Diacylglycerol kinases are essential for hepatocyte growth factor-dependent proliferation and motility of Kaposi's sarcoma cells.

Hepatocyte growth factor (HGF) is involved in the pathogenesis of Kaposi's sarcoma (KS), the most frequent neoplasia in patients with AIDS, characterized by proliferating spindle cells, infiltrating inflammatory cells, angiogenesis, edema, and invasiveness. In vitro, this factor sustains the biological behavior of KS derived cells, after activation of its receptor and the downstream MAPK and AKT signals. In other cell types, namely endothelial and epithelial cells, movement, proliferation, and survival stimulated by HGF and other growth factors and cytokines depend on diacylglycerol kinases (DGK). In an effort to identify new intracellular transducers operative in KS cells, which could represent therapeutic targets, we investigated the role of DGK in KS cell movement and proliferation by treating cells with the DGK pharmacological inhibitor R59949. We report that R59949 strongly inhibits HGF-induced KS motility, proliferation, and anchorage-independent growth with only a partial effect on cell adhesion and spreading. R59949 does not affect cell survival, HGF receptor activation, or the classical MAPK and AKT signalling pathways. Furthermore, we carried out an siRNA screen to characterize the DGK isoforms involved in KS motility and anchorage independent growth. Our data indicate a strong involvement of DGK-δ in KS motility and of DGK-ι in anchorage-independent growth. These results indicate that DGK inhibition is sufficient to impair in vitro KS cell proliferation and movement and suggest that selected DGK represent new pharmacological targets to interfere with the malignant properties of KS, independently from the well-known RAS/MAPK and PI3K/AKT pathways.

Agonist monoclonal antibodies against HGF receptor protect cardiac muscle cells from apoptosis.

Hepatocyte growth factor (HGF), a pleiotropic cytokine with mitogenic, motogenic, morphogenic, and antiapoptotic effects in various cell types, is a cardioprotective growth factor that can counteract the loss of cardiomyocytes usually observed in cardiac diseases. HGF is a quite unstable molecule in its biologically active heterodimeric form. Since all HGF-induced biological responses are mediated by its high-affinity tyrosine kinase receptor (Met/HGF-R) encoded by the Met gene, we asked whether a monoclonal antibody (MAb) that displays receptor full agonist activity could protect cardiac muscle cell lines from hydrogen peroxide-induced apoptosis. We report that the MAb efficiently inhibited hydrogen peroxide-induced cell shrinkage, DNA fragmentation, annexin V positivity, mitochondrial translocation of bax, and caspase activation. The MAb was thus able to counteract apoptosis evaluated by both morphological and biochemical criteria. The agonist activity of the MAb was mediated by Met/HGF-R, since a Met/HGF-R-specific short hairpin RNA (shRNA) inhibited both activation of transduction pathways and motility triggered by MAb DO-24. The protective antiapoptotic effect of MAb DO-24 was dependent on activation of the ras-MAPK Erk1/2 and phosphatidylinositol 3-kinase (PI3-kinase)-Akt transduction pathways, since it was abrogated by treatments with their specific pharmacological inhibitors, PD-98059 and wortmannin. Moreover, the MAb induced a motogenic, but not mitogenic, response in these cells, mimicking in all aspects the natural ligand HGF but displaying a significant higher stability than HGF in culture. This MAb may thus be a valuable substitute for HGF, being more easily available in a biologically active, highly stable, and purified form.

Deletion of the ectodomain unleashes the transforming, invasive, and tumorigenic potential of the MET oncogene.

The c-MET proto-oncogene, encoding the p190 hepatocyte growth factor tyrosine kinase receptor, can acquire oncogenic potential by multiple mechanisms, such as gene rearrangement, amplification and overexpression, point mutation, and ectopic expression, all resulting in its constitutive activation. Hepatocyte growth factor receptor truncated forms are generated by post-translational cleavage: p140 and p130 lack the kinase domain and are inactive. Their C-terminal remnant fragments are generally undetectable in normal cells, but a membrane-associated truncated form is recognized by anti-C-terminus antibodies in some human tumors, suggesting that a hepatocyte growth factor receptor lacking the ectodomain, but retaining the transmembrane and intracellular domains (Met-EC-), could acquire oncogenic properties. Herein we show that NIH-3T3 cells transduced with MET-EC- expressed a membrane-associated constitutively tyrosine-phosphorylated 60-kDa protein and, similarly to NIH-3T3 cells expressing the cytosolic oncoprotein Tpr-Met, showed activated extracellular regulated kinase 1/2 mitogen-activated protein kinase and Akt downstream transducers. Compared to control NIH-3T3 cells, NIH-3T3-Met-EC- cells grew faster and showed anchorage-independent growth and invasive properties in all aspects similar to cells expressing the transforming TPR-MET. Nude female mice injected subcutaneously with NIH-3T3-Met-EC- cells developed visible tumors, displaying the typical morphology of carcinomas with polygonal cells, in contrast to sarcomas with spindle-shaped cells induced by the injection of NIH-3T3-Tpr-Met cells. It is suggested that the different subcellular localization of the oncoproteins, more than differences in signal transduction, could be responsible for the tumor phenotype. All together, these data show that deletion of the ectodomain activates the hepatocyte growth factor receptor and its downstream signaling pathways, unleashing its transforming, invasive, and tumorigenic potential.

Functionalized nanomaterials for diagnosis and therapy of cancer.

Recent developments in nanotechnology have provided new tools for both cancer imaging and treatment. The functionalization of the nanocarrier based drugs using biological ligands allows increasing specificity of drug targeting enhancing efficacy and reducing side effects. In this paper some current nanocarrier based drugs are described and the principles for their functionalization in cancer treatment and imaging are reviewed.