Roger Abounader

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.

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

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

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

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

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.

Glycolytic glioma cells with active glycogen synthase are sensitive to PTEN and inhibitors of PI3K and gluconeogenesis

Increased glycolysis is characteristic of malignancy. Previously, with a mitochondrial inhibitor, we demonstrated that glycolytic ATP production was sufficient to support migration of melanoma cells. Recently, we found that glycolytic enzymes were abundant and some were increased in pseudopodia formed by U87 glioma (astrocytoma) cells. In this study, we examined cell migration, adhesion (a step in migration), and Matrigel invasion of U87 and LN229 glioma cells when their mitochondria were inhibited with sodium azide or limited by 1% O2. Cell migration, adhesion, and invasion were comparable, with and without mitochondrial inhibition. Upon discovering that glycolysis alone can support glioma cell migration, unique features of glucose metabolism in astrocytic cells were investigated. The ability of astrocytic cells to remove lactate, the inhibitor of glycolysis, via gluconeogenesis and incorporation into glycogen led to consideration of supportive genetic mutations. Loss of phosphatase and tensin homolog (PTEN) releases glycogenesis from constitutive inhibition by glycogen synthase kinase-3 (GSK3). We hypothesize that glycolysis in gliomas can support invasive migration, especially when aided by loss of PTENs regulation on the phosphatidylinositol-3 kinase (PI3K)/Akt pathway leading to inhibition of GSK3. Migration of PTEN-mutated U87 cells was studied for release of extracellular lactic acid and support by gluconeogenesis, loss of PTEN, and active PI3K. Lactic acid levels plateaued and phosphorylation changes confirmed activation of the PI3K/Akt pathway and glycogen synthase when cells relied only on glycolysis. Glycolytic U87 cell migration and phosphorylation of GSK3 were inhibited by PTEN transfection. Glycolytic migration was also suppressed by inhibiting PI3K and gluconeogenesis with wortmannin and metformin, respectively. These findings confirm that glycolytic glioma cells can migrate invasively and that the loss of PTEN is supportive, with activated glycogenic potential included among the relevant downstream effects.

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

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

CD44-Independent Hepatocyte Growth Factor/c-Met Autocrine Loop Promotes Malignant Peripheral Nerve Sheath Tumor Cell Invasion In Vitro

Malignant peripheral nerve sheath tumors (MPNSTs) are invasive peripheral nerve neoplasms that express both the receptor tyrosine kinase c‐Met and its ligand hepatocyte growth factor (HGF). The combined expression of these proteins has been implicated in tumor cell growth and metastasis. However, HGF/c‐Met autocrine activity requires the presence of a serine protease, the HGF activator (HGFA), and, in some cells, the CD44 transmembrane glycoprotein. Here, we found that HGFA, HGF, c‐Met, and CD44 are coexpressed in MPNSTs but their localization did not correlate with increased cell proliferation. The ST8814 MPNST cell line also expresses all of these proteins, can convert pro‐HGF to active HGF, and exhibits constitutive c‐Met phosphorylation. Blocking c‐Met activity or expression inhibits the invasive behavior of these cells but not their proliferation. Interestingly, although a CD44 splice variant contributes to MPNST cell invasion and interacts with c‐Met and HGF in ST8814 cells, it is not required for c‐Met activation. These data indicate that an HGF/c‐Met autocrine loop can promote MPNST invasion through a CD44‐independent mechanism and suggest that c‐Met, HGFA, and HGF are potential molecular targets to inhibit MPNST metastasis.