Antibodies targeting hepatoma-derived growth factor as a novel strategy in treating lung cancer.

Hepatoma-derived growth factor (HDGF) is overexpressed in lung cancer and the overexpression correlates with aggressive biological behaviors and poor clinical outcomes. We developed anti-HDGF monoclonal antibodies and tested their antitumor activity in lung cancer xenograft models. We also determined biological effects in tumors treated with the antibody alone or in combination with bevacizumab/avastin (an anti-vascular endothelial growth factor antibody) and/or gemcitabine (a chemotherapeutic agent). We found the anti-HDGF was effective to inhibit tumor growth in non-small cell lung cancer xenograft models. In the A549 model, compared with control IgG, tumor growth was substantially inhibited in animals treated with anti-HDGF antibodies, particularly HDGF-C1 (P = 0.002) and HDGF-H3 (P = 0.005). When HDGF-H3 was combined with either bevacizumab or gemcitabine, we observed enhanced tumor growth inhibition, particularly when the three agents were used together. HDGF-H3-treated tumors exhibited significant reduction of microvessel density with a pattern distinctive from the microvessel reduction pattern observed in bevacizumab-treated tumors. HDGF-H3-treated but not bevacizumab-treated tumors also showed a significant increase of apoptosis. Interestingly, many of the apoptotic cells in HDGF-H3-treated tumors are stroma cells, suggesting that the mechanism of the antitumor activity is, at least in part, through disrupting formation of tumor-stroma structures. Our results show that HDGF is a novel therapeutic target for lung cancer and can be effectively targeted by an antibody-based approach.

Localized diacylglycerol-dependent stimulation of Ras and Rap1 during phagocytosis.

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcgamma receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcgamma receptor-mediated phagocytosis to enhance the innate immune response.

Structural and functional analysis of p84N5

Normal development and tissue homeostasis requires the carefully orchestrated balance between cell proliferation and cell death. Cell cycle checkpoints control the extent of cell proliferation. Cell death is coordinated through the activation of a cell suicide pathway that results in the morphologically recognizable form of death, apoptosis. Tumorigenesis requires that the balance between these two pathways be disrupted. The tumor suppressor protein Rb has not only been shown to be involved in the enforcement of cell cycle checkpoints, but has also been implicated in playing a role in the regulation of apoptosis. The manner in which Rb enforces cell cycle checkpoints has been well studied; however, its involvement in the regulation of apoptosis is still very unclear. p84N5 is a novel nuclear death domain containing protein that has been shown to interact with the N-terminus of Rb. The fact that it contains a death domain and the fact that it is nuclear localized possibly provides the first known mechanism for apoptotic signaling from the nucleus. The following study tested the hypothesis that the novel exclusively nuclear death domain containing protein p84N5 is an important mediator of programmed cell death and that its apoptotic function is reliant upon its nuclear localization and is regulated by unique functional domains within the p84N5 protein. We identified the p84N5 nuclear localization signal (NLS), eliminated it, and tested the functional significance of nuclear localization by using wild type and mutant sequences fused to EGFP-C1 (Clontech) to create wild type GFPN5 and subsequent mutants. The results of these assays demonstrated exclusive nuclear localization of GFPN5 is required for normal p84N5 induced apoptosis. We further conducted large-scale mutagenesis of the GFPN5 construct to identify a minimal region within p84N5 capable of interacting with Rb. We were able to identify a minimal sequence containing p84N5 amino acids 318 to 464 that was capable of interacting with Rb in co-immunoprecipitation assays. We continued by conducting a structural and functional analysis to identify the region or regions within p84N5 responsible for inducing apoptosis. Point mutations and small-scale deletions within the death domain of p84N5 lessened the effect but did not eliminate p84N5-induced cytotoxicity. Further analysis revealed that the minimal sequence of 318 to 464 of p84N5 was capable of inducing apoptosis to a similar degree as wild-type GFPN5 protein. Since amino acids 318 to 464 of p84N5 are capable of inducing apoptosis and interacting with Rb, we propose possible mechanisms whereby p84N5 may function in a Rb regulated manner. These results demonstrate that p84N5 induced apoptosis is reliant upon its nuclear localization and is regulated by unique functional domains within the p84N5 protein. ^