisms by which subclonal alterations have an impact on tumour biology and influence cancer progression are extremely important in order to define the best treatment strategy.

The stabilization of endothelial cell (EC) barrier function within newly formed capillaries is a critical feature of angiogenesis. We examined human lung EC barrier regulation elicited by hepatocyte growth factor (HGF), a recognized angiogenic factor and EC chemoattractant. HGF rapidly and dose-dependently elevated transendothelial electrical resistance (TER) of EC monolayers (>50% increase at 100 ng/ml), with immunofluorescence microscopic evidence of both cytoplasmic actin stress fiber dissolution and strong augmentation of the cortical actin ring. HGF rapidly stimulated phosphatidylinositol 3'-kinase, ERK, p38 mitogen-activated protein kinase, and protein kinase C activities. Pharmacological inhibitor studies demonstrated each pathway to be intimately involved in HGF-induced increases in TER, cortical actin thickening, and phosphorylation of the Ser/Thr glycogen synthase kinase-3beta (GSK-3beta), a potential target for the HGF barrier-promoting response. GSK-3beta phosphorylation was strongly correlated with reductions in both HGF-induced TER and enhanced beta-catenin immunoreactivity observed at cell-cell junctions. Our data suggest a model in which HGF-mediated EC cytoskeletal rearrangement and barrier enhancement depend critically on the activation of a complex kinase cascade that converges at GSK-3beta to increase the availability of beta-catenin, thereby enhancing endothelial junctional integrity and vascular barrier function.

Fibroblasts or their conditioned medium stimulated invasion by squamous cell carcinoma cells. The fibroblast-derived activity responsible for increased invasion is the hepatocyte growth factor/scatter factor (HGF/SF), a ligand for the c-Met receptor. HGF/SF stimulated migration of the cells on various extracellular matrix substrates but did not alter their adhesion efficiency nor integrin expression. HGF/SF stimulated motility in a two step process: initially cells spread rapidly and formed focal adhesions, and then they disassembled these condensations, which was followed by increased cell locomotion. The focal adhesions contained vinculin, p125FAK, beta 1 integrin, and phosphotyrosine. Within minutes after exposure of cells to HGF/SF, proteins of 125 and 145 kDa showed elevated tyrosine phosphorylation and were identified as p125FAK and c-Met, respectively. Gradual loss of tyrosine phosphorylation coincided with disruption of focal adhesions and conversion to a motile phenotype. HGF/SF-mediated tyrosine phosphorylation of p125FAK was inhibited by the tyrosine kinase inhibitor, herbimycin A, which also blocked spreading and the migratory response. These results indicate that fibroblast-derived HGF/SF triggers migration through the initial recruiting of integrins, cytoskeletal proteins, and p125FAK into focal adhesions that is dependent on tyrosine kinase activity.

Hepatocyte growth factor (HGF) is a paracrine inducer of morphogenesis and invasive growth in epithelial and endothelial cells. HGF is secreted by mesenchymal cells as an inactive precursor (pro-HGF). The crucial step for HGF activation is the extracellular hydrolysis of the Arg494-Val495 bond, which converts pro-HGF into alpha beta-HGF, the high-affinity ligand for the Met receptor. We previously reported that the urokinase-type plasminogen activator (uPA) activates pro-HGF in vitro. We now show that this is a stoichiometric reaction, and provide evidence for its occurrence in tissue culture. Activation involves the formation of a stable complex between pro-HGF and uPA. This complex was isolated from the in vitro reaction of pure uPA with recombinant pro-HGF, as well as from the membrane of target cells, after sequential addition of uPA and pro-HGF. On the cell membrane, the uPA-HGF complex was bound to the Met receptor. Monocytic cell lines, and primary monocytes after adhesion, activated efficiently pro-HGF both on their surface and in the culture medium. This activation was inhibited by anti-catalytic anti-uPA antibodies, and occurred by a stoichiometric reaction. The stoichiometry of the activation reaction suggests that the biological effects of HGF can be titrated in vivo by the level of uPA activity. Adequate amounts of uPA can be locally provided by the macrophages, which would condition the tissue microenvironment by rendering HGF bioavailable to its target cells.

OBJECTIVE

The heparin-binding growth factors fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) are potent mitogens for hepatocellular carcinomas (HCCs). Heparin-binding growth factor signaling is regulated by sulfation of cell-surface heparan sulfate proteoglycans (HSPGs). We hypothesized that hSulf1, a recently described sulfatase, regulates growth signaling in HCCs.

METHODS

Expression of hSulf1 in human HCC tumors was determined by real-time PCR. Down-regulation of hSulf1 expression was investigated by analyzing loss of heterozygosity (LOH) at the hSulf1 locus and the effect of the DNA methylation inhibitor 5-aza-deoxycytidine on hSulf1 expression. The subcellular location of hSulf1 and sulfation state of cell-surface HSPGs were assessed by immunocytochemistry. FGF and HGF signaling was examined by phospho-specific immunoblot analysis. Cell growth was measured by trypan blue exclusion, and the MTT assay and apoptosis were quantitated by fluorescence microscopy.

RESULTS

hSulf1 expression was decreased in 29% of HCCs and 82% of HCC cell lines. There was LOH at the hSulf1 locus in 42% of HCCs. Treatment with 5-aza-deoxycytidine reactivated hSulf1 expression in hSulf1-negative cell lines. Low hSulf1-expressing cells showed increased sulfation of cell-surface HSPGs, enhanced FGF and HGF-mediated signaling, and increased HCC cell growth. Conversely, forced expression of hSulf1 decreased sulfation of cell-surface HSPGs and abrogated growth signaling. HCC cells with high-level hSulf1 expression were sensitive to staurosporine- or cisplatin-induced apoptosis, whereas low expressing cells were resistant. Transfection of hSulf1 into hSulf1-negative cells restored staurosporine and cisplatin sensitivity.

CONCLUSIONS

Down-regulation of hSulf1 contributes to hepatocarcinogenesis by enhancing heparin-binding growth factor signaling and resistance to apoptosis.

Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF β-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not β-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.

The Wnt/β-catenin signalling pathway shares a key component, β-catenin, with the cadherin-based adhesion system. The signalling function of β-catenin is conferred by a soluble cytoplasmic pool that is unstable in the absence of a Wnt signal, whilst the adhesion function is based on a cadherin-bound, stable pool at the membrane. The cadherin complex is dynamic, allowing for cell-cell rearrangements such as epithelial-mesenchymal transition (EMT), where the complex turns over through internalisation. Potential interplay between the two pools remains poorly understood, but cadherins are generally considered negative regulators of Wnt signalling because they sequester cytoplasmic β-catenin. Here we explore how cellular changes at EMT affect the signalling capacity of β-catenin using two models of EMT: hepatocyte growth factor (HGF) treatment of MDCK cells, and gastrulation in embryonic development. We show that EMT not only provides a pool of signalling-competent β-catenin following internalisation of cadherin, but also significantly facilitates activation of the Wnt pathway in response to both Wnt signals and exogenous β-catenin. We further demonstrate that availability of β-catenin in the cytoplasm does not necessarily correlate with Wnt/β-catenin pathway activity, since blocking endocytosis or depleting endogenous cadherin abolishes pathway activation despite the presence of β-catenin in the cytoplasm. Lastly we present data suggesting that cadherins are required for augmented activation of the Wnt/β-catenin pathway in vivo. This suggests that cadherins play a crucial role in β-catenin-dependent transcription.

Rhabdomyosarcomas (RMSs) are frequently characterized by bone marrow involvement. Recently, we reported that human RMS cells express the CXC chemokine receptor-4 (CXCR4) and postulated a role for the CXCR4 stromal-derived factor (SDF)-1 axis in the metastasis of RMS cells to bone marrow. Because RMS cells also express the tyrosine kinase receptor c-MET, the specific ligand hepatocyte growth factor (HGF) that is secreted in bone marrow and lymph node stroma, we hypothesized that the c-MET-HGF axis modulates the metastatic behavior of RMS cells as well. Supporting this concept is our observation that conditioned media harvested from expanded ex vivo human bone marrow fibroblasts chemoattracted RMS cells in an HGF- and SDF-1-dependent manner. Six human alveolar and three embryonal RMS cell lines were examined. We found that although HGF, similar to SDF-1, did not affect the proliferation of RMS cells, it induced in several of them: (a) locomotion; (b) stress fiber formation; (c) chemotaxis; (d) adhesion to human umbilical vein endothelial cells; (e) trans-Matrigel invasion and matrix metalloproteinase secretion; and (f) phosphorylation of mitogen-activated protein kinase p42/44 and AKT. Moreover HGF, but not SDF-1, increased the survival of RMS cells exposed to radio- and chemotherapy. We also found that the more aggressive alveolar RMS cells express higher levels of c-MET than embryonal RMS cell lines and "home/seed" better into bone marrow after i.v. injection into immunocompromised mice. Because we could not find any activating mutations in the kinase region of c-MET or any evidence for HGF autocrine stimulation, we suggest that the increased response of RMS cell lines depends on overexpression of functional c-MET. We conclude that HGF regulates the metastatic behavior of c-MET-positive RMS cells, directing them to the bone marrow and lymph nodes. Signaling from the c-MET receptor may also contribute to the resistance of RMS cells to conventional treatment modalities.

HER2 amplification is found in more than 15% of gastric cancers and is associated with poor clinical outcome. Lapatinib, a dual HER2 and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has shown promising in vitro results in treating HER2(+) cancer cells. However, several studies have shown that activation of alternative receptor tyrosine kinases can mediate resistance to HER-targeted therapy. Here, we investigated whether activated MET can confer resistance to lapatinib inhibition of gastric cancer cells. A panel of gastric cancer cell lines was treated with lapatinib, and we observed that cell proliferation was reduced by 70% and that the degree of HER2 amplification corresponds to sensitivity to lapatinib. Immunoblotting analysis indicated that phosphorylation of HER2, EGFR, MET, AKT, and extracellular signal-regulated kinase was inhibited by lapatinib and presumably led to cell-cycle arrest as observed with flow cytometry. Hepatocyte growth factor (HGF) activation of MET receptors rescued cells from lapatinib-induced growth inhibition by restimulating the downstream pathways and restoring normal cell-cycle progression. This rescue effect could be abrogated by inhibiting MET with PHA-665752 (a highly specific MET inhibitor) or downregulating MET expression with short interfering RNA. No synergy in growth inhibition was observed when cells were treated with a combination of lapatinib and PHA-665752. Repeat studies using insulin-like growth factor 1 and fibroblast growth factor 3 could not uniformly rescue the lapatinib-treated gastric cancer cells. In conclusion, HGF/MET-mediated resistance to lapatinib is a novel mechanism of resistance to HER2-targeted agents in gastric cancer cells. Development of inhibitors targeting multiple receptors or common downstream signaling proteins merits further investigation.

The primary structure of rat hepatocyte growth factor (HGF) was elucidated by determining the base sequence of the complementary DNA (cDNA) of HGF. The cDNA for rat HGF was isolated by screening a liver cDNA library with oligonucleotides based on the partial N-terminal amino acid sequence of the beta subunit of purified rat HGF. HGF is encoded in an mRNA of about 6 kilobases. Both alpha and beta subunits of HGF are specified in a single open reading frame for a 728-amino acid protein with a calculated molecular weight of 82,904. The N-terminal part of HGF has a signal sequence and a prosequence with 30 and 25 amino acid residues, respectively. The mature heterodimer structure is derived proteolytically from this single pre-pro precursor polypeptide. The calculated molecular weights of the alpha and beta subunits are 50,664 and 25,883, respectively, and each subunit has two potential N-linked glycosylation sites. The amino acid sequence of HGF is 38% identical with that of plasminogen. The alpha subunit of HGF contains four "kringle" structures, and the beta subunit has 37% amino acid identity with the serine protease domain of plasmin. Northern blot analysis revealed that HGF mRNA was expressed in rat various tissues, including the liver, kidney, lung, and brain.

A transforming growth factor-beta (TGF-beta) found in platelets strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin plus EGF or by hepatocyte growth factor (HGF) from rat platelets, but not the syntheses of secretory and intracellular proteins by the cells. TGF-beta had no cytotoxic effect, as judged by phase-contrast microscopic examination of the cell morphology. The inhibition of DNA synthesis by TGF-beta was correlated with marked decrease in the labeling index. TGF-beta did not inhibit growth of hepatoma cell line. These findings indicate that TGF-beta is a strong growth inhibitor of adult rat hepatocytes and may block their shift from the G1 phase to the S phase. The physiological role of TGF-beta in inhibiting growth of adult hepatocytes during liver regeneration is discussed.

Interleukin 6 (IL6) is the new definition of a group of cytokines previously named according to their biological activity, e.g. B cell stimulatory factor 2 (BSF-2), hybridoma plasmocytoma-growth factor (HGF), interferon-beta 2 (IFN-beta 2), hepatocyte stimulating factor (HSF). It has recently been suggested that IL6 may represent the major mediator of acute-phase protein response whereas IL1 beta and TNF-alpha could play a minor role. We compared the effect of the three cytokines on hepatic protein synthesis by performing in vitro as well as in vivo experiments. Human hepatoma cells (PLC/PRF5) were exposed to each cytokine separately for 20 h, and the effect was then studied at the protein and RNA level. All three cytokines reduced albumin and increased C3 and ceruloplasmin biosynthesis. The cytokines induced the same effect at the RNA level indicating that the modulation was pretranslational. The effect of the cytokines was specific since actin gene expression was not changed; furthermore the effect was blocked by specific antibodies against the cytokines. The effect of the single cytokines was dose and time dependent, and quantitatively comparable. None of the cytokines was able to alter alpha 1-anti-trypsin synthesis. In vivo experiments with mice showed that IL1 beta and TNF-alpha both induce serum amyloid A (SAA) mRNA in the mouse liver and increase factor B (Bf) gene expression. Human recombinant IL6 induced SAA gene expression and it also had a weak positive effect on Bf gene expression after i.p. injection. These data demonstrate that the three cytokines studied are quantitatively and qualitatively comparable, and that all three are probably involved in acute-phase protein response.

Hepatocyte growth factor (HGF) plays an important role in many biological events such as angiogenesis, cell proliferation, anti-fibrosis and antiapoptosis. It is well known that HGF promotes tumor progression and suppresses development of fibrosis after tissue injury. In contrast, its role in immune-mediated disorders has not been fully clarified. In the present study, we examined the role of HGF in Ag-specific immune response using in vitro studies and an experimental model of allergic airway inflammation. We first confirmed that dendritic cells (DCs) expressed the receptor for HGF, c-met, which was not expressed in T cells. Treatment with HGF both in vitro and in vivo potently suppressed DC functions such as Ag-presenting capacity, thus down-regulating Ag-induced Th1- and Th2-type immune responses. Exogenous administration of the HGF expression plasmid into Ag-primed mice markedly suppressed the development of airway eosinophilia and airway hyperresponsiveness, which was induced by Ag inhalation, with suppression of the Ag-presenting capacity of DCs in the lung. HGF exhibited these immunosuppressive effects without up-regulation of IL-10 or TGF-beta. We also found that expression of endogenous HGF in the lung significantly increased following Ag sensitization and inhalation challenges. Finally, neutralization of endogenous HGF in vivo significantly increased airway eosinophilia and airway hyperresponsiveness with up-regulation of the Ag-presenting capacity of DCs in the lung. These results demonstrated a novel, significant, and possibly therapeutic role of HGF as a potent regulator in immune-mediated disorders such as asthma.

Application of mechanical stretch to cultured adult rat muscle satellite cells results in release of hepatocyte growth factor (HGF) and accelerated entry into the cell cycle. Stretch activation of cultured rat muscle satellite cells was observed only when medium pH was between 7.1 and 7.5, even though activation of satellite cells was accelerated by exogenous HGF over a pH range from 6.9 to 7.8. Furthermore, HGF was only released in stretched cultures when the pH of the medium was between 7.1 and 7.4. Conditioned medium from stretched satellite cell cultures stimulated activation of unstretched satellite cells, and the addition of anti-HGF neutralizing antibodies to stretch-conditioned medium inhibited the stretch activation response. Conditioned medium from satellite cells that were stretched in the presence of nitric-oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester hydrochloride did not accelerate activation of unstretched control satellite cells, and HGF was not released into the medium. Conditioned medium from unstretched cells that were treated with a nitric oxide donor, sodium nitroprusside dihydrate, was able to accelerate the activation of satellite cells in vitro, and HGF was found in the conditioned medium. Immunoblot analysis indicated that both neuronal and endothelial NOS isoforms were present in satellite cell cultures. Furthermore, assays of NOS activity in stretched satellite cell cultures demonstrated that NOS is stimulated when satellite cells are stretched in vitro. These experiments indicate that stretch triggers an intracellular cascade of events, including nitric oxide synthesis, which results in HGF release and satellite cell activation.

We used 2D-cocultures employing fibroblasts of different genetic backgrounds and MCF10A-derived human breast epithelial cells of increasingly malignant potential to investigate tumor-stroma interactions in breast cancer and to identify possible signaling pathways involved. Tumor cells induced expression of matrix-metalloproteinase 9 (MMP-9) in fibroblasts in a pattern dependent on the degree of their malignancy. In-situ zymography localized the main gelatinolytic activity around stromal cells in cocultures and xenografted tumors. Use of Smad3 knockout fibroblasts, small molecule inhibitors, and neutralizing antibodies showed that MMP-9 expression was induced by tumor cell-derived TNF-alpha and TGF-beta, dependent on Smad-, Ras-, and PI3-kinase-signaling, and likewise modulated by subsequent HGF- and EGF-signaling. Together, our results indicate that MMP-9 levels in tumor fibroblasts are regulated by a complex tumor-stroma cross-talk, involving multiple ligands and cellular signaling pathways.

c-Met is a receptor tyrosine kinase belonging to the MET (MNNG HOS transforming gene) family, and is expressed on the surfaces of various cells. Hepatocyte growth factor (HGF) is the ligand for this receptor. The binding of HGF to c-Met initiates a series of intracellular signals that mediate embryogenesis and wound healing in normal cells. However, in cancer cells, aberrant HGF/c-Met axis activation, which is closely related to c-Met gene mutations, overexpression, and amplification, promotes tumor development and progression by stimulating the PI3K/AKT, Ras/MAPK, JAK/STAT, SRC, Wnt/β-catenin, and other signaling pathways. Thus, c-Met and its associated signaling pathways are clinically important therapeutic targets. In this review, we elaborate on the molecular structure of c-Met and HGF and the mechanism through which their interaction activates the PI3K/AKT, Ras/MAPK, and Wnt signaling pathways. We also summarize the connection between c-Met and RON and EGFR, which are also receptor tyrosine kinases. Finally, we introduce the current therapeutic drugs that target c-Met in primary tumors, and their use in clinical research.

Increased numbers of tumor-infiltrating neutrophils are linked to poorer outcome in patients with adenocarcinoma of the bronchioloalveolar carcinoma (BAC) subtype. Hepatocyte growth factor (HGF) is a pleiotropic cytokine operating through activation of the proto-oncogene c-met and is a factor of poor prognosis in various cancers. Reports that neutrophils produce HGF led us to investigate their participation in the aerogenous spread of tumor cells and the prognosis of BAC, through the effect of HGF on c-met-expressing tumor cells. Immunoreactive HGF was detected in bronchoalveolar lavage fluid (BALF) supernatants from 34 of 36 patients, whereas it was undetectable in BALF from healthy controls. The HGF thus detected was locally produced, because HGF mRNA was expressed by the patients' fresh alveolar cells, and HGF protein was detected in 24-h culture supernatants. In immunocytochemical studies of BALF cytospin preparations and tumor specimens from the patients, neutrophils were always HGF-positive, whereas alveolar macrophages and tumor cells gave inconsistent results. Alveolar neutrophil-derived HGFs induced significant, concentration-dependent migration of BAC-derived tumor cells in vitro, and this effect was inhibited by anti-HGF neutralizing antibodies. Granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha (present in the lung tumor microenvironment) provoked HGF release from neutrophil intracellular stocks, and the capacity of blood neutrophils from BAC patients to produce HGF was unaltered. Immunochemical studies of c-met expression in BALF cytospin preparations and tumor sections showed that most HGF receptor-bearing cells were tumor cells. High HGF levels in BALF supernatants were significantly associated with poorer outcome in patients with BAC and were an independent predictor of clinical outcome in multivariate analysis. Altogether, our results support the notion that BAC generates a local environment that attracts functionally normal neutrophils from peripheral blood and leads to neutrophil release of biologically active HGF on contact with HGF receptor-expressing tumor cells, thereby contributing to poorer patient outcome.

Signaling pathways have become a major source of targets for novel therapies in hepatocellular carcinoma (HCC). Survival benefits achieved with sorafenib, a multikinase inhibitor, are unprecedented and underscore the importance of improving our understanding of how signaling networks interact in transformed cells. Numerous signaling modules are de-regulated in HCC, including some related to growth factor signaling (e.g., IGF, EGF, PDGF, FGF, HGF), cell differentiation (WNT, Hedgehog, Notch), and angiogenesis (VEGF). Intracellular mediators such as RAS and AKT/MTOR may also play a role in HCC development and progression. Different molecular mechanisms have been shown to induce aberrant pathway activation. These include point mutations, chromosomal aberrations, and epigenetically driven down-regulation. The use of novel molecular technologies such as next-generation sequencing in HCC research has enabled the identification of novel pathways previously underexplored in the HCC field, such as chromatin remodeling and autophagy. Considering recent failures of molecular therapies in advanced clinical trials (e.g., sunitinib, brivanib), survey of these and other new pathways may provide alternative therapeutic targets.

BACKGROUND

Under normal conditions, hepatocyte growth factor (HGF)-induced activation of its cell surface receptor, the Met tyrosine kinase (TK), is tightly regulated by paracrine ligand delivery, ligand activation at the target cell surface, and ligand-activated receptor internalization and degradation. Despite these controls, HGF/Met signaling contributes to oncogenesis and tumor progression in several cancers and promotes aggressive cellular invasiveness that is strongly linked to tumor metastasis.

METHODS

The prevalence of HGF/Met pathway activation in human malignancies has driven rapid growth in cancer drug development programs. The authors review Met structure and function, the basic properties of HGF/Met pathway antagonists now in preclinical and clinical development, as well as the latest clinical trial results.

CONCLUSIONS

Clinical trials with HGF/Met pathway antagonists show that as a class these agents are well tolerated. Although widespread efficacy was not seen in several completed Phase II studies, promising results have been reported in lung, gastric, prostate and papillary renal cancer patients treated with these agents. The main challenges facing the effective use of HGF/Met-targeted antagonists for cancer treatment are optimal patient selection, diagnostic and pharmacodynamic biomarker development, and the identification and testing of optimal therapy combinations. The wealth of basic information, analytical reagents, and model systems available concerning HGF/Met oncogenic signaling will continue to be invaluable in meeting these challenges and moving expeditiously toward more effective disease control.

Immune-mediated diseases of the CNS, such as multiple sclerosis and its animal model, experimental autoimmune encephalitis (EAE), are characterized by the activation of antigen-presenting cells and the infiltration of autoreactive lymphocytes within the CNS, leading to demyelination, axonal damage, and neurological deficits. Hepatocyte growth factor (HGF) is a pleiotropic factor known for both neuronal and oligodendrocytic protective properties. Here, we assess the effect of a selective overexpression of HGF by neurons in the CNS of C57BL/6 mice carrying an HGF transgene (HGF-Tg mice). EAE induced either by immunization with myelin oligodendrocyte glycoprotein peptide or by adoptive transfer of T cells was inhibited in HGF-Tg mice. Notably, the level of inflammatory cells infiltrating the CNS decreased, except for CD25(+)Foxp3(+) regulatory T (T(reg)) cells, which increased. A strong T-helper cell type 2 cytokine bias was observed: IFN-gamma and IL-12p70 decreased in the spinal cord of HGF-Tg mice, whereas IL-4 and IL-10 increased. Antigen-specific response assays showed that HGF is a potent immunomodulatory factor that inhibits dendritic cell (DC) function along with differentiation of IL-10-producing T(reg) cells, a decrease in IL-17-producing T cells, and down-regulation of surface markers of T-cell activation. These effects were reversed fully when DC were pretreated with anti-cMet (HGF receptor) antibodies. Our results suggest that, by combining both potentially neuroprotective and immunomodulatory effects, HGF is a promising candidate for the development of new treatments for immune-mediated demyelinating diseases associated with neurodegeneration such as multiple sclerosis.

Hepatocyte growth factor (HGF) has been demonstrated to be synthesized and secreted by non-parenchymal liver cells for liver regeneration after hepatic injury. We performed in situ hybridization to identify HGF-producing cell types in rat liver hepatitis induced by administrating carbon tetrachloride as a hepatotoxin. We found that transcripts of the HGF gene are localized in the Kupffer and endothelial cells in normal livers and increased remarkably in the Kupffer cells of the damaged livers. Thus, HGF is concluded to be synthesized in the Kupffer and endothelial cells to repair the liver tissue in paracrine fashion. No significant increase in the transcripts of the HGF gene was observed in livers after partial hepatectomy, indicating that a mechanism on liver regeneration after the hepatectomy differs from that on liver repairs. Since the HGF gene expression was also found in lung and kidney, HGF may be a ubiquitous factor for tissue repairs.

MicroRNA (miR)-26a can suppress tumor growth and metastasis of hepatocellular carcinoma (HCC). Since angiogenesis is important for tumor growth and metastasis, we investigated the possible roles of miR-26a in tumor angiogenesis. Down-regulation of miR-26a was found to correlate with an increased angiogenic potential of HCC. Through gain- and loss-of-function studies, miR-26a was demonstrated to significantly inhibit vascular endothelial growth factor A (VEGFA) expression in HCC cells and then suppress the promoting effects of HCC cells on in vitro proliferation, migration, and capillary tube formation of endothelial cells, as well as in vivo tumor angiogenesis of HCC. Hepatocyte growth factor (HGF) was identified as a target of miR-26a. HGF simulation antagonized the effects induced by miR-26a up-regulation. In contrast, silencing HGF induced similar effects to miR-26a. We further found that miR-26a exerted its antiangiogenesis function, at least in part, by inhibiting HGF-hepatocyte growth factor receptor (cMet) and its downstream signaling pathway, in turn, suppressing VEGFA production in HCC cells and impairing VEGFR2-signaling in endothelial cells. HCC patients who had high miR-26a, low HGF, low VEGFA, or low microvessel density (MVD) in tumor tissues had a better prognosis with longer overall survival (OS) and time to recurrence (TTR). In multivariate analysis, miR-26a, or in combination with HGF, was demonstrated to be an independent prognostic indicator for OS and TTR of HCC patients.

CONCLUSIONS

miR-26a could suppress tumor angiogenesis of HCC through HGF-cMet signaling, and it is a new hopeful therapeutic target and prognostic marker for HCC.

Hepatic fibrosis is a common outcome of a variety of chronic liver diseases. Here we evaluated the therapeutic efficacy of hepatocyte growth factor (HGF) on liver fibrosis induced by bile duct ligation (BDL) and investigated potential mechanisms. Mice underwent BDL, followed by intravenous injections of naked HGF expression plasmid or control vector. HGF gene therapy markedly ameliorated hepatic fibrotic lesions, as demonstrated by reduced alpha-smooth muscle actin (alphaSMA) expression, attenuated deposition of type I and type III collagen, and normalized total hydroxyproline content. HGF also suppressed transforming growth factor-beta1 (TGF-beta1) expression. Interestingly, colocalization of alphaSMA and cytokeratin-19 in bile duct epithelium was observed, suggesting the possibility of biliary epithelial to myofibroblast transition after BDL. Cells that were still positive for cytokeratin-19 but actively producing type I collagen were found in the biliary epithelia and periductal region. Laminin staining revealed an impaired basement membrane of the bile duct epithelium in diseased liver. These lesions were largely prevented by HGF administration. In vitro, treatment of human biliary epithelial cells with TGF-beta1 induced alphaSMA and fibronectin expression and suppressed cytokeratin-19. HGF abolished the phenotypic conversion of biliary epithelial cells induced by TGF-beta1. These results suggest that HGF ameliorates hepatic biliary fibrosis in part by blocking bile duct epithelial to mesenchymal transition.

Hepatocyte growth factor (HGF) is important for cell proliferation, differentiation, and related activities. HGF acts through its receptor c-Met, which activates downstream signaling pathways. HGF binds to c-Met at the plasma membrane, where it is generally believed that c-Met signaling is initiated. Here we report that c-Met rapidly translocates to the nucleus upon stimulation with HGF. Ca(2+) signals that are induced by HGF result from phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate formation within the nucleus rather than within the cytoplasm. Translocation of c-Met to the nucleus depends upon the adaptor protein Gab1 and importin beta1, and formation of Ca(2+) signals in turn depends upon this translocation. HGF may exert its particular effects on cells because it bypasses signaling pathways in the cytoplasm to directly activate signaling pathways in the nucleus.