We have shown that Wnt5A increases the motility of melanoma cells. To explore cellular pathways involving Wnt5A, we compared gain-of-function (WNT5A stable transfectants) versus loss-of-function (siRNA knockdown) of WNT5A by microarray analysis. Increasing WNT5A suppressed the expression of several genes, which were re-expressed after small interference RNA-mediated knockdown of WNT5A. Genes affected by WNT5A include KISS-1, a metastasis suppressor, and CD44, involved in tumor cell homing during metastasis. This could be validated at the protein level using both small interference RNA and recombinant Wnt5A (rWnt5A). Among the genes up-regulated by WNT5A was the gene vimentin, associated with an epithelial to mesenchymal transition (EMT), which involves decreases in E-cadherin, due to up-regulation of the transcriptional repressor, Snail. rWnt5A treatment increases Snail and vimentin expression, and decreases E-cadherin, even in the presence of dominant-negativeTCF4, suggesting that this activation is independent of Wnt/␤-catenin signaling. Because Wnt5A can signal via protein kinase C (PKC), the role of PKC in Wnt5A-mediated motility and EMT was also assessed using PKC inhibition and activation studies. Treating cells expressing low levels of Wnt5A with phorbol ester increased Snail expression inhibiting PKC in cells expressing high levels of Wnt5A decreased Snail. Furthermore, inhibition of PKC before Wnt5A treatment blocked Snail expression, implying that Wnt5A can potentiate melanoma metastasis via the induction of EMT in a PKC-dependent manner.The molecular mechanisms that govern the motility and metastasis of melanoma cells are not well understood. The prognosis for patients with recurrent melanoma has shown no improvement over the past 50 years. Many of these tumors are histopathologically quite similar but can be subclassified based upon their gene expression profiles (1, 2). In a study by Bittner et al. (1), the gene that best separated highly aggressive tumors from less aggressive tumors was WNT5A, which was consistently underexpressed in the less motile tumors. Wnt5A is a member of the Wnt family of proteins, which were first identified during studies of development in Drosophila (3) and in studies of the mouse mammary tumor virus (4). Unlike its family members Wnt1 and Wnt3A, which signal via the canonical Wnt pathway, resulting in the nuclear translocation of ␤-catenin, Wnt5A acts via G-protein-coupled receptors to activate protein kinase C (PKC) 6 and intracellular calcium (5, 6). The interplay between these two pathways is not well understood, but it does appear that the non-canonical Wnt pathway can inhibit ␤-catenin stabilization both in vitro in human HEK293 cells and in vivo in zebrafish (7,8).In melanoma cells with low motility and low expression of WNT5A, overexpressing WNT5A resulted in an increase in both the activation of PKC and an increase in motility (9). High expression of WNT5A in melanoma patients also correlated to poor outcome in this study. In addition, many studies have highligh...

The apoptotic initiator caspase-2 has been implicated in oocyte death, in DNA damage-and heat shock-induced death, and in mitotic catastrophe. We show here that the mitosis-promoting kinase, cdk1-cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase-2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase-2 interdomain, prevents caspase-2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase-2 detected during interphase was lost in mitosis. Expression of S340A non-phosphorylatable caspase-2 abrogated mitotic suppression of caspase-2 and apoptosis in various settings, including oocytes induced to undergo cdk1-dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase-2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase-2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase-2 and suggest that under conditions of mitotic arrest, cdk1-cyclin B1 activity must be overcome for apoptosis to occur.

The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds to many proteins, some of which trigger signal transduction. Receptor-recognized forms of ␣ 2 -Macroglobulin (␣ 2 M*) bind to LRP, but the pattern of signal transduction differs significantly from that observed with other LRP ligands. For example, neither Ni 2؉ nor the receptor-associated protein, which blocks binding of all known ligands to LRP, block ␣ 2 M*-induced signal transduction. In the current study, we employed ␣ 2 -macroglobulin (␣ 2 M)-agarose column chromatography to purify cell surface membrane binding proteins from 1-LN human prostate cancer cells and murine macrophages. The predominant binding protein purified from 1-LN prostate cancer cells was Grp 78 with small amounts of LRP, a fact that is consistent with our previous observations that there is little LRP present on the surface of these cells. The ratio of LRP:Grp 78 is much higher in macrophages. Flow cytometry was employed to demonstrate the presence of Grp 78 on the cell surface of 1-LN cells. Purified Grp 78 binds to ␣ 2 M* with high affinity (K d ϳ150 pM). A monoclonal antibody directed against Grp 78 both abolished ␣ 2 M*-induced signal transduction and co-precipitated LRP. Ligand blotting with ␣ 2 M* showed binding to both Grp 78 and LRP heavy chains in these preparations. Use of RNA interference to silence LRP expression had no effect on ␣ 2 M*-mediated signaling. We conclude that Grp 78 is essential for ␣ 2 M*-induced signal transduction and that a "co-receptor" relationship exists with LRP like that seen with several other ligands and receptors such as the uPA/uPAR (urinary type plasminogen activator or urokinase/uPA receptor) system.1 is a plasma proteinase inhibitor with broad specificity. Upon binding to proteinases, it undergoes a major conformational change that exposes receptor recognition sites on the molecule (1). This activated form of ␣ 2 M is designated ␣ 2 M*. Though difficult to reconcile, accumulating evidence has demonstrated that ␣ 2 M*-induced signaling occurs via a receptor that is functionally unique from the previously characterized ␣ 2 M* receptor, the low density lipoprotein receptor-related protein (LRP). LRP is a scavenger receptor (1) that binds to a variety of proteins, many of which trigger signal transduction (2-5). This pathway requires the activation of a pertussis toxin-sensitive G protein (2-5). The receptor-associated protein (RAP) that blocks the binding of all known ligands to LRP is also an antagonist for this signaling pathway (2-3). Whereas ␣ 2 M* binds to LRP, when cells are exposed to ␣ 2 M* a distinct set of signaling events is observed that differ from those induced by other ligands for this receptor (2-6). These include activation of a different G protein and the lack of antagonism by RAP or Ni 2ϩ (2-7). In addition, binding studies with a variety of cells in culture demonstrate two classes of binding sites, one of very high affinity (K d ϳ100 pM and 1600 sites/cell) and one of lower affinity (K d ...

Human plasminogen contains structural domains that are termed kringles. Proteolytic cleavage of plasminogen yields kringles 1-3 or 4 and kringle 5 (K5), which regulate endothelial cell proliferation. The receptor for kringles 1-3 or 4 has been identified as cell surfaceassociated ATP synthase; however, the receptor for K5 is not known. Sequence homology exists between the plasminogen activator streptokinase and the human voltagedependent anion channel (VDAC); however, a functional relationship between these proteins has not been reported. A streptokinase binding site for K5 is located between residues Tyr 252 -Lys 283 , which is homologous to the primary sequence of VDAC residues Tyr 224 -Lys 255 . Antibodies against these sequences react with VDAC and detect this protein on the plasma membrane of human endothelial cells. K5 binds with high affinity (K d of 28 nM) to endothelial cells, and binding is inhibited by these antibodies. Purified VDAC binds to K5 but only when reconstituted into liposomes. K5 also interferes with mechanisms controlling the regulation of intracellular Ca 2؉ via its interaction with VDAC. K5 binding to endothelial cells also induces a decrease in intracellular pH and hyperpolarization of the mitochondrial membrane. These studies suggest that VDAC is a receptor for K5.

Brain tumors are typically resistant to conventional chemotherapeutics, most of which initiate apoptosis upstream of mitochondrial cytochrome c release. In this study, we demonstrate that directly activating apoptosis downstream of the mitochondria, with cytosolic cytochrome c, kills brain tumor cells but not normal brain tissue. Specifically, cytosolic cytochrome c is sufficient to induce apoptosis in glioblastoma and medulloblastoma cell lines. In contrast, primary neurons from the cerebellum and cortex are remarkably resistant to cytosolic cytochrome c. Importantly, tumor tissue from mouse models of both high-grade astrocytoma and medulloblastoma display hypersensitivity to cytochrome c when compared with surrounding brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in the tumor tissue compared with low Apaf-1 levels in the adjacent brain tissue. These differences in Apaf-1 abundance correlate with differences in the levels of E2F1, a previously identified activator of Apaf-1 transcription. ChIP assays reveal that E2F1 binds the Apaf-1 promoter specifically in tumor tissue, suggesting that E2F1 contributes to the expression of Apaf-1 in brain tumors. Together, these results demonstrate an unexpected sensitivity of brain tumors to postmitochondrial induction of apoptosis. Moreover, they raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancer cells while sparing the surrounding brain parenchyma.astrocytoma ͉ caspase ͉ cell death ͉ medulloblastoma ͉ neurons

Many pro‐apoptotic signals trigger mitochondrial cytochrome c release, leading to caspase activation and ultimate cellular breakdown. Cell survival pathways, including the mitogen‐activated protein kinase (MAPK) cascade, promote cell viability by impeding mitochondrial cytochrome c release and by inhibiting subsequent caspase activation. Here, we describe a mechanism for the inhibition of cytochrome c‐induced caspase activation by MAPK signalling, identifying a novel mode of apoptotic regulation exerted through Apaf‐1 phosphorylation by the 90‐kDa ribosomal S6 kinase (Rsk). Recruitment of 14‐3‐3ε to phosphorylated Ser268 impedes the ability of cytochrome c to nucleate apoptosome formation and activate downstream caspases. High endogenous levels of Rsk in PC3 prostate cancer cells or Rsk activation in other cell types promoted 14‐3‐3ε binding to Apaf‐1 and rendered the cells insensitive to cytochrome c, suggesting a potential role for Rsk signalling in apoptotic resistance of prostate cancers and other cancers with elevated Rsk activity. Collectively, these results identify a novel locus of apoptosomal regulation wherein MAPK signalling promotes Rsk‐catalysed Apaf‐1 phosphorylation and consequent binding of 14‐3‐3ε, resulting in decreased cellular responsiveness to cytochrome c.

The antiangiogenic protein angiostatin inhibits ATP synthase on the endothelial cell surface, blocking cellular proliferation. To examine the specificity of this interaction, we generated monoclonal antibodies (mAb) directed against ATP synthase. mAb directed against the B-catalytic subunit of ATP synthase (MAb3D5AB1) inhibits the activity of the F 1 domain of ATP synthase and recognizes the catalytic B-subunit of ATP synthase. We located the antibody recognition site of MAb3D5AB1 in domains containing the active site of the Bsubunit. MAb3D5AB1 also binds to purified Escherichia coli F 1 with an affinity 25-fold higher than the affinity of angiostatin for this protein. MAb3D5AB1 inhibits the hydrolytic activity of F 1 ATP synthase at lower concentrations than angiostatin. Like angiostatin, MAb3D5AB1 inhibits ATP generation by ATP synthase on the endothelial cell surface in acidic conditions, the typical tumor microenvironment where cell surface ATP synthase exhibits greater activity. MAb3D5AB1 disrupts tube formation and decreases intracellular pH in endothelial cells exposed to low extracellular pH. Neither angiostatin nor MAb3D5AB1 showed an antiangiogenic effect in the corneal neovascularization assay; however, both were effective in the low-pH environment of the chicken chorioallantoic membrane assay. Thus, MAb3D5AB1 shows angiostatin-like properties superior to angiostatin and may be exploited in cancer chemotherapy. [Cancer Res 2007;67(10):4716-24]

During apoptosis, caspases cleave cellular substrates to break down and package the apoptotic cell for removal. Reporting in Cell, Mahrus et al. (2008) and Dix et al. (2008) use new approaches that identify hundreds of previously unrecognized caspase substrates, many of which appear to produce polypeptide fragments with potentially new functional activities.