Dasatinib inhibits the development of metastases in a mouse model of pancreatic ductal adenocarcinoma.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive and metastatic disease for which conventional treatments are of limited efficacy. A number of agents in development are potential anti-invasive and antimetastatic agents, including the Src kinase inhibitor dasatinib. The aim of this study was to assess the importance of Src in human PDAC and to use a genetically engineered mouse model of PDAC to determine the effects of dasatinib on PDAC progression. METHODS: Src expression and activity was measured by immunohistochemistry in 114 human PDACs. Targeting expression of Trp53(R172H) and Kras(G12D) to the mouse pancreas results in the formation of invasive and metastatic PDAC. These mice were treated with dasatinib, and disease progression monitored. Cell lines were derived from mouse PDACs, and in vitro effects of dasatinib assessed. RESULTS: Src expression and activity were up-regulated in human PDAC and this correlated with reduced survival. Dasatinib inhibited the migration and invasion of PDAC cell lines, although no effects on proliferation were seen at concentrations that inhibited Src kinase activity. In addition, dasatinib significantly inhibited the development of metastases in Pdx1-Cre, Z/EGFP, LSL-Kras(G12D/+), LSL-Trp53(R172H/+) mice. However, there was no survival advantage in the dasatinib-treated animals owing to continued growth of the primary tumor. CONCLUSIONS: This study confirms the importance of Src in human PDAC and shows the usefulness of a genetically engineered mouse model of PDAC for assessing the activity of potential antimetastatic agents and suggests that dasatinib should be evaluated further as monotherapy after resection of localized invasive PDAC.

Identification of the insulin-responsive tyrosine phosphorylation sites on IRSp53.

The 53-kDa insulin receptor substrate protein (IRSp53) is part of a regulatory network that organises the actin cytoskeleton in response to stimulation by small GTPases, promoting formation of actin-rich cell protrusions such as filopodia and lamellipodia. It had been established earlier that IRSp53 is tyrosine phosphorylated in response to stimulation of the insulin and insulin-related growth factor receptors, but the consequences of tyrosine phosphorylation for IRSp53 function are unknown. Here, we have used a variety of IRSp53 truncation and point mutants to identify insulin-responsive tyrosine phosphorylation sites on IRSp53. We have found that the C-terminal half of IRSp53 (residues 251-521) undergoes tyrosine phosphorylation in response to insulin stimulation of the insulin beta receptor or epidermal growth factor stimulation via the epidermal growth factor receptor, and that the key residue for insulin receptor-mediated phosphorylation is tyrosine 310, located in a region between the N-terminal IRSp53/MIM homology domain (IMD, residue 1-250) and the central SH3 domain (residues 374-438) that is predicted to be natively unstructured. Mutation of tyrosine 310 to phenylalanine or glutamic acid abrogates the phosphorylation in response to insulin stimulation, but not in response to stimulation of the epidermal growth factor receptor. The N-terminal IMD, which mediates dimerisation of IRSp53, is required for efficient tyrosine phosphorylation downstream of either the insulin or epidermal growth factor receptor stimulation, yet does not appear to include a tyrosine-phosphorylated site itself. Thus, we have identified tyrosine 310 as a primary site of tyrosine phosphorylation in response to insulin signalling and we have shown that although IRSp53 is tyrosine phosphorylated in response to epidermal growth factor receptor signalling, tyrosine 310 is not crucial. Furthermore, the tyrosine phosphorylation status does not appear to affect the cell morphology and production of filopod-like structures upon expression of IRSp53.

Glial cell line-derived neurotrophic factor protects astrocytes from staurosporine- and ischemia- induced apoptosis.

Glial cell line-derived neurotrophic factor (GDNF) promotes the survival and functions of neurons. It has been shown to be a promising candidate in the treatment of ischemia and other neurodegenerative diseases. We transfected mouse astrocytes in primary cultures with a human GDNF gene and found that their conditioned medium could not only support the growth and survival of cultured dopaminergic neurons but also protect astrocytes from staurosporine- and ischemia-induced apoptosis. This indicated that these transfected astrocytes could release GDNF. A similar protective effect on astrocytes against apoptosis was evident when recombinant human GDNF was used. Moreover, GDNF reduced caspase-3 activity but not that of caspase-1 in cultured astrocytes after ischemia treatment. Thus, GDNF protects astrocytes from apoptosis by inhibiting the activation of caspase-3.

Structural basis of filopodia formation induced by the IRSp53/MIM homology domain of human IRSp53.

The scaffolding protein insulin receptor tyrosine kinase substrate p53 (IRSp53), a ubiquitous regulator of the actin cytoskeleton, mediates filopodia formation under the control of Rho-family GTPases. IRSp53 comprises a central SH3 domain, which binds to proline-rich regions of a wide range of actin regulators, and a conserved N-terminal IRSp53/MIM homology domain (IMD) that harbours F-actin-bundling activity. Here, we present the crystal structure of this novel actin-bundling domain revealing a coiled-coil domain that self-associates into a 180 A-long zeppelin-shaped dimer. Sedimentation velocity experiments confirm the presence of a single molecular species of twice the molecular weight of the monomer in solution. Mutagenesis of conserved basic residues at the extreme ends of the dimer abrogated actin bundling in vitro and filopodia formation in vivo, demonstrating that IMD-mediated actin bundling is required for IRSp53-induced filopodia formation. This study promotes an expanded view of IRSp53 as an actin regulator that integrates scaffolding and effector functions.