Neuropeptide Y/Y5 Receptor Pathway Stimulates Neuroblastoma Cell Motility Through RhoA Activation

Neuropeptide Y (NPY) has been implicated in the regulation of cellular motility under various physiological and pathological conditions, including cancer dissemination. Yet, the exact signaling pathways leading to these effects remain unknown. In a pediatric malignancy, neuroblastoma (NB), high NPY release from tumor tissue associates with metastatic disease. Here, we have shown that NPY stimulates NB cell motility and invasiveness and acts as a chemotactic factor for NB cells. We have also identified the Y5 receptor (Y5R) as the main NPY receptor mediating these actions. In NB tissues and cell cultures, Y5R is highly expressed in migratory cells and accumulates in regions of high RhoA activity and dynamic cytoskeleton remodeling. Y5R stimulation activates RhoA and results in Y5R/RhoA-GTP interactions, as shown by pull-down and proximity ligation assays, respectively. This is the first demonstration of the role for the NPY/Y5R axis in RhoA activation and the subsequent cytoskeleton remodeling facilitating cell movement. These findings implicate Y5R as a target in anti-metastatic therapies for NB and other cancers expressing this receptor.

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PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility

The Journal of Cell Biology ◽

10.1083/jcb.200708194 ◽

2008 ◽

Vol 180 (1) ◽

pp. 187-203 ◽

Cited By ~ 141

Author(s):

Yangmi Lim ◽

Ssang-Taek Lim ◽

Alok Tomar ◽

Margaret Gardel ◽

Joie A. Bernard-Trifilo ◽

...

Keyword(s):

Cell Motility ◽

Focal Adhesion ◽

Adhesion Formation ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Rhoa Activity ◽

Rhoa Activation ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Cellular Contact

Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK−/− or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK–p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility.

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Actinin-4, a Novel Actin-bundling Protein Associated with Cell Motility and Cancer Invasion

The Journal of Cell Biology ◽

10.1083/jcb.140.6.1383 ◽

1998 ◽

Vol 140 (6) ◽

pp. 1383-1393 ◽

Cited By ~ 308

Author(s):

Kazufumi Honda ◽

Tesshi Yamada ◽

Ritsuko Endo ◽

Yoshinori Ino ◽

Masahiro Gotoh ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Cell Motility ◽

Cell Movement ◽

Metastatic Potential ◽

Cancer Invasion ◽

Cytoplasmic Localization ◽

Cellular Motility ◽

Phosphatidylinositol 3 Kinase ◽

Antigenic Protein ◽

Histological Phenotype

Regulation of the actin cytoskeleton may play a crucial role in cell motility and cancer invasion. We have produced a monoclonal antibody (NCC- Lu-632, IgM, k) reactive with an antigenic protein that is upregulated upon enhanced cell movement. The cDNA for the antigen molecule was found to encode a novel isoform of nonmuscle α-actinin. This isoform (designated actinin-4) was concentrated in the cytoplasm where cells were sharply extended and in cells migrating and located at the edge of cell clusters, but was absent from focal adhesion plaques or adherens junctions, where the classic isoform (actinin-1) was concentrated. Actinin-4 shifted steadily from the cytoplasm to the nucleus upon inhibition of phosphatidylinositol 3 kinase or actin depolymerization. The cytoplasmic localization of actinin-4 was closely associated with an infiltrative histological phenotype and correlated significantly with a poorer prognosis in 61 cases of breast cancer. These findings suggest that cytoplasmic actinin-4 regulates the actin cytoskeleton and increases cellular motility and that its inactivation by transfer to the nucleus abolishes the metastatic potential of human cancers.

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Aggregation of Burkitt Lymphoma Cells in Stationary Culture: Experimental and Theoretical Analysis

Journal of Cell Science ◽

10.1242/jcs.10.3.749 ◽

1972 ◽

Vol 10 (3) ◽

pp. 749-758

Author(s):

E. MAYHEW ◽

L. E. BLUMENSON

Keyword(s):

Computer Simulation ◽

Cell Motility ◽

Burkitt Lymphoma ◽

Cell Movement ◽

Experimental System ◽

Cell Aggregation ◽

Cellular Motility ◽

Final Size ◽

Lymphoma Cells ◽

Aggregation Behaviour

Cellular motility plays an important role in natural aggregative phenomena but previously has been difficult to quantitate. We here describe a general method which can be used to determine the importance of cell motility in cell aggregation behaviour. When actively moving Burkitt lymphoma cells cultured in microtest plate wells come into contact they adhere to one another forming an aggregate. The aggregate increases in size when more cells come in contact with it. The final size and number of aggregates per well was found to be dependent on the number of cells added per well. With increasing cell numbers added per well the number of aggregates formed increased until it reached a peak of 47-57 aggregates per well 20 h after the addition of 180-710 cells per well. At higher cell concentrations the number of aggregates formed decreased. The system was analysed theoretically by programming a computer to simulate the experimental system. This simulation showed it was probable that the experimental results obtained were due to (a) random dropping of the cells at zero time and (b) the adhesion of the cells when they made contact and (c) unrestricted random movement of cells in the well when they reached the well surface. The computer simulation is such that given the experimentally determined cell concentration per well and rate of cell movement we can predict the number of aggregates formed for different probabilities that the adhesions formed after the cells come in contact are permanent. This experimental approach along with the computer simulation can be used to quantitate the role of cell motility and permanence of contact in cell aggregation.

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2D or 3D? How in vitro cell motility is conserved across dimensions, and predicts in vivo invasion

10.1101/627281 ◽

2019 ◽

Author(s):

Sualyneth Galarza ◽

Hyuna Kim ◽

Naciye Atay ◽

Shelly R Peyton ◽

Jennifer M Munson

Keyword(s):

Cell Migration ◽

Cell Motility ◽

Cell Movement ◽

Complex Model ◽

Model Systems ◽

Cellular Motility ◽

Statistical Tool ◽

3D Environments

AbstractCell motility is a critical aspect of wound healing, the immune response, and is deregulated in cancer. Current limitations in imaging tools make it difficult to study cell migration in vivo. To overcome this, and to identify drivers from the microenvironment that regulate cell migration, bioengineers have developed 2D and 3D tissue model systems in which to study cell motility in vitro, with the aim of mimicking the environments in which cells move in vivo. However, there has been no systematic study to explicitly relate and compare cell motility measurements between these geometries/systems. Here, we provide such analysis on our own data, as well as across data in existing literature to understand whether, and which, in vitro models are predictive of in vivo cell motility. To our surprise, many metrics of cell movement on 2D surfaces significantly and positively correlate with cell migration in 3D environments, and cell invasion in 3D is negatively correlated with glioblastoma invasion in vivo. Finally, to best compare across complex model systems, in vivo data, and data from different labs, we suggest that groups report an effect size, a statistical tool that is most translatable across experiments and labs, when conducting experiments that affect cellular motility.

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Y2 receptors for neuropeptide Y are coupled to three intracellular signal transduction pathways in a human neuroblastoma cell line.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)37045-x ◽

1994 ◽

Vol 269 (12) ◽

pp. 8842-8848

Author(s):

Y. Shigeri ◽

M. Fujimoto

Keyword(s):

Signal Transduction ◽

Neuropeptide Y ◽

Cell Line ◽

Neuroblastoma Cell ◽

Neuroblastoma Cell Line ◽

Signal Transduction Pathways ◽

Human Neuroblastoma Cell ◽

Human Neuroblastoma ◽

Human Neuroblastoma Cell Line ◽

Intracellular Signal

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Binding of Monoiodinated Neuropeptide Y to Hippocampal Membranes and Human Neuroblastoma Cell Lines

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)83476-7 ◽

1989 ◽

Vol 264 (12) ◽

pp. 6648-6654 ◽

Cited By ~ 6

Author(s):

S P Sheikh ◽

M M O'Hare ◽

O Tortora ◽

T W Schwartz

Keyword(s):

Neuropeptide Y ◽

Cell Lines ◽

Neuroblastoma Cell ◽

Human Neuroblastoma Cell ◽

Human Neuroblastoma ◽

Neuroblastoma Cell Lines

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Calcium-Sensing Receptor Stimulation in Cultured Glomerular Podocytes Induces TRPC6-Dependent Calcium Entry and RhoA Activation

Cellular Physiology and Biochemistry ◽

10.1159/000484064 ◽

2017 ◽

Vol 43 (5) ◽

pp. 1777-1789 ◽

Cited By ~ 7

Author(s):

Lei Zhang ◽

Tianrong Ji ◽

Qin Wang ◽

Kexin Meng ◽

Rui Zhang ◽

...

Keyword(s):

Protective Action ◽

Focal Adhesions ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fibers ◽

Dependent Manner ◽

Calcium Sensing Receptor ◽

Rhoa Activity ◽

Rhoa Activation ◽

Calcium Sensing

Background/Aims: Recent studies provided compelling evidence that stimulation of the calcium sensing receptor (CaSR) exerts direct renoprotective action at the glomerular podocyte level. This protective action may be attributed to the RhoA-dependent stabilization of the actin cytoskeleton. However, the underlying mechanisms remain unclear. Methods: In the present study, an immortalized human podocyte cell line was used. Fluo-3 fluorescence was utilized to determine intracellular Ca2+ concentration ([Ca2+]i), and western blotting was used to measure canonical transient receptor potential 6 (TRPC6) protein expression and RhoA activity. Stress fibers were detected by FITC-phalloidin. Results: Activating CaSR with a high extracellular Ca2+ concentration ([Ca2+]o) or R-568 (a type II CaSR agonist) induces an increase in the [Ca2+]i in a dose-dependent manner. This increase in [Ca2+]i is phospholipase C (PLC)-dependent and is smaller in the absence of extracellular Ca2+ than in the presence of 0.5 mM [Ca2+]o. The CaSR activation-induced [Ca2+]i increase is attenuated by the pharmacological blockage of TRPC6 channels or siRNA targeting TRPC6. These data suggest that TRPC6 is involved in CaSR activation-induced Ca2+ influx. Consistent with a previous study, CaSR stimulation results in an increase in RhoA activity. However, the knockdown of TRPC6 significantly abolished the RhoA activity increase induced by CaSR stimulation, suggesting that TRPC6-dependent Ca2+ entry is required for RhoA activation. The activated RhoA is involved in the formation of stress fibers and focal adhesions in response to CaSR stimulation because siRNA targeting RhoA attenuated the increase in the stress fiber mediated by CaSR stimulation. Moreover, this effect of CaSR activation on the formation of stress fibers is also abolished by the knockdown of TRPC6. Conclusion: TRPC6 is involved in the regulation of stress fiber formation and focal adhesions via the RhoA pathway in response to CaSR activation. This may explain the direct protective action of CaSR agonists.

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Modulation of Cellular Migration and Survival by c-Myc through the Downregulation of Urokinase (uPA) and uPA Receptor

Molecular and Cellular Biology ◽

10.1128/mcb.01442-09 ◽

2010 ◽

Vol 30 (7) ◽

pp. 1838-1851 ◽

Cited By ~ 17

Author(s):

Daniela Alfano ◽

Giuseppina Votta ◽

Almut Schulze ◽

Julian Downward ◽

Mario Caputi ◽

...

Keyword(s):

Cell Motility ◽

Cellular Transformation ◽

Tumor Formation ◽

Cellular Migration ◽

Epithelial Cell Line ◽

Cellular Motility ◽

Upa Receptor ◽

Upar Expression ◽

Repressive Effect ◽

Cell Invasiveness

ABSTRACT It has been proposed that c-Myc proapoptotic activity accounts for most of its restraint of tumor formation. We established a telomerase-immortalized human epithelial cell line expressing an activatable c-Myc protein. We found that c-Myc activation induces, in addition to increased sensitivity to apoptosis, reductions in cell motility and invasiveness. Transcriptome analysis revealed that urokinase (uPA) and uPA receptor (uPAR) were strongly downregulated by c-Myc. Evidence is provided that the repression of uPA and uPAR may account for most of the antimigratory and proapoptotic activities of c-Myc. c-Myc is known to cooperate with Ras in cellular transformation. We therefore investigated if this cooperation could converge in the control of uPA/uPAR expression. We found that Ras is able to block the effects of c-Myc activation on apoptosis and cellular motility but not on cell invasiveness. Accordingly, the activation of c-Myc in the context of Ras expression had only minor influence on uPAR expression but still had a profound repressive effect on uPA expression. Thus, the differential regulation of uPA and uPAR by c-Myc and Ras correlates with the effects of these two oncoproteins on cell motility, invasiveness, and survival. In conclusion, we have discovered a novel link between c-Myc and uPA/uPAR. We propose that reductions of cell motility and invasiveness could contribute to the inhibition of tumorigenesis by c-Myc and that the regulation of uPA and uPAR expression may be a component of the ability of c-Myc to reduce motility and invasiveness.

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