Excellent translational research in oncology: A journey towards novel and more effective anti-cancer therapies.

Comprehensive Cancer Centres (CCCs) serve as critical drivers for improving cancer survival. In Europe, we have developed an Excellence Designation System (EDS) consisting of criteria to assess "excellence" of CCCs in translational research (bench to bedside and back), with the expectation that many European CCCs will aspire to this status.

[Mouse models of K-ras-initiated oncogenesis]. / Apport des modèles animaux pour l'étude du rôle de K-ras dans l'oncogenèse.

Activating mutations of the oncogene K-ras are found in one third of all human cancers. Much of our knowledge on K-ras signal transduction and its influence on tumor initiation and progression come from in vitro studies with cell lines. However, mouse models of human cancer allow a much more faithful recapitulation of the human disease, and the in vivo perspective is crucial for our understanding of neoplasia. In recent years, several new murine models for K-ras-induced tumorigenesis have been described. They allow new insights into the specific role that oncogenic K-ras proteins play in different solid tumors, and they permit the molecular dissection of the pathways that are initiated by somatic mutations in subsets of cells. Key advances have been made by the use of tissue-specific and inducible control of expression, which is achieved by the Cre/loxP technology or the tetracycline system. From these sophisticated models, a common picture emerges: the effects of K-ras on tumor initiation depend strongly on the cellular context, and different tissues vary in their susceptibility to K-ras transformation.

Conditional mouse models of cancer.

The development of inducible and conditional technologies allowed us to generate transgenic mouse models that faithfully recapitulate human tumorigenesis. It is possible to control, in time and space, the development of tumors in almost every mouse tissue. The result is that now we have available mouse models for all major human cancers. Novel noninvasive approaches to tumor imaging will enable us to follow tumor development and metastasis in vivo, as well as the effects of candidate therapeutic drugs. Such new generation tumor models, which accurately emulate the disease state in situ, should provide a useful platform with which to experimentally test drugs targeted to specific gene products, or combinations of genes that control rate-limiting steps of tumor development. In this review, we focus on the different mouse models for colon cancer.

Src-dependent ezrin phosphorylation in adhesion-mediated signaling.

In addition to providing a regulated linkage between the membrane and the actin cytoskeleton, ezrin participates in signal transduction pathways. Here we describe that expression of the ezrin Y145F mutant delays epithelial cell spreading on fibronectin by inhibiting events leading to FAK activation. The defect in spreading was rescued by the overexpression of catalytically functional Src. We demonstrate that ezrin Y145 is phosphorylated in A431 cells stimulated with epidermal growth factor (EGF) and in v-Src-transformed cells. Moreover in cells devoid of Src, SYF-/- fibroblasts, ezrin Y145 phosphorylation could only be detected upon the introduction of an active form of Src. The phosphorylation of ezrin at Y145 required prior binding of the Src SH2 domain to ezrin. Our results further show that Src activity influences its binding to ezrin and a positive feedback mechanism for Src-mediated Y145 phosphorylation is implied. Interestingly, cells expressing ezrin Y145F did not proliferate when cultured in a 3D collagen gel. Collectively, our results demonstrate a key signaling input of Src-dependent ezrin phosphorylation in adhesion-mediated events in epithelial cells.

Na+-H+ exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3.

1. Rabbit ileal Na+-absorbing cell Na+-H+ exchanger 3 (NHE3) was shown to exist in three pools in the brush border (BB), including a population in lipid rafts. Approximately 50% of BB NHE3 was associated with Triton X-100-soluble fractions and the other approximately 50% with Triton X-100-insoluble fractions; approximately 33% of the detergent-insoluble NHE3 was present in cholesterol-enriched lipid microdomains (rafts). 2. The raft pool of NHE3 was involved in the stimulation of BB NHE3 activity with epidermal growth factor (EGF). Both EGF and clonidine treatments were associated with a rapid increase in the total amount of BB NHE3. This EGF- and clonidine-induced increase of BB NHE3 was associated with an increase in the raft pool of NHE3 and to a smaller extent with an increase in the total detergent-insoluble fraction, but there was no change in the detergent-soluble pool. In agreement with the rapid increase in the amount of NHE3 in the BB, EGF also caused a rapid stimulation of BB Na+-H+ exchange activity. 3. Disrupting rafts by removal of cholesterol with methyl-beta-cyclodextrin (MbetaCD) or destabilizing the actin cytoskeleton with cytochalasin D decreased the amount of NHE3 in early endosomes isolated by OptiPrep gradient fractionation. Specifically, NHE3 was shown to associate with endosomal vesicles immunoisolated by anti-EEA1 (early endosomal autoantigen 1) antibody-coated magnetic beads and the endosome-associated NHE3 was decreased by cytochalasin D and MbetaCD treatment. 4. We conclude that: (i) a pool of ileal BB NHE3 exists in lipid rafts; (ii) EGF and clonidine increase the amount of BB NHE3; (iii) lipid rafts and to a lesser extent, the cytoskeleton, but not the detergent-soluble NHE3 pool, are involved in the EGF- and clonidine-induced acute increase in amount of BB NHE3; (iv) lipid rafts and the actin cytoskeleton play important roles in the basal endocytosis of BB NHE3.

Actin filaments and myosin I alpha cooperate with microtubules for the movement of lysosomes.

An earlier report suggested that actin and myosin I alpha (MMIalpha), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIalpha were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIalpha. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMIalpha impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMIalpha contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors.

Immunocytochemical detection of bone marrow micrometastases in colorectal carcinoma patients, using a monoclonal antibody to villin.

The search continues to find methods to more effectively distinguish colorectal carcinoma patients who could be separated into high-risk and low-risk categories. Investigators have reported on the detection of occult micrometastases in bone marrow using antibodies to cytokeratin, which is a marker of epithelial cells but which has no tissue specificity, as opposed to villin, a cytoskeletal protein that is specifically involved in the formation of brush-border microvilli in the small intestine and colon epithelium. Specificity and sensitivity of antibody to villin (ID2C3) and antibody to cytokeratin (A45-B/B3) were first studied in normal bone marrow and in a test system in which cancer cell lines were mixed in normal bone marrow. In a preliminary study including 16 colorectal carcinoma patients, we compared the number of villin-positive cells with cytokeratin-presenting cells. As A45-B/B3, ID2C3 was determined to be sensitive enough to detect one cancer cell in 10(6) hematopoietic cells. Staining of hematopoietic cells with irrelevant antibody and a light staining of megakaryocytes with ID2C3 limited the specificity of the method. In colorectal carcinoma patients, correlation between ID2C3 and A45-B/B3 was 94%. Sensitivity and specificity of ID2C3 antibody to villin were satisfactory. Its clinical relevance must be investigated in further studies.

ActA and human zyxin harbour Arp2/3-independent actin-polymerization activity.

The actin cytoskeleton is a dynamic network that is composed of a variety of F-actin structures. To understand how these structures are produced, we tested the capacity of proteins to direct actin polymerization in a bead assay in vitro and in a mitochondrial-targeting assay in cells. We found that human zyxin and the related protein ActA of Listeria monocytogenes can generate new actin structures in a vasodilator-stimulated phosphoprotein-dependent (VASP) manner, but independently of the Arp2/3 complex. These results are consistent with the concept that there are multiple actin-polymerization machines in cells. With these simple tests it is possible to probe the specific function of proteins or identify novel molecules that act upon cellular actin polymerization.

Ezrin interacts with focal adhesion kinase and induces its activation independently of cell-matrix adhesion.

Ezrin, a membrane-cytoskeleton linker, is required for cell morphogenesis, motility, and survival through molecular mechanisms that remain to be elucidated. Using the N-terminal domain of ezrin as a bait, we found that p125 focal adhesion kinase (FAK) interacts with ezrin. We show that the two proteins coimmunoprecipitate from cultured cell lysates. However, FAK does not interact with full-length ezrin in vitro, indicating that the FAK binding site on ezrin is cryptic. Mapping experiments showed that the entire N-terminal domain of FAK (amino acids 1-376) is required for optimal ezrin binding. While investigating the role of the ezrin-FAK interaction, we observed that, in suspended kidney-derived epithelial LLC-PK1 cells, overproduction of ezrin promoted phosphorylation of FAK Tyr-397, the major autophosphorylation site, creating a docking site for FAK signaling partners. Treatment of the cells with a Src family kinase inhibitor reduced the phosphorylation of Tyr-577 but not that of Tyr-397, indicating that ezrin-mediated FAK activation does not require the activity of Src kinases. Altogether, these observations indicate that ezrin is able to trigger FAK activation in signaling events that are not elicited by cell-matrix adhesion.

Targeting of zyxin to sites of actin membrane interaction and to the nucleus.

The localization of proteins to particular intracellular compartments often regulates their functions. Zyxin is a LIM protein found prominently at sites of cell adhesion, faintly in leading lamellipodia, and transiently in cell nuclei. Here we have performed a domain analysis to identify regions in zyxin that are responsible for targeting it to different subcellular locations. The N-terminal proline-rich region of zyxin, which harbors binding sites for alpha-actinin and members of the Ena/VASP family, concentrates in lamellipodial extensions and weakly in focal adhesions. The LIM region of zyxin displays robust targeting to focal adhesions. When overexpressed in cells, the LIM region of zyxin causes displacement of endogenous zyxin from focal adhesions. Upon mislocalization of full-length zyxin, at least one member of the Ena/VASP family is also displaced, and the organization of the actin cytoskeleton is perturbed. Zyxin also has the capacity to shuttle between the nucleus and focal adhesion sites. When nuclear export is inhibited, zyxin accumulates in cell nuclei. The nuclear accumulation of zyxin occurs asynchronously with approximately half of the cells exhibiting nuclear localization of zyxin within 2.3 h of initiating leptomycin B treatment. Our results provide insight into the functions of different zyxin domains.

A common exocytotic mechanism mediates axonal and dendritic outgrowth.

Outgrowth of the dendrites and the axon is the basis of the establishment of the neuronal shape, and it requires addition of new membrane to both growing processes. It is not yet clear whether one or two exocytotic pathways are responsible for the respective outgrowth of axons and dendrites. We have previously shown that tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) defines a novel network of tubulovesicular structures present both at the leading edge of elongating dendrites and axons of immature hippocampal neurons developing in primary culture and that TI-VAMP is an essential protein for neurite outgrowth in PC12 cells. Here we show that the expression of the N-terminal domain of TI-VAMP inhibits the outgrowth of both dendrites and axons in neurons in primary culture. This effect is more prominent at the earliest stages of the development of neurons in vitro. Expression of the N-terminal domain deleted form of TI-VAMP has the opposite effect. This constitutively active form of TI-VAMP localizes as the endogenous protein, particularly concentrating at the leading edge of growing axons. Our results suggest that a common exocytotic mechanism that relies on TI-VAMP mediates both axonal and dendritic outgrowth in developing neurons.

Expression patterns of L-plastin isoform in normal and carcinomatous breast tissues.

Plastins are members of a family of actin-binding proteins which exhibit a tissue-specific expression pattern. L-plastin, which is specifically expressed in hematopoietic cell lineage, has been proposed to be involved in the control of cell adhesion and motility. This protein is also frequently expressed in cell lines derived from mammary solid tumors and therefore might be involved in cancer invasion and metastasis. We have analysed plastin expression in normal and carcinomatous breast tissues in vivo by immunohistochemistry and immunoblotting approaches using specific plastin isoform antibodies. L-plastin was not detected in normal epithelial cells of the mammary gland whereas a staining of myoepithelial cells was observed in 50% of the cases. In breast carcinomas, a significant immunostaining of malignant epithelial cells was observed in 4 of the 29 cases analysed (13.8%). No correlation between L-plastin expression and tumor size, histological grade or lymph node status was observed. In contrast, L-plastin was found expressed in 4 of the 11 estrogen and progesterone receptors negative tumors (p = 0.039). The potential role of plastin expression in the tumor process is discussed.

Differentiation of the epithelial apical junctional complex during mouse preimplantation development: a role for rab13 in the early maturation of the tight junction.

We have investigated the mechanisms by which the epithelial apicolateral junctional complex (AJC) is generated during trophectoderm differentiation in the mouse blastocyst using molecular, structural and functional analyses. The mature AJC comprises an apical tight junction (TJ), responsible for intercellular sealing and blastocoel formation, and subjacent zonula adherens E-cadherin/catenin adhesion complex which also extends along lateral membrane contact sites. Dual labelling confocal microscopy revealed that the AJC derived from a single 'intermediate' complex formed following embryo compaction at the 8-cell stage in which the TJ-associated peripheral membrane protein, ZO-1alpha- isoform, was co-localized with both alpha- and beta-catenin. However, following assembly of the TJ transmembrane protein, occludin, from the early 32-cell stage when blastocoel formation begins, ZO-1alpha- and other TJ proteins (ZO-1alpha+ isoform, occludin, cingulin) co-localized in an apical TJ which was separate from a subjacent E-cadherin/catenin zonula adherens complex. Thin-section electron microscopy confirmed that a single zonula adherens-like junctional complex present at the AJC site following compaction matured into a dual TJ and zonula adherens complex at the blastocyst stage. Embryo incubation in the tracer FITC-dextran 4 kDa showed that a functional TJ seal was established coincident with blastocoel formation. We also found that rab13, a small GTPase previously localized to the TJ, is expressed at all stages of preimplantation development and relocates from the cytoplasm to the site of AJC biogenesis from compaction onwards with rab13 and ZO-1alpha- co-localizing precisely. Our data indicate that the segregation of the two elements of the AJC occurs late in trophectoderm differentiation and likely has functional importance in blastocyst formation. Moreover, we propose a role for rab13 in the specification of the AJC site and the formation and segregation of the TJ.

Vimentin filaments in fibroblasts are a reservoir for SNAP23, a component of the membrane fusion machinery.

Soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors (SNAREs) are core machinery for membrane fusion during intracellular vesicular transport. Synaptosome-associated protein of 23 kDa (SNAP23) is a target SNARE previously identified at the plasma membrane, where it is involved in exocytotic membrane fusion. Here we show that SNAP23 associates with vimentin filaments in a Triton X-100 insoluble fraction in fibroblasts in primary culture and HeLa cells. Upon treatment of human fibroblasts with N-ethyl-maleimide, SNAP23 dissociates from vimentin filaments and forms a protein complex with syntaxin 4, a plasma membrane SNARE. The vimentin-associated pool of SNAP23 can therefore be a reservoir, which would supply the plasma membrane fusion machinery, in fibroblasts. Our observation points to a yet unexplored role of intermediate filaments.

Morphogenic effects of ezrin require a phosphorylation-induced transition from oligomers to monomers at the plasma membrane.

ERM (ezrin, radixin, moesin) proteins act as linkers between the plasma membrane and the actin cytoskeleton. An interaction between their NH(2)- and COOH-terminal domains occurs intramolecularly in closed monomers and intermolecularly in head-to-tail oligomers. In vitro, phosphorylation of a conserved threonine residue (T567 in ezrin) in the COOH-terminal domain of ERM proteins disrupts this interaction. Here, we have analyzed the role of this phosphorylation event in vivo, by deriving stable clones producing wild-type, T567A, and T567D ezrin from LLC-PK1 epithelial cells. We found that T567A ezrin was poorly associated with the cytoskeleton, but was able to form oligomers. In contrast, T567D ezrin was associated with the cytoskeleton, but its distribution was shifted from oligomers to monomers at the membrane. Moreover, production of T567D ezrin induced the formation of lamellipodia, membrane ruffles, and tufts of microvilli. Both T567A and T567D ezrin affected the development of multicellular epithelial structures. Collectively, these results suggest that phosphorylation of ERM proteins on this conserved threonine regulates the transition from membrane-bound oligomers to active monomers, which induce and are part of actin-rich membrane projections.

Characterization of the interaction between zyxin and members of the Ena/vasodilator-stimulated phosphoprotein family of proteins.

Zyxin contains a proline-rich N-terminal domain that is similar to the C-terminal domain in the ActA protein of the bacteria, Listeria monocytogenes. We screened the entire amino acid sequence of human zyxin for Mena-interacting peptides and found that, as with ActA, proline-rich sequences were the sole zyxin sequences capable of binding to Ena/vasodilator-stimulated phosphoprotein (VASP) family members in vitro. From this information, we tested zyxin mutants in which the proline-rich sequences were altered. The reduction in Mena/VASP binding was confirmed by peptide tests, immunoprecipitation, and ectopic expression of zyxin variants at the surface of mitochondria. By transfection assays we showed that zyxin interaction with Mena/VASP in vivo enhances the production of actin-rich structures at the apical surface of cells. Microinjection into cells of peptides corresponding to the first proline-rich sequence of zyxin caused the loss of Mena/VASP from focal contacts. Furthermore, these peptides reduced the degree of spreading of cells replated after trypsinization. We conclude that zyxin and proteins that harbor similar proline-rich repeats contribute to the positioning of Mena/VASP proteins. The positioning of Ena/VASP family members appears to be important when the actin cytoskeleton is reorganized, such as during spreading.

Role of tetanus neurotoxin insensitive vesicle-associated membrane protein (TI-VAMP) in vesicular transport mediating neurite outgrowth.

How vesicular transport participates in neurite outgrowth is still poorly understood. Neurite outgrowth is not sensitive to tetanus neurotoxin thus does not involve synaptobrevin-mediated vesicular transport to the plasma membrane of neurons. Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicle-SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor), involved in transport to the apical plasma membrane in epithelial cells, a tetanus neurotoxin-resistant pathway. Here we show that TI-VAMP is essential for vesicular transport-mediating neurite outgrowth in staurosporine-differentiated PC12 cells. The NH(2)-terminal domain, which precedes the SNARE motif of TI-VAMP, inhibits the association of TI-VAMP with synaptosome-associated protein of 25 kD (SNAP25). Expression of this domain inhibits neurite outgrowth as potently as Botulinum neurotoxin E, which cleaves SNAP25. In contrast, expression of the NH(2)-terminal deletion mutant of TI-VAMP increases SNARE complex formation and strongly stimulates neurite outgrowth. These results provide the first functional evidence for the role of TI-VAMP in neurite outgrowth and point to its NH(2)-terminal domain as a key regulator in this process.

Growing an actin gel on spherical surfaces.

Inspired by the motility of the bacteria Listeria monocytogenes, we have experimentally studied the growth of an actin gel around spherical beads grafted with ActA, a protein known to be the promoter of bacteria movement. On ActA-grafted beads F-actin is formed in a spherical manner, whereas on the bacteria a "comet-like" tail of F-actin is produced. We show experimentally that the stationary thickness of the gel depends on the radius of the beads. Moreover, the actin gel is not formed if the ActA surface density is too low. To interpret our results, we propose a theoretical model to explain how the mechanical stress (due to spherical geometry) limits the growth of the actin gel. Our model also takes into account treadmilling of actin. We deduce from our work that the force exerted by the actin gel on the bacteria is of the order of 10 pN. Finally, we estimate from our theoretical model possible conditions for developing actin comet tails.