Tiam1-deficiency impairs mammary tumor formation in MMTV-c-neu but not in MMTV-c-myc mice.

BACKGROUND: Rho-like small GTPases, including RhoA, Rac1 and Cdc42, are crucial for the regulation of a large variety of biological processes such as the cytoskeletal organization and gene transcription. The activities of Rho GTPases are predominantly controlled by guanine nucleotide exchange factors (GEFs), which activate GTPases by catalyzing the exchange of bound GDP for GTP. Earlier, we have identified the Tiam1 gene as an invasion-inducing gene that encodes a specific activator (GEF) of the Rac GTPase. We found that Tiam1-mediated Rac signaling functions in various aspects of tumorigenicity including the formation and progression of Ras-induced skin tumors and Wnt-induced intestinal tumors. Here, we further distinguish the oncogenic pathways that depend on Tiam1 signaling in the mammary gland. MATERIAL AND METHODS: We crossed Tiam1 knockout mice with MMTV-c-myc and MMTV-c-neu transgenic mice, in which the expression of both oncogenes is targeted to the mammary gland leading to mammary tumorigenesis. RESULTS: We found Tiam1 important for Neu-induced tumor formation and progression but not for Myc-induced tumors. Tiam1-deficiency delayed Neu-induced tumor initiation and reduced metastasis but had no effect on the growth of the MMTV-c-neu tumors. CONCLUSION: Our data indicate that the Rac activator Tiam1 contributes to tumorigenicity induced by specific oncogenic signaling pathways only.

Prognostic relevance of Tiam1 protein expression in prostate carcinomas.

The Rac-specific guanine nucleotide exchange factor, Tiam1, plays a major role in oncogenicity, tumour invasion and metastasis but its usefulness as a prognostic marker in human cancer has not been tested yet. In the present study, Tiam1 expression was analysed in benign secretory epithelium, pre-neoplastic high-grade prostatic intraepithelium neoplasia (HG-PIN) and prostate carcinomas of 60 R0-resected radical prostatectomy specimens by semiquantitative immunohistochemistry. Tiam1 proved significantly overexpressed in both HG-PIN (P<0.001) and prostate carcinomas (P<0.001) when compared to benign secretory epithelium. Strong Tiam1 overexpression (i.e. > or =3.5-fold) in prostate carcinomas relative to the respective benign prostatic epithelium was statistically significantly associated with disease recurrence (P=0.016), the presence of lymph vessel invasion (P=0.031) and high Gleason scores (GS) (i.e. > or =7) (P=0.044). Univariate analysis showed a statistically significant association of strong Tiam1 overexpression with decreased disease-free survival (DFS) (P=0.03). This prognostic effect of strong Tiam1 overexpression remained significant in multivariate analysis including preoperative prostate-specific antigen levels, pT stage, and GS (relative risk= 3.75, 95% confidence interval=1.06-13.16; P=0.04). Together, our data suggest that strong Tiam1 overexpression relative to the corresponding benign epithelial cells is a new and independent predictor of decreased DFS for patients with prostate cancer.

An essential role for ARF6-regulated membrane traffic in adherens junction turnover and epithelial cell migration.

We describe a novel role for the ARF6 GTPase in the regulation of adherens junction (AJ) turnover in MDCK epithelial cells. Expression of a GTPase-defective ARF6 mutant, ARF6(Q67L), led to a loss of AJs and ruffling of the lateral plasma membrane via mechanisms that were mutually exclusive. ARF6-GTP-induced AJ disassembly did not require actin remodeling, but was dependent on the internalization of E-cadherin into the cytoplasm via vesicle transport. ARF6 activation was accompanied by increased migratory potential, and treatment of cells with hepatocyte growth factor (HGF) induced the activation of endogenous ARF6. The effect of ARF6(Q67L) on AJs was specific since ARF6 activation did not perturb tight junction assembly or cell polarity. In contrast, dominant-negative ARF6, ARF6(T27N), localized to AJs and its expression blocked cell migration and HGF-induced internalization of cadherin-based junctional components into the cytoplasm. Finally, we show that ARF6 exerts its role downstream of v-Src activation during the disassembly of AJs. These findings document an essential role for ARF6- regulated membrane traffic in AJ disassembly and epithelial cell migration.

Rac affects invasion of human renal cell carcinomas by up-regulating tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 expression.

Rho-like GTPases, including Cdc42, Rac1, and RhoA, regulate distinct actin cytoskeleton changes required for adhesion, migration, and invasion of cells. Tiam1 specifically activates Rac, and earlier studies have demonstrated that Tiam1-Rac signaling affects migration and invasion in a cell type- and cell substrate-specific manner. In the present study, we examined the role of Tiam1-Rac signaling in migration and invasion of human renal cell carcinomas. Stable overexpression of Tiam1 or constitutively active V12-Rac1 in a human renal cell carcinoma cell line (clearCa-28) strongly inhibited cell migration by promoting E-cadherin-mediated cell-cell adhesion. Blocking E-cadherin-mediated adhesion by E-cadherin-specific HAV peptides allowed cells to migrate, but was not sufficient to antagonize Tiam1- and V12-Rac1-induced inhibition of Matrigel invasion, suggesting that Rac may influence invasion also through other mechanisms. Indeed, Tiam1-mediated Rac activation induced transcriptional up-regulation of tissue inhibitor of metalloproteinases-1 (TIMP-1) and post-transcriptional up-regulation of TIMP-2, whereas secretion and activity levels of their counterparts, matrix metalloproteinase-9 and matrix metalloproteinase-2, respectively, were not affected. Application of recombinant TIMP-1 and TIMP-2 proteins significantly inhibited invasion of mock-transfected clearCa-28 cells, supporting a role of TIMPs in Rac-mediated inhibition of invasion. To our knowledge, this is the first evidence that increased Rac signaling may inhibit invasion of epithelial tumor cells by up-regulation of TIMP-1 and TIMP-2.

Regulation of the cytoskeleton by Rho-family GTPases: implications for tumour cell invasion.

Rho-like GTPases regulate both cell-cell and cell-matrix adhesions and can influence the motile and invasive properties of tumour cells in vitro. Increased Rac signalling can promote either cell-cell adhesion or motility depending on the cell type and composition of the extracellular matrix. Ultimately, the balance of Rac and Rho activities and intracellular localization appears to be critical in determining the cellular phenotype.

Differential effect of simvastatin on various signal transduction intermediates in cultured human smooth muscle cells.

The underlying mechanism of the antiproliferative effect of S (simvastatin), a HMG-CoA reductase inhibitor, in vascular smooth muscle cells (SMC) is still poorly understood. In the present study, we used synchronized human SMC, isolated from left interior mammary artery, as an in vitro model to test the effects of S on platelet-derived growth factor (PDGF)-induced DNA synthesis, extracellular-regulated kinase 1/2 (ERK1/2), p38/stress-activated protein kinase 2 (SAPK2), RhoA and Rac1 activation. ERK1/2 phosphorylation was triggered within 2 min of PDGF stimulation (early G1 phase) and was blocked by PD98059, a specific inhibitor of the ERK1/2 pathway, which also strongly inhibited PDGF-induced DNA synthesis (IC(50) = 10 micromol/L). PDGF quickly induced p38 phosphorylation (early G1 phase) and SB203580, a specific inhibitor of the p38/SAPK2 pathway, also blocked PDGF-induced DNA synthesis (IC(50) = 0.3 micromol/L). Translocation to the plasma membrane of small GTPases, such as RhoA and Rac1, could not be detected within 15 min of stimulation with PDGF or lysophosphatidic acid (LPA) (early G1 phase), but occurred after 24 hr of PDGF stimulation (late G1/S phase). S inhibited PDGF-induced DNA synthesis (IC(50) = 3.5 micromol/L), and this effect was dependent on intracellular mevalonate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate availability. The critical time period for the reversal of the S effect by mevalonate comprised both the early and late G1 phase of the SMC cycle. PDGF-induced ERK1/2 phosphorylation and PDGF-induced p38 phosphorylation were not markedly affected by S during the whole G1 phase. However, S treatment blocked the PDGF- and LPA-induced membrane translocation of RhoA that occurred during the late G1/S phase. In the case of Rac1, the same process was also inhibited by S treatment. We concluded from these results that, in SMC, the early events associated with ERK1/2 and p38 signal transduction pathways, recruited for PDGF-mediated DNA synthesis, were insensitive to S action, whereas the mevalonate-dependent, posttranslational modification of RhoA and Rac1 molecules, required for PDGF-induced membrane translocation, was blocked by this drug. These results suggest that the antiproliferative effect of S can be explained not only by the blockage of RhoA-mediated signaling events but also by Rac1-mediated signaling events.

Activation of RhoA by thrombin in endothelial hyperpermeability: role of Rho kinase and protein tyrosine kinases.

Endothelial cells (ECs) actively regulate the extravasation of blood constituents. On stimulation by vasoactive agents and thrombin, ECs change their cytoskeletal architecture and small gaps are formed between neighboring cells. These changes partly depend on a rise in [Ca(2+)](i) and activation of the Ca(2+)/calmodulin-dependent myosin light chain kinase. In this study, mechanisms that contribute to the thrombin-enhanced endothelial permeability were further investigated. We provide direct evidence that thrombin induces a rapid and transient activation of RhoA in human umbilical vein ECs. Under the same conditions, the activity of the related protein Rac was not affected. This was accompanied by an increase in myosin light chain phosphorylation, the generation of F-actin stress fibers, and a prolonged increase in endothelial permeability. Inhibition of the RhoA target Rho kinase with the specific inhibitor Y-27632 reduced all of these effects markedly. In the presence of Y-27632, the thrombin-enhanced permeability was additionally reduced by chelation of [Ca(2+)](i) by BAPTA. These data indicate that RhoA/Rho kinase and Ca(2+) represent 2 pathways that act on endothelial permeability. In addition, the protein tyrosine kinase inhibitor genistein reduced thrombin-induced endothelial permeability without affecting activation of RhoA by thrombin. Our data support a model of thrombin-induced endothelial permeability that is regulated by 3 cellular signal transduction pathways.

Rho family proteins in cell adhesion and cell migration.

Cell migration and the regulation of cadherin-mediated homotypic cell-cell interactions are critical events during development, morphogenesis and wound healing. Aberrations in signalling pathways involved in the regulation of cell migration and cadherin-mediated cell-cell adhesion contribute to tumour invasion and metastasis. The rho family proteins, including cdc42, rac1 and rhoA, regulate signalling pathways that mediate the distinct actin cytoskeleton changes required for both cellular motility and cell-cell adhesion. Recent studies indicate that rac directly influences rho activity at the GTPase level and that the reciprocal balance between rac and rho activity can determine epithelial or mesenchymal cell morphology and migratory behaviour of epithelial (tumour) cells.

Oncogenic Ras downregulates Rac activity, which leads to increased Rho activity and epithelial-mesenchymal transition.

Proteins of the Rho family regulate cytoskeletal rearrangements in response to receptor stimulation and are involved in the establishment and maintenance of epithelial cell morphology. We recently showed that Rac is able to downregulate Rho activity and that the reciprocal balance between Rac and Rho activity is a major determinant of cellular morphology and motility in NIH3T3 fibroblasts. Using biochemical pull-down assays, we analyzed the effect of transient and sustained oncogenic Ras signaling on the activation state of Rac and Rho in epithelial MDCK cells. In contrast to the activation of Rac by growth factor-induced Ras signaling, we found that sustained signaling by oncogenic RasV12 permanently downregulates Rac activity, which leads to upregulation of Rho activity and epithelial-mesenchymal transition. Oncogenic Ras decreases Rac activity through sustained Raf/MAP kinase signaling, which causes transcriptional downregulation of Tiam1, an activator of Rac in epithelial cells. Reconstitution of Rac activity by expression of Tiam1 or RacV12 leads to downregulation of Rho activity and restores an epithelial phenotype in mesenchymal RasV12- or RafCAAX-transformed cells. The present data reveal a novel mechanism by which oncogenic Ras is able to interfere with the balance between Rac and Rho activity to achieve morphological transformation of epithelial cells.

Induction of cell scattering by expression of beta1 integrins in beta1-deficient epithelial cells requires activation of members of the rho family of GTPases and downregulation of cadherin and catenin function.

Adhesion receptors, which connect cells to each other and to the surrounding extracellular matrix (ECM), play a crucial role in the control of tissue structure and of morphogenesis. In this work, we have studied how intercellular adhesion molecules and beta1 integrins influence each other using two different beta1-null cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of beta1A or the cytoplasmic splice variant beta1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated with the redistribution of zonula occluden (ZO)-1 from tight junctions to adherens junctions at high cell confluency. In addition, the expression of beta1 integrins caused a dramatic reorganization of the actin cytoskeleton and of focal contacts. Interaction of beta1 integrins with their respective ligands was required for a complete morphological transition towards the spindle-shaped fibroblast-like phenotype. The expression of an interleukin-2 receptor (IL2R)-beta1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM-cell contacts, but failed to promote cell migration on fibronectin, in contrast to full-length beta1A. This indicates that the disruption of cell-cell adhesion is not simply the consequence of the stimulated cell migration. Expression of beta1 integrins in GE11 cells resulted in a decrease in cadherin and alpha-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction. Regulation of alpha-catenin protein levels by beta1 integrins is likely to play a role in the morphological transition, since overexpression of alpha-catenin in GE11 cells before beta1 prevented the disruption of intercellular adhesions and cell scattering. In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of beta1A, beta1D, or IL2R-beta1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42. Moreover, dominant negative Rac1 (N17Rac1) inhibited the disruption of cell-cell adhesions when expressed before beta1. However, all three GTPases might be involved in the morphological transition, since expression of either N19RhoA, N17Rac1, or N17Cdc42 reversed cell scattering and partially restored cadherin-based adhesions in GE11-beta1A cells. Our results indicate that beta1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of alpha-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.

Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior.

Using biochemical assays to determine the activation state of Rho-like GTPases, we show that the guanine nucleotide exchange factor Tiam1 functions as a specific activator of Rac but not Cdc42 or Rho in NIH3T3 fibroblasts. Activation of Rac by Tiam1 induces an epithelial-like morphology with functional cadherin-based adhesions and inhibits migration of fibroblasts. This epithelial phenotype is characterized by Rac-mediated effects on Rho activity. Transient PDGF-induced as well as sustained Rac activation by Tiam1 or V12Rac downregulate Rho activity. We found that Cdc42 also downregulates Rho activity. Neither V14Rho or N19Rho affects Rac activity, suggesting unidirectional signaling from Rac towards Rho. Downregulation of Rho activity occurs independently of Rac- induced cytoskeletal changes and cell spreading. Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho. Restoration of Rho activity in Tiam1-expressing cells by expression of V14Rho results in reversion of the epithelioid phenotype towards a migratory, fibroblastoid morphology. We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.

Identification and characterization of hPEM-2, a guanine nucleotide exchange factor specific for Cdc42.

Guanine nucleotide exchange factors of the Dbl family regulate the actin cytoskeleton through activation of Rho-like GTPases. At present the Dbl family consists of more than thirty members; many have not been phenotypically or biochemically characterized. Guanine nucleotide exchange factors universally feature a Dbl homology domain followed by a pleckstrin homology domain. Employing data base screening we identified a recently cloned cDNA, KIAA0424, showing substantial sequence homology with Rac activators such as Tiam1, Sos, Vav, and PIX within the catalytic domain. This cDNA appears to be the human homologue of the Ascidian protein Posterior End Mark-2 (PEM-2). We refer to this exchanger as hPEM-2. hPEM-2 encodes a protein of 70 kDa and features an N-terminal src homology 3 domain, followed by tandem Dbl homology and pleckstrin homology domains. The gene is highly expressed in brain and is localized on the human X-chromosome. Employing biochemical activity assays for Rho-like GTPases we found that hPEM-2 specifically activates Cdc42 and not Rac or RhoA. Ectopic expression of hPEM-2 in NIH3T3 fibroblasts revealed a Cdc42 phenotype featuring filopodia formation, followed by cortical actin polymerization and cell rounding. hPEM-2 represents an exchange factor, which may have a role in the regulation of a number of cellular processes through Cdc42.

Rac regulates phosphorylation of the myosin-II heavy chain, actinomyosin disassembly and cell spreading.

GTPases of the Rho family regulate actinomyosin-based contraction in non-muscle cells. Activation of Rho increases contractility, leading to cell rounding and neurite retraction in neuronal cell lines. Activation of Rac promotes cell spreading and interferes with Rho-mediated cell rounding. Here we show that activation of Rac may antagonize Rho by regulating phosphorylation of the myosin-II heavy chain. Stimulation of PC12 cells or N1E-115 neuroblastoma cells with bradykinin induces phosphorylation of threonine residues in the myosin-II heavy chain; this phosphorylation is Ca2+ dependent and regulated by Rac. Both bradykinin-mediated and constitutive activation of Rac promote cell spreading, accompanied by a loss of cortical myosin II. Our results identify the myosin-II heavy chain as a new target of Rac-regulated kinase pathways, and implicate Rac as a Rho antagonist during myosin-II-dependent cell-shape changes.

Regulation of p21rac activation in human neutrophils.

The small guanosine triphosphate (GTPase) p21rac is highly expressed in human neutrophils where it is thought to play a role in cytoskeletal reorganization and superoxide production. Using the p21rac binding domain of PAK (PAK-RBD) as an activation-specific probe, we have investigated agonist-stimulated activation of p21rac. Stimulation of neutrophils with the chemoattractants fMet-Leu-Phe (fMLP) or platelet-activating factor (PAF) induced an extremely rapid and transient p21rac activation, being optimal within 5 seconds. This activation correlates with the rapid changes of intracellular free Ca(2+) ([Ca(2+)](i)) stimulated by fMLP; however, changes in [Ca(2+)](i) were neither sufficient nor required for p21rac activation. Furthermore, fMLP-induced p21rac activation was not inhibited by broad tyrosine kinase inhibitors or specific inhibitors of ERK, p38 mitogen activated protein kinase, Src, or phosphatidylinositol 3-kinases. Surprisingly, the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha did not cause p21rac activation or modulate fMLP-induced p21rac activation. AlF(-), a potent activator of heterotrimeric G-protein alpha-subunits, however, was found to activate p21rac. Stimulation of neutrophils with phorbol myristate acetate (PMA) strongly activated the respiratory burst, but did not induce p21rac activation, suggesting that superoxide production per se can occur independently of p21rac activation. These data suggest that in human granulocytes, G-protein coupled receptors, but not cytokine receptors, activate p21rac via a rapid, novel exchange-mechanism independently of changes in [Ca(2+)](i), tyrosine phosphorylation, or PI3K.

Rho-like GTPases: their role in cell adhesion and invasion.

Metastasis formation is the leading cause of death in cancer patients. Using an in vitro model system, we have identified Tiam1 (T-lymphoma invasion and metastasis 1) as a gene that can induce invasion by and metastasis of mouse T-lymphoma cells. Subsequent studies showed that Tiam1 is a guanine nucleotide exchange factor for the Rho-like GTPase Rac1, a member of the Ras superfamily of small GTP-binding proteins. Rho-like GTPases play a pivotal role in the orchestration of changes in the actin cytoskeleton in response to receptor stimulation, but have also been shown to be involved in transcriptional activation and cell cycle regulation. Moreover, they can induce oncogenic transformation in fibroblast cells. In this chapter, we first summarize what is known about the signalling pathways that are activated by Tiam1 and Rho-like GTPases, and discuss the putative effectors that may mediate the effects in different cell types. In the latter part, we will more tentatively discuss the role of Tiam1 and Rho-like GTPases in invasion by and metastasis of tumour cells.

Rho-like GTPases: their role in epithelial cell-cell adhesion and invasion.

The family members of small Rho-like GTPases, RhoA, Rac1 and Cdc42Hs, are regulators of diverse cellular signalling pathways, including cytoskeletal organisation, transcription and cell-cycle progression. Recent research has given insight into the complex regulation of cell-cell adhesion and migratory responses of epithelial cells. The Rho-like GTPases RhoA, Rac1 and Cdc42Hs as major determinants of cytoskeletal organisation have been identified as key regulators of epithelial architecture, as well as of cell migration. These findings highlight the complex regulation and cross-talk of GTPase-dependent signalling pathways arising from cell-cell and cell-matrix interactions. The molecular mechanism of how Rho-like GTPases couple to molecules mediating either cell-cell adhesion or cell migration will be of particular interest to understand the invasive phenotype of epithelial tumours.

Rho-like GTPases: their role in epithelial cell-cell adhesion and invasion.

The family members of small Rho-like GTPases, RhoA, Rac1 and Cdc42Hs, are regulators of diverse cellular signalling pathways, including cytoskeletal organisation, transcription and cell-cycle progression. Recent research has given insight into the complex regulation of cell-cell adhesion and migratory responses of epithelial cells. The Rho-like GTPases RhoA, Rac1 and Cdc42Hs as major determinants of cytoskeletal organisation have been identified as key regulators of epithelial architecture, as well as of cell migration. These findings highlight the complex regulation and cross-talk of GTPase-dependent signalling pathways arising from cell-cell and cell-matrix interactions. The molecular mechanism of how Rho-like GTPases couple to molecules mediating either cell-cell adhesion or cell migration will be of particular interest to understand the invasive phenotype of epithelial tumours.