Similar Datasets
Project description:SOX4/11 Enhances NGN2 Reprogramming Glioblastoma Cells into Neuron Cells by Modulating RhoA and CDC42/RAC1 Pathway Activity
| PRJNA448602 | ENA
Project description:Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.
| S-EPMC2677995 | biostudies-literature
Project description:Mesenchymal stem cells (MSC) are suggested to be important progenitors of myofibroblasts in fibrosis. To understand the role of Rho GTPase signaling in TGFβ-induced myofibroblast differentiation of MSC, we generated a novel MSC line and its descendants lacking functional Rho GTPases and Rho GTPase signaling components. Unexpectedly, our data revealed that Rho GTPase signaling is required for TGFβ-induced expression of α-smooth muscle actin (αSMA) but not of collagen I α1 (col1a1). Whereas loss of RhoA and Cdc42 reduced αSMA expression, ablation of the Rac1 gene had the opposite effect. Although actin polymerization and MRTFa were crucial for TGFβ-induced αSMA expression, neither Arp2/3-dependent actin polymerization nor cofilin-dependent severing and depolymerization of F-actin were required. Instead, F-actin levels were dependent on cell contraction, and TGFβ-induced actin polymerization correlated with increased cell contraction mediated by RhoA and Cdc42. Finally, we observed impaired collagen I secretion in MSC lacking RhoA or Cdc42. These data give novel molecular insights into the role of Rho GTPases in TGFβ signaling and have implications for our understanding of MSC function in fibrosis.
| S-EPMC6005422 | biostudies-literature
Project description:To identify novel proteins that control melanoma development and progression, we integrated high-throughput proteomics, mRNA and miRNAs datasets from isogenic primary (WM115) and metastatic (WM266-4) melanoma cell-lines. We focused on differentially expressed proteins that had non-differential mRNA levels and were putatively targeted by a high number of miRNAs. SNX18, a sub-family member of sorting-nexins that participates in endocytosis and autophagy was down-regulated by 6-fold in WM266-4, and predicted to be targeted by 17 miRNAs. Over-expression of SNX18 in several melanoma cell-lines significantly reduced migration rate, while silencing of SNX18 resulted in the opposite effect. Notably, progression tissue microarrays demonstrated a trend for SNX18 downregulation in early metastasis. Luciferase assays confirmed the direct regulation of SNX18 by multiple miRNAs. Indeed, over-expression of these miRNAs repressed endogenous SNX18 expression at the protein but not mRNA level. Further, protein but not mRNA expression levels correlated with better overall survival as determined in a cohort of 48 metastatic melanoma patients and in the TCGA database. Mechanistically, SNX18-mediated reduced migration coincided with RhoA activation and Cdc42 inactivation. Both play a role, as RhoA inhibitor and constitutively activated Cdc42 mutants abrogated this effect. Moreover, phosphorylation-defective SNX18 mutants (S233A,S233D) migrated faster, with S233A inactivating RhoA and activating Cdc42, while S233D activated both. The downstream phosphorylation of the p38>MAPKAPK2>HSP27 signaling pathway was concordant. Both mutants were more abundant in the plasma membrane. Taken together, phosphorylation of S233 is crucial for SNX18 activation, but de-phosphorylation dynamics, which may occur at the membrane, is required for functional effect. In this study we compared the proteome, transcriptome and miRnome of primary and metastatic melanoma isogenic cell-lines and identified SNX18 as a novel inhibitor of melanoma migration.
2021-09-15 | GSE120029 | GEO
Project description:To identify novel proteins that control melanoma development and progression, we integrated high-throughput proteomics, mRNA and miRNAs datasets from isogenic primary (WM115) and metastatic (WM266-4) melanoma cell-lines. We focused on differentially expressed proteins that had non-differential mRNA levels and were putatively targeted by a high number of miRNAs. SNX18, a sub-family member of sorting-nexins that participates in endocytosis and autophagy was down-regulated by 6-fold in WM266-4, and predicted to be targeted by 17 miRNAs. Over-expression of SNX18 in several melanoma cell-lines significantly reduced migration rate, while silencing of SNX18 resulted in the opposite effect. Notably, progression tissue microarrays demonstrated a trend for SNX18 downregulation in early metastasis. Luciferase assays confirmed the direct regulation of SNX18 by multiple miRNAs. Indeed, over-expression of these miRNAs repressed endogenous SNX18 expression at the protein but not mRNA level. Further, protein but not mRNA expression levels correlated with better overall survival as determined in a cohort of 48 metastatic melanoma patients and in the TCGA database. Mechanistically, SNX18-mediated reduced migration coincided with RhoA activation and Cdc42 inactivation. Both play a role, as RhoA inhibitor and constitutively activated Cdc42 mutants abrogated this effect. Moreover, phosphorylation-defective SNX18 mutants (S233A,S233D) migrated faster, with S233A inactivating RhoA and activating Cdc42, while S233D activated both. The downstream phosphorylation of the p38>MAPKAPK2>HSP27 signaling pathway was concordant. Both mutants were more abundant in the plasma membrane. Taken together, phosphorylation of S233 is crucial for SNX18 activation, but de-phosphorylation dynamics, which may occur at the membrane, is required for functional effect. miRNA expression profile of primary (WM115) and metastatic (WM266-4) melanoma cell lines.
2021-09-17 | GSE120033 | GEO
Project description:To identify novel proteins that control melanoma development and progression, we integrated high-throughput proteomics, mRNA and miRNAs datasets from isogenic primary (WM115) and metastatic (WM266-4) melanoma cell-lines. We focused on differentially expressed proteins that had non-differential mRNA levels and were putatively targeted by a high number of miRNAs. SNX18, a sub-family member of sorting-nexins that participates in endocytosis and autophagy was down-regulated by 6-fold in WM266-4, and predicted to be targeted by 17 miRNAs. Over-expression of SNX18 in several melanoma cell-lines significantly reduced migration rate, while silencing of SNX18 resulted in the opposite effect. Notably, progression tissue microarrays demonstrated a trend for SNX18 downregulation in early metastasis. Luciferase assays confirmed the direct regulation of SNX18 by multiple miRNAs. Indeed, over-expression of these miRNAs repressed endogenous SNX18 expression at the protein but not mRNA level. Further, protein but not mRNA expression levels correlated with better overall survival as determined in a cohort of 48 metastatic melanoma patients and in the TCGA database. Mechanistically, SNX18-mediated reduced migration coincided with RhoA activation and Cdc42 inactivation. Both play a role, as RhoA inhibitor and constitutively activated Cdc42 mutants abrogated this effect. Moreover, phosphorylation-defective SNX18 mutants (S233A,S233D) migrated faster, with S233A inactivating RhoA and activating Cdc42, while S233D activated both. The downstream phosphorylation of the p38>MAPKAPK2>HSP27 signaling pathway was concordant. Both mutants were more abundant in the plasma membrane. Taken together, phosphorylation of S233 is crucial for SNX18 activation, but de-phosphorylation dynamics, which may occur at the membrane, is required for functional effect. mRNA expression profile of primary (WM115) and metastatic (WM266-4) melanoma cell lines.
2021-09-21 | GSE120295 | GEO
Project description:The three canonical Rho GTPases RhoA, Rac1 and Cdc42 co-ordinate cytoskeletal dynamics. Recent studies indicate that all three Rho GTPases are activated at the leading edge of motile fibroblasts, where their activity fluctuates at subminute time and micrometer length scales. Here, we use a microfluidic chip to acutely manipulate fibroblast edge dynamics by applying pulses of platelet-derived growth factor (PDGF) or the Rho kinase inhibitor Y-27632 (which lowers contractility). This induces acute and robust membrane protrusion and retraction events, that exhibit stereotyped cytoskeletal dynamics, allowing us to fairly compare specific morphodynamic states across experiments. Using a novel Cdc42, as well as previously described, second generation RhoA and Rac1 biosensors, we observe distinct spatio-temporal signaling programs that involve all three Rho GTPases, during protrusion/retraction edge dynamics. Our results suggest that Rac1, Cdc42 and RhoA regulate different cytoskeletal and adhesion processes to fine tune the highly plastic edge protrusion/retraction dynamics that power cell motility.
| S-EPMC4766498 | biostudies-literature
Project description:Human neurons engineered from induced pluripotent stem cells (iPSCs) through neurogenin 2 (NGN2) overexpression are widely used to study neuronal differentiation mechanisms and to model neurological diseases. However, the differentiation paths and heterogeneity of emerged neurons have not been fully explored. Here, we used single-cell transcriptomics to dissect the cell states that emerge during NGN2 overexpression across a time course from pluripotency to neuron functional maturation. We find a substantial molecular heterogeneity in the neuron types generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to NGN2 expression level and the duration of NGN2-forced expression. Our data reveal that NGN2 dosage can regulate neuron fate acquisition, and that NGN2-iN heterogeneity can confound results that are sensitive to neuron type.
| S-EPMC8452516 | biostudies-literature
Project description:Epithelial-to-mesenchymal transition (EMT) is a crucial process for the invasion and metastasis of epithelial tumors. However, the molecular mechanisms underlying this transition are poorly understood. In this study, we demonstrate that interferon regulatory factor 4 binding protein (IBP) regulates EMT and the motility of breast cancer cells through Rac1, RhoA and Cdc42 signaling pathways. We found that increased expression of IBP was associated with the progression of breast cancer and that IBP protein levels were significantly elevated in matched distant metastases. High IBP levels also predict shorter overall survival of breast cancer patients. Furthermore, the forced expression of IBP decreased the expression of the epithelial marker E-cadherin but increased the mesenchymal markers in breast cancer cells. In contrast, silencing IBP in metastatic breast tumor cells promoted a shift toward an epithelial morphology concomitant with increased expression of E-cadherin and decreased expression of mesenchymal markers. IBP silencing also reduced the expression of EMT-inducing transcription factors (Snail, Slug, ZEB1 and ZEB2). Moreover, we identified a role for IBP in endogenous EMT induced by epidermal growth factor (EGF) and deletion of IBP attenuated EGF receptor (EGFR) signaling in breast cancer cells. Furthermore, IBP regulates the migration, invasion and matrix metalloprotease production in breast cancer cells as well as actin cytoskeleton rearrangement and the activation of GTP-Rac1, GTP-RhoA and GTP-Cdc42. Taken together, our findings demonstrate an oncogenic property for IBP in promoting the metastatic potential of breast cancer cells.
| S-EPMC4078416 | biostudies-literature
Project description:G-protein-coupled receptor kinase interacting protein 1 (GIT1) is participated in cell movement activation, which is a fundamental process during tissue development and cancer progression. GIT1/PIX forming a functional protein complex that contributes to Rac1/Cdc42 activation, resulting in increasing cell mobility. Although the importance of Rac1/Cdc42 activation is well documented in cancer aggressiveness, the clinical importance of GIT1 remains largely unknown. Here, we investigated the clinical significance of GIT1 expression in non-small-cell lung cancer (NSCLC) and also verified the importance of GIT1-Rac1/Cdc42 axis in stimulating NSCLC cell mobility. The result indicated higher GIT1 expression patients had significantly poorer prognoses in disease-free survival (DFS) and overall survival (OS) compared with lower GIT1 expression patients. Higher GIT1 expression was an independent prognostic factor by multivariate analysis and associated with migration/invasion of NSCLC cells in transwell assay. In vivo studies indicated that GIT1 promotes metastasis of NSCLC cells. Finally, GIT1 was found to stimulate migration/invasion by altering the activity of Rac1/Cdc42 in NSCLC cells. Together, the GIT1 expression is associated with poor prognosis in patients with NSCLC. GIT1 is critical for the invasiveness of NSCLC cells through stimulating the activity of Rac1/Cdc42.
| S-EPMC4742177 | biostudies-literature