Tiam1 interaction with the PAR complex promotes talin-mediated Rac1 activation during polarized cell migration.

Migrating cells acquire front-rear polarity with a leading edge and a trailing tail for directional movement. The Rac exchange factor Tiam1 participates in polarized cell migration with the PAR complex of PAR3, PAR6, and atypical protein kinase C. However, it remains largely unknown how Tiam1 is regulated and contributes to the establishment of polarity in migrating cells. We show here that Tiam1 interacts directly with talin, which binds and activates integrins to mediate their signaling. Tiam1 accumulated at adhesions in a manner dependent on talin and the PAR complex. The interactions of talin with Tiam1 and the PAR complex were required for adhesion-induced Rac1 activation, cell spreading, and migration toward integrin substrates. Furthermore, Tiam1 acted with talin to regulate adhesion turnover. Thus, we propose that Tiam1, with the PAR complex, binds to integrins through talin and, together with the PAR complex, thereby regulates Rac1 activity and adhesion turnover for polarized migration.

Tumor type-dependent function of the par3 polarity protein in skin tumorigenesis.

Cell polarization is crucial during development and tissue homeostasis and is regulated by conserved proteins of the Scribble, Crumbs, and Par complexes. In mouse skin tumorigenesis, Par3 deficiency results in reduced papilloma formation and growth. Par3 mediates its tumor-promoting activity through regulation of growth and survival, since Par3 deletion increases apoptosis and reduces growth in vivo and in vitro. In contrast, Par3-deficient mice are predisposed to formation of keratoacanthomas, cutaneous tumors thought to originate from different cellular origin and frequently observed in humans. Par3 expression is reduced in both mouse and human keratoacanthomas, indicating tumor-suppressive properties of Par3. Our results identify a dual function of Par3 in skin cancer, with both pro-oncogenic and tumor-suppressive activity depending on the tumor type.

The Rac activator Tiam1 is required for polarized protrusional outgrowth of primary astrocytes by affecting the organization of the microtubule network.

Polarized cell migration is a crucial process in the development and repair of tissues, as well as in pathological conditions, including cancer. Recent studies have elucidated important roles for Rho GTPases in the establishment and maintenance of polarity prior to and during cell migration. Here, we show that Tiam1, a specific activator of the small GTPase Rac, is required for the polarized outgrowth of protrusions in primary astrocytes during the initial phase of cell polarization after scratch-wounding monolayers of cells. Tiam1 deficiency delays closure of wounds in confluent monolayers. Lack of Tiam1 impairs adoption of an asymmetrical cell shape as well as microtubule organization within protrusions. Positioning of the centrosome and Golgi apparatus, however, are independent of Tiam1-Rac signaling. We speculate that the function of Tiam1 in polarized outgrowth of astrocyte protrusions involves regulation of microtubule organization, possibly by stabilizing the microtubule cytoskeleton. Our results add Tiam1 as a player to the growing list of proteins involved in polarized outgrowth of protrusions and further elucidate the signaling pathways leading to cell polarization.

Cell polarity proteins and cancer.

Cell polarity is essential in many biological processes and required for development as well as maintenance of tissue integrity. Loss of polarity is considered both a hallmark and precondition for human cancer. Three conserved polarity protein complexes regulate different modes of polarity that are conserved throughout numerous cell types and species. These complexes are the Crumbs, Par and Scribble complex. Given the importance of cell polarity for normal tissue homeostasis, aberrant polarity signaling is suggested to contribute to the multistep processes of human cancer. Most human cancers are formed from epithelial cells. Evidence confirming the roles for polarity proteins in different phases of the oncogenic trajectory comes from functional studies using mammalian cells as well as Drosophila and zebrafish models. Furthermore, several reports have revealed aberrant expression and localization of polarity proteins in different human tumors. In this review we will give an overview on the current data available that couple polarity signaling to tumorigenesis, particularly in epithelial cells.

Concepts of metastasis in flux: the stromal progression model.

The ability of tumor cells to leave a primary tumor, to disseminate through the body, and to ultimately seed new secondary tumors is universally agreed to be the basis for metastasis formation. An accurate description of the cellular and molecular mechanisms that underlie this multistep process would greatly facilitate the rational development of therapies that effectively allow metastatic disease to be controlled and treated. A number of disparate and sometimes conflicting hypotheses and models have been suggested to explain various aspects of the process, and no single concept explains the mechanism of metastasis in its entirety or encompasses all observations and experimental findings. The exciting progress made in metastasis research in recent years has refined existing ideas, as well as giving rise to new ones. In this review we survey some of the main theories that currently exist in the field, and show that significant convergence is emerging, allowing a synthesis of several models to give a more comprehensive overview of the process of metastasis. As a result we postulate a stromal progression model of metastasis. In this model, progressive modification of the tumor microenvironment is equally as important as genetic and epigenetic changes in tumor cells during primary tumor progression. Mutual regulatory interactions between stroma and tumor cells modify the stemness of the cells that drive tumor growth, in a manner that involves epithelial-mesenchymal and mesenchymal-epithelial-like transitions. Similar interactions need to be recapitulated at secondary sites for metastases to grow. Early disseminating tumor cells can progress at the secondary site in parallel to the primary tumor, both in terms of genetic changes, as well as progressive development of a metastatic stroma. Although this model brings together many ideas in the field, there remain nevertheless a number of major open questions, underscoring the need for further research to fully understand metastasis, and thereby identify new and effective ways of treating metastatic disease.

Rac3 inhibits adhesion and differentiation of neuronal cells by modifying GIT1 downstream signaling.

Rac1 and Rac3 are highly homologous regulatory proteins that belong to the small GTPases of the Rho family. Previously, we showed that Rac3 induces cell rounding and prevents neuronal differentiation, in contrast to its close relative Rac1, which stimulates cell spreading and neuritogenesis. To explain these opposing effects, we investigated whether Rac1 and Rac3 interact with different proteins. Here, we show that both Rac1 and Rac3 interact with GIT1, a multifunctional Arf-GAP protein, which regulates cell-matrix adhesion, cell spreading and endocytosis. However, in contrast to Rac1, the Rac3-GIT1 interaction is not mediated by betaPix. Interestingly, Rac3 expression severely attenuates the interaction between GIT1 and paxillin, accompanied by defective paxillin distribution, focal adhesion formation and disturbed cell spreading. Moreover, in Rac3-expressing cells, Arf6 activity is strongly reduced and the Arf6-GAP activity of GIT1 is required for Rac3 downstream signaling. Indeed, expression of wild-type Arf6 or the Arf6-GEF ARNO induced cell spreading in the otherwise rounded Rac3-expressing cells. Our data suggest that Rac3 and Rac1 oppose each other's function by differently modulating GIT1 signaling. Rac1 induces adhesion and differentiation by activating PAK1 and stimulating the GIT1-paxillin interaction, whereas Rac3 blocks this interaction and inactivates Arf6 by stimulating the GAP function of GIT1, thereby preventing cell spreading and differentiation.

The Rac activator Tiam1 controls efficient T-cell trafficking and route of transendothelial migration.

Migration toward chemoattractants is a hallmark of T-cell trafficking and is essential to produce an efficient immune response. Here, we have analyzed the function of the Rac activator Tiam1 in the control of T-cell trafficking and transendothelial migration. We found that Tiam1 is required for chemokine- and S1P-induced Rac activation and subsequent cell migration. As a result, Tiam1-deficient T cells show reduced chemotaxis in vitro, and impaired homing, egress, and contact hypersensitivity in vivo. Analysis of the T-cell transendothelial migration cascade revealed that PKCzeta/Tiam1/Rac signaling is dispensable for T-cell arrest but is essential for the stabilization of polarization and efficient crawling of T cells on endothelial cells. T cells that lack Tiam1 predominantly transmigrate through individual endothelial cells (transcellular migration) rather than at endothelial junctions (paracellular migration), suggesting that T cells are able to change their route of transendothelial migration according to their polarization status and crawling capacity.

Crosstalk between small GTPases and polarity proteins in cell polarization.

Cell polarization is crucial for the development of multicellular organisms, and aberrant cell polarization contributes to various diseases, including cancer. How cell polarity is established and how it is maintained remain fascinating questions. Conserved proteins of the partitioning defective (PAR), Scribble and Crumbs complexes guide the establishment of cell polarity in various organisms. Moreover, GTPases that regulate actin cytoskeletal dynamics have been implicated in cell polarization. Recent findings provide insights into polarization mechanisms and show intriguing crosstalk between small GTPases and members of polarity complexes in regulating cell polarization in different cellular contexts and cell types.

The Rac activator Tiam1 prevents keratinocyte apoptosis by controlling ROS-mediated ERK phosphorylation.

Tiam1 is a ubiquitously expressed activator of the small GTPase Rac. Previously, we found that Tiam1 knockout (KO) mice are resistant to DMBA-induced skin tumorigenicity, which correlated with increased apoptosis in keratinocytes of the skin epidermis. Here, we have studied the mechanisms by which Tiam1 protects against apoptosis. We found that Tiam1-KO keratinocytes show increased apoptosis in response to apoptotic stimuli, including growth factor deprivation and heat-shock treatment. Expression of catalytically active Tiam1, but not inactive Tiam1, rescues the apoptosis susceptibility of Tiam1-KO keratinocytes, indicating that this defect is caused by impaired Tiam1-mediated Rac activation. Apoptosis induced by growth factor starvation correlates with impaired ERK phosphorylation in Tiam1-KO keratinocytes. Moreover, Tiam1-KO keratinocytes contain lower levels of intracellular reactive oxygen species (ROS) when compared with wild-type cells. The ROS content of keratinocytes is dependent on both Tiam1 and the activity of NADPH oxidase (Nox), and is required for ERK-mediated survival signaling. Indeed, Tiam1 deficiency or the inhibition of intracellular ROS production blocks ERK phosphorylation and sensitizes wild-type keratinocytes to apoptotic stimuli. Our results indicate that the Rac activator Tiam1 controls the intracellular redox balance by Nox-mediated ROS production, which regulates ERK phosphorylation and the susceptibility of keratinocytes to apoptotic signaling.

Increased Rac activity is required for the progression of T-lymphomas induced by Pten-deficiency.

Mutation of the tumor suppressor PTEN results in loss of its PI3-kinase counteracting function. PI3-kinase stimulates tumor formation by PKB/Akt-mediated cell proliferation and prevention of apoptosis. PI3-kinase may also activate Rho-GTPases and their regulatory GEFs to promote invasion. Here we have analyzed the function of the Rac-specific activator, Tiam1, in PI3-kinase-induced T-lymphomagenesis. Mice with a T cell-specific Pten deletion developed T-lymphomas with enhanced PKB/Akt phosphorylation. However, these T-lymphomas infiltrated more frequently into various organs in Tiam1-deficient mice compared to wild type mice. Surprisingly, Tiam1-deficient lymphomas showed increased Rac activity, suggesting that the lack of Tiam1 is compensated by alternative Rac-activating mechanisms that lead to increased progression of PI3-kinase-induced T-lymphomas.

Rho GTPases: functions and association with cancer.

Rho GTPases are small proteins that act as binary molecular switches in a wide range of signalling pathways upon stimulation of cell surface receptors. Three different classes of regulatory proteins control their activity. In the activated state small GTPases are able to bind a variety of effector proteins and initiate downstream signalling. Rho GTPases regulate important cellular processes ranging from cytoskeletal remodelling and gene expression to cell proliferation and membrane trafficking. Therefore it is not surprising that deregulated Rho signalling can contribute to disturbed cellular phenotypes in a wide range of diseases. The main focus of this review will be the diversity of functions of Rho GTPases and the effects of aberrant Rho GTPase signalling in various aspects of cancer.

The Par-Tiam1 complex controls persistent migration by stabilizing microtubule-dependent front-rear polarity.

BACKGROUND: The establishment and maintenance of cell polarity is crucial for many biological functions and is regulated by conserved protein complexes. The Par polarity complex consisting of Par3, Par6, and PKCzeta, in conjunction with Tiam1-mediated Rac signaling, controls apical-basal cell polarity in contacting epithelial cells. Here we tested the hypothesis that the Par complex, in conjunction with Tiam1, controls "front-rear" polarity during the persistent migration of freely migrating keratinocytes. RESULTS: Wild-type (WT) epidermal keratinocytes lacking cell-cell contacts are stably front-rear polarized and migrate persistently. In contrast, Tiam1-deficient (Tiam1 KO) and (si)Par3-depleted keratinocytes are generally unpolarized and migrate randomly because front-rear polarity is short lived. Immunoprecipitation experiments show that in migrating keratinocytes, Tiam1 associates with Par3 and PKCzeta. Moreover, Par3, PKCzeta, and Tiam1 proteins are enriched at the leading edges of polarized keratinocytes. Tiam1 KO keratinocytes are impaired in chemotactic migration toward growth factors, whereaes haptotactic migration is similar to WT. Par3 depletion or the blocking of PKCzeta signaling in WT keratinocytes impairs chemotaxis but has no additional effect on Tiam1 KO cells. The migratory and morphological defects in keratinocytes with impaired Par-Tiam1 function closely resemble cells with pharmacologically destabilized microtubules (MTs). Indeed, MTs in Tiam1 KO keratinocytes and WT cells treated with a PKCzeta inhibitor are unstable, thereby negatively influencing directional but not random migration. CONCLUSIONS: We conclude that the Par-Tiam1 complex stabilizes front-rear polarization of noncontacting migratory cells, thereby stimulating persistent and chemotactic migration, whereas in contacting keratinocytes, the same complex controls the establishment of long-lasting apical-basal polarity. These findings underscore a remarkable flexibility of the Par polarity complex that, depending on the biological context, controls distinct forms of cellular polarity.

The Par polarity complex regulates Rap1- and chemokine-induced T cell polarization.

Cell polarization is required for virtually all functions of T cells, including transendothelial migration in response to chemokines. However, the molecular pathways that establish T cell polarity are poorly understood. We show that the activation of the partitioning defective (Par) polarity complex is a key event during Rap1- and chemokine-induced T cell polarization. Intracellular localization and activation of the Par complex are initiated by Rap1 and require Cdc42 activity. The Rac activator Tiam1 associates with both Rap1 and components of the Par complex, and thereby may function to connect the Par polarity complex to Rap1 and to regulate the Rac-mediated actin remodelling required for T cell polarization. Consistent with these findings, Tiam1-deficient T cells are impaired in Rap1- and chemokine-induced polarization and chemotaxis. Our studies implicate Tiam1 and the Par polarity complex in polarization of T cells, and provide a mechanism by which chemokines and Rap1 regulate T cell polarization and chemotaxis.

Rac1 and Rac3 have opposing functions in cell adhesion and differentiation of neuronal cells.

Rac1 and Rac3 are highly homologous members of the Rho small GTPase family. Rac1 is ubiquitously expressed and regulates cell adhesion, migration and differentiation in various cell types. Rac3 is primarily expressed in brain and may therefore have a specific function in neuronal cells. We found that depletion of Rac1 by short interference RNA leads to decreased cell-matrix adhesions and cell rounding in neuronal N1E-115 cells. By contrast, depletion of Rac3 induces stronger cell adhesions and dramatically increases the outgrowth of neurite-like protrusions, suggesting opposite functions for Rac1 and Rac3 in neuronal cells. Consistent with this, overexpression of Rac1 induces cell spreading, whereas overexpression of Rac3 results in a contractile round morphology. Rac1 is mainly found at the plasma membrane, whereas Rac3 is predominantly localized in the perinuclear region. Residues 185-187, present in the variable polybasic rich region at the carboxyl terminus are responsible for the difference in phenotype induced by Rac1 and Rac3 as well as for their different intracellular localization. The Rac1-opposing function of Rac3 is not mediated by or dependent on components of the RhoA signaling pathway. It rather seems that Rac3 exerts its function through negatively affecting integrin-mediated cell-matrix adhesions. Together, our data reveal that Rac3 opposes Rac1 in the regulation of cell adhesion and differentiation of neuronal cells.

The Rac activator Tiam1 and Ras-induced oncogenesis.

The Tiam1 gene encodes a guanine nucleotide exchange factor (GEF) that specifically activates the Rho-like GTPase Rac. In vitro studies indicate that Tiam1 localizes to adherens junctions and plays a role in the formation and maintenance of cadherin-based cell adhesions, thereby regulating migration of epithelial cells. In vivo studies implicate Tiam1 in various aspects of tumorigenesis. In this chapter, we discuss the use of the DMBA/TPA chemical carcinogenesis protocol in Tiam1-deficient mice to study the role of Tiam1 in Ras-induced skin tumors. This two-stage carcinogenesis protocol allows us to study initiation, promotion, and progression of tumors in a Tiam1-positive and Tiam1-negative background. Moreover, we describe methods to study the role of Tiam1 in susceptibility to apoptosis, cell growth, and Ras transformation by in vivo and in vitro experiments. The latter makes use of tumor cells and primary embryonic fibroblasts and keratinocytes isolated from mice.

Tiam1 takes PARt in cell polarity.

Cell polarity is an essential requirement for the proper tissue development of complex organisms. This is underscored by in vivo studies showing that loss of cell polarity contributes to the formation and progression of tumours. Evolutionary conserved multiprotein complexes, such as the Par3-Par6-aPKC or, in short, the Par polarity complex, regulate the establishment of cell polarity. The small Rho GTPases CDC42 and Rac control the activation of the Par polarity complex. Evidence now implicates the Rac activator Tiam1 as a crucial component of the Par complex in regulating neuronal (axonal) and epithelial (apical-basal) polarity. Our current knowledge places Tiam1 at the centre of a pivotal biological process, the establishment and maintenance of cell polarity, and suggests that deregulation of the Tiam1-Par complex contributes to tumourigenicity.

Interaction between Tiam1 and the Arp2/3 complex links activation of Rac to actin polymerization.

The Rac-specific GEF (guanine-nucleotide exchange factor) Tiam1 (T-lymphoma invasion and metastasis 1) regulates migration, cell-matrix and cell-cell adhesion by modulating the actin cytoskeleton through the GTPase, Rac1. Using yeast two-hybrid screening and biochemical assays, we found that Tiam1 interacts with the p21-Arc [Arp (actin-related protein) complex] subunit of the Arp2/3 complex. Association occurred through the N-terminal pleckstrin homology domain and the adjacent coiled-coil region of Tiam1. As a result, Tiam1 co-localizes with the Arp2/3 complex at sites of actin polymerization, such as epithelial cell-cell contacts and membrane ruffles. Deletion of the p21-Arc-binding domain in Tiam1 impairs its subcellular localization and capacity to activate Rac1, suggesting that binding to the Arp2/3 complex is important for the function of Tiam1. Indeed, blocking Arp2/3 activation with a WASP (Wiskott-Aldrich syndrome protein) inhibitor leads to subcellular relocalization of Tiam1 and decreased Rac activation. Conversely, functionally active Tiam1, but not a GEF-deficient mutant, promotes activation of the Arp2/3 complex and its association with cytoskeletal components, indicating that Tiam1 and Arp2/3 are mutually dependent for their correct localization and signalling. Our data suggests a model in which the Arp2/3 complex acts as a scaffold to localize Tiam1, and thereby Rac activity, which are both required for activation of the Arp2/3 complex and further Arp2/3 recruitment. This 'self-amplifying' signalling module involving Tiam1, Rac and the Arp2/3 complex could thus drive actin polymerization at specific sites in cells that are required for dynamic morphological changes.

The rac activator Tiam1 is a Wnt-responsive gene that modifies intestinal tumor development.

Mutations in the canonical Wnt signaling pathway leading to its activation are known to cause the majority of intestinal tumors. However, few genes targeted by this pathway have been demonstrated to affect tumor development in vivo. Here we show that Tiam1, a selective Rac GTPase activator, is a Wnt-responsive gene expressed in the base of intestinal crypts and up-regulated in mouse intestinal tumors and human colon adenomas. Moreover, by comparing tumor development in APC mutant Min (multiple intestinal neoplasia) mice expressing or lacking Tiam1, we found that Tiam1 deficiency significantly reduces the formation and growth of polyps in vivo. However, invasion of malignant intestinal tumors is enhanced by a lack of Tiam1. In line with this, knock-down of Tiam1 reduced the growth potential of human colorectal cancer cells and their ability to form E-cadherin-based adhesions, a prerequisite for local invasion of tumor cells. Our data indicate a novel cross-talk between Tiam1-Rac and canonical Wnt-signaling pathways that influences intestinal tumor formation and progression.

The Rac activator Tiam1 is required for (alpha)3(beta)1-mediated laminin-5 deposition, cell spreading, and cell migration.

The Rho-like guanosine triphosphatase Rac1 regulates various signaling pathways, including integrin-mediated adhesion and migration of cells. However, the mechanisms by which integrins signal toward Rac are poorly understood. We show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis 1) is required for the integrin-mediated laminin (LN)-5 deposition, spreading, and migration of keratinocytes. In contrast to wild-type keratinocytes, Tiam1-deficient (Tiam1-/-) keratinocytes are unable to adhere to and spread on a glass substrate because they are unable to deposit their own LN5 substrate. Both Tiam1 and V12Rac1 can rescue the defects of Tiam1-/- keratinocytes, indicating that these deficiencies are caused by impaired Tiam1-mediated Rac activation. Tiam1-/- cells are unable to activate Rac upon alpha3beta1-mediated adhesion to an exogenous LN5 substrate. Moreover, Tiam1 deficiency impairs keratinocyte migration in vitro and reepithelialization of excision wounds in mouse skin. Our studies indicate that Tiam1 is a key molecule in alpha3beta1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for the spreading and migration of keratinocytes.

The Rac activator Tiam1 controls tight junction biogenesis in keratinocytes through binding to and activation of the Par polarity complex.

The GTPases Rac and Cdc42 play a pivotal role in the establishment of cell polarity by stimulating biogenesis of tight junctions (TJs). In this study, we show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis) controls the cell polarity of epidermal keratinocytes. Similar to wild-type (WT) keratinocytes, Tiam1-deficient cells establish primordial E-cadherin-based adhesions, but subsequent junction maturation and membrane sealing are severely impaired. Tiam1 and V12Rac1 can rescue the TJ maturation defect in Tiam1-deficient cells, indicating that this defect is the result of impaired Tiam1-Rac signaling. Tiam1 interacts with Par3 and aPKCzeta, which are two components of the conserved Par3-Par6-aPKC polarity complex, and triggers biogenesis of the TJ through the activation of Rac and aPKCzeta, which is independent of Cdc42. Rac is activated upon the formation of primordial adhesions (PAs) in WT but not in Tiam1-deficient cells. Our data indicate that Tiam1-mediated activation of Rac in PAs controls TJ biogenesis and polarity in epithelial cells by association with and activation of the Par3-Par6-aPKC polarity complex.