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Supplemental Figures S1-S12. Figure S1: Metformin does not affect mouse weight an insulinemia; Figure S2: Metformin does not induce apoptosis in prostate cancer cells; Figure S3: Metformin does not affect P69 cell migration; Figure S4: Metformin blocks cell migration out of the spheroid; Figure S5: Metformin inhibits cell motility; Figure S6: Metformin effects on actin and fascin levels; Figure S7: Metformin does not affect Rho GTP levels; Figure S8: A rac1 inhibitor affects cytoskeletal organisation; Figure S9: Metformin does not affect cAMP levels in PC3 cells; Figure S10: Metformin increases P CREB in DU145 cells; Figure S11: dbcAMP decreases Rac1GTP levels; Figure S12: Metformin decreases CXCR4 at the cell surface

The cellular heterogeneity within a variety of solid tumors is influenced by several type of tumor-associated bacteria through yet poorly understood mechanisms. Unbiased genomic analysis revealed that right-sided colorectal tumors with poorer prognosis are preferentially populated with species related toEscherichia coli, including strains that are producing colibactin. By applying metabolomic profilingin situ, the presence of colibactin-producingEscherichia coli(CoPEC) was identified to establish a high-glycerophospholipid microenvironment within right-sided colorectal tumors that are bearing mutations in either APC or KRAS. Using spatial approaches, we revealed that bacterial microniches are poorly infiltrated with CD8+T cells. The aforementioned alterations in lipid metabolism negatively correlated with immunomodulatory and neurotrophic factors among which the human regenerating family member 3 alpha gene (REG3A). Accordingly, the loss of its ortholog enhances growth at the proximal colon of tumors with a microenvironment that impedes immune surveillance and shares similarities to human right-sided colorectal tumors. This work clarifies how the presence of colibactin-producing bacteria may locally establish tumor heterogeneity for lipid metabolism to promote cancer progression and provide unique insights both for therapeutic intervention and enabling basic research into the mechanisms of microbiota-host interaction.STATEMENT OF SIGNIFICANCEApplyingin situmetabolic imaging to patients’ tumors revealed that the accumulation of colibactin-producing bacteria that locally creates a potential vulnerability to cancer treatments through lipid metabolic reprogramming of tumor cells.

SummaryRecently, an intestinal dysbiotic microbiota with enrichment in oral cavity bacteria has been described in colorectal cancer (CRC) patients. Here we characterized and investigated one of these oral pathobionts, the Gram-positive anaerobic coccusParvimonas micra.We identified two phylotypes (A and B) exhibiting different phenotypes and adhesion capabilities. We observed a strong association of phylotype A with CRC, with its higher abundance in feces and in tumoral tissue compared with the normal homologous colonic mucosa, which was associated with a distinct methylation status of patients. By developing anin vitrohypoxic co-culture system of human primary colonic cells with anaerobic bacteria, we showed thatP. micraphylotype A alters the DNA methylation profile promoters of key tumor-suppressor genes, oncogenes, and genes involved in epithelial-mesenchymal transition. In colonic mucosa of CRC patients carryingP. micraphylotype A, we found similar DNA methylations alterations, together with significant enrichment of differentially expressed genes in pathways involved in inflammation, cell adhesion, and regulation of actin cytoskeleton, providing evidence ofP. micrapossible role in the carcinogenic process.

Extracellular matrix (ECM) elasticity is perceived by cells via focal adhesion structures, which transduce mechanical cues into chemical signalling to conform cell behavior. Although the contribution of ECM compliance to the control of cell migration or division is extensively studied, little is reported regarding infectious processes. We study this phenomenon with the extraintestinal Escherichia coli pathogen UTI89. We show that UTI89 takes advantage, via its CNF1 toxin, of integrin mechanoactivation to trigger its invasion into cells. We identify the HACE1 E3 ligase-interacting protein Optineurin (OPTN) as a protein regulated by ECM stiffness. Functional analysis establishes a role of OPTN in bacterial invasion and integrin mechanical coupling and for stimulation of HACE1 E3 ligase activity towards the Rac1 GTPase. Consistent with a role of OPTN in cell mechanics, OPTN knockdown cells display defective integrin-mediated traction force buildup, associated with limited cellular invasion by UTI89. Nevertheless, OPTN knockdown cells display strong mechanochemical adhesion signalling, enhanced Rac1 activation and increased cyclin D1 translation, together with enhanced cell proliferation independent of ECM stiffness. Together, our data ascribe a new function to OPTN in mechanobiology. Uropathogenic strains of Escherichia coli (UPEC) are a leading cause of urinary tract infections (UTIs) and invasion involves Rho GTPase members, notably Rac1, to drive actin cytoskeleton rearrangement leading to engulfment. Here, Petracchini et al. provide evidence of an ECM stiffnessmodulated role of Optineurin (OPTN), which regulates HACE1-dependant Rac1 activity and thus controls integrinmediated mechanotransduction and bacterial invasion.

A challenge for the development of host-targeted anti-infectives against a large spectrum of AB-like toxin-producing bacteria encompasses the identification of chemical compounds corrupting toxin transport through both endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of small chemical compounds blocking active Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, followed by orthogonal screens against two AB toxins hijacking defined endolysosomal (Diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule N-(3,3-diphenylpropyl)-1-propyl-4-piperidinamine, referred to as C910. This compound induces the swelling of EEA1-positive early endosomes, in absence of PIKfyve kinase inhibition, and disturbs the trafficking of CNF1 and the B-subunit of Shiga toxin along the endolysosomal or retrograde pathways, respectively. Together, we show that C910 protects cells against 8 bacterial AB toxins including large clostridial glucosylating toxins from Clostridium difficile. Of interest, C910 also reduced viral infection in vitro including influenza A virus subtype H1N1 and SARS-CoV-2. Moreover, parenteral administration of C910 to the mice resulted in its accumulation in lung tissues and reduced lethal influenza infection.

Metabolic studies and animal knockout models point to the critical role of polyunsaturated docosahexaenoic acid (22:6, DHA)-containing phospholipids (DHA-PLs) in physiology. Here, we investigated the impact of DHA-PLs on the dynamics of transendothelial cell macroapertures (TEMs) triggered by RhoA inhibition-associated cell spreading. Lipidomic analyses showed that human umbilical vein endothelial cells (HUVECs) subjected to a DHA diet undergo a 6-fold enrichment in DHA-PLs at the plasma membrane (PM) at the expense of monounsaturated oleic acid-containing PLs (OA-PLs). Consequently, DHA-PL enrichment at the PM induces a reduction in cell thickness and shifts cellular membranes towards a permissive mode of membrane fusion for transcellular tunnel initiation. We provide evidence that a global homeostatic control of membrane tension and cell cortex rigidity minimizes overall changes of TEM area through a decrease of TEM size and lifetime. Conversely, low DHA-PL levels at the PM lead to the opening of unstable and wider TEMs. Together, this provides evidence that variations of DHA-PL levels in membranes affect cell biomechanical properties.

Metabolic studies and animal knockout models point to the critical role of polyunsaturated docosahexaenoic acid (22:6, DHA)-containing phospholipids (PLs) in physiology. Here, we study the impact of DHA-PLs on the dynamics of transendothelial cell macroapertures (TEMs) tunnels triggered by the RhoA GTPase inhibitory exotoxin C3 from Clostridium botulinum. Through lipidomic analyses, we show that primary human umbilical vein endothelial cells (HUVECs) subjected to DHA-diet undergo a 6-fold DHA-PLs enrichment in plasma membrane at the expense of monounsaturated OA-PLs. In contrast, OA-diet had almost no effect on PLs composition. Consequently, DHA treatment increases the nucleation rate of TEMs by 2-fold that we ascribe to a reduction of cell thickness. We reveal that the global transcellular area of cells remains conserved through a reduction of the width and lifetime of TEMs. Altogether, we reveal a homeostasis between plasma membrane DHA-PLs content and large-scale membrane dynamics.

The presence of aberrant number of centrioles is a recognized cause of aneuploidy and hallmark of cancer. Hence, centriole duplication needs to be tightly regulated. It has been proposed that centriole separation limits centrosome duplication. The mechanism driving centriole separation is poorly understood and little is known on how this is linked to centriole duplication. Here, we propose that actin-generated forces regulate centriole separation. By imposing geometric constraints via micropatterns, we were able to prove that precise acto-myosin force arrangements control direction, distance and time of centriole separation. Accordingly, inhibition of acto-myosin contractility impairs centriole separation. Alongside, we observed that organization of acto-myosin force modulates specifically the length of S-G2 phases of the cell cycle, PLK4 recruitment at the centrosome and centriole fidelity. These discoveries led us to suggest that acto-myosin forces might act in fundamental mechanisms of aneuploidy prevention., Centriolar separation is thought to be crucial for centriole duplication, but the mechanism behind separation is poorly understood. Here, using micropatterning, the authors report that actomyosin forces influence the direction, distance, and time of centriole separation.

International audience; Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionary conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system. However, its biological role and the functional significance of this interaction remain largely unknown. Here, we demonstrate that depletion of UBTD1 drastically affects the mechanical properties of epithelial cancer cells via RhoA activation and strongly promotes their aggressiveness. On a stiff matrix, UBTD1 expression is regulated by cell-cell contacts, and the protein is associated with β-catenin at cell junctions. Yes-associated protein (YAP) is a major cell mechano-transducer, and we show that UBTD1 is associated with components of the YAP degradation complex. Interestingly, UBTD1 promotes the interaction of YAP with its E3 ubiquitin ligase β-TrCP Consequently, in cancer cells, UBTD1 depletion decreases YAP ubiquitylation and triggers robust ROCK2-dependent YAP activation and downstream signaling. Data from lung and prostate cancer patients further corroborate the in cellulo results, confirming that low levels of UBTD1 are associated with poor patient survival, suggesting that biological functions of UBTD1 could be beneficial in limiting cancer progression.

The regulation of Rac1 by HACE1-mediated ubiquitination and proteasomal degradation is emerging as an essential element in the maintenance of cell homeostasis. However, how the E3 ubiquitin ligase activity of HACE1 is regulated remains undetermined. Using a proteomic approach, we identified serine 385 as a target of group-I PAK kinases downstream Rac1 activation by CNF1 toxin from pathogenic E. coli. Moreover, cell treatment with VEGF also promotes Ser-385 phosphorylation of HACE1. We have established in vitro that HACE1 is a direct target of PAK1 kinase activity. Mechanistically, we found that the phospho-mimetic mutant HACE1(S385E), as opposed to HACE1(S385A), displays a lower capacity to ubiquitinate Rac1 in cells. Concomitantly, phosphorylation of Ser-385 plays a pivotal role in controlling the oligomerization state of HACE1. Finally, Ser-385 phosphorylated form of HACE1 localizes in the cytosol away from its target Rac1. Together, our data point to a feedback inhibition of HACE1 ubiquitination activity on Rac1 by group-I PAK kinases.

Alterations of the cellular proteome over time due to spontaneous or toxin-mediated enzymatic deamidation of glutamine (Gln) and asparagine (Asn) residues contribute to bacterial infection and might represent a source of aging-related diseases. Here, we put into perspective what is known about the mode of action of the CNF1 toxin from pathogenic Escherichia coli, a paradigm of bacterial deamidases that activate Rho GTPases, to illustrate the importance of determining whether exposure to these factors are risk factors in the etiology age-related diseases, such as cancer. In particular, through in silico analysis of the distribution of the CNF1-like deamidase active site Gly-Cys-(Xaa)n-His sequence motif in bacterial genomes, we unveil the wide distribution of the super-family of CNF-like toxins and CNF-like deamidase domains among members of the Enterobacteriacae and in association with a large variety of toxin delivery systems. We extent our discussion with recent findings concerning cellular systems that control activated Rac1 GTPase stability and provide protection against cancer. These findings point to the urgency for developing holistic approaches toward personalized medicine that include monitoring for asymptomatic carriage of pathogenic toxin-producing bacteria and that ultimately might lead to improved public health and increased lifespans.

International audience

It remains a challenge to decode the molecular basis of the long-term actin cytoskeleton rearrangements that are governed by the reprogramming of gene expression. Bacillus anthracis lethal toxin (LT) inhibits mitogen-activated protein kinase (MAPK) signaling, thereby modulating gene expression, with major consequences for actin cytoskeleton organization and the loss of endothelial barrier function. Using a laser ablation approach, we characterized the contractile and tensile mechanical properties of LT-induced stress fibers. These actin cables resist pulling forces that are transmitted at cell-matrix interfaces and at cell-cell discontinuous adherens junctions. We report that treating the cells with trichostatin A (TSA), a broad range inhibitor of histone deacetylases (HDACs), or with MS-275, which targets HDAC1, 2 and 3, induces stress fibers. LT decreased the cellular levels of HDAC1, 2 and 3 and reduced the global HDAC activity in the nucleus. Both the LT and TSA treatments induced Rnd3 expression, which is required for the LT-mediated induction of actin stress fibers. Furthermore, we reveal that treating the LT-intoxicated cells with garcinol, an inhibitor of histone acetyl-transferases (HATs), disrupts the stress fibers and limits the monolayer barrier dysfunctions. These data demonstrate the importance of modulating the flux of protein acetylation in order to control actin cytoskeleton organization and the endothelial cell monolayer barrier.

The E3 ubiquitin ligase HACE1 is a potent tumor suppressor that controls cell proliferation and ubiquitylates the small GTPase Rac1 to target it to proteasomal degradation. Whether and how the activity of HACE1 is regulated by the N-terminal ankyrin (ANK) and the middle (MID) domains is ill defined. Here, we identified in the version 64 of the Catalogue of Somatic Mutations in Cancer (COSMIC) 13 missense mutations of hace1 located outside the HECT domain, and found that all lead to defective control of cell proliferation. In addition, several mutations located in the ankyrin domain displayed a dramatic reduction in Rac1 ubiquitylation associated with a decrease of colony formation in soft agar. 3D structure modelling of the 7 ankyrin-repeats coupled to functional analysis identified a surface epitope centered on one of the mutated residue, Gly-175, which is critical for controlling Rac1 binding and ubiquitylation. We also identified a role for the MID domain in conferring the specificity of association of HACE1 to the active form of Rac1. Our study of the functional interplay between HACE1 and Rac1 in cancer thus sheds a new light on the molecular mechanism of Rac1 ubiquitylation by HACE1 and the impact of its cancer-associated mutations in cell proliferation.

Angiogenesis requires the development of a hierarchically branched network of vessels, which undergoes radial expansion and anastomosis to form a close circuit. Branching is achieved by coordinated behavior of endothelial cells that organize into leading “tip” cells and trailing “stalk” cells. Such organization is under control of the Dll4-Notch signaling pathway, which sets a hierarchy in receptiveness of cells to VEGF-A. Recent studies have shed light on a control of the Notch pathway by basement membrane proteins and integrin signaling, disclosing that extracellular matrix exerts active control on vascular branching morphogenesis. We will survey in the present review how extracellular matrix is a multifaceted substrate, which behind a classical structural role hides a powerful conductor function to shape the branching pattern of vessels.

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.

Rationale: Integrins play a crucial role in controlling endothelial cell proliferation and migration during angiogenesis. The Delta-like 4 (Dll4)/Notch pathway establishes an adequate ratio between stalk and tip cell populations by restricting tip cell formation through “lateral inhibition” in response to a vascular endothelial growth factor gradient. Because angiogenesis requires a tight coordination of these cellular processes, we hypothesized that adhesion, vascular endothelial growth factor, and Notch signaling pathways are interconnected. Objective: This study was aimed at characterizing the cross-talk between integrin and Notch signaling in endothelial cells. Methods and Results: Adhesion of primary human endothelial cells to laminin-111 triggers Dll4 expression, leading to subsequent Notch pathway activation. SiRNA-mediated knockdown of α2β1 and α6β1 integrins abolishes Dll4 induction, which discloses a selective integrin signaling acting upstream of Notch pathway. The increase in Foxc2 transcription, triggered by α2β1 binding to laminin, is required but not sufficient per se for Dll4 expression. Furthermore, vascular endothelial growth factor stimulates laminin γ1 deposition, which leads to integrin signaling and Dll4 induction. Interestingly, loss of integrins α2 or α6 mimics the effects of Dll4 silencing and induces excessive network branching in an in vitro sprouting angiogenesis assay on three-dimensional matrigel. Conclusions: We show that, in endothelial cells, ligation of α2β1 and α6β1 integrins induces the Notch pathway, and we disclose a novel role of basement membrane proteins in the processes controlling tip vs stalk cell selection.

We have investigated how Bacillus anthracis lethal toxin (LT) triggers caspase-3 activation and the formation of thick actin cables in human endothelial cells. By DNA array analysis we show that LT has a major impact on the cell transcriptome and we identify key host genes involved in LT cytotoxic effects. Indeed, upregulation of TRAIL and downregulation of XIAP both participate in LT-induced caspase-3 activation. LT induces a downregulation of the immediate early gene and master regulator of transcription egr1. Importantly, its re-expression in LT-intoxicated cells blocks caspase-3 activation. In parallel, we found that the formation of actin cables induced by LT occurs in the absence of direct activation of RhoA/ROCK signalling. We show that knock-down of cortactin and rhophilin-2 under conditions of calponin-1 expression defines the minimal set of genes regulated by LT for actin cable formation. Together our data establish that the modulation of the cell transcriptome by LT plays a key role in triggering human endothelial cell toxicity.

International audience; It remains a challenge to decode the molecular basis of the long-term actin cytoskeleton rearrangements that are governed by the reprogramming of gene expression. Bacillus anthracis lethal toxin (LT) inhibits mitogen-activated protein kinase (MAPK) signaling, thereby modulating gene expression, with major consequences for actin cytoskeleton organization and the loss of endothelial barrier function. Using a laser ablation approach, we characterized the contractile and tensile mechanical properties of LT-induced stress fibers. These actin cables resist pulling forces that are transmitted at cell-matrix interfaces and at cell-cell discontinuous adherens junctions. We report that treating the cells with trichostatin A (TSA), a broad range inhibitor of histone deacetylases (HDACs), or with MS-275, which targets HDAC1, 2 and 3, induces stress fibers. LT decreased the cellular levels of HDAC1, 2 and 3 and reduced the global HDAC activity in the nucleus. Both the LT and TSA treatments induced Rnd3 expression, which is required for the LT-mediated induction of actin stress fibers. Furthermore, we reveal that treating the LT-intoxicated cells with garcinol, an inhibitor of histone acetyl-transferases (HATs), disrupts the stress fibers and limits the monolayer barrier dysfunctions. These data demonstrate the importance of modulating the flux of protein acetylation in order to control actin cytoskeleton organization and the endothelial cell monolayer barrier.

Cell migration is a critical step in the progression of prostate cancer to the metastatic state, the lethal form of the disease. The antidiabetic drug metformin has been shown to display antitumoral properties in prostate cancer cell and animal models; however, its role in the formation of metastases remains poorly documented. Here, we show that metformin reduces the formation of metastases to fewer solid organs in an orthotopic metastatic prostate cancer cell model established in nude mice. As predicted, metformin hampers cell motility in PC3 and DU145 prostate cancer cells and triggers a radical reorganization of the cell cytoskeleton. The small GTPase Rac1 is a master regulator of cytoskeleton organization and cell migration. We report that metformin leads to a major inhibition of Rac1 GTPase activity by interfering with some of its multiple upstream signaling pathways, namely P-Rex1 (a Guanine nucleotide exchange factor and activator of Rac1), cAMP, and CXCL12/CXCR4, resulting in decreased migration of prostate cancer cells. Importantly, overexpression of a constitutively active form of Rac1, or P-Rex, as well as the inhibition of the adenylate cyclase, was able to reverse the antimigratory effects of metformin. These results establish a novel mechanism of action for metformin and highlight its potential antimetastatic properties in prostate cancer. Mol Cancer Ther; 14(2); 586–96. ©2014 AACR.

The GTPase RhoA is a major regulator of the assembly of actin stress fibers and the contractility of the actomyosin cytoskeleton. The epidermal cell differentiation inhibitor (EDIN) and EDIN-like ADP-ribosyltransferases of Staphylococcus aureus catalyze the inactivation of RhoA, producing actin cable disruption. We report that purified recombinant EDIN and EDIN-producing S. aureus provoke large transcellular tunnels in endothelial cells that we have named macroapertures (MAs). These structures open transiently, followed by the appearance of actin-containing membrane waves extending over the aperture. Disruption of actin cables, either directly or indirectly, through rhoA RNAi knockdown also triggers the formation of MAs. Intoxication of endothelial monolayers by EDIN produces a loss of barrier function and provides direct access of the endothelium basement membrane to S. aureus.

39 m/s n° 1, vol. 28, janvier 2012 DOI : 10.1051/medsci/2012281014 > La petite GTPase Rac1 controle des processus cellulaires essentiels, au nombre desquels la migration, la phagocytose, le cycle et l’apoptose [1]. La deregulation de l’activite de Rac1 est impliquee dans des pathologies humaines graves comme certains deficits immunitaires, cancers et maladies mentales. Rac1 est aussi la cible de nombreux facteurs de virulence bacteriens [2]. L’etude d’un de ces facteurs, CNF1 (cytotoxic necrotizing factor 1), a permis a notre equipe de decouvrir un nouveau mode de regulation des proteines Rho, notamment Rac1, par ubiquitinylation et degradation proteasomique, et d’identifier certains des acteurs de cette regulation cellulaire, avec la decouverte aujourd’hui de l’activite E3 ubiquitine ligase de HACE1 (HECT domain and ankyrin repeat containing E3 ubiquitin-protein ligase 1) vis-a-vis de Rac1 [3]. Ces observations posent la question de la relation entre cette activite biochimique de HACE1 et ses proprietes de suppresseur de tumeur.

CNF1 toxin is a virulence factor produced by uropathogenic Escherichia coli. Upon cell binding and introduction into the cytosol, CNF1 deamidates glutamine 63 of RhoA (or 61 of Rac and Cdc42), rendering constitutively active these GTPases. Unexpectedly, we measured in bladder cells a transient CNF1-induced activation of Rho GTPases, maximal for Rac. Deactivation of Rac correlated with the increased susceptibility of its deamidated form to ubiquitin/proteasome-mediated degradation. Sensitivity to ubiquitylation could be generalized to other permanent-activated forms of Rac and to its sustained activation by Dbl. Degradation of the toxin-activated Rac allowed both host cell motility and efficient cell invasion by uropathogenic bacteria. CNF1 toxicity thus results from a restricted activation of Rho GTPases through hijacking the host cell proteasomal machinery.

International audience; Dugesia japonica planarian flatworms are naturally exposed to various microbes but typically survive this challenge. We show that planarians eliminate bacteria pathogenic to Homo sapiens, Caenorhabditis elegans, and/or Drosophila melanogaster and thus represent a model to identify innate resistance mechanisms. Whole-transcriptome analysis coupled with RNAi screening of worms infected with Staphylococcus aureus or Legionella pneumophila identified 18 resistance genes with nine human orthologs, of which we examined the function of MORN2. Human MORN2 facilitates phagocytosis-mediated restriction of Mycobacterium tuberculosis, L. pneumophila, and S. aureus in macrophages. MORN2 promotes the recruitment of LC3, an autophagy protein also involved in phagocytosis, to M. tuberculosis-containing phagosomes and subsequent maturation to degradative phagolysosomes. MORN2-driven trafficking of M. tuberculosis to single-membrane, LC3-positive compartments requires autophagy-related proteins Atg5 and Beclin-1, but not Ulk-1 and Atg13, highlighting the importance of MORN2 in LC3-associated phagocytosis. These findings underscore the value of studying planarian defenses to identify immune factors.

Results We show here that metformin reduces the occurrence of metastases in an orthotopic metastatic prostate cancer cell model established in nude mice. As predicted, metformin hampers cell motility in PC3 and DU145 prostate cancer cells and triggers a radical reorganization of the cell cytoskeleton. The small GTPase Rac1 is a master regulator of cytoskeleton organization and cell migration. We report that metformin inhibits Rac1 GTPase activity by interfering with some of its multiple upstream signaling pathways, namely P-Rex1 (a Guanine nucleotide exchange factor and activator of Rac1), cyclic AMP and CXCL12/CXCR4, resulting in decreased migration of prostate cancer cells. Importantly, overexpression of a constitutively active form of Rac1 (Rac1-Q61L or Rac1-V12), or P-Rex1 as well as the inhibition of the adenylate cyclase were able to reverse the anti-migratory effects of metformin.

Plant water homeostasis is maintained by the phytohormone abscisic acid (ABA), which triggers stomatal pore closure in response to drought stress. We identified the Arabidopsis small guanosine triphosphatase (GTPase) protein AtRac1 as a central component in the ABA-mediated stomatal closure process. ABA treatment induced inactivation of AtRac GTPases and disruption of the guard cell actin cytoskeleton. In contrast, in the ABA-insensitive mutantabi1-1, which is impaired in stomatal closure, neither AtRac inactivation nor actin cytoskeleton disruption was observed on ABA treatment. These observations indicate that AtRac1 inactivation is a limiting step in the ABA-signaling cascade leading to stomatal closure. Consistent with these findings, expression of a dominant-positive mutant of AtRac1 blocked the ABA-mediated effects on actin cytoskeleton and stomatal closure in wild-type plants, whereas expression of a dominant-negative AtRac1 mutant recapitulated the ABA effects in the absence of the hormone. Moreover, the dominant-negative form of AtRac1 could also restore stomatal closure inabi1-1. These results define AtRac1 as a central element for plant adaptation to drought.

Adhesion to fibronectin through the alpha5beta1 integrin enables endothelial cells to proliferate in response to growth factors, whereas adhesion to laminin through alpha2beta1 results in growth arrest under the same conditions. On laminin, endothelial cells fail to translate Cyclin D1 mRNA and activate CDK4 and CDK6. Activated Rac, but not MEK1, PI-3K, or Akt, rescues biosynthesis of cyclin D1 and progression through the G(1) phase. Conversely, dominant negative Rac prevents these events on fibronectin. Mitogens promote activation of Rac on fibronectin but not laminin. This process is mediated by SOS and PI-3K and requires coordinate upstream signals through Shc and FAK. These results indicate that Rac is a crucial mediator of the integrin-specific control of cell cycle in endothelial cells.

Thrombospondin 1 (TSP1) is known for its significant anti-angiogenic properties. In a previous study, we have shown that transient or stable overexpression of the transcription factor c-Jun, in rat fibroblasts, leads to repression of TSP1. We now demonstrate that the c-Jun-induced repression of TSP1 does not occur directly and does not require binding of c-Jun to the TSP1 promoter. Instead, repression involves a factor secreted by c-Jun-overexpressing cells. This secreted factor triggers a signal transduction pathway from the membrane to the nucleus, and these signals lead to the binding of the product of the Wilms' tumor suppressor gene, WT1, to the -210 region of the TSP1 promoter. This region binds WT1 and SP1, but not EGR1, although its sequence fits the consensus binding site for this transcription factor. WT1 overexpression in transfected cells inhibits endogenous TSP1 gene expression and TSP1 transcription in experiments using TSP1 promoter-reporter constructs. The WT1 - KTS isoform is more active in repressing TSP1 transcription than WT1 + KTS, while EGR1 is inactive. Enhancement of WT1 binding to DNA in response to c-Jun does not require de novo protein synthesis. The above mechanism for TSP1 repression could apply to other genes, thus coordinating their regulation in the vicinity of a c-Jun-overexpressing cell. We conclude that WT1, which was discovered as a result of its tumor suppressor properties, may also possess oncogenic characteristics in the c-Jun transformation process, and thus repress the anti-angiogenic protein, TSP1.

In cooperation with an activated ras oncogene, the site-dependent AP-1 transcription factor c-Jun transforms primary rat embryo fibroblasts (REF). Although signal transduction pathways leading to activation of c-Jun proteins have been extensively studied, little is known about c-Jun cellular targets. We identified c-Jun-upregulated cDNA clones homologous to the tenascin-C gene by differential screening of a cDNA library from REF. This tightly regulated gene encodes a rare extracellular matrix protein involved in cell attachment and migration and in the control of cell growth. Transient overexpression of c-Jun induced tenascin-C expression in primary REF and in FR3T3, an established fibroblast cell line. Surprisingly, tenascin-C synthesis was repressed after stable transformation by c-Jun compared to that in the nontransformed parental cells. As assessed by using the tenascin-C (-220 to +79) promoter fragment cloned in a reporter construct, the c-Jun-induced transient activation is mediated by two binding sites: one GCN4/AP-1-like site, at position -146, and one NF-kappaB site, at position -210. Furthermore, as demonstrated by gel shift experiments and cotransfections of the reporter plasmid and expression vectors encoding the p65 subunit of NF-kappaB and c-Jun, the two transcription factors bind and synergistically transactivate the tenascin-C promoter. We previously described two other extracellular matrix proteins, SPARC and thrombospondin-1, as c-Jun targets. Thus, our results strongly suggest that the regulation of the extracellular matrix composition plays a central role in c-Jun-induced transformation.

The aim of this study was to identify the molecular signals produced in human endothelial cells (ECs) by the interaction of α5β1 integrin with soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) present in the extracellular matrix. We generated a gene expression profile of ECs adhering to sVEGFR-1 or to fibronectin, the classic extracellular matrix ligand for α5β1 integrin or in a nonadhering condition. Several biological pathways were differently modulated, 3 protein kinase C substrates [adducin, myristoylated alanine-rich protein kinase C substrate (MARCKS), and radixin] were differently expressed and phosphorylated when cells adhering to sVEGFR-1 were compared with those adhering to fibronectin. Rac1 activation and Gα13 protein involvement through the interaction with radixin were also detected after attachment to sVEGFR-1, and these responses depended on active VEGFR-2 signaling. On sVEGFR-1, ECs exhibited a motile phenotype that was consistent with the abundant presence of MARCKS, a stabilizer of dynamic adhesions. Moreover, ECs silenced for radixin expression no longer responded to the proangiogenic VEGFR-1-derived peptide 12. We propose that the presence of sVEGFR-1 in the EC microenvironment directs α5β1 integrin signaling to generate a dynamic, motile phenotype. Our findings also provide new insights into the mechanism of action of proangiogenic peptide 12, relevant to a therapeutic perspective.

The Hace1-HECT E3 ligase is a tumor suppressor that ubiquitylates the activated GTP-bound form of the Rho family GTPase Rac1, leading to Rac1 proteasomal degradation. Here we show that, in vertebrates, Hace1 targets Rac1 for degradation when Rac1 is localized to the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase holoenzyme. This event blocks de novo reactive oxygen species generation by Rac1-dependent NADPH oxidases, and thereby confers cellular protection from reactive oxygen species-induced DNA damage and cyclin D1-driven hyper-proliferation. Genetic inactivation of Hace1 in mice or zebrafish, as well as Hace1 loss in human tumor cell lines or primary murine or human tumors, leads to chronic NADPH oxidase-dependent reactive oxygen species elevation, DNA damage responses and enhanced cyclin D1 expression. Our data reveal a conserved ubiquitin-dependent molecular mechanism that controls the activity of Rac1-dependent NADPH oxidase complexes, and thus constitutes the first known example of a tumor suppressor protein that directly regulates reactive oxygen species production in vertebrates., HACE1 is an E3 ligase that promotes degradation of the GTPase Rac1. Here Daugaard et al. show that HACE1 inhibits the activity of Rac1-dependent NADPH oxidase complexes, which reduces the production of cellular reactive oxygen species and cell cycle progression.

Rho GTPases undergo ubiquitylation and degradation via the ubiquitin-proteasome pathway. We now report in the November issue of Developmental Cell that the E3 ubiquitin-ligase HACE1 catalyzes the ubiquitylation of GTP-bound Rac1. Depletion of HACE1 leads to an increase of Rac1 activity. We have proposed that HACE1 limits Rac1 activity in cells, a regulation that is usurped by some pathogenic bacteria for efficient invasion of host cell monolayers. We here review these findings in parallel with the regulation of RhoA by the ubiquitin and proteasome system (UPS) and discuss the impact of these regulations on the capacity of Rho GTPases to signal.

In culture, cell confluence generates signals that commit actively growing keratinocytes to exit the cell cycle and differentiate to form a stratified epithelium. Using a comparative proteomic approach, we studied this ‘confluence switch’ and identified a new pathway triggered by cell confluence that regulates basement membrane (BM) protein composition by suppressing the uPA/uPAR/plasmin pathway. Indeed, confluence triggers adherens junction maturation and enhances TGF-β/activin A activity, resulting in increased deposition of PAI-1 and perlecan in the BM. Extracellular matrix (ECM)-accumulated PAI-1 suppresses uPA/uPAR/plasmin pathway and further enhances perlecan deposition by inhibiting its plasmin-dependent proteolysis. We show that perlecan deposition in the ECM strengthens cell adhesion, inhibits keratinocyte motility and promotes additional accumulation of PAI-1 in the ECM at confluence. In agreement, during wound-healing, perlecan concentrates at the wound-margin, where BM matures to stabilize keratinocyte adhesion. Our results demonstrate that confluence dependent-signaling orchestrates not only growth-inhibition and differentiation but also controls ECM-proteolysis and BM formation. These data suggest that uncontrolled integration of confluence-dependent signaling, may favor skin disorders, including tumorigenesis, not only by promoting cell hyperproliferation but also by altering protease activity and deposition of ECM components.

The sequence-specific transcription factor c-Jun displays oncogenic potential in mammalian cells either in cooperation with activated Ras in primary embryonic fibroblasts or alone in established cell lines. Although pathways for signal transduction leading to activation of c-Jun proteins have been extensively studied, little is known about the events downstream of c-Jun stimulation. We isolated cellular genes that are targets of c-Jun by differential screening of a cDNA library from primary rat embryo fibroblasts. Two transcripts with sequences similar to known genes were repressed following transitory expression of a c-Jun-encoding vector. They correspond to the SPARC and thrombospondin 1 (TS1) genes, encoding extracellular matrix proteins. These genes are tightly regulated during embryogenesis and in adult tissues and are involved in the control of cell growth. c-Jun transitory repression of these two genes was demonstrated both in primary cells and in FR3T3, an established fibroblast cell line. The repression was also detected in FR3T3 derivatives stably transformed by c-Jun or Ras. Although c-Jun regulation of the TS1 gene was found at the promoter level, preliminary results strongly suggest that repression of SPARC and TS1 gene expression are mediated by a secreted factor. In contrast, expression of these genes was unaffected by transformation with oncogenes from DNA viruses. Our results identify new, specific, probably indirect c-Jun target genes and suggest previously unsuspected regulatory roles for SPARC and thrombospondin in the control of cell growth.

Rho GTPases including RhoA, Cdc42, and Rac1 are master regulators of cell cytoskeleton dynamic, thus controlling essential cellular processes notably cell polarity, migration and cytokinesis. These GTPases undergo a spatiotemporal regulation primarily controlled by cellular factors inducing both the exchange of GDP for GTP and the hydrolysis of GTP into GDP. Recent findings have unveiled another layer of complexity in the regulation of Rho proteins consisting in their ubiquitylation followed by their proteasomal degradation. Here, we describe how to assess the level of ubiquitylation of Rho proteins in cells, taking Rac1 as an example.

Rho GTPases including RhoA, Cdc42, and Rac1 are master regulators of cell cytoskeleton dynamic, thus controlling essential cellular processes notably cell polarity, migration and cytokinesis. These GTPases undergo a spatiotemporal regulation primarily controlled by cellular factors inducing both the exchange of GDP for GTP and the hydrolysis of GTP into GDP. Recent findings have unveiled another layer of complexity in the regulation of Rho proteins consisting in their ubiquitylation followed by their proteasomal degradation. Here, we describe how to assess the level of ubiquitylation of Rho proteins in cells, taking Rac1 as an example.

International audience; Rho GTPases, which are master regulators of both the actin cytoskeleton and membrane trafficking, are often hijacked by pathogens to enable their invasion of host cells. Here we report that the cytotoxic necrotizing factor-1 (CNF1) toxin of uropathogenic Escherichia coli (UPEC) promotes Rac1-dependent entry of bacteria into host cells. Our screen for proteins involved in Rac1-dependent UPEC entry identifies the Toll-interacting protein (Tollip) as a new interacting protein of Rac1 and its ubiquitinated forms. We show that knockdown of Tollip reduces CNF1-induced Rac1-dependent UPEC entry. Tollip depletion also reduces the Rac1-dependent entry of Listeria monocytogenes expressing InlB invasion protein. Moreover, knockdown of Tollip, Tom1 and clathrin, decreases CNF1 and Rac1-dependent internalization of UPEC. Finally, we show that Tollip, Tom1 and clathrin associate with Rac1 and localize at the site of bacterial entry. Collectively, these findings reveal a new link between Rac1 and Tollip, Tom1 and clathrin membrane trafficking components hijacked by pathogenic bacteria to allow their efficient invasion of host cells.

Integrin receptors and their extracellular matrix ligands provide cues to cell proliferation, survival, differentiation and migration. Here, we show that alpha2beta1 integrin, when ligated to the basement membrane component laminin-1, triggers a proliferation arrest in primary endothelial cells. Indeed, in the presence of strong growth signals supplied by growth factors and fibronectin, alpha2beta1 engagement alters assembly of mature focal adhesions by alpha5beta1 and leads to impairment of downstream signaling and cell-cycle arrest in the G1 phase. Although the capacity of alpha5beta1 to signal for GTP loading of Rac is preserved, the joint engagement of alpha2beta1 interferes with membrane anchorage of Rac. Adapting the 'split-ubiquitin' sensor to screen for membrane-proximal alpha2 integrin partners, we identified the CD9 tetraspanin and further establish its requirement for destabilization of focal adhesions, control of Rac subcellular localization and growth arrest induced by alpha2beta1 integrin. Altogether, our data establish that alpha2beta1 integrin controls endothelial cell commitment towards quiescence by triggering a CD9-dependent dominant signaling.

We have investigated how Bacillus anthracis lethal toxin (LT) triggers caspase-3 activation and the formation of thick actin cables in human endothelial cells. By DNA array analysis we show that LT has a major impact on the cell transcriptome and we identify key host genes involved in LT cytotoxic effects. Indeed, upregulation of TRAIL and downregulation of XIAP both participate in LT-induced caspase-3 activation. LT induces a downregulation of the immediate early gene and master regulator of transcription egr1. Importantly, its re-expression in LT-intoxicated cells blocks caspase-3 activation. In parallel, we found that the formation of actin cables induced by LT occurs in the absence of direct activation of RhoA/ROCK signalling. We show that knock-down of cortactin and rhophilin-2 under conditions of calponin-1 expression defines the minimal set of genes regulated by LT for actin cable formation. Together our data establish that the modulation of the cell transcriptome by LT plays a key role in triggering human endothelial cell toxicity.

Integrins are transmembrane receptors which bind extracellular matrix proteins and enable not only cell adhesion and cytoskeleton organization but also transduction of critical signals into the cells to promote survival, proliferation, differentiation, or migration programs. Integrins participate in many aspects of vascular biology. The past few years have experienced a sustained interest in the implication of integrin receptors in tumor angiogenesis. We will focus our review on studies giving concrete evidence to a role of the beta1 class of integrins in angiogenesis, and we will provide an overview of the molecular mechanisms involved in their action.

The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli. CNF1 catalyzes the constitutive activation of Rho proteins by deamidation. The threshold of activation of Rho proteins by CNF1 is, however, attenuated because of a concomitant decrease of their cellular levels. Depletion of activated-Rac1 is catalyzed by ubiquitin-mediated proteasomal degradation. Consequently, we show by effector-binding pull-down that co-treatment of intoxicated cells with the MG132 proteasome-inhibitor results in a higher level of activation of Rac, as well as RhoA and Cdc42. We show that CNF1 induces the transient recruitment of Rho proteins to cellular membranes. Interestingly, at the difference of Rac and Cdc42, the inhibition of the proteasome during CNF1 treatment does not result in a significant accumulation of RhoA to cellular membranes. Using an in vivo ubiquitylation assay, we evidence that mutation of the geranylgeranyl acceptor cysteine of Rac1 (Rac1C189G) abolished the sensitivity of permanently activated-Rac1 to ubiquitylation, whereas Rac1C189G remained able to bind to the effector-binding domain of p21-PAK. Collectively, these results indicate that association with the cellular membranes is a necessary step for activated-Rac1 ubiquitylation.

International audience; The Rep protein of geminiviruses is the sole viral protein required for their DNA replication. The amino acid sequence of Rep protein contains an NTP binding consensus motif (P-loop). Here we show that purified Rep protein of tomato yellow leaf curl virus expressed in Escherichia coli exhibits an ATPase activity in vitro. Amino acid exchanges in the P-loop sequence of Rep causes a substantial decrease or loss of the ATPase activity. In vivo, mutant viruses carrying these Rep mutations do not replicate in plant cells. These results show that ATP binding by the Rep protein of geminiviruses is required for its function in viral DNA replication.

SummaryRac1 small GTPase controls essential aspects of cell biology and is a direct target of numerous bacterial virulence factors. The CNF1 toxin of pathogenic Escherichia coli addresses Rac1 to ubiquitin-proteasome system (UPS). We report the essential role of the tumor suppressor HACE1, a HECT-domain containing E3 ubiquitin-ligase, in the targeting of Rac1 to UPS. HACE1 binds preferentially GTP-bound Rac1 and catalyzes its polyubiquitylation. HACE1 expression increases the ubiquitylation of Rac1, when the GTPase is activated by point mutations or by the GEF-domain of Dbl. RNAi-mediated depletion of HACE1 blocks the ubiquitylation of active Rac1 and increases GTP-bound Rac1 cellular levels. HACE1 antagonizes cell isotropic spreading, a hallmark of Rac1 activation, and is required for endothelial cell monolayer invasion by bacteria. Together, these data establish the role of the HACE1 E3 ubiquitin-ligase in controlling Rac1 ubiquitylation and activity.