Hepatocyte Growth Factor/scatter Factor Research Articles

A phase II study of the efficacy and safety of AMG 102 in patients with metastatic renal cell carcinoma

• To evaluate the efficacy and safety of single-agent AMG 102, an investigational, fully human monoclonal antibody to hepatocyte growth factor/scatter factor (HGF/SF), in renal cell carcinoma (RCC). • This open-label phase II study included patients ≥ 18 years old with histologically confirmed, advanced or metastatic RCC (mRCC) and Eastern Cooperative Oncology Group performance status 0 to 2. AMG 102 was administered i.v. at 10 or 20 mg/kg once every 2 weeks. • A two-stage design was used at each dose level and the primary endpoint was objective best confirmed response (by Response Evaluation Criteria in Solid Tumours) at any time. • Sixty-one patients with mRCC enrolled and received AMG 102 (40 at 10 mg/kg; 21 at 20 mg/kg). Overall, 70.5% were men, median age was 59 years (range, 39 to 84 years), and 92% had received previous anti-vascular endothelial growth factor therapy. RCC histologies were: clear cell (75.4%), papillary (11.5%), chromophobe (4.9%) and unclassified (8.2%). • One confirmed partial response occurred at 10 mg/kg, maintained for over 2.5 years; 26 patients (43%) had stable disease, 10 (16%) for ≥ 32 weeks. The median profression-free survival was 3.7 months at 10 mg/kg and 2.0 months at 20 mg/kg. The commonest adverse events were oedema (45.9%), fatigue (37.7%) and nausea (27.9%). Grade 3 or 4 adverse events occurred in 33% of patients, the most common being oedema (9.8%). • Baseline levels of plasma HGF/SF and soluble c-Met as well as archival-tumour c-Met did not correlate with measures of efficacy. • Single-agent AMG 102 was tolerable, but it is unclear if AMG 102 was growth inhibitory in this population of patients with mRCC.

Ligand-independent activation of c-Met by fibronectin and α5β1-integrin regulates ovarian cancer invasion and metastasis

The role of the fibronectin receptor, α5β1-integrin, as an adhesion receptor and in angiogenesis, is well established. However, its role in cancer cell invasion and metastasis is less clear. We describe a novel mechanism by which fibronectin regulates ovarian cancer cell signaling and promotes metastasis. Fibronectin binding to α5β1-integrin led to a direct association of α5-integrin with the receptor tyrosine kinase, c-Met, activating it in a hepatocyte growth factor/scatter factor (HGF/SF) independent manner. Subsequently, c-Met associated with Src and activated Src and focal adhesion kinase (FAK). Inhibition of α5β1-integrin decreased the phosphorylation of c-Met, FAK and Src, both in vitro and in vivo. Independent activation of c-Met by its native ligand, HGF/SF, or overexpression of a constitutively active FAK in HeyA8 cells could overcome the effect of α5β1-integrin inhibition on tumor cell invasion, indicating that α5β1-integrin is upstream of c-Met, Src and FAK. Inhibition of α5β1-integrin on cancer cells in two xenograft models of ovarian cancer metastasis resulted in a significant decrease of tumor burden, which was independent of the effect of α5β1-integrin on angiogenesis. These data suggest that fibronectin promotes ovarian cancer invasion and metastasis through an α5β1-integrin/c-Met/FAK/Src dependent signaling pathway, transducing signals through c-Met in a HGF/SF independent manner.

C-Met and Its Ligand Hepatocyte Growth Factor/Scatter Factor Regulate Mature B Cell Survival in a Pathway Induced by CD74

The signals regulating the survival of mature splenic B cells have become a major focus in recent studies of B cell immunology. Durable B cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism involved in mature B cell homeostasis, the hepatocyte growth factor/scatter factor (HGF)/c-Met pathway. We demonstrate that c-Met activation by HGF leads to a survival cascade, whereas its blockade results in induction of mature B cell death. Our results emphasize a unique and critical function for c-Met signaling in the previously described macrophage migration inhibitory factor/CD74-induced survival pathway. Macrophage migration inhibitory factor recruits c-Met to the CD74/CD44 complex and thereby enables the induction of a signaling cascade within the cell. This signal results in HGF secretion, which stimulates the survival of the mature B cell population in an autocrine manner. Thus, the CD74-HGF/c-Met axis defines a novel physiologic survival pathway in mature B cells, resulting in the control of the humoral immune response.

Biosensors coated with sulfated polysaccharides for the detection of hepatocyte growth factor/scatter factor in cell culture medium

Process control methods for cell culture bioreactors include on-line monitoring of protein concentrations. Bioreactor samples typically contain high amounts of different proteins. The direct detection of a single protein in this complex medium is a challenging task within the development of biosensors with label-free detection. We introduce the development of a mass-sensitive biosensor based on surface acoustic waves (SAW) for the detection of hepatocyte growth factor/scatter factor (HGF/SF) in the serum containing medium of a miniaturized bioreactor for culturing hepatocytes. The specificity of the biosensor was obtained following two approaches. In the first approach, antibodies against HGF (anti-HGF) were immobilized covalently via an intermediate layer of dicarboxy polyethylene glycol on the biosensor surface. In the second approach, dextran sulfate and fucoidan were used as sensor coatings exploiting the fact that HGF binds specifically to those sulfated polysaccharides. Performing HGF assays, similar results were obtained using biosensors coated with dextran sulfate and biosensors coated with anti-HGF. Even higher sensor signals were obtained using biosensors coated with fucoidan, particularly at 37 °C. Therefore, biosensor coatings based on biospecific sulfated polysaccharides offer a simple and cost-saving alternative compared to the commonly used coating with antibodies.

Inhibitory effects of Lang-du extract on the in vitro and in vivo growth of melanoma cells and its molecular mechanisms of action

The purpose of this study is to investigate the effects of Lang-du extract (LDE) from Traditional Chinese Medicine (TCM) Euphorbia fischeriana Steud on the in vitro and in vivo growth of melanoma cells and its molecular mechanisms of action. Our present results have shown that LDE significantly suppressed the in vitro melanoma cell growth in dose- and time-dependent manners. LDE also displayed the synergistic effect with γ-radiation on the reduction of the cell viability in melanoma cells. The animal experimental results further confirmed that compared with the control group without drug treatment, the tumor volume in mice was significantly and time-dependently less in LDE group. The absolute weight of solid tumor in the LDE group was 7-fold lower than that in the control group. Western blot analysis indicated that LDE markedly down-regulated the expression of anti-apoptotic protein Bcl-2 and up-regulated the level of pro-apoptotic protein Bax, eventually leading the reduction of Bcl-2/Bax protein ratios both in the cultured melanoma cells and in the tumors from melanoma-bearing mice. In addition, LDE significantly reduced the tumor progression-associated protein levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor/scatter factor (HGF/SF), and osteopontin (OPN) in tumors from the LDE-treated mice. Our findings suggest that LDE may have a wide therapeutic and/or adjuvant therapeutic application in the treatment of melanoma and other cancer.

Trekking through the Telencephalon: Hepatocyte Growth Factor-Mediated Guidance for Parvalbumin-Expressing Interneurons

Hepatocyte Growth Factor (HGF) Modulates GABAergic Inhibition and Seizure Susceptibility. Bae MH, Bissonette GB, Mars WM, Michalopoulos GK, Achim CL, Depireux DA, Powell EM. Exp Neurol 2010;221(1):129–135. Disrupted ontogeny of forebrain inhibitory interneurons leads to neurological disorders, including epilepsy. Adult mice lacking the urokinase plasminogen activator receptor ( Plaur) have decreased numbers of neocortical GABAergic interneurons and spontaneous seizures, attributed to a reduction of hepatocyte growth factor/scatter factor (HGF/SF). We report that by increasing endogenous HGF/SF concentration in the postnatal Plaur null mouse brain maintains the interneuron populations in the adult, reverses the seizure behavior and stabilizes the spontaneous electroencephalogram activity. The perinatal intervention provides a pathway to reverse potential birth defects and ameliorate seizures in the adult.

Characterization and binding activity of the chondroitin/dermatan sulfate chain from Endocan, a soluble endothelial proteoglycan

Endocan is a recently identified soluble chondroitin/dermatan sulfate (CS/DS) proteoglycan. Synthesized by endothelial cells, it has been found to be over-expressed in the vasculature surrounding a number of tumors, and by promoting growth factor mitogenic activities, hepatocyte growth factor/scatter factor (HGF/SF) in particular, it supports cellular proliferation. In this work, we characterized the glycosaminoglycan (GAG) chain of Endocan, purified either from the naturally producing human umbilical vein endothelial cells (HUVEC) or from a recombinant over-expression system in human embryonic kidney cells (HEK). Compositional analysis using different chondroitinases as well as nuclear magnetic resonance studies revealed that the GAG chains from both sources share many characteristics, with the exception of size (15 and 40kDa, respectively, for HUVEC and HEK-293 cells). The DS-specific, IdoA-containing disaccharides contribute 30% of the chain (15% of which are 2-O-sulfated) and are mostly clustered in tetra- (35%), hexa- (12%), and octa- (5%) saccharide domains. Highly sulfated D, E, and B disaccharide units (HexA2S-GalNAc6S, HexA-GalNAc4S6S, and HexA2S-GalNAc4S) were also detected in significant amounts in both chains and may account for the HGF/SF-binding activity of the CS/DS. This work establishes that HEK-293 cells can be engineered to provide a valuable source of Endocan with authentic CS/DS chains, enabling the purification of sufficient amounts for structural and/or binding analysis and providing a possible model of Endocan CS/DS chain organization.

Inhibition of c-Met with the Specific Small Molecule Tyrosine Kinase Inhibitor SU11274 Decreases Growth and Metastasis Formation of Experimental Human Melanoma

The hepatocyte growth factor/scatter factor (HGF/SF) tyrosine kinase (TK) receptor c-Met plays a crucial role in the development of the invasive phenotype of tumors and thus represents an attractive candidate for targeted therapies in a variety of malignancies, including human malignant melanoma (MM). In contrast to what has been shown previously, we were not able to detect any genetic alterations, either in the juxtamembrane- or in the TK-domain of c-Met, in the studied MM cell lines. Nevertheless, c-Met was constitutively active in these cell lines without exogenous HGF/SF stimulation. The active receptor was localized to the adhesion sites of the cells. Addition of the c-Met TK inhibitor SU11274 specifically decreased the phosphotyrosine signal at the focal adhesions sites, which was accompanied by a decrease in cell proliferation as well as an increase in apoptotic cells. In addition, non-apoptotic concentrations of SU11274 significantly reduced the in vitro migratory capacity of MM cells in the modified Boyden-chamber assay. Administration of SU11274 significantly decreased primary tumor growth as well as the capacity for liver colony formation of MM cells in SCID mice. Our study provides the first evidence for an in vivo antitumor activity of SU11274 in a human melanoma xenograft model, and suggests c-Met as a valid target for the therapy of MM. Consequently, SU11274 treatment might represent a useful strategy for controlling melanoma progression and metastasis in patients with MM.

A Phase Ib Study of AMG 102 in Combination with Bevacizumab or Motesanib in Patients with Advanced Solid Tumors

This phase Ib study evaluated the safety, pharmacokinetics, pharmacodynamics, and antitumor activity of AMG 102, a fully human monoclonal antibody against hepatocyte growth factor/scatter factor (HGF/SF), in combination with bevacizumab or motesanib in patients with advanced solid tumors. Patients with treatment-refractory advanced solid tumors were sequentially enrolled into four cohorts (3, 10, or 20 mg/kg AMG 102 plus 10 mg/kg bevacizumab i.v. every 2 weeks, or 3 mg/kg AMG 102 i.v. every 2 weeks plus 75 mg motesanib orally once daily). Fourteen patients were enrolled and received AMG 102. The combination of AMG 102 with bevacizumab (n = 12) seemed to have acceptable toxicity. The number of patients (n = 2) who received AMG 102 plus motesanib was insufficient to adequately assess safety. No dose-limiting toxicities were reported. Enrollment in the motesanib cohort was suspended because of reports of cholecystitis in other motesanib studies. Treatment-emergent adverse events among patients receiving AMG 102 plus bevacizumab were generally mild and included fatigue (75%), nausea (58%), constipation (42%), and peripheral edema (42%). No anti-AMG 102 antibodies were detected. Bevacizumab did not seem to affect AMG 102 pharmacokinetics. Circulating total HGF/SF increased from baseline throughout the study. Eight of 10 evaluable patients had reductions in tumor dimensions, and stable disease at > or =8, > or =16, and > or =24 weeks occurred in 9, 7, and 4 patients, respectively. Progression-free survival ranged from 7.9 to 121.9 weeks. AMG 102 in combination with bevacizumab was well tolerated. Further evaluation of AMG 102 in combination with antiangiogenic agents is warranted.

Comment on “Effect of transferred NK4 gene on proliferation, migration, invasion, and apoptosis of human prostate cancer DU145 cells” by Dan Yue et al. in Asian Journal of Andrology

Comment on “Effect of transferred NK4 gene on proliferation, migration, invasion, and apoptosis of human prostate cancer DU145 cells” by Dan Yue et al. in Asian Journal of Andrology

Abstract LB-305: Why two are better than one: Suppression of carcinoma cell growth in vitro and in human HGF/SF transgenic SCID by inhibitors of MET (SGX523) and EGFR (erlotinib)

Abstract The signaling cascade originating from hepatocyte growth factor/scatter factor (HGF/SF)-MET ligand-receptor pair is frequently observed in a broad spectrum of human cancers. Inappropriate activation of this pathway has been shown to occur via: paracrine/autocrine activation; by amplification or mutation in the MET gene; or through ligand independent mechanisms (www.vai.org/met). Various MET drugs targeting this pathway are under development and in need of relevant in vivo tumor models to allow assessment of drug efficacy alone or in combination against human MET expressing tumors. Conventional mouse models post a significant limitation on fulfilling such demand as mouse HGF/SF has low affinity/activity on the human MET. Such limitations are abrogated in human HGF/SF transgenic SCIDs (designated hHGFtg-SCID), which, unlike its non-transgenic littermates, produce human HGF/SF to allow human MET signaling on cells of human cancer xenografts. Here, we show that the hHGFtg-SCID mouse significantly enhances tumor xenograft growth of many MET-expressing human carcinoma cell lines including lung, breast, kidney, colon, gastric and pancreatic cancers. Using the hHGFtg-SCID mouse model systems, we demonstrated that SGX523, a MET small molecule kinase inhibitor which suppressed MET phosphorylation and downstream signaling activation in vitro, partially inhibits the growth of xenografts derived from NCI-H596 lung cancer cells, HCC1954 breast cancer cells and HPAF II pancreatic cancer cells. Furthermore, we found that the partial tumor growth inhibition by SGX523 was significantly enhanced by co-administration of erlotinib, the EGFR small molecule kinase inhibitor. In vitro, erlotinib also enhanced SGX523 inhibitory activity in cell proliferation and G1/S cell cycle progression assays. Our data suggest that the hHGFtg-SCID mouse is a valuable system for evaluating MET drugs, and simultaneously targeting MET and EGFR pathways may provide a better treatment for cancers harboring inappropriate activation of both pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-305.

Integrating Novel Protein Engineering and Nanotechnology for Therapeutic Angiogenesis

Therapeutic angiogenesis is an emerging concept that can potentially be harnessed for management of ischemic pathologies. Hepatocyte growth factor/scatter factor (HGF/SF) promotes angiogenesis but the native protein has limited therapeutic potential due to its complex multidomain structure with glycosylation sites. To address this translational challenge, a novel spliced HGF/SF variant, 1K1 encoding the N‐terminal domain and first kringle (K1) domain was engineered with reverse mutations (K132E:R134E) introduced in the K1 domain. To address the clinical requirement of sustained delivery, 1K1 was formulated in a polymeric nanoparticle of 60–140 nm size and with the total loading efficiency 54.27±7.12%. 1K1‐nanoparticle (NP) induced significantly greater angiogenesis both in vitro and in vivo than free 1K1 (P<0.05). 1K1‐NP induced angiogenesis were blocked by PHA665752, LY294002 and PD98059 inhibitors in a concentration‐dependent manner (P<0.05), suggesting that 1K1‐NP induces angiogenesis through MET receptor and downstream MAPK and PI3K pathways. Integrating novel protein engineering strategy and nanotechnology offer stimulating revascularization at the site of ischemic pathology.

Hepatocyte growth factor protects hepatoblastoma cells from chemotherapy-induced apoptosis by AKT activation

Hepatocyte growth factor/scatter factor (HGF) is a ubiquitously expressed molecule that elicits pleiotropic functions on epithelial cells, including mitogenic, motogenic, differentiating, angiogenic and morphogenic effects. In hepatoblastoma (HB), post-operative residual tumor growth and tumor recurrences are often associated with markedly elevated serum levels of HGF, suggesting a link between this molecule and tumor malignancy. Here, we demonstrate that HGF has no impact on overall cell viability and proliferation of HB cells, although signal transduction occurs downstream of HGF, such as c-Met phosphorylation, activation of phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK)/ERK-1/2 signaling. Instead of being mitogenic, HGF confers anti-apoptotic properties upon serum starvation and moreover protects HB cells against strong apoptotic inducers such as cisplatin and camptothecin, thereby contributing to chemotherapeutic resistance. This effect is mainly dependent on the PI3K/AKT signaling pathway, since inhibition by wortmannin resulted in abrogation of HGF-mediated survival, whereas inhibition of the MAPK pathway had no effect. Together, these findings highlight the importance of HGF in tumor cell survival and suggest that HGF and its cognate receptor c-Met should be considered as a candidate for combined therapeutic strategies of advanced pediatric liver tumors.

Biochemical Characterization of AMG 102: A Neutralizing, Fully Human Monoclonal Antibody to Human and Nonhuman Primate Hepatocyte Growth Factor

AMG 102 is a fully human monoclonal antibody that selectively targets and neutralizes hepatocyte growth factor/scatter factor (HGF/SF). A detailed biochemical and functional characterization of AMG 102 was done to support its clinical development for the treatment of cancers dependent on signaling through the HGF/SF:c-Met pathway. In competitive equilibrium binding experiments, AMG 102 bound to human and cynomolgus monkey HGF with affinities of approximately 19 pmol/L and 41 pmol/L, respectively. However, AMG 102 did not detect mouse or rabbit HGF on immunoblots. Immunoprecipitation experiments showed that AMG 102 preferentially bound to the mature, active form of HGF, and incubation of AMG 102/HGF complexes with kallikrein protease indicated that AMG 102 had no apparent effect on proteolytic processing of the inactive HGF precursor. AMG 102 inhibited human and cynomolgus monkey HGF-induced c-Met autophosphorylation in PC3 cells with IC(50) values of 0.12 nmol/L and 0.24 nmol/L, respectively. AMG 102 also inhibited cynomolgus monkey HGF-induced migration of human MDA-MB-435 cells but not rat HGF-induced migration of mouse 4T1 cells. Epitope-mapping studies of recombinant HGF molecules comprising human/mouse chimeras and human-to-mouse amino acid substitutions showed that amino acid residues near the NH(2)-terminus of the beta-chain are critical for AMG 102 binding. Bound AMG 102 protected one trypsin protease cleavage site near the NH(2)-terminus of the beta-chain of human HGF, further substantiating the importance of this region for AMG 102 binding. Currently, AMG 102 is in phase II clinical trials in a variety of solid tumor indications. Mol Cancer Ther; 9(2); 400-9.

Application of Biological Study for Met Expression to Cancer Therapy

Metastasization is an undesirable process in cancer development and may represent the most critical factor in deciding patient prognosis. Organ specificity of the metastasis process suggests the importance of the paracrine factors: one of the most potent paracrine regulators of tumor cell migration is hepatocyte growth factor/scatter factor (HGF/SF). Because the liver-specific growth factor is HGF, its receptor c-Met expression might play a critical role in metastasization to the liver. Activation of HGF/c-Met signaling has been shown to promote cancer cell invasiveness and trigger metastasis though direct involvement of the angiogenic pathway. Given the importance of aberrant HGF/c-Met signaling, several different therapeutic strategies aimed at inhibiting the pathway have been developed and are currently being evaluated in clinical trials. Among these agents, NK4 and AM102 were introduced as HGF inhibitors, and PHA-665752 and Su11274 as c-Met inhibitors and are under study in clinical trials. Further, clinical experience-based study to apply the accumulation of biological knowledge concerning HGF/c-Met to the surgical field is presented.

Abstract A249: Potent protein kinase inhibitors block Pim kinase-mediated increase in prostate epithelial cell migration, regulation of p27 protein half-life, and secretion of hepatocyte growth factor

Abstract Pim-1 proto-oncogene encodes a serine/threonine protein kinase that regulates apoptosis, cell cycle progression, and transcription. The expression of this protein kinase is elevated in both prostate intraepithelial neoplasia (PIN) and prostatic adenocarcinoma, suggesting an important role for Pim-1 kinase in prostate cancer development and growth. To investigate the role of Pim-1 in controlling tumor growth we have synthesized novel benzylidene-thiazolidine2, 4-dione (J. Med. Chem. (2009) 52:74) inhibitors of this kinase. The most potent members of this chemotype have IC50s of 13 nM for Pim-1. To examine the activity of these agents in prostate cancer we have first set out to define the biochemical activity of Pim-1 in epithelial cells. We have expressed Pim-1 in a mouse prostate epithelial cell (MPECs) line that demonstrates stem cell characteristics. We find that Pim-1 expressing cells produce and secrete markedly increased levels of hepatocyte growth factor/scatter factor (HGF/SF), and this protein kinase stimulates increases in HGF/SF mRNA. Additionally, expression of Pim-1 markedly increases HGF/SF induced migration of MPECs. The contribution of Pim-1 kinase to this biochemical pathway is confirmed in murine embryonic fibroblasts (MEFs) that are deficient for all three Pim protein kinases (TKO) and evidence reduced level of HGF mRNA in TKO versus wild type MEFs. The Pim-1-stimulated migration of MPECs is inhibited by two benzylidene-thiazolidine-2, 4-diones, SMI-4a and 16a, as well as known inhibitors of the HGF receptor, c-Met. HGF treatment of MPECs induced p27 upregulation that could be inhibited by the expression of Pim-1, thus allowing cell cycle progression. Further studies showed that expression of Pim-1 did not alter the mRNA level of p27, but enhanced the cell cycle-dependent degradation and thus decreased the half-life of the p27 protein. Consistent with this finding, p27 ubiquitination assays showed that Pim-1 increases this modification in vivo. We found that the Pim-1-mediated ubiquitination is regulated by complex formation between Pim-1 and Skp2, a protein component of the SCF complex, which is known to regulate the ubiquitination and degradation of p27. Pim-1 does not affect Skp2's E3 ligase activity, but appears to inhibit the degradation of Skp2 through phosphorylation. Incubation of these cells with the Pim protein kinase inhibitor, SMI-4a, decreased Skp2 expression and increased p27 and cyclin E expression. Together our data demonstrates the complex pathway by which Pim-1 protein kinase regulates HGF/SF and p27 levels, thus controlling cell migration, proliferation, and potentially transformation. Potent Pim kinase inhibitors block these two signaling pathways thus inhibiting prostate epithelial cell migration and growth. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A249.

PHA665752, a small-molecule inhibitor of c-Met, inhibits hepatocyte growth factor-stimulated migration and proliferation of c-Met-positive neuroblastoma cells

Backgroundc-Met is a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), and both c-Met and its ligand are expressed in a variety of tissues. C-Met/HGF/SF signaling is essential for normal embryogenesis, organogenesis, and tissue regeneration. Abnormal c-Met/HGF/SF signaling has been demonstrated in different tumors and linked to aggressive and metastatic tumor phenotypes. In vitro and in vivo studies have demonstrated inhibition of c-Met/HGF/SF signaling by the small-molecule inhibitor PHA665752. This study investigated c-Met and HGF expression in two neuroblastoma (NBL) cell lines and tumor tissue from patients with NBL, as well as the effects of PHA665752 on growth and motility of NBL cell lines. The effect of the tumor suppressor protein PTEN on migration and proliferation of tumor cells treated with PHA665752 was also evaluated.MethodsExpression of c-Met and HGF in NBL cell lines SH-EP and SH-SY5Y and primary tumor tissue was assessed by immunohistochemistry and quantitative RT-PCR. The effect of PHA665752 on c-Met/HGF signaling involved in NBL cell proliferation and migration was evaluated in c-Met-positive cells and c-Met-transfected cells. The transwell chemotaxis assay and the MTT assay were used to measure migration and proliferation/cell-survival of tumor cells, respectively. The PPAR-γ agonist rosiglitazone was used to assess the effect of PTEN on PHA665752-induced inhibition of NBL cell proliferation/cell-survival and migrationResultsHigh c-Met expression was detected in SH-EP cells and primary tumors from patients with advanced-stage disease. C-Met/HGF signaling induced both migration and proliferation of SH-EP cells. Migration and proliferation/cell-survival were inhibited by PHA665752 in a dose-dependent manner. We also found that induced overexpression of PTEN following treatment with rosiglitazone significantly enhanced the inhibitory effect of PHA665752 on NBL-cell migration and proliferation.Conclusionc-Met is highly expressed in most tumors from patients with advanced-stage, metastatic NBL. Furthermore, using the NBL cell line SH-EP as a model, PHA665752 was shown to inhibit cMet/HGF/SF signaling in vitro, suggesting c-Met inhibitors may have efficacy for blocking local progression and/or metastatic spread of c-Met-positive NBL in vivo. These are novel findings for this disease and suggest that further studies of agents targeting the c-Met/HGF axis in NBL are warranted

Hepatocyte growth factor (HGF) modulates GABAergic inhibition and seizure susceptibility

Disrupted ontogeny of forebrain inhibitory interneurons leads to neurological disorders, including epilepsy. Adult mice lacking the urokinase plasminogen activator receptor ( Plaur) have decreased numbers of neocortical GABAergic interneurons and spontaneous seizures, attributed to a reduction of hepatocyte growth factor/scatter factor (HGF/SF). We report that by increasing endogenous HGF/SF concentration in the postnatal Plaur null mouse brain maintains the interneuron populations in the adult, reverses the seizure behavior and stabilizes the spontaneous electroencephalogram activity. The perinatal intervention provides a pathway to reverse potential birth defects and ameliorate seizures in the adult.

Bone marrow mesenchymal stem cells undergo nemosis and induce keratinocyte wound healing utilizing the HGF/c‐Met/PI3K pathway

We previously showed cell-cell contacts of human dermal fibroblasts to induce expression of the hepatocyte growth factor/scatter factor (HGF) in a process designated as nemosis. Now we report on nemosis initiation in bone marrow mesenchymal stem cells (BMSCs). Because BMSCs are being used increasingly in cell transplantation therapy we aimed to demonstrate a functional effect and benefit of BMSC nemosis for wound healing. Nemotic and monolayer cells were used to stimulate HaCaT keratinocyte migration in a scratch-wound healing assay. Both indicators of nemosis, HGF production and cyclooxygenase-2 expression, were induced in BMSC spheroids. When compared with a similar amount of cells as monolayer, nemotic cells induced keratinocyte in vitro scratch-wound healing in a concentration-dependent manner. The HGF receptor, c-Met, was rapidly phosphorylated in the nemosis-stimulated keratinocytes. Nemosis-induced in vitro scratch-wound healing was inhibited by an HGF-neutralizing antibody as well as the small molecule c-Met inhibitor, SU11274. HGF-induced in vitro scratch-wound healing was inhibited by PI3K inhibitors, wortmannin and LY294002, while LY303511, an inactive structural analogue of LY294002, had no effect. Inhibitors of the mitogen-activated protein kinases MEK/ERK1/2 (PD98059 and U0126), and p38 (SB203580) attenuated HGF-induced keratinocyte in vitro scratch-wound healing. We conclude that nemosis of BMSCs can induce keratinocyte in vitro scratch-wound healing, and that in this effect signaling via HGF/c-Met is involved.

Cancer cells harboring MET gene amplification activate alternative signaling pathways to escape MET inhibition but remain sensitive to Hsp90 inhibitors

Hepatocyte growth factor/scatter factor (HGF/SF) receptor c-Met is implicated in growth, invasion and metastasis of many tumors. Tumor cells harboring MET gene amplification are initially sensitive to c-Met tyrosine kinase inhibitors (TKI), but escape from long-term treatment has not been investigated. C-Met is a client of heat shock protein 90 (Hsp90) and is destabilized by Hsp90 inhibitors, suggesting that these drugs may inhibit tumors driven by MET amplification, although tumor escape under these conditions also has not been explored. Here, we evaluated the initial inhibitory effects of, and the likelihood of escape from, the Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin (17-AAG) and the c-Met TKI SU11274, using two cell lines harboring MET gene amplification. 17-AAG inhibited cell growth in both cell lines and induced substantial apoptosis, whereas SU11274 was only growth inhibitory in one cell line. In both cell lines, c-Met-dependent Akt, Erk and/or STAT3 signaling, as well as activation of the EGFR family, resumed shortly after treatment with c-Met TKI despite sustained c-Met inhibition. PKC δ upregulation may participate in reactivation of c-Met downstream signaling in both cell lines. In contrast to c-Met TKI, 17-AAG destabilized c-Met protein and durably blocked reactivation of downstream signaling pathways and EGFR family members. Our data demonstrate that downstream signaling in tumor cells over-expressing c-Met is not stably suppressed by c-Met TKI, even though c-Met remains fully inhibited. In contrast, Hsp90 inhibitors provide long-lasting suppression of c-Met-dependent signaling, and these drugs should be further evaluated in tumors driven by MET gene amplification.