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Journal ArticleDOI

Positive regulation of migration and invasion by vasodilator-stimulated phosphoprotein via Rac1 pathway in human breast cancer cells

01 Jan 1994-Oncology Reports (Oncol Rep)-Vol. 20, Iss: 4, pp 929-939

TL;DR: The data showed that the higher expression level of VASP contributed to a higher invasive migration capacity of human breast cancer cells which was controlled by the Rac1 pathway.

AbstractThis study aimed to investigate the role of the cytoskeleton-associated protein vasodilator-stimulated phosphoprotein (VASP) on the migration and invasion of human breast cancer cells and its relationship to Rac1 which is a member of the Rho family and has been found to be implicated in tumorigenesis, invasion and metastasis. We detected the mRNA and protein expression levels of VASP and Rac1 of the non-invasive breast cancer cell line MCF-7 as well as the invasive cell line MDA-MB-231 by RT-PCR and Western blotting. GST pull-down assay was used to examine the activity of Rac1. Accordingly, the cell invasive migration ability was analyzed in a wound-healing assay (2D) and transwell assays (3D migration and invasion). We then used VASP-siRNA to inhibit the expression of VASP in breast cancer cells in order to study the relationship between the VASP expression level and the invasive migration ability of breast cancer cells. Rac1-siRNA was used to decrease the expression of Rac1, and observe its effect on the VASP expression level together with the migration and invasion ability of MCF-7 and MDA-MB-231 cells. Our results revealed that the invasive breast cancer cell line MDA-MB-231 showed a higher Rac1 activity and VASP expression level compared with the non-invasive MCF-7. Inhibition of Rac1 or VASP by siRNA, respectively, decreased the migration and invasion ability of breast cancer cells and the transfection of Rac1 siRNA-mediated reduction of VASP expression at mRNA and protein levels. Collectively, our data showed that the higher expression level of VASP contributed to a higher invasive migration capacity of human breast cancer cells which was controlled by the Rac1 pathway.

Topics: Vasodilator-stimulated phosphoprotein (58%), Cancer cell (54%), Breast disease (54%), Cancer (53%), Cell migration (53%)

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Journal ArticleDOI
Abstract: Under hypoxia, tumor cells produce a secretion that modulates their microenvironment to facilitate tumor angiogenesis and metastasis. Here, we observed that hypoxic or reoxygenated A431 carcinoma cells exhibited enhanced angiogenic and metastatic potential such as reduced cell-cell and cell-extracellular matrix adhesion, increased invasiveness, and production of a secretion with increased chorioallantoic membrane angiogenic activity. Consistent with these observations, quantitative proteomics revealed that under hypoxia the tumor cells secreted proteins involved in angiogenesis, focal adhesion, extracellular matrix-receptor interaction, and immune cell recruitment. Unexpectedly, the secreted proteins were predominantly cytoplasmic and membrane proteins. Ultracentrifugation at 100,000 × g precipitated 54% of the secreted proteins and enriched for many exosome-associated proteins such as the tetraspanins and Alix and also proteins with the potential to facilitate angiogenesis and metastasis. Two tetraspanins, CD9 and CD81, co-immunoprecipitated. Together, these data suggested that tumor cells secrete proteins and exosomes with the potential to modulate their microenvironment and facilitate angiogenesis and metastasis.

422 citations


Cites background from "Positive regulation of migration an..."

  • ...Up-regulated proteins include filamins (FLNA and FLNB), actin cross-linkers that anchor membrane proteins to the actin cytoskeleton (33); F11 receptor, an important regulator of tight junction assemblies (34); plectin-1, a cross-linker of actin, microtubules, and intermediate filaments (35); VASP (vasodilator-stimulated phosphoprotein), involved in invasive migration of cancer cells (36); S100A4, a metastasis promoter involved in up-regulation of matrix metalloproteases (MMPs) and down-regulation of tissue inhibitors of matrix metalloprotease (TIMPs) (37); VCL (vinculin), involved in anchoring F-actin to the membrane (38); and LGALS3 (lectin, galactoside-binding, soluble-3), a protein that cross-links Mgat-5 to the cell surface....

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Journal ArticleDOI
TL;DR: The role of GPCR mediated signal transduction and their importance in the regulation of actin remodeling leading to cell migration are reviewed.
Abstract: Cell migration is a fundamental biological process involved in normal physiology. Altered motile phenotypes are however often associated with the development and progression of diseases such as cancer and atherosclerosis. Remodeling of the actin cytoskeleton is required for cell shape changes and is controlled by a broad variety of cellular proteins. Interestingly, several extracellular stimuli can promote actin reorganization and result in enhanced cell migration. Namely, G protein-coupled receptors (GPCRs), which are activated by factors ranging from small amines, to hormones, and chemokines, initiate signalling cascades resulting in cell shape changes, formation of a migrating front (leading edge) and altered adhesion. GPCRs are heptahelical membrane proteins, which classically transmit signal via the activation of heterotrimeric G proteins. Sustained stimulation leads to the activation of G protein-coupled receptor kinases (GRKs) and the recruitment of arrestin proteins, which engage alternative signalling pathways. In this review, we will discuss the role of GPCR mediated signal transduction and review their importance in the regulation of actin remodeling leading to cell migration.

230 citations


Journal ArticleDOI
TL;DR: It is demonstrated that EHop-016 inhibits Rac activity in the MDA-MB-435 metastatic cancer cells that overexpress Rac and exhibits high endogenous Rac activity, and holds promise as a targeted therapeutic agent for the treatment of metastatic cancers with high Rac activity.
Abstract: The Rho GTPase Rac regulates actin cytoskeleton reorganization to form cell surface extensions (lamellipodia) required for cell migration/invasion during cancer metastasis. Rac hyperactivation and overexpression are associated with aggressive cancers; thus, interference of the interaction of Rac with its direct upstream activators, guanine nucleotide exchange factors (GEFs), is a viable strategy for inhibiting Rac activity. We synthesized EHop-016, a novel inhibitor of Rac activity, based on the structure of the established Rac/Rac GEF inhibitor NSC23766. Herein, we demonstrate that EHop-016 inhibits Rac activity in the MDA-MB-435 metastatic cancer cells that overexpress Rac and exhibits high endogenous Rac activity. The IC(50) of 1.1 μM for Rac inhibition by EHop-016 is ∼100-fold lower than for NSC23766. EHop-016 is specific for Rac1 and Rac3 at concentrations of ≤5 μM. At higher concentrations, EHop-016 inhibits the close homolog Cdc42. In MDA-MB-435 cells that demonstrate high active levels of the Rac GEF Vav2, EHop-016 inhibits the association of Vav2 with a nucleotide-free Rac1(G15A), which has a high affinity for activated GEFs. EHop-016 also inhibits the Rac activity of MDA-MB-231 metastatic breast cancer cells and reduces Rac-directed lamellipodia formation in both cell lines. EHop-016 decreases Rac downstream effects of PAK1 (p21-activated kinase 1) activity and directed migration of metastatic cancer cells. Moreover, at effective concentrations (<5 μM), EHop-016 does not affect the viability of transformed mammary epithelial cells (MCF-10A) and reduces viability of MDA-MB-435 cells by only 20%. Therefore, EHop-016 holds promise as a targeted therapeutic agent for the treatment of metastatic cancers with high Rac activity.

166 citations


Journal ArticleDOI
TL;DR: This review of recent literature focuses on aspects of cell biology related to motility and metastasis, and suggests some directions for future breast cancer research.
Abstract: Cell motility is a critical step in cancer invasion and metastasis that must be unravelled to gain an appropriate understanding of the behaviour of cancer cells. A broad spectrum of motility mechanisms that facilitate invasion of extramammary tissues and metastasis exists in breast cancer cells (e.g. reorganization of the actin cytoskeleton, regulation of focal adhesion, changes in response to a different microenvironment, epithelial mesenchymal transition, and control of membrane proteins through endocytosis). These cellular responses are tightly regulated by intracellular signalling pathways evoked by humoral factors that include growth factors, chemokines, and cytokines. Learning more about the cellular and molecular basis of these different motility programmes will aid in the development of treatments for breast cancer invasion and metastasis. This review of recent literature focuses on aspects of cell biology related to motility and metastasis, and suggests some directions for future breast cancer research.

145 citations


Journal ArticleDOI
Jun Du1, Rui Xu1, Zhenzhen Hu1, Yinhui Tian1, Yichao Zhu1, Luo Gu1, Lei Zhou1 
27 Sep 2011-PLOS ONE
TL;DR: It is demonstrated that hypoxia-induced HIF-1α expression involves a cascade of signaling events including ROS generation, activation of PI3K and ERK signaling, and subsequent activation of Rac1.
Abstract: Background Hypoxia-inducible factor 1 (HIF-1α) expression induced by hypoxia plays a critical role in promoting tumor angiogenesis and metastasis. However, the molecular mechanisms underlying the induction of HIF-1α in tumor cells remain unknown. Methodology/Principal Findings In this study, we reported that hypoxia could induce HIF-1α and VEGF expression accompanied by Rac1 activation in MCF-7 breast cancer cells. Blockade of Rac1 activation with ectopic expression of an inactive mutant form of Rac1 (T17N) or Rac1 siRNA downregulated hypoxia-induced HIF-1α and VEGF expression. Furthermore, Hypoxia increased PI3K and ERK signaling activity. Both PI3K inhibitor LY294002 and ERK inhibitor U0126 suppressed hypoxia-induced Rac1 activation as well as HIF-1α expression. Moreover, hypoxia treatment resulted in a remarkable production of reactive oxygen species (ROS). N-acetyl-L-cysteine, a scavenger of ROS, inhibited hypoxia-induced ROS generation, PI3K, ERK and Rac1 activation as well as HIF-1α expression. Conclusions/Significance Taken together, our study demonstrated that hypoxia-induced HIF-1α expression involves a cascade of signaling events including ROS generation, activation of PI3K and ERK signaling, and subsequent activation of Rac1.

86 citations


Cites background from "Positive regulation of migration an..."

  • ...Rac1 activation promotes angiogenesis of various types of vascular endothelial cells [20,38], and mediates hypoxia-stimulated breast cancer cell migration [39,40]....

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References
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Journal ArticleDOI
24 May 2001-Nature
TL;DR: 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.
Abstract: RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.

10,155 citations


"Positive regulation of migration an..." refers background in this paper

  • ...It has been found that 21 short nucleotide (nt) ds RNA molecules, known as short interfering RNAs (siRNAs), can mediate RNAi in mammalian cell lines (19)....

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Journal ArticleDOI
TL;DR: This work challenges previous assumptions about how the G1/S transition of the mammalian cell cycle is governed, helps explain some enigmatic features of cell cycle control that also involve the functions of the retinoblastoma protein (Rb) and the INK4 proteins, and changes the thinking about how either p16 loss or overexpression of cyclin D-dependent kinases contribute to cancer.
Abstract: Mitogen-dependent progression through the first gap phase (G1) and initiation of DNA synthesis (S phase) during the mammalian cell division cycle are cooperatively regulated by several classes of cyclin-dependent kinases (CDKs) whose activities are in turn constrained by CDK inhibitors (CKIs). CKIs that govern these events have been assigned to one of two families based on their structures and CDK targets. The first class includes the INK4 proteins (inhibitors of CDK4), so named for their ability to specifically inhibit the catalytic subunits of CDK4 and CDK6. Four such proteins [p16 (Serrano et al. 1993), p15 (Hannon and Beach 1994), p18 (Guan et al. 1994; Hirai et al. 1995), and p19 (Chan et al. 1995; Hirai et al. 1995)] are composed of multiple ankyrin repeats and bind only to CDK4 and CDK6 but not to other CDKs or to D-type cyclins. The INK4 proteins can be contrasted with more broadly acting inhibitors of the Cip/Kip family whose actions affect the activities of cyclin D-, E-, and A-dependent kinases. The latter class includes p21 (Gu et al. 1993; Harper et al. 1993; El-Deiry et al. 1993; Xiong et al. 1993a; Dulic et al. 1994; Noda et al. 1994), p27 (Polyak et al. 1994a,b; Toyoshima and Hunter 1994), and p57 (Lee et al. 1995; Matsuoka et al. 1995), all of which contain characteristic motifs within their amino-terminal moieties that enable them to bind both to cyclin and CDK subunits (Chen et al. 1995, 1996; Nakanishi et al. 1995; Warbrick et al. 1995; Lin et al. 1996; Russo et al. 1996). Based largely on in vitro experiments and in vivo overexpression studies, CKIs of the Cip/Kip family were initially thought to interfere with the activities of cyclin D-, E-, and A-dependent kinases. More recent work has altered this view and revealed that although the Cip/Kip proteins are potent inhibitors of cyclin Eand A-dependent CDK2, they act as positive regulators of cyclin Ddependent kinases. This challenges previous assumptions about how the G1/S transition of the mammalian cell cycle is governed, helps explain some enigmatic features of cell cycle control that also involve the functions of the retinoblastoma protein (Rb) and the INK4 proteins, and changes our thinking about how either p16 loss or overexpression of cyclin D-dependent kinases contribute to cancer. Here we focus on the biochemical interactions that occur between CKIs and cyclin Dand E-dependent kinases in cultured mammalian cells, emphasizing the manner by which different positive and negative regulators of the cell division cycle cooperate to govern the G1-to-S transition. To gain a more comprehensive understanding of the biology of CDK inhibitors, readers are encouraged to refer to a rapidly emerging but already extensive literature (for review, see Elledge and Harper 1994; Sherr and Roberts 1995; Chellappan et al. 1998; Hengst and Reed 1998a; Kiyokawa and Koff 1998; Nakayama 1998; Ruas and Peters 1998).

5,881 citations


"Positive regulation of migration an..." refers background in this paper

  • ...As a significant member of the Rho family, Rac1 has been implicated in tumorigenesis (2), tumor angiogenesis (3), invasion and metastasis (4), cell-cycle control and apoptosis (5)....

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Journal ArticleDOI
TL;DR: In this review, functions of small G proteins and their modes of activation and action are described.
Abstract: Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.

2,437 citations


Additional excerpts

  • ...progression and cell-cell adhesion (1)....

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Journal ArticleDOI
07 Oct 1999-Nature
TL;DR: Pure components of the actin cytoskeleton are used to reconstitute sustained movement in Listeria and Shigella in vitro and have implications for the understanding of the mechanism of actin-based motility in cells.
Abstract: Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, alpha-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.

935 citations


"Positive regulation of migration an..." refers background in this paper

  • ...Furthermore, the addition of VASP resulted in an increase in bacterial speed in the in vitro reconstitution assays of Listeria motility (34)....

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  • ...That VASP can affect the cell movements of bacteria, mouse melanoma cells, human platelet, fibroblasts and neurons by regulating cytoskeleton has been detected (14,15,34,35)....

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Journal ArticleDOI
TL;DR: A spatial inventory of the many molecular players in this dynamic domain of the actin cytoskeleton is given in order to highlight the open questions and the challenges ahead.
Abstract: Lamellipodia, filopodia and membrane ruffles are essential for cell motility, the organization of membrane domains, phagocytosis and the development of substrate adhesions. Their formation relies on the regulated recruitment of molecular scaffolds to their tips (to harness and localize actin polymerization), coupled to the coordinated organization of actin filaments into lamella networks and bundled arrays. Their turnover requires further molecular complexes for the disassembly and recycling of lamellipodium components. Here, we give a spatial inventory of the many molecular players in this dynamic domain of the actin cytoskeleton in order to highlight the open questions and the challenges ahead.

907 citations


"Positive regulation of migration an..." refers background in this paper

  • ...During cell migration, activated Rac and Cdc42 induce reorganization of the actin cytoskeleton at the leading edge (6)....

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