Scatter Factor/hepatocyte Growth Factor Research Articles

23 Invited lecture: Tumor–stroma interactions promote operational flexibility in cancer progression

Malignant cancer cells do not act as lone wolves to achieve metastasis, as they exist within a complex ecosystem consisting of an extracellular matrix scaffold populated by carcinoma-associated fibroblasts (CAFs), endothelial cells and immune cells (De Wever and Mareel, J Pathol, 2003; De Wever et al., Int J Cancer, 2008; De Wever et al., Semin Cancer Biol., 2014). Paracrine interactions between CAFs, also termed myofibroblasts, and cancer cells stimulate invasion of colon cancer cells in matrix scaffolds. The pro-invasive activity was found also with the secretome and we identified the combined action of Scatter Factor/Hepatocyte Growth Factor (SF/HGF) and tenascin-C (TNC) to promote invasion (De Wever et al., FASEBJ, 2004). Secretome proteomics revealed the presence of multiple other pro-metastatic proteins suggesting that the molecular characteristics of a CAF remain in evolution (De Boeck et al., Proteomics, 2013). CAFs show operational flexibility; eg their ability to respond dynamically to a cancer cell’s fluctuating demands by shifting profitable signals necessary in metastasis. Local, tissue-resident fibroblasts and mesenchymal stem cells (MSCs) coming from reservoir sites such as bone marrow (BM) are the main progenitor cells of CAFs (De Boeck et al., Oral Oncol, 2010). A successful invader must survive in an ectopic environment and CAFs provide advantageous signals to cancer cells to accomplish survival through AKT activation. Neuregulin (NRG)-1 secreted by CAFs increase cancer cell number through HER2/HER3-dependent activation of the PI3K/AKT pathway. Translational data demonstrate transmembrane NRG-1 expression in CAF in primary colorectal cancer and in liver metastasis, with the strongest expression in the immediate vicinity of HER2/HER3 expressing cancer cells. Moreover, high transmembrane NRG-1 expression is a poor prognosis marker in colorectal cancer (De Boeck et al., GUT, 2013). The odds that an aggressive cancer cell will form a metastatic colony in some organ are vanishingly small. Nevertheless, metastasis is responsible for more than 90% of cancer-associated mortality; thus the clinical need to prevent or target metastasis is high. Viewing cancer within the context of ecosystems may provide opportunities to exploit CAFs to create tumor-environment bio-mimetics to deceive and redirect metastatic cancer cells.

MMP-13 Plays a Role in Keratinocyte Migration, Angiogenesis, and Contraction in Mouse Skin Wound Healing

Matrix metalloproteinases (MMPs) have been implicated in wound healing. To analyze the roles of MMP-9 and MMP-13 in wound healing, we generated full-thickness cutaneous wounds in MMP-9 knockout (KO), MMP-13 KO, MMP-9/13 double KO, and wild-type mice. Macroscopic wound closure was delayed in all of the KO mice, as compared with wild-type mice. The rate of re-epithelialization was significantly delayed in MMP-9 KO and MMP-13 KO mice and remarkably delayed in MMP-9/13 double KO mice, as compared with wild-type mice. Both MMP-9 and MMP-13 were expressed by the leading edges of epidermal cells in wild-type mice, and the migration of keratinocytes was suppressed by treatment with an MMP inhibitor or transfection of small interfering RNAs for MMP-9 or MMP-13, as compared with controls. The vascular density in wound granulation was significantly lower in both MMP-13 KO and MMP-9/13 double KO mice than in wild-type mice. Degradation of connective tissue growth factor in wound tissue was transiently prevented in MMP-13 KO mice. Morphometric analyses demonstrated a reduction in both wound contraction and myofibroblast formation in both MMP-13 KO and MMP-9/13 double KO mice. Proliferation and transforming growth factor-beta1-induced myofibroblast differentiation of dermal fibroblasts from MMP-13 KO mice were decreased, as compared with wild-type dermal fibroblasts. These data suggest that MMP-13 plays a role in keratinocyte migration, angiogenesis, and contraction in wound healing, while MMP-9 functions in keratinocyte migration.

Stroma reactions and cancer invasion

Stroma reactions and cancer invasion

Hypoxia can induce c‐Met expression in glioma cells and enhance SF/HGF‐induced cell migration

The c-Met receptor and its ligand scatter factor/hepatocyte growth factor (SF/HGF) are strongly overexpressed in malignant gliomas. Signaling through c-Met as well as exposure to hypoxia can stimulate glioma cell migration and invasion. In several cancer cell types, hypoxia was shown to activate the c-met promoter, which contains hypoxia inducible factor-1 (HIF-1) binding sites. We hypothesized that hypoxia might upregulate c-Met also in glioma cells. Analyzing 18 different glioblastoma cell lines and 10 glioblastoma primary cultures, we found that in 50% of both the cell lines and the primary cultures c-Met protein levels were increased following exposure to hypoxia. Upregulation of c-met in response to hypoxia was also detected at the transcriptional level. In all primary cultures and in 16 of the 18 cell lines (89%), HIF-1 alpha levels were increased by hypoxia. Transfection of siRNA against HIF-1 alpha abgrogated the hypoxic induction of c-Met, suggesting that c-Met expression is upregulated by a HIF-1 alpha-dependent mechanism. Hypoxia sensitized glioblastoma cell lines which showed hypoxic induction of c-Met to the motogenic effects of SF/HGF. These findings suggest that approximately half of all human glioblastomas respond to hypoxia with an induction of c-Met, which can enhance the stimulating effect of SF/HGF on tumor cell migration.

A Novel One-Armed Anti-c-Met Antibody Inhibits Glioblastoma Growth In vivo

Expression of the receptor tyrosine kinase c-Met and its ligand scatter factor/hepatocyte growth factor (SF/HGF) are strongly increased in glioblastomas, where they promote tumor proliferation, migration, invasion, and angiogenesis. We used a novel one-armed anti-c-Met antibody to inhibit glioblastoma growth in vivo. U87 glioblastoma cells (c-Met and SF/HGF positive) or G55 glioblastoma cells (c-Met positive and SF/HGF negative) were used to generate intracranial orthotopic xenografts in nude mice. The one-armed 5D5 (OA-5D5) anti-c-Met antibody was infused intratumorally using osmotic minipumps. Following treatment, tumor volumes were measured and tumors were analyzed histologically for extracellular matrix (ECM) components and proteases relevant to tumor invasion. Microarray analyses were done to determine the effect of the antibody on invasion-related genes. U87 tumor growth, strongly driven by SF/HGF, was inhibited > 95% with OA-5D5 treatment. In contrast, G55 tumors, which are not SF/HGF driven, did not respond to OA-5D5, suggesting that the antibody can have efficacy in SF/HGF-activated tumors. In OA-5D5-treated U87 tumors, cell proliferation was reduced > 75%, microvessel density was reduced > 90%, and apoptosis was increased > 60%. Furthermore, OA-5D5 treatment decreased tumor cell density > 2-fold, with a consequent increase in ECM deposition and increased immunoreactivity for laminin, fibronectin, and tenascin. Microarray studies showed no increase in these ECM factors, rather down-regulation of urokinase-type plasminogen activator and matrix metalloproteinase 16 in glioblastoma cells treated with OA-5D5. Local treatment with OA-5D5 can almost completely inhibit intracerebral glioblastoma growth when SF/HGF is driving tumor growth. The mechanisms of tumor inhibition include antiproliferative, antiangiogenic, and proapoptotic effects.

BMPs restrict the position of premuscle masses in the limb buds by influencing Tcf4 expression

Previous studies have shown the distally retreating source of Scatter factor/Hepatocyte growth factor (SF/HGF) can account for the distal migration of myogenic precursor cells in the limb bud mesenchyme. However, the normal expression pattern of Sf/Hgf alone does not explain the distribution of muscle precursor cells. Hence, the position of the dorsal and ventral premuscle masses suggests the presence of additional patterning factors. We present evidence that BMP2 and 4 can act as such factors by inhibiting the expression of Tcf4, a downstream element of the canonical Wnt pathway. The normal position of muscle cells depends on the correct distribution of BMP and SF/HGF throughout the limb bud mesenchyme. Removal or inhibition of the BMP signals within the limb margins leads to a shift in position resulting in the fusion of the dorsal and ventral premuscle masses towards the manipulated areas. In the absence of BMPs, mispositioning requires the presence of SF/HGF. Consequently, ectopic application of exogenous SF/HGF in the presence of BMP signals does not change muscle positioning. We conclude that correct positioning of the premuscle masses in the limb buds is controlled by the combined influence of SF/HGF signals – guiding cells mainly in the proximo-distal axis – and BMP signals that restrict the positioning to the dorsal and ventral central portions of the limb buds.

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.

Scatter factor/hepatocyte growth factor in brain tumor growth and angiogenesis.

The multifunctional growth factor scatter factor/hepatocyte growth factor (SF/HGF) and its receptor tyrosine kinase c-Met have emerged as key determinants of brain tumor growth and angiogenesis. SF/HGF and c-Met are expressed in brain tumors, the expression levels frequently correlating with tumor grade, tumor blood vessel density, and poor prognosis. Overexpression of SF/HGF and/or c-Met in brain tumor cells enhances their tumorigenicity, tumor growth, and tumor-associated angiogenesis. Conversely, inhibition of SF/HGF and c-Met in experimental tumor xenografts leads to inhibition of tumor growth and tumor angiogenesis. SF/HGF is expressed and secreted mainly by tumor cells and acts on c-Met receptors that are expressed in tumor cells and vascular endothelial cells. Activation of c-Met leads to induction of proliferation, migration, and invasion and to inhibition of apoptosis in tumor cells as well as in tumor vascular endothelial cells. Activation of tumor endothelial c-Met also induces extracellular matrix degradation, tubule formation, and angiogenesis in vivo. SF/HGF induces brain tumor angiogenesis directly through only partly known mechanisms and indirectly by regulating other angiogenic pathways such as VEGF. Different approaches to inhibiting SF/HGF and c-Met have been recently developed. These include receptor antagonism with SF/HGF fragments such as NK4, SF/HGF, and c-Met expression inhibition with U1snRNA/ribozymes; competitive ligand binding with soluble Met receptors; neutralizing antibodies to SF/HGF; and small molecular tyrosine kinase inhibitors. Use of these inhibitors in experimental tumor models leads to inhibition of tumor growth and angiogenesis. In this review, we summarize current knowledge of how the SF/HGF:c-Met pathway contributes to brain tumor malignancy with a focus on glioma angiogenesis.

Neural stem cell migration toward gliomas in vitro

Various in vivo studies demonstrated a migration tendency of neural stem cells (NSCs) toward gliomas, making these cells a potential carrier for delivery of therapeutic genes to disseminated glioma cells. We analyzed which factors determine NSC migration and invasion in vitro. Conditioned media prepared from 10 different human glioma cell lines, as well as 13 different tumor-associated growth factors, were analyzed for their chemotactic effects on murine C17.2 NSCs. The growth factor receptor status was analyzed by reverse transcriptase-polymerase chain reaction. Invasion of NSCs into multicellular tumor spheroids generated from 10 glioma cell lines was quantified. NSCs displayed a heterogeneous migration pattern toward glioma spheroids as well as toward glioma-cell-conditioned medium. Chemotactic migration was stimulated up to fivefold by conditioned medium as compared to controls. In coculture assays, NSC invasion varied from single cell invasion into glioma spheroids to complete dissemination of NSCs into glioma spheroids of different cell lines. Among 13 different growth factors, scatter factor/hepatocyte growth factor (SF/HGF) was the most powerful chemoattractant for NSCs, inducing a 2.5-fold migration stimulation. An antibody against SF/HGF inhibited migratory stimulation induced by conditioned media. NSC migration can be stimulated by various growth factors, similar to glioma cell migration. The extent to which NSCs infiltrate three-dimensional glioma cell aggregates appears to depend on additional factors, which are likely to include cell-to-cell contacts and interaction with extracellular matrix proteins.

Targeting the c-Met Pathway Potentiates Glioblastoma Responses to γ-Radiation

Resistance to current cytotoxic therapies limits the treatment of most solid malignancies. This results, in part, from the overactivation of receptor tyrosine kinases and their downstream pathways in tumor cells and their associated vasculature. In this report, we ask if targeting the multifunctional mitogenic, cytoprotective, and angiogenic scatter factor/hepatocyte growth factor (SF/HGF)/c-Met pathway potentiates antitumor responses to gamma-radiation. Endogenous expression of SF/HGF and c-Met was targeted in U87 MG human malignant glioma cells and xenografts using chimeric U1/ribozymes. The effects of U1/ribozymes +/- gamma-radiation on glioma cell proliferation, apoptosis, xenograft growth, and animal survival were examined. U1/ribozymes knocked down SF/HGF and c-Met mRNA and protein levels, sensitized cells to gamma-radiation (P < 0.005), and enhanced radiation-induced caspase-dependent cytotoxicity in vitro (P < 0.005). Intravenous U1/ribozyme therapy as liposome/DNA complexes or radiation alone modestly and transiently inhibited the growth of s.c. U87 xenografts. Combining the therapies caused tumor regression and a 40% tumor cure rate. In animals bearing intracranial xenografts, long-term survival was 0% in response to radiation, 20% in response to intratumoral adenoviral-based U1/ribozyme delivery, and 80% (P < 0.0005) in response to combining U1/ribozymes with radiation. This apparent synergistic antitumor response was associated with a approximately 70% decrease in cell proliferation (P < 0.001) and a approximately 14- to 40-fold increase in apoptosis (P < 0.0001) within xenografts. Targeting the SF/HGF/c-Met pathway markedly potentiates the anti-glioma response to gamma-radiation. Clinical trials using novel SF/HGF/c-Met pathway inhibitors in glioma and other malignancies associated with c-Met activation should ultimate include concurrent radiation and potentially other cytotoxic therapeutics.

Regulation of c-Met-dependent gene expression by PTEN

Receptor tyrosine kinases (RTK) and the tumor suppressor PTEN co-regulate oncogenic cell signaling pathways. How these interactions influence gene transcription is inadequately understood. We used expression microarrays to investigate the effects of PTEN on gene expression changes caused by activating c-Met in human glioblastoma cells. c-Met activation by scatter factor/hepatocyte growth factor (SF/HGF) altered the expression of 27-fold more genes in PTEN-null U-373MG cells than in PTEN homozygous primary normal human astrocytes (523 vs 19 genes). Restoring wt-PTEN in U-373MG cells dramatically altered patterns of c-Met regulated gene expression. This effect was varied depending on the specific gene in question. PTEN reduced the number of c-Met regulated transcripts from 931 to 502, decreased the relative number of genes upregulated by c-Met from 46 to 25%, and increased the relative number of downregulated genes from 54 to 75%. PTEN and c-Met co-regulated many genes involved in cell growth regulation such as oncogenes, growth factors, transcription factors, and constituents of the ubiquitin pathway. c-Met activation in PTEN-null (but not PTEN reconstituted) cells led to upregulation of the EGFR agonist TGFalpha and subsequently to EGFR activation. Using PTEN mutants, we found that PTEN's transcriptional effects were either lipid-phosphatase dependent, protein-phosphatase dependent, or phosphatase-independent. These results show that PTEN has critical and mechanistically complex effects on RTK-regulated gene transcription. These findings expand our understanding of tumor promoter/suppressor inter-relationships and downstream transcriptional effects of PTEN loss and c-Met overexpression in malignant gliomas.

Tenascin‐C and SF/HGF produced by myofibroblasts in vitro provide convergent proinvasive signals to human colon cancer cells through RhoA and Rac

Myofibroblasts are present at the invasion front in colon cancer. In an attempt to understand their putative proinvasive activity, we have developed an in vitro model. Myofibroblasts isolated from colon cancer tissue or obtained through transdifferentiation of colon fibroblasts by transforming growth factor (TGF)-beta stimulate invasion of colon cancer cells into collagen type I and Matrigel. We identified two convergent proinvasive agents secreted by myofibroblasts: namely scatter factor/hepatocyte growth factor (SF/HGF) and the TGF-beta-upregulated extracellular matrix glycoprotein tenascin-C (TNC), each of which is necessary though not sufficient for invasion. Myofibroblast-stimulated invasion into collagen type I is characterized by a change from a round, nonmigratory morphotype with high RhoA and low Rac activity to an elongated, migratory morphotype with low RhoA and high Rac activity. RhoA inactivation is determined by the epidermal growth factor (EGF)-like repeats of TNC through EGF-receptor signaling that confers a permissive and priming signal for the proinvasive activity of SF/HGF that activates Rac via c-Met. We confirmed the validity of this mechanism by using pharmacological modulators and dominant negative or constitutive active mutants that interfere with RhoA-Rho kinase and Rac signaling. Our in vitro results point to a new putative proinvasive signal for colon cancer cells provided by myofibroblasts in the tumor stroma.

Interleukin 6 Indirectly Induces Keratinocyte Migration

IL-6-deficient transgenic mice (IL-6 KO) display significantly delayed cutaneous wound healing. To further elucidate the role of IL-6 in skin wound healing, epidermal keratinocyte and dermal fibroblast cells were isolated from neonatal IL-6 KO mice and treated with rmIL-6. It was found that rmIL-6 alone did not significantly modulate the proliferation or migration of cultured IL-6 KO keratinocytes. rmIL-6, however, significantly induced the migration of IL-6 KO keratinocytes (up to 5-fold) when co-cultured with dermal fibroblasts. Culture supernatants from IL-6-treated fibroblasts were also found to induce the migration of keratinocytes to a similar degree. Genomics analysis of treated fibroblasts indicated that rmIL-6 does not induce any known soluble keratinocyte migratory factors. rmIL-6 treatment of fibroblast, however, induced a rapid and sustained phosphorylation of STAT3 protein. These data indicate that IL-6 could influence wound healing by inducing keratinocyte migration through the production of a soluble fibroblast-derived factor, and its activity may be associated with STAT3 activation.

Microscopic analysis of the cellular events during scatter factor/hepatocyte growth factor‐induced epithelial tubulogenesis

Scatter factor/hepatocyte growth factor (SF/HGF), a large multifunctional polypeptide growth and motility factor, is known to play important roles during embryonic development, adult tissue growth and repair. In an established three-dimensional type I collagen model, SF/HGF induces Madin-Darby canine kidney (MDCK) epithelial cysts to form long, branching tubules (tubulogenesis). In addition, the composition of the surrounding extracellular matrix (ECM) has been shown to modulate SF/HGF-induced morphogenesis, where tubulogenesis was completely abrogated in Matrigel basement membrane. Many cellular events that occur during SF/HGF-mediated remodelling, and its modulation by the ECM, remain unclear. We have investigated these mechanisms through microscopic examination of the time-course of SF/HGF-induced responses in MDCK cysts cultured in type I collagen or Matrigel. We found that early responses to SF/HGF were matrix-independent. Changes included increased paracellular spacing between normally closely apposed lateral membranes, and the formation of filopodial processes, indicating a partial motile response. Cell-cell contact was maintained, with the persistence of cell junctions. Therefore, while one or a number of ECM components are preventing SF/HGF-primed cells from undergoing an invasive and/or migratory programme, non-permissive matrices are not preventing SF/HGF signalling to the cell. Later matrix-dependent responses, which occurred in type I collagen but not Matrigel, included the formation of basal protrusions that comprise two or more neighbouring cells, which extend to form nascent tubules. Modified polarity of cells comprising the basal protrusions was evident, with a marker for the apical membrane being found in the same region as adherens junctions and desmosomes, typically localized at lateral membranes. We propose a model for SF/HGF-induced tubulogenesis in which tubules form from basal protrusions of adjacent cells. This mechanism of in vitro tubule formation has many similarities to reported in vivo epithelial tubulogenesis.

Glioblastoma and cerebral microvascular endothelial cell migration in response to tumor-associated growth factors.

Glioma cell migration is determined by a complex interplay between soluble motogens and extracellular matrix components. Several growth factors are thought to be involved in glioma cell migration; however, little is known about their motogenic potency relative to one another. Using modified Boyden chamber assays, we compared the chemotactic effects of scatter factor/hepatocyte growth factor (SF/HGF), transforming growth factor (TGF)-alpha, TGF-beta1, TGF-beta2, epidermal growth factor (EGF), fibroblast growth factor (FGF)-1, FGF-2, insulin-like growth factor (IGF)-1, IGF-2, platelet-derived growth factor (PDGF)-AA, PDGF-BB, vascular endothelial growth factor (VEGF), pleiotrophin (PTN), and midkine (MK) in concentrations ranging from 1 pmol/L to 50 nmol/L on three different human glioblastoma cell lines. Checkerboard analyses distinguished between chemotaxis and chemokinesis. We further investigated the motogenic effects on human cerebral microvascular endothelial cells and analyzed receptor expression profiles. SF/HGF was the most potent chemotactic factor for all three glioblastoma cell lines, inducing up to 33-fold stimulation of migration. TGF-alpha showed the second strongest effect (up to 17-fold stimulation), and FGF-1 was also chemotactic for all three glioblastoma cell lines analyzed (maximal 4-fold effect). EGF, FGF-2, IGF-1, IGF-2, TGF-beta1, and TGF-beta2 were chemotactic for one or two of the cell lines (2- to 4-fold effects), whereas PDGF-AA, PDGF-BB, VEGF, PTN, and MK had no effect. In contrast, the most potent stimulators of cerebral microvascular endothelial cell migration were PDGF-AA (4-fold) and PDGF-BB (6-fold). The expression levels of SF/HGF and TGF-alpha as well as their respective receptors, MET and EGFR, are known to correlate with glioma malignancy grade. The particularly strong motogenic effects of these two growth factors suggest that they could be promising targets for an antimigratory component of glioma therapy, at least in comparison with the 12 other factors that were analyzed.

Neuroprotection by scatter factor/hepatocyte growth factor and FGF-1 in cerebellar granule neurons is phosphatidylinositol 3-kinase/akt-dependent and MAPK/CREB-independent.

Neuroprotective actions of scatter factor/hepatocyte growth factor (SF/HGF) have not been described. We examined the effects of SF/HGF in comparison to acidic fibroblast growth factor-1 (FGF-1) on N-methyl-D-aspartate (NMDA) and quinolinic acid (QUIN)-induced excitotoxicity in primary cerebellar granule neurons. Exposure to NMDA or QUIN for 24 h resulted in concentration-dependent cell death (p < 0.001) that was completely attenuated (p < 0.001) by pre-treatment of cells with SF/HGF (50 ng/mL) or FGF-1 (40 ng/mL). SF/ HGF and FGF-1 activated both Akt and MAP-kinase > threefold (p < 0.001). Neither SF/HGF nor FGF-1 activated cyclic AMP-response element binding protein (CREB), a downstream target of MAP-kinase, whereas brain-derived neurotrophic factor (BDNF) activated both MAP-kinase and CREB in granule neurons. Neuroprotection against NMDA or QUIN by SF/HGF and FGF-1 was negated by the addition of LY294002 (10 microM) or wortmannin (100 microM), two distinct inhibitors of phosphatidylinositol 3-kinase (P13-K), but not by the MAP-kinase kinase (MEK) inhibitor PD98059 (33 microm). Likewise, expression of a dominant-negative mutant of Akt (Akt-kd) completely prevented the neuroprotective actions of SF/HGF and FGF-1. Overexpression of a constitutively activated Akt (Akt-myr) or wild-type Akt (wtAkt) attenuated excitotoxic cell death. These data show that both SF/HGF and FGF-1 protect cerebellar granule neurons against excitotoxicity with similar potency in a P13-K/Akt-dependent and MAP-kinase/CREB-independent manner.

In vivo targeting of SF/HGF and c-met expression via U1snRNA/ribozymes inhibits glioma growth and angiogenesis and promotes apoptosis.

The multifunctional growth factor scatter factor/hepatocyte growth factor (SF/HGF) and its receptor c-met have been implicated in the genesis, malignant progression, and chemo/radioresistance of multiple human malignancies, including gliomas. We examined the antitumor effects of targeting SF/HGF and c-met expression in pre-established glioma xenografts by using novel chimeric U1snRNA/ribozymes. Transient expression of anti-SF/HGF and anti-c-met U1snRNA/ribozymes inhibited SF/HGF and c-met expression, c-met receptor activation, tumor cell migration, and anchorage-independent colony formation in vitro. Delivery of U1snRNA/ribozymes to established subcutaneous glioma xenografts via liposome-DNA complexes significantly inhibited tumor growth as well as tumor SF/HGF and c-met expression levels. Histologic analysis of tumors treated with U1snRNA/ribozymes showed a significant decrease in blood vessel density, an increase in activation of the pro-apoptotic enzyme caspase-3, and an increase in tumor cell apoptosis. Treatment of animals bearing intracranial glioma xenografts with anti-SF/HGF and anti-c-met U1snRNA/ribozymes by either intratumoral injections of adenoviruses expressing the transgenes or intravenous injections of U1snRNA/ribozyme-liposome complexes substantially inhibited tumor growth and promoted animal survival. We demonstrate that SF/HGF and/or c-met expression can be targeted in vivo to inhibit tumor growth. In addition, our findings represent the first in vivo application of chimeric U1snRNA/ribozymes, which have numerous potential therapeutic gene-targeting applications.

Motility factors identified in supernatants of human cholangiocarcinoma cell lines

Motility factors, e.g. SF/HGF (scatter factor/hepatocyte growth factor) or AMF (autocrine motility factor) can influence the migration of tumor cells in vitro and may facilitate invasive growth and metastases in vivo. The production of motility factors was studied in cell lines derived from human cholangiocarcinomas. Culture supernatants from 5 different cholangiocarcinoma cell lines (EGI-1, RPMI 7451, MZ CHA-1, MZ CHA-2 and MZ CHA-3) were analyzed in scatter assays with NRK and MDCK cells as indicator cells which react with cellular migration in the presence of motility factors. Culture supernatants from 4 of the 5 cell lines investigated induced migration of the indicator cells thus demonstrating the production of motility factors. Three of the cell lines (MZ CHA-1, MZ CHA-2, RPMI 7451) produced a factor with a molecular weight ranging between 50 and 100 kDa, EGI-1 cells secreted a factor with a molecular weight >100 kDa. None of the factors was identical to HGF as demonstrated by the lacking reactivity in a HGF specific ELISA and by the inability to induce scattering of HPAF indicator cells like HGF. Similar to SF/HGF, the activity of the EGI-1 factor was inhibited by the proteoglycan heparin and stimulated the chemotactic cell migration, but in contrast to SF/HGF it could not induce invasive growth of NRK cells. The production of scatter factors could be involved in tumor progression and formation of metastases of cholangiocarcinomas.

Signaling pathways in the induction of c-met receptor expression by its ligand scatter factor/hepatocyte growth factor in human glioblastoma.

Scatter factor/hepatocyte growth factor (SF/HGF) and its tyrosine kinase receptor c-met are developmentally expressed, neuroprotective, and tumorigenic within the CNS. In the present study SF/HGF is shown to induce the expression of c-met in two human glioblastoma cell lines, U-373 MG and T98G, and the signaling pathways involved in this induction are dissected. SF/HGF activated mitogen-activated protein kinase (MAPK) and inhibition of either Ras or MAPK-kinase completely inhibited SF/HGF-mediated c-met induction. Inhibition of phospholipase-C (PLC) did not affect c-met induction in either cell line. Inhibition of phosphoinositide 3-kinase (PI3-kinase) substantially reduced c-met induction by SF/HGF in T98G cells but had no effect in U-373 MG cells. Protein kinase C (PKC) inhibition reduced c-met induction in T98G cells but not in U-373 MG cells. SF/HGF induced the expression of c-fos and c-jun mRNA and increased the levels of AP-1 transcription factor in both cells lines as determined by AP-1-luciferase reporter expression. Transfection of either cell line with TAM-67, a dominant negative for the jun transactivation domain, completely inhibited AP-1 and c-met induction by SF/HGF. These results support a model of c-met induction by SF/HGF in human glioma cells that uniformly involves Ras, MAPK, and AP-1 and additionally involves PI3-kinase and PKC in some cell lines.

Sequential activation of ERK and repression of JNK by scatter factor/hepatocyte growth factor in madin-darby canine kidney epithelial cells.

The scattering of Madin-Darby canine kidney (MDCK) epithelial cells by scatter factor/hepatocyte growth factor (SF/HGF) is associated with transcriptional induction of the urokinase gene, which occurs essentially through activation of an EBS/AP1 response element. We have investigated the signal transduction pathways leading to this transcriptional response. We found that SF/HGF induces rapid and sustained phosphorylation of the extracellular signal-regulated kinase (ERK) MAPK while stimulating weakly and then repressing phosphorylation of the JUN N-terminal kinase (JNK) MAPK for several hours. This delayed repression of JNK was preceded by phosphorylation of the MKP2 phosphatase, and both MKP2 induction and JNK dephosphorylation were under the control of MEK, the upstream kinase of ERK. ERK and MKP2 stimulate the EBS/AP1-dependent transcriptional response to SF/HGF, but not JNK, which inhibits this response. We further demonstrated that depending on cell density, the RAS-ERK-MKP2 pathway controls this transrepressing effect of JNK. Together, these data demonstrate that in a sequential manner SF/HGF activates ERK and MKP2, which in turn dephosphorylates JNK. This sequence of events provides a model for efficient cell scattering by SF/HGF at low cell density.