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.     

Neuropilin-1 promotes human glioma progression through potentiating the activity of the HGF/SF autocrine pathway

Neuropilin-1 (NRP1) functions as a coreceptor through interaction with plexin A1 or vascular endothelial growth factor (VEGF) receptor during neuronal development and angiogenesis. NRP1 potentiates the signaling pathways stimulated by semaphorin 3A and VEGF-A in neuronal and endothelial cells, respectively. In this study, we investigate the role of tumor cell-expressed NRP1 in glioma progression. Analyses of human glioma specimens (WHO grade I-IV tumors) revealed a significant correlation of NRP1 expression with glioma progression. In tumor xenografts, overexpression of NRP1 by U87MG gliomas strongly promoted tumor growth and angiogenesis. Overexpression of NRP1 by U87MG cells stimulated cell survival through the enhancement of autocrine hepatocyte growth factor/scatter factor (HGF/SF)/c-Met signaling. NRP1 not only potentiated the activity of endogenous HGF/SF on glioma cell survival but also enhanced HGF/SF-promoted cell proliferation. Inhibition of HGF/SF, c-Met and NRP1 abrogated NRP1-potentiated autocrine HGF/SF stimulation. Furthermore, increased phosphorylation of c-Met correlated with glioma progression in human glioma biopsies in which NRP1 is upregulated and in U87MG NRP1-overexpressing tumors. Together, these data suggest that tumor cell-expressed NRP1 promotes glioma progression through potentiating the activity of the HGF/SF autocrine c-Met signaling pathway, in addition to enhancing angiogenesis, suggesting a novel mechanism of NRP1 in promoting human glioma progression.     

Epidermal growth factor receptor plays a significant role in hepatocyte growth factor mediated biological responses in mammary epithelial cells

Breast cancers often have deregulated hepatocyte growth factor (HGF) and c-Met signaling that results in increased tumor growth and invasion. Elucidating the mechanism responsible for HGF/c-Met action in breast cancer progression has been difficult as c-Met communicates with a number of secondary receptors that can lead to various pathological outcomes. Understanding how these secondary receptors facilitate HGF/c-Met cellular responses will aid in the development of better therapeutic treatment options for breast cancer patients with elevated HGF signaling. In the present study it was shown that the epidermal growth factor receptor (EGFR) plays a significant role in HGF/c-Met mediated biological activities indicative of advanced tumor pathology, including enhanced proliferation and invasion. The clinically relevant EGFR inhibitor gefitinib was used to determine the role of EGFR in HGF-induced proliferation and motility in several mammary carcinoma cells including PyVmT, MDA-MB-231 and 4T1. Our analyses indicated that EGFR inhibition significantly blocked HGF activation of c-Met and EGFR and that inhibition of these pathways mitigated HGF induced proliferation and motility. The data indicate that this inhibition was not through a direct effect of gefitinib on c-Met, but that EGFR is necessary for c-Met activation in the assays performed. These results provide a novel mechanism of action for EGFR as a mediator of HGF signaling thereby linking EGFR to the oncogenic potential of c-Met in mammary carcinomas cells.     

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.     

Mitogenic synergy through multilevel convergence of hepatocyte growth factor and interleukin-4 signaling pathways

Hepatocyte growth factor (HGF) regulates various physiological and developmental processes in concert with other growth factors, cytokines and hormones. We examined interactions between cell signaling events elicited by HGF and the cytokine interleukin (IL)-4, in the IL-3-dependent murine myeloid cell line 32D transfected with the human HGF receptor, c-Met. HGF was a potent mitogen in these cells, and prevented apoptosis in response to IL-3 withdrawal. IL-4 showed modest anti-apoptotic activity, but no significant mitogenic activity. IL-4 synergistically enhanced HGF-stimulated DNA synthesis, whereas only additive prevention of apoptosis was observed. IL-4 did not enhance HGF-dependent tyrosine phosphorylation of c-Met or Shc. In contrast, HGF-stimulated activation of MAP kinases was enhanced by IL-4, suggesting that the IL-4 and HGF signaling pathways converge upstream of these events. Although phosphatidylinositol 3-kinase (PI3K) inhibitors diminished HGF-induced mitogenesis, anti-apoptosis, and MAP kinase activation, IL-4 enhanced HGF signaling persisted even in the presence of these inhibitors. IL-4 enhancement of HGF signaling was partially blocked in 32D/c-Met cells treated with inhibitors of MEK1 or c-Src kinases, completely blocked by expression of a catalytically inactive mutant of Janus kinase 3 (Jak3), and increased in 32D/c-Met cells overexpressing STAT6. Our results suggest that the IL-4 and HGF pathways converge at multiple levels, and that IL-4-dependent Jak3 and STAT6 activities modulate signaling events independent of PI3K to enhance HGF-dependent mitogenesis in myeloid cells, and possibly other common cellular targets.     

miR-199a-3p inhibits hepatocyte growth factor/c-Met signaling in renal cancer carcinoma

MicroRNAs (miRNAs) are a class of small non-coding RNAs that bind protein-coding mRNAs and negatively regulate protein expression by translation repression or mRNA cleavage. Accumulating evidence suggests that miRNAs are involved in cancer development and progression, acting as either tumor suppressors or oncogenes. It has been shown that miR-199a-3p was significantly down-regulated in several types of cancers. However, its role and relevance in renal cell carcinoma (RCC) are still largely unknown. Here, we show that miR-199a-3p is significantly down-regulated in human RCC primary tumors and cell lines compared to their non-tumor counterparts. Moreover, the down-regulation of miR-199a-3p is correlated with the histological grade and TNM (tumor–lymph node–metastasis) stage of RCC. Reintroducing miR-199a-3p in RCC cell lines 769-P and Caki-1 inhibited cell proliferation and caused G1 phase arrest. We found that c-Met was up-regulated in RCC cell lines and its expression could be repressed by miR-199a-3p. Moreover, c-Met was up-regulated in RCC primary tumors and reversely correlated with miR-199a-3p expression in the same paired RCC tissues. Reintroducing miR-199a-3p inhibited c-Met expression and led to attenuated activation of c-Met downstream signaling pathways including STAT3, mTOR and ERK1/2. We found that the concentrations of serum hepatocyte growth factor (HGF), the ligand of c-Met receptor, were significantly elevated in RCC patients compared to healthy persons. In addition, HGF treatment could promote proliferation of RCC cells, and the increased cell proliferation was abrogated by miR-199a-3p. Our findings indicated that miR-199a-3p target HGF/c-Met signaling pathway which is crucial for RCC development and suggest that miR-199a-3p may serve as a potential target miRNA for RCC therapy.     

Reversion of human glioblastoma malignancy by U1 small nuclear RNA/ribozyme targeting of scatter factor/hepatocyte growth factor and c-met expression.

BACKGROUND: Expression of scatter factor (SF), also known as hepatocyte growth factor (HGF), and its receptor, c-met, is often associated with malignant progression of human tumors, including gliomas. Overexpression of SF/HGF in experimental gliomas enhances tumorigenicity and tumor-associated angiogenesis (i.e., growth of new blood vessels). However, the role of endogenous SF/HGF or c-met expression in the malignant progression of gliomas has not been examined directly. In this study, we tested the hypothesis that human glioblastomas can be SF/HGF-c-met dependent and that a reduction in endogenous SF/HGF or c-met expression can lead to inhibition of tumor growth and tumorigenicity. METHODS: Expression of the SF/HGF and c-met genes was inhibited by transfecting glioblastoma cells with chimeric transgenes consisting of U1 small nuclear RNA, a hammerhead ribozyme, and antisense sequences. The effects of reduced SF/HGF and c-met expression on 1) SF/HGF-dependent induction of immediate early genes (c-fos and c-jun), indicative of signal transduction; 2) anchorage-independent colony formation (clonogenicity), an in vitro correlate of solid tumor malignancy; and 3) intracranial tumor formation in immunodeficient mice were quantified. Statistical tests were two-sided. RESULTS: Introduction of the transgenes into glioblastoma cells reduced expression of the SF/HGF and c-met genes to as little as 2% of control cell levels. Reduction in c-met expression specifically inhibited SF/HGF-dependent signal transduction (P<.01). Inhibition of SF/HGF or c-met expression in glioblastoma cells possessing an SF/HGF-c-met autocrine loop reduced tumor cell clonogenicity (P =.005 for SF/HGF and P=.009 for c-met) and substantially inhibited tumorigenicity (P<.0001) and tumor growth in vivo (P<.0001). CONCLUSIONS: To our knowledge, this is the first successful inhibition of SF/HGF and c-met expression in a tumor model directly demonstrating a role for endogenous SF/HGF and c-met in human glioblastoma. Our results suggest that targeting the SF/HGF-c-met signaling pathway may be an important approach in controlling tumor progression.     

Induction of hepatocyte growth factor/scatter factor by fibroblast clustering directly promotes tumor cell invasiveness

For determining the malignant behavior of a tumor, paracrine interactions between stromal and cancer cells are crucial. We previously reported that fibroblast clustering induces cyclooxygenase-2 (COX-2), plasminogen activation, and programmed necrosis, all of which were significantly reduced by nonsteroidal anti-inflammatory drugs (NSAID). We have now found that tumor cell-conditioned medium induces similar fibroblast clustering. Activation of the necrotic pathway in clustering fibroblasts, compared with control monolayer cultures, induced a massive >200-fold production of bioactive hepatocyte growth factor/scatter factor (HGF/SF), which made human carcinoma cells spread and invade a collagen lattice. This response occurred only if a functional, properly processed c-Met receptor was present, which was then rapidly phosphorylated. The invasion-promoting activity was inhibited by a neutralizing HGF/SF antibody. NSAIDs, if added early during fibroblast aggregation, inhibited HGF/SF production effectively but had no effect at later stages of cell aggregation. Our results thus provide the first evidence that aggravated progression of tumors with necrotic foci may involve paracrine reciprocal signaling leading to stromal activation by direct cell-cell contact (i.e., nemosis).     

A selective c-met inhibitor blocks an autocrine hepatocyte growth factor growth loop in ANBL-6 cells and prevents migration and adhesion of myeloma cells.

PURPOSE: We wanted to examine the role of the hepatocyte growth factor (HGF) receptor c-Met in multiple myeloma by applying a novel selective small molecule tyrosine kinase inhibitor, PHA-665752, directed against the receptor. EXPERIMENTAL DESIGN: Four biological sequels of HGF related to multiple myeloma were studied: (1) proliferation of myeloma cells, (2) secretion of interleukin-11 from osteogenic cells, (3) migration of myeloma cells, and (4) adhesion of myeloma cells to fibronectin. We also examined effects of the c-Met inhibitor on intracellular signaling pathways in myeloma cells. RESULTS: PHA-665752 effectively blocked the biological responses to HGF in all assays, with 50% inhibition at 5 to 15 nmol/L concentration and complete inhibition at around 100 nmol/L. PHA-665752 inhibited phosphorylation of several tyrosine residues in c-Met (Tyr(1003), Tyr(1230/1234/1235), and Tyr(1349)), blocked HGF-mediated activation of Akt and p44/42 mitogen-activated protein kinase, and prevented the adaptor molecule Gab1 from complexing with c-Met. In the HGF-producing myeloma cell line ANBL-6, PHA-665752 revealed an autocrine HGF-c-Met-mediated growth loop. The inhibitor also blocked proliferation of purified primary myeloma cells, suggesting that autocrine HGF-c-Met-driven growth loops are important for progression of multiple myeloma. CONCLUSIONS: Collectively, these findings support the role of c-Met and HGF in the proliferation, migration, and adhesion of myeloma cells and identify c-Met kinase as a therapeutic target for treatment of patients with multiple myeloma.     

Targeting of urokinase plasminogen activator receptor in human pancreatic carcinoma cells inhibits c-Met- and insulin-like growth factor-I receptor-mediated migration and invasion and orthotopic tumor growth in mice

Pancreatic carcinomas express high levels of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), both of which mediate cell migration and invasion. We investigated the hypotheses that (a) insulin-like growth factor-I (IGF-I)- and hepatocyte growth factor (HGF)-mediated migration and invasion of human pancreatic carcinoma cells require uPA and uPAR function and (b) inhibition of uPAR inhibits tumor growth, retroperitoneal invasion, and hepatic metastasis of human pancreatic carcinomas in mice. Using transwell assays, we investigated the effect of IGF-I and HGF on L3.6pl migration and invasion. We measured the induction of uPA and uPAR following treatment of cells with IGF-I and HGF using immunoprecipitation and Western blot analysis. The importance of uPA and uPAR on L3.6pl cell migration and invasion was studied by inhibiting their activities with amiloride and antibodies before cytokine treatment. In an orthotopic mouse model of human pancreatic carcinoma, we evaluated the effect of anti-uPAR monoclonal antibodies with and without gemcitabine on primary tumor growth, retroperitoneal invasion, and hepatic metastasis. IGF-I and HGF mediated cell migration and invasion in L3.6pl cells. In addition, IGF-I and HGF induced uPA and uPAR expression in L3.6pl cells. In vitro, blockade of uPA and uPAR activity inhibited IGF-I- and HGF-mediated cell migration and invasion. Treatment of mice with anti-uPAR monoclonal antibody significantly decreased pancreatic tumor growth and hepatic metastasis and completely inhibited retroperitoneal invasion. Our study shows the importance of the uPA/uPAR system in pancreatic carcinoma cell migration and invasion. These findings suggest that uPAR is a potential target for therapy in patients with pancreatic cancer.     

Co-expression of hepatocyte growth factor and c-Met predicts peritoneal dissemination established by autocrine hepatocyte growth factor/c-Met signaling in gastric cancer

Epithelial-mesenchymal transition (EMT) promotes and facilitates migration and invasion of epithelial tumor cells. EMT is induced by factors such as hepatocyte growth factor (HGF). This study aimed to establish whether the HGF/c-Met pathway is associated with gastric cancer metastasis; especially peritoneal dissemination. HGF and c-Met expression and EMT-related molecules were evaluated using real-time PCR and immunohistochemistry. The role of the HGF/c-Met pathway in EMT and anoikis was determined, and kinase inhibitor SU11274 was tested for its ability to block HGF-induced biological effects. In HGF(-) /c-Met(+) gastric cancer cells, recombinant HGF promoted an EMT phenotype that was characterized by morphology, impaired E-cadherin and induction of vimentin. HGF promoted cell growth, invasiveness and migration and inhibition of anoikis. SU11274 blocked HGF-induced EMT and biological effects in vitro. In HGF(+) /c-Met(+) gastric cancer cells, HGF did not affect the biological outcome of EMT and anoikis, but SU11274 exerted the same inhibitory effects as in HGF(-) /c-Met(+) cells. In vivo, HGF(+) /c-Met(+) gastric cancer cells only established peritoneal dissemination and SU11274 inhibited tumor growth. Clinically, HGF expression was significantly correlated with c-Met expression in gastric cancer. Increased HGF and c-Met had a significant association with poor prognosis and predicted peritoneal dissemination. We demonstrated that the HGF/c-Met pathway induces EMT and inhibition of anoikis in gastric cancer cells. Co-expression of HGF and c-Met has the potential to promote peritoneal dissemination in gastric cancer. Blockade of the autocrine HGF/c-Met pathway could be clinically useful for the treatment of peritoneal dissemination in gastric cancer.     

Overexpression of c-met in oral SCC promotes hepatocyte growth factor-induced disruption of cadherin junctions and invasion

Hepatocyte growth factor (HGF), the ligand for the c-met proto-oncogene product, is a multifunctional protein that enhances tumor cell motility, extracellular matrix invasion, and mitogenic or morphogenic activities of various cell types. In this study we examined the expression of the c-Met receptor in human oral squamous cell carcinoma (SCC) in vivo and in vitro to explore its relationship to tumor progression and invasiveness. Biopsy specimens of human oral SCC were immunohistochemically stained for c-Met. Nearly all primary oral SCC lesions and lymph node metastases consistently showed intense staining for c-Met, whereas normal oral mucosa showed faint to negative staining only on basal cells. In a panel of human oral SCC cell lines, we found a strong correlation between the levels of c-Met expression and the cells' response to HGF in motility and invasion assays. Sensitivity to HGF also correlated with the expression of the c-Met 9-kb mRNA. When the non-invasive HOC-605 cell line, which expresses a low level of c-Met receptor, was transfected with an expression plasmid containing human c-met cDNA, the transfectant cells showed motile and invasive responses to HGF. Immunostaining and immunoprecipitation studies demonstrated that E-cadherin and c-Met were physically associated at SCC cell-cell junctions, suggesting a direct role for c-Met in induction of junctional integrity. Importantly, HGF caused a rapid elevation of unbound beta-catenin, suggesting its availability for nuclear signal transduction and triggering of cell motility and invasiveness. Thus, overexpression of c-Met may facilitate disruption of E-cadherin junctions. Collectively, these results suggest that HGF/c-Met signaling is a common event in oral SCC that may trigger phenotype modulation and enhanced invasion and metastasis.     

Radiation stimulates HGF receptor/c-Met expression that leads to amplifying cellular response to HGF stimulation via upregulated receptor tyrosine phosphorylation and MAP kinase activity in pancreatic cancer cells

Hepatocyte growth factor (HGF) is a stromal-derived cytokine that plays a crucial role in invasion and metastasis of tumor cells through the interaction with HGF receptor, c-Met, which is frequently overexpressed in pancreatic cancer. The present study was designed to investigate the change in HGF receptor and HGF-mediated signaling after irradiation in pancreatic cancer cells. Six cell lines from human pancreatic cancer were included in the study. Gamma-radiation was used for irradiation treatment. The changes in expression levels of c-Met were evaluated by immunoblot and confirmed morphologically by indirect immunofluorescence staining. Whether the resultant alteration in c-Met would cascade as biologically usable signals upon HGF ligation was traced by receptor tyrosine phosphorylation analysis and mitogen activated protein kinase (MAP kinase or MAPK) activity assay. The various biological responses to HGF (including cell proliferation, cell scattering, migration and invasion) were evaluated as well. We also used a 4-kringle antagonist of HGF, NK4, to block the HGF/c-Met signaling pathway. Both immunoblot and immunofluorescent analysis showed moderate increased expression of c-Met in 3 of 6 pancreatic cancer cell lines after irradiation. The actions seemed to be dose-responsible, which began at 3 hr and reached its peak value at 24 hr following irradiation. The radiation-increased expression of c-Met could transform into magnifying receptor tyrosine phosphorylation reaction and MAP kinase activity once the ligand was added, fairly corresponding with alteration in the receptor. Sequentially, the cellular responses to HGF, including scattering and invasion but not proliferation, were enhanced. Also, in the presence of HGF, the elevated receptor could help to recover the radiation-compromised cell migration. A recombinant HGF antagonist, NK4 could effectively block these aberrant effects activated by irradiation both in molecular and cellular levels, thus suggesting the deep involvement of the c-Met/HGF pathway in the enhanced malignant potential after irradiation. These results suggest that radiation may promote HGF-induced malignant biological behaviors of certain pancreatic cancer cells through the up-regulated HGF/c-Met signal pathway. Selectively targeted blockade of the HGF/c-Met pathway could help to abolish the enforced malignant behavior of tumor cells by irradiation and therefore may improve the efficacy of radiotherapy for pancreatic cancer.     

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

Background  

c-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.     

MetMAb, the one-armed 5D5 anti-c-Met antibody, inhibits orthotopic pancreatic tumor growth and improves survival

The hepatocyte growth factor (HGF) and its receptor, c-Met, have been implicated in driving proliferation, invasion, and poor prognosis in pancreatic cancer. Here, we investigated the expression of HGF and c-Met in primary pancreatic cancers and described in vitro and in vivo models in which MetMAb, a monovalent antibody against c-Met, was evaluated. First, expression of HGF and MET mRNA was analyzed in 59 primary pancreatic cancers and 51 normal samples, showing that both factors are highly expressed in pancreatic cancer. We next examined HGF responsiveness in pancreatic cancer lines to select lines that proliferate in response to HGF. Based on these studies, two lines were selected for further in vivo model development: BxPC-3 (c-Met(+), HGF(-)) and KP4 (c-Met(+), HGF(+)) cells. As BxPC-3 cells are responsive to exogenous HGF, s.c. tumor xenografts were grown in a paracrine manner with purified human HGF provided by osmotic pumps, wherein MetMAb treatment significantly inhibited tumor growth. KP4 cells are autocrine for HGF and c-Met, and MetMAb strongly inhibited s.c. tumor growth. To better model pancreatic cancer and to enable long-term survival studies, an orthotopic model of KP4 was established. MetMAb significantly inhibited orthotopic KP4 tumor growth in 4-week studies monitored by ultrasound and also improved survival in 90-day studies. MetMAb significantly reduced c-Met phosphorylation in orthotopic KP4 tumors with a concomitant decrease in Ki-67 staining. These data suggest that the HGF/c-Met axis plays an important role in the progression of pancreatic cancer and that targeting c-Met therein may have therapeutic value.     

Loss of hepatocyte growth factor/c-Met signaling pathway accelerates early stages of N-nitrosodiethylamine induced hepatocarcinogenesis

Hepatocyte growth factor (HGF) has been reported to have both positive and negative effects on carcinogenesis. Here, we show that the loss of c-Met signaling in hepatocytes enhanced rather than suppressed the early stages of chemical hepatocarcinogenesis. c-Met conditional knockout mice (c-metfl/fl, AlbCre+/-; MetLivKO) treated with N-nitrosodiethylamine developed significantly more and bigger tumors and with a shorter latency compared with control (w/w, AlbCre+/-; Cre-Ctrl) mice. Accelerated tumor development was associated with increased rate of cell proliferation and prolonged activation of epidermal growth factor receptor (EGFR) signaling. MetLivKO livers treated with N-nitrosodiethylamine also displayed elevated lipid peroxidation, decreased ratio of reduced glutathione to oxidized glutathione, and up-regulation of superoxide dismutase 1 and heat shock protein 70, all consistent with increased oxidative stress. Likewise, gene expression profiling done at 3 and 5 months after N-nitrosodiethylamine treatment revealed up-regulation of genes associated with cell proliferation and stress responses in c-Met mutant livers. The negative effects of c-Met deficiency were reversed by chronic p.o. administration of antioxidant N-acetyl-L-cysteine. N-acetyl-L-cysteine blocked the EGFR activation and reduced the N-nitrosodiethylamine-initiated hepatocarcinogenesis to the levels of Cre-Ctrl mice. These results argue that intact HGF/c-Met signaling is essential for maintaining normal redox homeostasis in the liver and has tumor suppressor effect(s) during the early stages of N-nitrosodiethylamine-induced hepatocarcinogenesis.     

Hepatocyte growth factor promotes cancer cell migration and angiogenic factors expression: a prognostic marker of human esophageal squamous cell carcinomas.

PURPOSE: Hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, c-Met, play important roles in tumor development and progression. In this study, we measured the serum HGF levels in patients with esophageal squamous cell carcinoma (ESCC) to evaluate its relationships with clinicopathologic features and the role of HGF in ESCC. EXPERIMENTAL DESIGN: One hundred and forty-nine patients with ESCC were studied. Pretherapy serum was collected and ELISA was used to detect the concentrations of HGF, vascular endothelial growth factor (VEGF), and interleukin 8 (IL-8). The function of HGF was shown by invasion chamber assay. RESULTS: Pretherapy serum HGF was found to be significantly higher in patients with ESCC than in control subjects. The levels of HGF correlated significantly with advanced tumor metastasis stage and survival. Multivariate analyses showed that serum HGF level in cell migration was an independent prognostic factor. Increased HGF serum levels correlated positively with serum levels of VEGF and IL-8. Our results also showed that HGF was overexpressed in ESCC tissues and cell lines. In vitro study showed that HGF could stimulate ESCC cell to express VEGF and IL-8 and markedly enhance invasion and migration of ESCC cells. Furthermore, HGF-induced IL-8 and VEGF expression was dependent on extracellular signal-regulated kinase signaling pathways. The inhibition of extracellular signal-regulated kinase activation reduced HGF-mediated IL-8 and VEGF expression. CONCLUSIONS: Our results suggest that serum HGF may be a useful biomarker of tumor progression and a valuable independent prognostic factor in patients with ESCC. HGF may be involved in the progression of ESCC as an autocrine/paracrine factor via enhancing angiogenesis and tumor cell invasion and migration.     

Glutamate Promotes Cell Growth by EGFR Signaling on U-87MG Human Glioblastoma Cell Line

Accumulating evidences suggest that glutamate plays a key role in the proliferation and invasion of malignant glioblastoma (GBM) tumors. It has been shown that GBM cells release and exploit glutamate for proliferation and invasion through AMPA glutamate receptors. Additionally, amplification of the epidermal growth factor receptor (EGFR) gene occurs in 40–50% of GBM. Since, PI3K/Akt is considered one of the main intracellular pathways involved in EGFR activation, AKT functions could trigger EGFR signaling. Thus, we investigated whether EGFR-phospho-Akt pathway is involved on the glutamate inducing U-87MG human GBM cell line proliferation. For these purpose, we treated the U-87MG cell line with 5 to 200 mM of glutamate and assessed the number of viable cells by trypan blue dye exclusion test. An increase in cell number (50%) was found at 5 mM glutamate, while the addition of DNQX (500 μM), an antagonist of AMPA receptor, inhibited the effect of glutamate on the U87-MG cells proliferation. Also, at 5 mM glutamate we observed an increase on the EGFR and phospho-Akt contents evaluated by immunohistochemistry. Moreover, U-87MG cells treated with glutamate exhibited an increase about 2 times in the EGFR mRNA expression. While, in the presence of the anti-EGFR gefitinib (50 μM) or the PI3K inhibitor wortmannin (5 μM), the U-87MG proliferation was restored to control levels. Together, our data suggest that glutamate signaling mediated by AMPA receptor induces U-87MG human GBM cell line proliferation via EGFR-phospho-Akt pathway.     

Identification of a novel recepteur d'origine nantais/c-met small-molecule kinase inhibitor with antitumor activity in vivo

Recepteur d'origine nantais (RON) is a receptor tyrosine kinase closely related to c-Met. Both receptors are involved in cell proliferation, migration, and invasion, and there is evidence that both are deregulated in cancer. Receptor overexpression has been most frequently described, but other mechanisms can lead to the oncogenic activation of RON and c-Met. They include activating mutations or gene amplification for c-Met and constitutively active splicing variants for RON. We identified a novel inhibitor of RON and c-Met, compound I, and characterized its in vitro and in vivo activities. Compound I selectively and potently inhibited the kinase activity of RON and c-Met with IC(50)s of 9 and 4 nmol/L, respectively. Compound I inhibited hepatocyte growth factor-mediated and macrophage-stimulating protein-mediated signaling and cell migration in a dose-dependent manner. Compound I was tested in vivo in xenograft models that either were dependent on c-Met or expressed a constitutively active form of RON (RONDelta160 in HT-29). Compound I caused complete tumor growth inhibition in NIH3T3 TPR-Met and U-87 MG xenografts but showed only partial inhibition in HT-29 xenografts. The effect of compound I in HT-29 xenografts is consistent with the expression of the activating b-Raf V600E mutation, which activates the mitogen-activated protein kinase pathway downstream of RON. Importantly, tumor growth inhibition correlated with the inhibition of c-Met-dependent and RON-dependent signaling in tumors. Taken together, our results suggest that a small-molecule dual inhibitor of RON/c-Met has the potential to inhibit tumor growth and could therefore be useful for the treatment of patients with cancers where RON and/or c-Met are activated.     

The Plasticity of Oncogene Addiction: Implications for Targeted Therapies Directed to Receptor Tyrosine Kinases

A common mutation of the epidermal growth factor receptor (EGFR) in glioblastoma multiforme (GBM) is an extracellular truncation known as the de2-7 EGFR (or EGFRvIII). Hepatocyte growth factor (HGF) is the ligand for the receptor tyrosine kinase (RTK) c-Met, and this signaling axis is often active in GBM. The expression of the HGF/c-Met axis or de2-7 EGFR independently enhances GBMgrowth and invasiveness, particularly through the phosphatidylinositol-3 kinase/pAkt pathway. Using RTK arrays, we show that expression of de2-7 EGFR in U87MG GBM cells leads to the coactivation of several RTKs, including platelet-derived growth factor receptor β and c-Met. A neutralizing antibody to HGF (AMG102) did not inhibit de2-7 EGFR-mediated activation of c-Met, demonstrating that it is ligand-independent. Therapy for parental U87MG xenografts with AMG 102 resulted in significant inhibition of tumor growth, whereas U87MG.Δ2-7 xenografts were profoundly resistant. Treatment of U87MG.Δ2-7 xenografts with panitumumab, an anti-EGFR antibody, only partially inhibited tumor growth as xenografts rapidly reverted to the HGF/c-Met signaling pathway. Cotreatment with panitumumab and AMG 102 prevented this escape leading to significant tumor inhibition through an apoptotic mechanism, consistent with the induction of oncogenic shock. This observation provides a rationale for using panitumumab and AMG 102 in combination for the treatment of GBM patients. These results illustrate that GBM cells can rapidly change the RTK driving their oncogene addiction if the alternate RTK signals through the same downstream pathway. Consequently, inhibition of a dominant oncogene by targeted therapy can alter the hierarchy of RTKs resulting in rapid therapeutic resistance.