Hepatocyte growth factor (HGF) is associated with tumour progression and increases the invasiveness of prostate carcinoma cells. Migration and invasion require coordinated reorganisation of the actin cytoskeleton and regulation of cell-adhesion dynamics. Rho-family GTPases orchestrate both of these cellular processes. p21-activated kinase 4 (PAK4), a specific effector of the Rho GTPase Cdc42, is activated by HGF, and we have previously shown that activated PAK4 induces a loss of both actin stress fibres and focal adhesions. We now report that DU145 human prostate cancer cells with reduced levels of PAK4 expression are unable to successfully migrate in response to HGF, have prominent actin stress fibres, and an increase in the size and number of focal adhesions. Moreover, these cells have a concomitant reduction in cell-adhesion turnover rates. We find that PAK4 is localised at focal adhesions, is immunoprecipitated with paxillin and phosphorylates paxillin on serine 272. Furthermore, we demonstrate that PAK4 can regulate RhoA activity via GEF-H1. Our results suggest that PAK4 is a pluripotent kinase that can regulate both actin cytoskeletal rearrangement and focal-adhesion dynamics.

Plasminogen-related growth factors, a new family of polypeptide growth factors with the basic domain organization and mechanism of activation of the blood proteinase plasminogen, include hepatocyte growth factor/scatter factor (<em>HGF</em>/SF), a potent effector of the growth, movement, and differentiation of epithelia and endothelia, and hepatocyte growth factor-like/macrophage stimulating protein (<em>HGF</em>1/MSP), an effector of macrophage chemotaxis and phagocytosis. Phylogeny of the serine proteinase domains and analysis of intron-exon boundaries and kringle sequences indicate that <em>HGF</em>/SF, <em>HGF</em>1/MSP, plasminogen, and apolipoprotein (a) have evolved from a common ancestral gene that consisted of an N-terminal domain corresponding to plasminogen activation peptide (PAP), 3 copies of the kringle domain, and a serine proteinase domain. Models of the N domains of <em>HGF</em>/SF, <em>HGF</em>1/MSP, and plasminogen, characterized by the presence of 4 conserved Cys residues forming a loop in a loop, have been modeled based on disulfide-bond constraints. There is a distinct pattern of charged and hydrophobic residues in the helix-strand-helix motif proposed for the PAP domain of <em>HGF</em>/SF; these may be important for receptor interaction. Three-dimensional structures of the 4 kringle and the serine proteinase domains of <em>HGF</em>/SF were constructed by comparative modeling using the suite of programs COMPOSER and were energy minimized. Docking of a lysine analogue indicates a putative lysine-binding pocket within kringle 2 (and possibly another in kringle 4). The models suggest a mechanism for the formation of a noncovalent <em>HGF</em>/SF homodimer that may be responsible for the activation of the Met receptor. These data provide evidence for the divergent evolution and structural similarity of plasminogen, <em>HGF</em>/SF, and <em>HGF</em>1/MSP, and highlight a new strategy for growth factor evolution, namely the adaptation of a proteolytic enzyme to a role in receptor activation.

Hepatocyte growth factor (HGF), the ligand for the Met receptor tyrosine kinase, induces epithelial cell dispersal, invasion, and morphogenesis, events that require remodeling of the actin cytoskeleton. The scaffold protein Gab1 is essential for these biological responses downstream from Met. We have identified p21-activated kinase 4 (Pak4) as a novel Gab1-interacting protein. We show that in response to HGF, Gab1 and Pak4 associate and colocalize at the cell periphery within lamellipodia. The association between Pak4 and Gab1 is dependent on Gab1 phosphorylation but independent of Pak4 kinase activity. The interaction is mediated through a region in Gab1, which displays no homology to known Gab1 interaction motifs and through the guanine exchange factor-interacting domain of Pak4. In response to HGF, Gab1 and Pak4 synergize to enhance epithelial cell dispersal, migration, and invasion, whereas knockdown of Pak4 attenuates these responses. A Gab1 mutant unable to recruit Pak4 fails to promote epithelial cell dispersal and an invasive morphogenic program in response to HGF, demonstrating a physiological requirement for Gab1-Pak4 association. These data demonstrate a novel association between Gab1 and Pak4 and identify Pak4 as a key integrator of cell migration and invasive growth downstream from the Met receptor.

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering through the tyrosine kinase-type HGF/SF receptor c-Met. We have previously shown that Rho small G protein (Rho) is involved in the HGF/SF-induced scattering of Madin-Darby canine kidney (MDCK) cells by regulating at least the assembly and disassembly of stress fibers and focal adhesions, but it remains unknown how c-Met regulates Rho activity. We have found here a novel signaling pathway of c-Met consisting of SHP-2-Rho that regulates the assembly and disassembly of stress fibers and focal adhesions in MDCK cells. SHP-2 is a protein-tyrosine phosphatase that contains src homology-2 domains. Expression of a dominant negative mutant of SHP-2 (SHP-2-C/S) markedly increased the formation of stress fibers and focal adhesions in MDCK cells and inhibited their scattering. C3, a Clostridium botulinum ADP-ribosyltransferase, and Y-27632, a specific inhibitor for ROCK, reversed the stimulatory effect of SHP-2-C/S on stress fiber formation and the inhibitory effect on cell scattering. Vav2 is a GDP/GTP exchange protein for Rho. Expression of a dominant negative mutant of Vav2 blocked the stimulatory effect of SHP-2-C/S on stress fiber formation. Conversely, expression of mutants of Vav2 that increased stress fiber formation inhibited HGF/SF-induced cell scattering. These results indicate that SHP-2 physiologically modulates the activity of Rho to form stress fibers and focal adhesions and thereby regulates HGF/SF-induced cell scattering. In addition, Vav2 may be involved in the SHP-2-Rho pathway.

When experimental hepatitis was induced by administrating rats with hepatotoxins such as CCl4 and D-galactosamine, HGF mRNA increased dramatically in the injured liver. The increase of HGF mRNA was time- and dose-dependent. At 5 hr after CCl4-treatment, HGF mRNA was remarkably increased; it reached the maximum level at 10 hr and maintained at this level for 40 hr. On the contrary, in D-galactosamine-induced hepatitis, HGF mRNA started to increase from 24 hr after a long lag time. Moreover, HGF mRNA was expressed transiently, decreasing rapidly to the basal level after reaching the maximum level at 36 hr. The degree of induction of HGF mRNA correlates well to the degree of liver damage. In the liver, HGF mRNA could be detected in only non-parenchymal cells, not in parenchymal hepatocytes. These findings suggest that liver is a main producing organ of HGF for liver regeneration after hepatic injury, and HGF is synthesized and secreted by non-parenchymal liver cells so that it stimulates the growth of parenchymal hepatocytes to repair liver tissue in paracrine fashion.

Cell migration is a complex biological process playing a key role in physiological and pathological conditions. During central nervous system development, positioning and function of cortical neurons is tightly regulated by cell migration. Recently, signaling events involving the urokinase-type plasminogen activator receptor, which is a key regulator for the activation of hepatocyte growth factor (HGF), have been implicated in modulating cortical neuron migration. However, the intracellular pathways controlling neuronal migration triggered by the HGF receptor Met have not been elucidated. By combining pharmacological and genetic approaches, we show here that the Ras/ERK pathway and phosphatidylinositol 3-kinase (PI3K) are both required for cortical neuron migration. By dissecting the downstream signals necessary for this event, we found that Rac1/p38 and Akt are required, whereas the c-Jun N-terminal kinase (JNK) and mTOR/p70(s6k) pathways are dispensable. This study demonstrates that concomitant activation of the Ras/ERK, PI3K/Akt, and Rac1/p38 pathways is required to achieve full capacity of cortical neurons to migrate upon HGF stimulation.

For cancer metastasis, tumor cells present in the circulation must first adhere to the endothelium. Integrins lymphocyte function-associated antigen (LFA) 1 and very late antigen 4 play a central role in leukocyte adhesion to the endothelium and subsequent migration into tissues. The majority of tumor cells derived from solid cancers including colorectal cancer do not express suitable adhesion receptors, LFA-1 and very late antigen 4. We investigated the mechanisms of adhesion and transendothelial migration of cancer cells using colorectal carcinoma cell lines. Our results showed the following novel features of CD44 on the cells: (a) colon cancer cells express high levels of CD44; (b) stimulation of cancer cells by CD44 cross-linking or fragmented hyaluronan markedly induces the expression of LFA-1s, some of which reveal an activation epitope on the cells; (c) CD44 cross-linking induces F-actin polymerization in the cell cortex; (d) fragmented hyaluronan induces up-regulation of the activation epitope of LFA-1, which is mediated through protein kinase C; (e) stimulation of CD44 augments the LFA-1-mediated adhesion of cancer cells to endothelial cells and intercellular adhesion molecule 1-transfected cells and facilitates transendothelial migration; (f) stimulation of CD44 also induces expression of the hepatocyte growth factor (HGF) receptor c-Met on cancer cells; and (g) HGF further amplifies the LFA-1-mediated adhesion of cells prestimulated by CD44-derived signaling. Our results indicated that stimulation by CD44 induces "outside-in signaling," which consists of a direct pathway via CD44 and an alternate pathway through the induction of c-Met expression via HGF. Such stimuli augment the expression and trigger the function of integrins via "inside-out signaling" in colon cancer cells, which leads to amplification of integrin-mediated adhesion to the vessel wall and subsequent transendothelial migration.

OBJECTIVE

To compare the relative levels of connective tissue growth factor (CTGF), platelet-derived growth factor alpha (PDGF-AA), and hepatocyte growth factor (HGF) in glial and retinal pigment epithelial (RPE) cells of epiretinal membranes from proliferative vitreoretinopathy (PVR).

METHODS

A total of 37 PVR membranes, of various stages, underwent fluorescent immunohistochemisty and confocal laser scanning microscopy to localize CTGF, HGF, and PDGF-AA in RPE and glial cells.

RESULTS

Numerous RPE, and relatively fewer glial cells, were found in all stages of PVR. CTGF immunoreactivity increased from early to late stage PVR and was principally expressed by RPE cells in early stage, and by glial cells in late stage PVR. HGF, expressed by both RPE and glial cells, was principally expressed in mid-stage PVR. PDGF-AA, expressed by both cell types, demonstrated a uniform level of staining throughout all stages of PVR.

CONCLUSIONS

RPE and glial cells contribute to the expression of CTGF, HGF, and PDGF-AA during PVR, but with specific developmental patterns. PDGF-AA is expressed uniformly throughout all stages of PVR, while HGF expression peaks during mid stage, and CTGF expression is highest during late stage PVR. These results allow for the development of stage-specific therapeutics for PVR that may allow targeting of the early proliferative and/or the late tractional stages of PVR.

OBJECTIVE

To compare the mRNA expression of growth factor receptors in cultured human trabecular meshwork (HTM) cells with ex vivo HTM tissues and to determine whether HTM cells generate a physiologic response after exposure to exogenous growth factors.

METHODS

The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the expression of various growth factor receptor mRNAs using early passaged, cultured HTM cells from donors of several ages. RT-PCR on ex vivo HTM tissues from healthy donors and donors with glaucoma were also used to compare and contrast mRNA expression with cell culture results. After the exogenous administration of growth factors, cell proliferation and extracellular acidification rate studies were used to measure the functional responses of HTM cells to growth factors.

RESULTS

Amplification products of the expected size for 15 growth factor receptors were detected in cultured HTM cells and in ex vivo HTM tissues. The administration of exogenous growth factors showed that (a) hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulinlike growth factor (IGF)-1, tumor necrosis factor (TNF) alpha, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and basic fibroblast growth factor (FGF-2) stimulated cell proliferation, whereas FGF-1 (acidic), transforming growth factor (TGF) alpha, interleukin (IL)-1alpha, nerve growth factor (NGF), and FGF-7 (keratinocyte growth factor [KGF]) had no significant influence on cell proliferation; (b) TGF-beta isoforms significantly inhibited EGF-stimulated trabecular meshwork cell proliferation; and (c) FGF-1 (acidic), TGF-alpha, EGF, IL-1alpha, IL-1beta, HGF, TNF-alpha, PDGF-AA, and IGF-1 significantly stimulated extracellular acidification, whereas FGF-2 (basic), FGF-7 (KGF), TGF-beta1-beta3 and NGF had no significant influence on extracellular acidification.

CONCLUSIONS

These studies show that mRNA for numerous growth factor receptors can be detected in cultured HTM cells and in ex vivo HTM tissues. They also show that many of the receptors are functional, because exogenous growth factor administration elicits a physiologic response. In vivo, these receptors may be activated by growth factors present within the aqueous humor (aquecrine/paracrine) or by growth factors synthesized and released locally by trabecular meshwork cells themselves (autocrine). Specific growth factors acting through high-affinity receptors may be involved in maintaining the normal microenvironment of the HTM and also may be involved in the pathogenesis of primary open-angle glaucoma.

Hepatocyte growth factor (HGF) and beta-catenin both play a crucial role in stimulating hepatocyte proliferation, but whether these 2 pathways cooperate in inducing hepatocyte proliferation is unclear. We have previously reported that beta-catenin forms a complex with c-Met (HGF receptor) that undergoes dissociation because of beta-catenin tyrosine phosphorylation on stimulation by HGF. It is also known that delivery of the human HGF gene cloned in a plasmid under a CMV promoter results in hepatomegaly in mice. In addition, recently characterized beta-catenin transgenic mice also showed hepatomegaly. The present study was based on the hypothesis that HGF-induced hepatomegaly is mediated, at least in part, by activation of the Wnt/beta-catenin pathway. Here we report that delivery of the human HGF gene delivery in mice led to hepatomegaly via beta-catenin activation in the liver in 1- and 4-week studies. The mechanisms of beta-catenin activation in the 1-week study included loss of c-Met-beta-catenin association as well as canonical beta-catenin activation, leading to its nuclear translocation. In the 4-week study, beta-catenin activation was observed via canonical mechanisms, whereas the c-Met-beta-catenin complex remained unchanged. In both studies there was an associated increase in the E-cadherin-beta-catenin association at the membrane. In addition, we generated liver-specific beta-catenin knockout mice, which demonstrated significantly smaller livers. HGF gene delivery failed to induce hepatomegaly in these beta-catenin conditionally null mice. In conclusion, beta-catenin- and HGF-mediated signaling pathways cooperate in hepatocyte proliferation, which may be crucial in liver development, regeneration following partial hepatectomy, and pathogenesis of hepatocellular carcinoma.

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.

The purpose of this study was to evaluate the vasoformative response of isolated vascular explants to a variety of growth factors that have been shown to stimulate angiogenesis. Rings of rat aorta were cultured in collagen gels under serum-free conditions in the presence or absence of vascular endothelial growth factor (VEGF), natural platelet-derived growth factor (PDGF), PDGF-AA, PDGF-BB, insulin-like growth factor-1 (IGF-1), transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta 1 (TGF-beta 1), epidermal growth factor (EGF), interleukin-1 alpha (IL-1 alpha), or hepatocyte growth factor (HGF). The angiogenic response of the rat aorta was stimulated by VEGF, PDGF, PDGF-AA, PDGF-BB, and IGF-1. Maximum stimulatory effects were obtained with VEGF and PDGF-BB. By contrast, TGF-beta 1 and IL-1 alpha had inhibitory activity. No significant effects were observed with TGF-alpha, EGF, or HGF. The vascular outgrowth of VEGF-stimulated cultures was primarily composed of microvessels, whereas that of PDGF- and IGF-1-stimulated cultures contained an increased number of fibroblast-like cells. The inability of TGF-alpha, TGF-beta 1, IL-1 alpha, EGF, and HGF to stimulate rat aortic angiogenesis in serum-free culture suggests that either these factors require the mediatory activity of accessory cells that are not present in the rat aorta model or that blood vessels are heterogeneous in their capacity to respond to different angiogenic factors.

Hepatocyte growth facor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. Our previous study demonstrated that tumor-stromal interaction under hypoxia augments the aggressive invasive features of pancreatic cancer line PK8 through activated HGF/c-Met signaling. The present study investigated whether or not hypoxia increases HGFA expression in PK8 cells and promotes the processing of HGF, and leads to c-Met activation. Moreover, HGFA promoter assays were performed to define whether hypoxia inducible factor-1 alpha (HIF-1alpha) directly activates the HGFA promoter in a hypoxia-dependent fashion. As a result, hypoxia induced the HGFA mRNA and protein expression in PK8 and the elevation under hypoxia was inhibited by the transfection of HIF-1alpha siRNA, thus indicating HIF-1alpha-dependent induction of HGFA. The transfection of siRNA against HGFA to PK8 cells suppressed the conversion to the active HGF, which is secreted from fibroblast MRC5. Furthermore, the phosphorylation of c-Met and cancer invasion of PK8 cells were decreased by the transfection of HGFA siRNA under hypoxia. Using the luciferase reporter system, HIF-1alpha was shown to transactivate the HGFA promoter under hypoxia. These experiments demonstrated for the first time that HGFA is a novel HIF-1 target gene. Under hypoxia, HGFA might be overexpressed and secreted from pancreatic cancer cells, which contributes to accelerate processing of HGF from fibroblast, resulting in the activation of the c-Met pathway. HGF/HGFA/c-Met recruited between cancer-stromal fibroblasts is activated under hypoxic conditions and therefore might play a central role in the aggressive invasion of pancreatic cancer.

Hepatocyte growth factor receptor (HGFR), the product of the MET gene, plays an important role in normal cellular function and oncogenesis. In cancer, HGFR has been implicated in cellular proliferation, cell survival, invasion, cell motility, metastasis and angiogenesis. Activation of HGFR can occur through binding to its ligand, hepatocyte growth factor (HGF), overexpression/amplification, mutation, and/or decreased degradation. Amplification of HGFR can occur de novo or in resistance to therapy. Mutations of HGFR have been described in the tyrosine kinase domain, juxtamembrane domain, or semaphorin domain in a number of tumors. These mutations appear to have gain of function, and also reflect differential sensitivity to therapeutic inhibition. There have been various drugs developed to target HGFR, including antibodies to HGFR/HGF, small-molecule inhibitors against the tyrosine kinase domain of HGFR and downstream targets. Different HGFR inhibitors are currently in clinical trials in lung cancer and a number of solid tumors. Several phase I trials have already been completed, and two specific trials have been reported combining HGFR with epidermal growth factor receptor (EGFR) inhibition in non-small cell lung cancer. In particular, trials involving MetMAb and ARQ197 (tivantinib) have gained interest. Ultimately, as individualized therapies become a reality for cancers, HGFR will be an important molecular target.

BACKGROUND

Pancreatic cancer is one of the representative solid tumors, in which the hypoxic microenvironment plays a crucial role in malignant progression. We previously demonstrated that tumor-stromal interaction under hypoxia enhances the invasiveness of pancreatic cancer cells through hepatocyte growth factor (HGF)/c-Met signaling.

METHODS

We investigated the immunohistochemical expression of hypoxia inducible factor-1alpha (HIF-1alpha) c-Met, and HGF in both cancer and stromal cells using 41 pancreatic cancer tissue specimens, and tried to identify any correlations with the clinical features and survival.

RESULTS

Positive staining for HIF-1alpha was observed in both pancreatic cancer and the surrounding stromal cells in more than 30% of the cases, and it significantly correlated with lymph node metastasis (P < .05). A significant correlation was observed between the expression of HIF-1alpha and HGF in stromal cells (P < .05). In addition, the c-Met expression in cancer cells was found to significantly correlate with the HGF expression in not only cancer but also stromal cells. The disease-free survival rates of the patients with HIF-1alpha in cancer, stromal, c-Met in cancer, and an HGF expression in stromal cells was significantly worse than those without such expressions (P < .05).

CONCLUSIONS

These data suggest that the HGF/c-Met signaling via HIF-1alpha ?may therefore negatively affect the prognosis in patients with pancreatic cancer, and targeting tumor stroma under hypoxia might thus be potentially useful as a novel therapy for this cancer.

We have investigated the use of natural and synthetic collagenous matrices as carriers of exogenous growth factors. A bladder acellular matrix (BAM) was processed from rat bladder and compared with sponge matrix of porcine type 1 collagen. The lyophilized matrices were rehydrated by the aqueous solutions of basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), platelet derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), insulin like growth factor-1 (IGF-1) and heparin binding epidermal growth factor-like growth factor (HB-EGF), to obtain the matrix incorporating each growth factor. The rehydration method enabled the growth factor protein to distribute into the matrix homogeneously. In vivo release test in the mouse subcutis revealed that, the property of BAM for growth factor release was similar to that of collagen sponge. Among the growth factors examined, bFGF release was the most sustained, followed by HGF and PDGF-BB. bFGF released from the two matrices showed similar in vivo angiogenic activity at the mouse subcutis in a dose-dependent manner. These findings demonstrate that the collagenous matrices function as release carriers of growth factors. This feature is promising to create a scaffold, which has a nature to control the tissue regeneration actively.

Hepatocyte growth factor (HGF) is a potent mitogen for a variety of cells including hepatocytes. While rat oval cells are supposed to be one of hepatic stem cells, biological effects of HGF on oval cells and their relevant signal transduction pathways remain to be determined. We sought to investigate them on OC/CDE22 rat oval cells, which are established from the liver of rats fed a choline-deficient/DL-ethionine-supplemented diet. The oval cells were cultured on fibronectin-coated dishes and stimulated with recombinant HGF, transforming growth factor-alpha (TGF-alpha), and thrombopoietin (TPO) under the serum-free medium condition. HGF treatment enhanced [3H]thymidine incorporation into oval cells in a dose-dependent manner. On the contrary, treatment with TGF-alpha or TPO had no significant effects on [3H]thymidine incorporation into the oval cells. c-Met protein was phosphorylated at the tyrosine residues after the HGF treatment. AKT, extracellular signal-regulated kinase 1/2 (ERK1/2), and p70(s6k) were simultaneously activated after the HGF stimulation, peaking at 30min after the treatment. The activation of AKT, p70(s6k), and ERK1/2 induced by HGF was abolished by pre-treatment with LY294002, a phosphoinositide 3-OH kinase (PI3K) inhibitor, and U0126, a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, respectively. When the cells were pre-treated with LY294002 prior to the HGF stimulation, the proliferative action of HGF was completely abrogated, implying that the PI3K/AKT signaling pathway is responsible for the biological effect of HGF. These in vitro data indicate that HGF exerts a proliferative action on hepatic oval cells via activation of the PI3K/AKT signaling pathway.

BACKGROUND

We have previously demonstrated that peroxisome proliferator-activated receptor (PPARγ) activation inhibits hepatocarcinogenesis. We aim to investigate the effect of PPARγ on hepatocellular carcinoma (HCC) metastatic potential and explore its underlying mechanisms.

METHODS

Human HCC cells (MHCC97L, BEL-7404) were infected with adenovirus-expressing PPARγ (Ad-PPARγ) or Ad-lacZ and treated with or without PPARγ agonist (rosiglitazone). The effects of PPARγ on cell migration and invasive activity were determined by wound healing assay and Matrigel invasive model in vitro, and in an orthotopic liver tumour metastatic model in mice.

RESULTS

Pronounced expression of PPARγ was demonstrated in HCC cells (MHCC97L, BEL-7404) treated with Ad-PPARγ, rosiglitazone or Ad-PPARγ plus rosiglitazone, compared with control (Ad-LacZ). Such induction markedly suppressed HCC cell migration. Moreover, the invasiveness of MHCC97L and BEL-7404 cells infected with Ad-PPARγ, or treated with rosiglitazone was significantly diminished up to 60%. Combination of Ad-PPARγ and rosiglitazone showed an additive effect. Activation of PPARγ by rosiglitazone significantly reduced the incidence and severity of lung metastasis in an orthotopic HCC mouse model. Key mechanisms underlying the effect of PPARγ in HCC include upregulation of cell adhesion genes, E-cadherin and SYK (spleen tyrosine kinase), extracellular matrix regulator tissue inhibitors of metalloproteinase (TIMP) 3, tumour suppressor gene retinoblastoma 1, and downregulation of pro-metastatic genes MMP9 (matrix metallopeptidase 9), MMP13, HPSE (heparanase), and Hepatocyte growth factor (HGF). Direct transcriptional regulation of TIMP3, MMP9, MMP13, and HPSE by PPARγ was shown by ChIP-PCR.

CONCLUSIONS

Peroxisome proliferator-activated receptor-gamma exerts an inhibitory effect on the invasive and metastatic potential of HCC in vitro and in vivo, and is thus, a target for the prevention and treatment of HCC metastases.

The c-met proto-oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor (HGF), a potent mitogen and motogen for epithelial cells. Because of its profound effects on cell growth and motility, HGF may be important in the development of cancer metastases in hepatocellular carcinoma (HCC). In this study, we examined HGF concentration and expression of the c-met proto-oncogene product (c-Met) in 62 patients with HCC to determine the relationship between the level of expression and clinicopathological features, and patient outcome following hepatectomy. Western blotting was used to examine the c-Met expression, and HGF concentration in tumors was measured using an enzyme-linked immunosorbent assay. c-Met was found to be overexpressed in HCC compared with nontumorous liver tissue (P < .01), and correlated with an increased incidence of intrahepatic metastases (P = .039). Patients were divided into two groups: low c-Met HCC and high c-Met HCC. Patients with high c-Met HCC had a significantly shorter 5-year survival than patients with low c-Met HCC (33.5% vs. 80.3%, respectively; P < .05). However, there was no correlation between HGF concentration in the tumor tissue and clinicopathological factors and patient survival. These results indicate that the expression of c-Met played an important role in tumor growth and metastases in patients who underwent hepatectomy for HCC.

OBJECTIVE

To determine the role of hepatocyte growth factor (HGF)/c-Met on β-cell survival in diabetogenic conditions in vivo and in response to cytokines in vitro.

METHODS

We generated pancreas-specific c-Met-null (PancMet KO) mice and characterized their response to diabetes induced by multiple low-dose streptozotocin (MLDS) administration. We also analyzed the effect of HGF/c-Met signaling in vitro on cytokine-induced β-cell death in mouse and human islets, specifically examining the role of nuclear factor (NF)-κB.

RESULTS

Islets exposed in vitro to cytokines or from MLDS-treated mice displayed significantly increased HGF and c-Met levels, suggesting a potential role for HGF/c-Met in β-cell survival against diabetogenic agents. Adult PancMet KO mice displayed normal glucose and β-cell homeostasis, indicating that pancreatic c-Met loss is not detrimental for β-cell growth and function under basal conditions. However, PancMet KO mice were more susceptible to MLDS-induced diabetes. They displayed higher blood glucose levels, marked hypoinsulinemia, and reduced β-cell mass compared with wild-type littermates. PancMet KO mice showed enhanced intraislet infiltration, islet nitric oxide (NO) and chemokine production, and β-cell apoptosis. c-Met-null β-cells were more sensitive to cytokine-induced cell death in vitro, an effect mediated by NF-κB activation and NO production. Conversely, HGF treatment decreased p65/NF-κB activation and fully protected mouse and, more important, human β-cells against cytokines.

CONCLUSIONS

These results show that HGF/c-Met is critical for β-cell survival by attenuating NF-κB signaling and suggest that activation of the HGF/c-Met signaling pathway represents a novel strategy for enhancing β-cell protection.

The Wnt-beta-catenin pathway plays a role in liver growth and development. Here, we investigate the direct effect of Wnt-3A on ex vivo liver development. Livers from mouse embryos at day 10 were cultured in serum-free Wnt-3A-conditioned media alone or with HGF and insulin for 72 h and analyzed for histology, proliferation, apoptosis and lineage. Control cultures grown in serum-free conditions or Wnt-3A and sFRP-1 combination display loss of architecture and proliferation and increased apoptosis. In the presence of Wnt-3A, embryonic liver cultures show CK-19-positive cells (biliary phenotype) displaying proliferation, minimal apoptosis and duct-like histological arrangement. HGF and Wnt combination exhibited normal histology as seen in the presence of 10% serum displaying stem cells, hepatocytes and primitive bile ducts. HGF, insulin and Wnt combination provided no additional benefits rather had an overall deleterious effect. Thus, Wnt supports biliary differentiation by enhancing stem cell specification, hepatocyte trans-differentiation and promoting biliary survival. HGF and Wnt combination supports stem cells, hepatocytes and bile ducts. The addition of insulin to the combination of HGF and Wnt provided no growth or differentiation advantage. Our results indicate usefulness of Wnt and HGF in hepatocyte cultures and suggest their balance during normal liver development.

Exogenous HGF/SF converts subconfluent cultures of NBT-II epithelial carcinoma cells into mobile fibroblast-like cells while being only mitogenic for cells maintained at high density. To investigate the potential role of such factor in tumor progression, we generated HGF/SF-producing NBT-II cells by transfection with an expression plasmid containing human HGF/SF cDNA. HGF/SF-producing cells also exhibit a fibroblastic phenotype. Media conditioned by these cells are potent inducers of in vitro tubulogenesis which can be inhibited with specific anti-HGF/SF antibodies; these antibodies are also able to reverse the scattered phenotype of the HGF/SF-producing cells. In addition spheroids of HGF/SF-producing cells are dispersed into 3D collagen gels suggesting an increase of invasive properties of these cells. When injected in nude mice, these HGF/SF-producing cells induce tumors appearing more rapidly than did those obtained with untransfected cells. These results show that HGF/SF can promote motility and invasive properties of NBT-II bladder carcinoma cells and also confers a tumorigenic advantage when acting as an autocrine factor.

This study determines the effect of hepatocyte growth factor (HGF) on post-infarction left ventricular (LV) remodeling and cardiac function. In mice, on day 1 after myocardial infarction (MI), HGF (0.45 mg/kg per day) was injected into the tail vein for 7 days (n = 12). In the control mice (n = 12), 0.9% sodium chloride was injected instead of HGF. Hemodynamic data were obtained in vehicle treated control and HGF-treated hearts 4 weeks after the onset of MI. In the HGF-treated group, cardiac function was well preserved as indicated by LV pressure-volume relationship. These mice exhibited better LV systolic and diastolic function. The infarcted LV wall in HGF-treated heart was thicker as compared to vehicle treated group. Fibrosis and infarct size of the ventricular wall was significantly reduced in the HGF-treated hearts. 5-Bromo-2'-deoxy-uridine (BrdU) and Ki67 positive cardiomyocytes were observed in the border area of the HGF-treated infarcted hearts. c-Met and c-kit positive cardiomyocytes were observed in the border area and epicardium. Angiogenesis was significantly enhanced in HGF-treated hearts as determined by vessel density per unit area. A significant reduction in apoptosis in the HGF-treated hearts was observed compared with control hearts, and was strongly associated with increased Akt activation. Treatment with HGF improved heart function through angiogenesis, ventricular wall thickening, and hypertrophy of cardiomyocytes. The antiapoptotic effect of HGF was mediated by activation of PI3-kinase/Akt pathway.

Melanoma is one of the most devastating malignancies with a rising incidence and lack of effective treatments for advanced disease. Constitutive activation of the mitogen-activated protein kinase (MAPK) pathway and altered expression of alpha(v)beta(3) integrin are critical for melanoma development and progression. Ras-associated protein-1 (Rap1), a Ras family member of the small GTPases, has emerged as a key mediator in these two important processes. In this study, we have shown Rap1 activation in cells derived from two human metastatic melanomas and also in three of seven cutaneous metastatic melanoma tissues. We found increased extracellular signal-regulated kinase (ERK) activity in the tumors with detected Rap1 activity that interestingly harbored neither BRAF nor N-Ras mutation, suggesting a role for Rap1 in ERK activation in vivo. We also showed Rap1 and ERK activation by both hepatocyte growth factor (HGF) and 8CPT-2Me-cAMP (an activator of Epac, a Rap1 guanine nucleotide exchange factor) in two human melanoma cell lines. In addition, the activation of ERK by HGF was reduced, at least in part, by small interfering RNAs against Rap1 and a dominant-negative Rap1. Finally, a functional role for Rap1 activation was shown by Rap1-induced alpha(v)beta(3) integrin activation and consequent increased melanoma cell migration in vitro. Taken together, these results show that Rap1 is involved in the activation of MAPK pathway and integrin activation in human melanoma and suggest a potential role for Rap1 in melanoma tumorigenesis and metastasis.