HUWE1 ubiquitylates and degrades the RAC activator TIAM1 promoting cell-cell adhesion disassembly, migration, and invasion.
ABSTRACT: The E3 ubiquitin ligase HUWE1, deregulated in carcinoma, has been implicated in tumor formation. Here, we uncover a role for HUWE1 in cell migration and invasion through degrading the RAC activator TIAM1, implying an additional function in malignant progression. In MDCKII cells in response to HGF, HUWE1 catalyzes TIAM1 ubiquitylation and degradation predominantly at cell-cell adhesions, facilitating junction disassembly, migration, and invasion. Depleting HUWE1 or mutating the TIAM1 ubiquitylation site prevents TIAM1 degradation, antagonizing scattering, and invasion. Moreover, simultaneous depletion of TIAM1 restores migration and invasion in HUWE1-depleted cells. Significantly, we show that HUWE1 stimulates human lung cancer cell invasion through regulating TIAM1 stability. Finally, we demonstrate that HUWE1 and TIAM1 protein levels are inversely correlated in human lung carcinomas. Thus, we elucidate a critical role for HUWE1 in regulating epithelial cell-cell adhesion and provide additional evidence that ubiquitylation contributes to spatiotemporal control of RAC.
Project description:We previously demonstrated that both Tiam1, an activator of Rac, and constitutively active V12Rac promote E-cadherin-mediated cell-cell adhesion in epithelial Madin Darby canine kidney (MDCK) cells. Moreover, Tiam1 and V12Rac inhibit invasion of Ras-transformed, fibroblastoid MDCK-f3 cells by restoring E-cadherin-mediated cell-cell adhesion. Here we show that the Tiam1/Rac-induced cellular response is dependent on the cell substrate. On fibronectin and laminin 1, Tiam1/Rac signaling inhibits migration of MDCK-f3 cells by restoring E-cadherin-mediated cell- cell adhesion. On different collagens, however, expression of Tiam1 and V12Rac promotes motile behavior, under conditions that prevent formation of E-cadherin adhesions. In nonmotile cells, Tiam1 is present in adherens junctions, whereas Tiam1 localizes to lamellae of migrating cells. The level of Rac activation by Tiam1, as determined by binding to a glutathione-S-transferase- PAK protein, is similar on fibronectin or collagen I, suggesting that rather the localization of the Tiam1/Rac signaling complex determines the substrate-dependent cellular responses. Rac activation by Tiam1 requires PI3-kinase activity. Moreover, Tiam1- but not V12Rac-induced migration as well as E-cadherin-mediated cell- cell adhesion are dependent on PI3-kinase, indicating that PI3-kinase acts upstream of Tiam1 and Rac.
Project description: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.
Project description:The tumor microenvironment undergoes changes concurrent with neoplastic progression. Cancer incidence increases with aging and is associated with tissue accumulation of senescent cells. Senescent fibroblasts are thought to contribute to tumor development in aging tissues. We have shown that fibroblasts deficient in the Rac exchange factor Tiam1 promote invasion and metastasis of associated epithelial tumor cells. Here, we use a three-dimensional culture model of cellular invasiveness to outline several steps underlying this effect. We find that stress-induced senescence induces decreased fibroblast Tiam1 protein levels and increased osteopontin levels, and that senescent fibroblast lysates induce Tiam1 protein degradation in a calcium- and calpain-dependent fashion. Changes in fibroblast Tiam1 protein levels induce converse changes in osteopontin mRNA and protein. Senescent fibroblasts induce increased invasion and migration in co-cultured mammary epithelial cells. These effects in epithelial cells are ameliorated by either increasing fibroblast Tiam1 or decreasing fibroblast osteopontin. Finally, in seeded cell migration assays we find that either senescent or Tiam1-deficient fibroblasts induce increased epithelial cell migration that is dependent on fibroblast secretion of osteopontin. These findings indicate that one mechanism by which senescent fibroblasts promote neoplastic progression in associated tumors is through degradation of fibroblast Tiam1 protein and the consequent increase in secretion of osteopontin by fibroblasts.
Project description:The Rac exchange factor Tiam1 is involved in diverse cell functions and signaling pathways through multiple protein interactions, raising the question of how signaling and functional specificity are achieved. We have shown that Tiam1 interactions with different scaffold proteins activate different Rac-dependent pathways by recruiting specific Rac effector proteins, and reasoned that there must be regulatory mechanisms governing each interaction. Fibroblasts express at least two Tiam1-interacting proteins, insulin receptor substrate protein 53 kDa (IRSp53) and spinophilin. We used fluorescent resonance energy transfer (FRET) to measure localized Rac activation associated with IRSp53 and spinophilin complexes in individual fibroblasts to test this hypothesis. Pervanadate or platelet-derived growth factor induced localized Rac activation dependent on Tiam1 and IRSp53. Forskolin or epinephrine induced localized Rac activation dependent on Tiam1 and spinophilin. In spinophilin-deficient cells, Tiam1 co-localized with IRSp53 in response to pervanadate or platelet-derived growth factor. In IRSp53-deficient cells, Tiam1 co-localized with spinophilin in response to forskolin or epinephrine. Total cellular levels of activated Rac were affected only in cells with exogenous Tiam1, and were primarily increased in the membrane fraction. Downstream effects of Rac activation were also stimulus and scaffold-specific. Cell ruffling, spreading, and cell adhesion were dependent on IRSp53, but not spinophilin. Epinephrine decreased IRSp53-dependent adhesion and increased cell migration in a Rac and spinophilin-dependent fashion. These results support the idea that Tiam1 interactions with different scaffold proteins couple distinct upstream signals to localized Rac activation and specific downstream pathways, and suggest that manipulating Tiam1-scaffold interactions can modulate Rac-dependent cellular behaviors.
Project description:Rac1-GTPases serve as intermediary cellular switches, which conduct transient and constitutive signals from upstream cues, including those from Ras oncoproteins. Although the sirtuin1 (SIRT1) deacetylase is overexpressed in several human cancers and has recently been linked to cancer cell motility as a context-dependent regulator of multiple pathways, its role in Rac1 activation has not been reported. Similarly, SIRT2 has been demonstrated to be upregulated in some cancers; however, studies have also reported its role in tumor suppression. Here, we demonstrate that SIRT1 and SIRT2 positively regulate the levels of Rac1-GTP and the activity of T-cell lymphoma invasion and metastasis 1 (TIAM1), a Rac guanine nucleotide exchange factor (GEF). Transient inhibition of SIRT1 and SIRT2 resulted in increased acetylation of TIAM1, whereas chronic SIRT2 knockdown resulted in enhanced acetylation of TIAM1. SIRT1 regulates Dishevelled (DVL) protein levels in cancer cells, and DVL along with TIAM1 are known to augment Rac activation; however, SIRT1 or 2 has not been previously linked with TIAM1. We found that diminished sirtuin activity led to the disruption of the DVL1-TIAM1 interaction. We hence propose a model for Rac activation where SIRT1/2 positively modulates the DVL/TIAM1/Rac axis and promotes sustained pathway activation.
Project description:The lethality of pancreatic adenocarcinoma stems from an elevated incidence of tumor cell invasion and metastasis that are mediated by mechanisms not yet understood. Recent studies indicate that the proinvasive integrin alpha 6 beta 4 is highly upregulated in pancreatic adenocarcinomas. To assess the importance of this integrin in pancreatic cancer cell migration and invasion, cell lines were screened for integrin alpha 6 beta 4 expression by immunoblotting and fluorescence-activated cell sorting and their ability to migrate and invade toward hepatocyte growth factor (HGF). We found that cell surface expression of the alpha 6 beta 4 integrin correlated with the cells' ability to migrate and invade toward HGF. When cells expressing high levels of integrin alpha 6 beta 4 were treated with small interfering RNA targeting alpha 6 or beta 4 integrin subunits, we observed a reduction in cell migration and invasion. Furthermore, the activity of the small GTPase Rac1 was stimulated by alpha 6 beta 4 integrin expression and was necessary for HGF-stimulated chemotaxis. We discovered that expression of the Rac-specific nucleotide exchange factor, Tiam1 (T-lymphoma invasion and metastasis), was upregulated in cells overexpressing the integrin alpha 6 beta 4 and required for the elevated Rac1 activity in these cells. We conclude that the integrin alpha 6 beta 4 promotes the migratory and invasive phenotype of pancreatic carcinoma cells through the Tiam1-Rac1 pathway in part through the upregulation of Tiam1.
Project description:The co-evolution of tumors and their microenvironment involves bidirectional communication between tumor cells and tumor-associated stroma. Various cell types are present in tumor-associated stroma, of which fibroblasts are the most abundant. The Rac exchange factor Tiam1 is implicated in multiple signaling pathways in epithelial tumor cells and lack of Tiam1 in tumor cells retards tumor growth in Tiam1 knockout mouse models. Conversely, tumors arising in Tiam1 knockout mice have increased invasiveness. We have investigated the role of Tiam1 in tumor-associated fibroblasts as a modulator of tumor cell invasion and metastasis, using retroviral delivery of short hairpin RNA to suppress Tiam1 levels in three different experimental models. In spheroid co-culture of mammary epithelial cells and fibroblasts, Tiam1 silencing in fibroblasts led to increased epithelial cell outgrowth into matrix. In tissue-engineered human skin, Tiam1 silencing in dermal fibroblasts led to increased invasiveness of epidermal keratinocytes with pre-malignant features. In a model of human breast cancer in mice, co-implantation of mammary fibroblasts inhibited tumor invasion and metastasis, which was reversed by Tiam1 silencing in co-injected fibroblasts. These results suggest that stromal Tiam1 may have a role in modulating the effects of the tumor microenvironment on malignant cell invasion and metastasis. This suggests a set of pathways for further investigation, with implications for future therapeutic targets.
Project description:TGF-? promotes cell migration and invasion, an attribute that is linked to the pro-metastasis function of this cytokine in late stage cancers. The LIM 1863 colon carcinoma organoid undergoes epithelial-mesenchymal transition (EMT) in response to TGF-?. This process is markedly accelerated by TNF-?, and we found that the levels of miR-21 and miR-31 were prominently elevated under the synergistic actions of TGF-?/TNF-?. Consistent with this, overexpression of either miR-21 or miR-31 significantly enhanced the effect of TGF-? alone on LIM 1863 morphological changes. More importantly, transwell assays demonstrated the positive effects of both miR-21 and miR-31 in TGF-? regulation of LIM 1863 motility and invasiveness. Elevated levels of miR-21 and miR-31 also enhanced motility and invasiveness of other colon carcinoma cell lines. We present compelling evidence that TIAM1, a guanidine exchange factor of the Rac GTPase, is a direct target of both miR-21 and miR-31. Indeed in LIM 1863 cells, suppression of TIAM1 is required for miR-21/miR-31 to enhance cell migration and invasion. Therefore, we have uncovered miR-21 and miR-31 as downstream effectors of TGF-? in facilitating invasion and metastasis of colon carcinoma cells.
Project description:Normal cells acquire aggressive behavior by modifying signaling pathways. For instance, alteration of endocytosis profoundly impacts both proliferation and migration during tumorigenesis. Here we investigate the mechanisms that enable the endocytic machinery to coordinate these processes. We show that a membrane curvature-sensing protein, endophilin A3, promotes growth and migration of colon cancer cells through two competing mechanisms: an endocytosis pathway that is required for proliferation and a GTPase regulatory pathway that controls cell motility. EndoA3 stimulates cell migration by binding the Rac GEF TIAM1 leading to activation of small GTPases. Competing interactions of EndoA3 with membrane versus TIAM1 modulate hyperproliferative and metastatic phenotypes. Disruption of EndoA3-membrane interactions stimulates TIAM1 and small GTPases in vitro, and further promotes pro-metastatic phenotypes in vivo. Together, these results uncover a coupling mechanism, by which EndoA3 promotes growth and migration of colon cancers, by linking membrane dynamics to GTPase regulation.
Project description:The exchange factor Tiam1 regulates multiple cellular functions by activating the Rac GTPase. Active Rac has various effects in cells, including alteration of actin cytoskeleton and gene expression, via binding to and modulating the activity of diverse effector proteins. How individual Rac effectors are selected for activation and regulated in response to upstream signals is not well understood. We find that Tiam1 contributes to both of these processes by binding to IRSp53, an adaptor protein that is an effector for both Rac and Cdc42. Tiam1 directs IRSp53 to Rac signaling by enhancing IRSp53 binding to both active Rac and the WAVE2 scaffold. Moreover, Tiam1 promotes IRSp53 localization to Rac-induced lamellipodia rather than Cdc42-induced filopodia. Finally, IRSp53 depletion from cells prevents Tiam1-dependent lamellipodia induced by Tiam1 overexpression or platelet-derived growth factor stimulation. These findings indicate that Tiam1 not only activates Rac but also contributes to Rac signaling specificity through binding to IRSp53.