One of the key steps during tumour metastasis is tumour cell migration and invasion, which require actin cytoskeletal reorganization. Among the critical actin cytoskeletal protrusion structures are the filopodia, which act like cell sensory organs to communicate with the extracellular microenvironment and participate in fundamental cell functions such as cell adhesion, spreading and migration in the three-dimensional environment. Fascin is the main actin-bundling protein in filopodia. Using high-throughput screening, here we identify and characterize small molecules that inhibit the actin-bundling activity of fascin. Focusing on one such inhibitor, we demonstrate that it specifically blocks filopodial formation, tumour cell migration and invasion in vitro, and metastasis in vivo. Hence, target-specific anti-fascin agents have a therapeutic potential for cancer treatment.

A B S T R A C TTumor metastasis is the major cause of mortality of cancer patients, being responsible for w90% of all cancer deaths. One of the key steps during tumor metastasis is tumor cell migration which requires actin cytoskeletal reorganization. Among the critical actin cytoskeletal protrusion structures are antenna-like filopodia. Fascin protein is the main actinbundling protein in filopodia. Here we report the development of fascin-specific small-molecules that inhibit the interaction between fascin and actin. These inhibitors block the in vitro actin-binding and actin-bundling activities of fascin, tumor cell migration and tumor metastasis in mouse models. Mechanistically, these inhibitors likely occupy one of the actin-binding sites, reduce the binding of actin filaments, and thus lead to the inhibition of the bundling activity of fascin. At the cellular level, these inhibitors impair actin cytoskeletal reorganization. Our data indicate that target-specific anti-fascin agents will have great potential for treating metastatic tumors.

Heterotrimeric G proteins are critical transducers of cellular signaling. In addition to their classic roles in relaying signals from G protein-coupled receptors (GPCRs), heterotrimeric G proteins also mediate physiological functions from non-GPCRs. Previously, we have shown that G␣ 13 , a member of the heterotrimeric G proteins, is essential for growth factor receptor-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. These G␣ 13 -mediated dorsal ruffle turnover and cell migration by growth factors acting on their receptor tyrosine kinases (RTKs) are independent of GPCRs. However, the mechanism by which RTKs signal to G␣ 13 is not known. Here, we show that cholinesterase-8A (Ric-8A), a nonreceptor guanine nucleotide exchange factor for some heterotrimeric G proteins, is critical for coupling RTKs to G␣ 13 . Down-regulation of Ric-8A protein levels in cells by RNA interference slowed down platelet-derived growth factor (PDGF)-induced dorsal ruffle turnover and inhibited PDGF-initiated cell migration. PDGF was able to increase the activity of Ric-8A in cells. Furthermore, purified Ric-8A proteins interact directly with purified G␣ 13 protein in a nucleotide-dependent manner. Deficiency of Ric-8A prevented the translocation of G␣ 13 to the cell cortex. Hence, Ric-8A is critical for growth factor receptorinduced actin cytoskeletal reorganization.Heterotrimeric G proteins are essential for the transmembrane signaling by G protein-coupled receptors (GPCRs).2 A structurally diverse repertoire of ligands activates GPCRs to elicit their physiological functions (1). Ligand-bound GPCRs function as guanine nucleotide exchange factors (GEFs) catalyzing the exchange of GDP bound on the G␣ subunit with GTP in the presence of G␤␥. This leads to the dissociation of the G␣ subunit from the G␤␥ dimer to form two functional units (G␣ and G␤␥) (2). Both G␣ and G␤␥ subunits signal to various cellular pathways. Based on the sequence and functional homologies, G proteins are grouped into four families: G s , G i , G q , and G 12 (3). Among these four subfamilies of G proteins, the physiological function of the G 12 subfamily is less well understood. In this family, there are two members, G 12 and G 13 . G␣ 12 knock-out mice appeared normal (4). G␣ 13 knock-out mice displayed embryonic lethality (ϳE9.5) (5). The molecular basis that underlies the vascular defect observed in G␣ 13 Ϫ/Ϫ mouse embryos has not been defined.In addition to their classic roles in GPCR signaling, heterotrimeric G proteins have been genetically demonstrated to play important roles in GPCR-independent signaling (6). The best examples are in the mitotic spindle positioning and orientation (in the establishment of cell polarity) during asymmetric division in Caenorhabditis elegans embryos and in Drosophila neuroblasts (7-11). In these processes, G␣ i/o -GDP binds to a protein with the tetratricopeptide-GoLoco domain (such as GPR-1/2 in C. elegans and Pins in Drosophila) and disrupts intramolecular tetratricopeptide-...

Neuroendocrine tumors (NET) often harbor loss-of-function mutations in the MEN1 and DAXX tumor suppressor genes. Here we report that the products of these genes, menin and Daxx, interact directly with each other to suppress the proliferation of NET cells, to a large degree by inhibiting expression of the membrane metallo-endopeptidase MME. Menin and Daxx were required to enhance histone H3 lysine9 trimethylation (H3K9me3) at the MME promoter, as mediated partly by the histone H3 methyltransferase SUV39H1. Notably, the menin T429K mutation associated with a NET syndrome abolished Daxx binding, MME repression and proliferation of NET cells. Conversely, inhibition of MME in NET cells repressed proliferation and tumor growth in vivo. Our findings reveal a previously unappreciated crosstalk between two crucial tumor suppressor genes thought to work by independent pathways, focusing on MME as a common target of menin/Daxx to treat NET.

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