The mammalian cilium protrudes from the apical/lumenal surface of polarized cells and acts as a sensor of environmental cues. Numerous developmental disorders and pathological conditions have been shown to arise from defects in cilia-associated signaling proteins. Despite mounting evidence that cilia are essential sites for coordination of cell signaling, little is known about the cellular mechanisms controlling their formation and disassembly. Here, we show that interactions between the prometastatic scaffolding protein HEF1/Cas-L/NEDD9 and the oncogenic Aurora A (AurA) kinase at the basal body of cilia causes phosphorylation and activation of HDAC6, a tubulin deacetylase, promoting ciliary disassembly. We show that this pathway is both necessary and sufficient for ciliary resorption and that it constitutes an unexpected nonmitotic activity of AurA in vertebrates. Moreover, we demonstrate that small molecule inhibitors of AurA and HDAC6 selectively stabilize cilia from regulated resorption cues, suggesting a novel mode of action for these clinical agents.

The tumor suppressor p53 inhibits tumor growth primarily through its ability to induce apoptosis. Mutations in p53 occur in at least 50% of human tumors. We hypothesized that reactivation of mutant p53 in such tumors should trigger massive apoptosis and eliminate the tumor cells. To test this, we screened a library of low-molecular-weight compounds in order to identify compounds that can restore wild-type function to mutant p53. We found one compound capable of inducing apoptosis in human tumor cells through restoration of the transcriptional transactivation function to mutant p53. This molecule, named PRIMA-1, restored sequence-specific DNA binding and the active conformation to mutant p53 proteins in vitro and in living cells. PRIMA-1 rescued both DNA contact and structural p53 mutants. In vivo studies in mice revealed an antitumor effect with no apparent toxicity. This molecule may serve as a lead compound for the development of anticancer drugs targeting mutant p53.

Although HEF1 has a well-defined role in integrin-dependent attachment signaling at focal adhesions, it relocalizes to the spindle asters at mitosis. We report here that overexpression of HEF1 causes increase in centrosome numbers and multipolar spindles, resembling defects induced by manipulation of the mitotic regulatory kinase Aurora A (AurA). We show that HEF1 associates with and controls activation of AurA. We also show HEF1 depletion causes centrosomal splitting, monoastral spindles, and hyperactivation of Nek2, implying additional action earlier in cell cycle. These results provide new insights into the role of an adhesion protein in coordination of cell attachment and division. KeywordsHEF1; centrosome; spindle; mitosis; Aurora-A; Nek2 HEF1 is a member of a group of scaffolding proteins that includes p130Cas and Efs/Sin 1,2 . This group of Cas proteins localizes to focal adhesions in interphase cells, and acts as intermediates in a variety of integrin-dependent signaling processes, including establishment of cell attachments, migration, and cell survival signaling. In 1998, we proposed that HEF1 might have a previously unsuspected function in mitosis 3 , based on the observation that the HEF1 protein relocalized from focal adhesions to the mitotic spindle asters in M-phase. Since that time, reports have appeared suggesting of the association of other focal adhesion complex proteins, such as zyxin 4 , paxillin 5 , FAK, and Pyk2 6 with the mitotic spindle or other relevant structures such as the microtubule organizing center (MTOC) or centrosome. Recent work has emphasized the dual activity of centrosomes in contributing to control of cell polarization in interphase cell migration 7,8 , but also in coordinating assembly of the mitotic spindle in Mphase 9 . Centrosomally-associated signaling activities such as the Aurora-A (AurA) kinase also govern the timing of mitotic entry 10 , for instance by regulating the activation of cyclin B1 11 . In this study, we demonstrate an requirement for HEF1 in activation of AurA and Nek2 12 , a second kinase important for centrosome cohesion, and we provide additional data indicating HEF1 provides a crucial bridge coordinating cell attachment and cell division processes in mammalian cells. Results Cell cycle-regulated HEF1 localizationHEF1 localizes to the centrosome of MCF7 cells in a cell cycle regulated manner ( Figure 1A), with centrosomal signal lowest in G1, and strongest in G2/M cells. This corresponds to * corresponding author: Erica Golemis, W406, Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111, Phone: 215-728-2860, Fax: 215-728-3616 Figure 2A; 3). As HEF1 levels increase during G2, a slower migrating, hyperphosphorylated species becomes more apparent, as we have previously reported 3 . At mitotic entry, a significant fraction of HEF1 moves to the spindle, and HEF1 is no longer detectable at the centrosome at cytokinesis. The endogenous HEF1 localization pattern described here was lost following HEF1 depletion with siRNA, supporting si...

The highly invasive behavior of glioblastoma cells contributes to the morbidity and mortality associated with these tumors. The integrin-mediated adhesion and migration of glioblastoma cells on brain matrix proteins is enhanced by stimulation with growth factors, including platelet-derived growth factor (PDGF). As focal adhesion kinase (FAK), a nonreceptor cytoplasmic tyrosine kinase, has been shown to promote cell migration in various other cell types, we analysed its role in glioblastoma cell migration. Forced overexpression of FAK in serumstarved glioblastoma cells plated on recombinant (rec)-osteopontin resulted in a twofold enhancement of basal migration and a ninefold enhancement of PDGF-BBstimulated migration. Both expression of mutant FAK(397F) and the downregulation of FAK with small interfering (si) RNA inhibited basal and PDGF-stimulated migration. FAK overexpression and PDGF stimulation was found to increase the phosphorylation of the Crk-associated substrate (CAS) family member human enhancer of filamentation 1 (HEF1), but not p130CAS or Src-interacting protein (Sin)/Efs, although the levels of expression of these proteins was similar. Moreover downregulation of HEF1 with siRNA, but not p130CAS, inhibited basal and PDGF-stimulated migration. The phosphorylated HEF1 colocalized with vinculin and was associated almost exclusively with 0.1% Triton X-100 insoluble material, consistent with its signaling at focal adhesions. FAK overexpression promoted invasion through normal brain homogenate and siHEF1 inhibited this invasion. Results presented here suggest that HEF1 acts as a necessary and specific downstream effector of FAK in the invasive behavior of glioblastoma cells and may be an effective target for treatment of these tumors.

In the past 3 years, altered expression of the HEF1/CAS-L/ NEDD9 scaffolding protein has emerged as contributing to cancer metastasis in multiple cancer types. However, whereas some studies have identified elevated NEDD9 expression as prometastatic, other work has suggested a negative role in tumor progression. We here show that the Nedd9-null genetic background significantly limits mammary tumor initiation in the MMTV-polyoma virus middle T genetic model. Action of NEDD9 is tumor cell intrinsic, with immune cell infiltration, stroma, and angiogenesis unaffected. The majority of the late-appearing mammary tumors of MMTV-polyoma virus middle T;Nedd9 À/À mice are characterized by depressed activation of proteins including AKT, Src, FAK, and extracellular signal-regulated kinase, emphasizing an important role of NEDD9 as a scaffolding protein for these prooncogenic proteins. Analysis of cells derived from primary Nedd9 +/+ and Nedd9 À/À tumors showed persistently reduced FAK activation, attachment, and migration, consistent with a role for NEDD9 activation of FAK in promoting tumor aggressiveness. This study provides the first in vivo evidence of a role for NEDD9 in breast cancer progression and suggests that NEDD9 expression may provide a biomarker for tumor aggressiveness.