Oncogenic Ras Downregulates Rac Activity, Which Leads to Increased Rho Activity and Epithelial–Mesenchymal Transition

The Netherlands Cancer Institute, Division of Cell Biology, 1066 CX Amsterdam, The Netherlands.
The Journal of Cell Biology (Impact Factor: 9.83). 06/2000; 149(4):775-82.
Source: PubMed


Proteins of the Rho family regulate cytoskeletal rearrangements in response to receptor stimulation and are involved in the establishment and maintenance of epithelial cell morphology. We recently showed that Rac is able to downregulate Rho activity and that the reciprocal balance between Rac and Rho activity is a major determinant of cellular morphology and motility in NIH3T3 fibroblasts. Using biochemical pull-down assays, we analyzed the effect of transient and sustained oncogenic Ras signaling on the activation state of Rac and Rho in epithelial MDCK cells. In contrast to the activation of Rac by growth factor-induced Ras signaling, we found that sustained signaling by oncogenic RasV12 permanently downregulates Rac activity, which leads to upregulation of Rho activity and epithelial-mesenchymal transition. Oncogenic Ras decreases Rac activity through sustained Raf/MAP kinase signaling, which causes transcriptional downregulation of Tiam1, an activator of Rac in epithelial cells. Reconstitution of Rac activity by expression of Tiam1 or RacV12 leads to downregulation of Rho activity and restores an epithelial phenotype in mesenchymal RasV12- or RafCAAX-transformed cells. The present data reveal a novel mechanism by which oncogenic Ras is able to interfere with the balance between Rac and Rho activity to achieve morphological transformation of epithelial cells.

Download full-text


Available from: Gerben C M Zondag, Oct 01, 2015
11 Reads
  • Source
    • "Ras along with rac1 or rho has been implicated in tumourigenesis [35]. Point mutations or overexpression can make these GTPases constitutively active [36]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Success of imatinib in chronic myeloid leukemia (CML) therapy has undoubtedly proved utility of signalling molecules as therapeutic targets. However, development of imatinib resistance and progression to blastic crisis are the current challenges in clinics. To develop therapeutic alternatives for CML, understanding of signalling events downstream of bcr-abl might be helpful. Current CML cell lines do not give comprehensive picture of signalling events involved in pathogenesis of CML. Hence, there is a major unmet need for a better preclinical model for CML. Here, we report on development of RIN9815/bcr-abl, a novel cell line model that mimics signalling events in CML PMNL. Studies on crucial signalling molecules i.e., ras, rac, rhoA and actin in this cell line identified rhoA as the key regulator involved in CML cell function as well as proliferation of both, imatinib sensitive and resistant cells. Hence, RIN9815/bcr-abl could serve as the unique preclinical model in understanding pathogenesis of CML and in drug development. Copyright © 2015 Elsevier Masson SAS. All rights reserved.
    Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 06/2015; 73. DOI:10.1016/j.biopha.2015.06.004 · 2.02 Impact Factor
  • Source
    • "This indicates that RhoA may be directly involved in renal tubular epithelial EMT. Furthermore, in contrast to the putative roles of Rac1 and Cdc42, which are believed to be involved in the establishment and maintenance of epithelial intercellular adhesions [35]–[37], activation of these proteins can also induce EMT accompanied by breakdown of cell-cell adhesion and rearrangement of the actin cytoskeleton [38]–[40]. Similar to these cells, EMT-like changes caused by small GTPases can occur in PMCs. A recent study by Zhang et al. [20] found that activation of RhoA in rat PMCs by TGF-β1 up-regulated α-SMA, vimentin, and collagen expression and down-regulated E-cadherin expression, suggesting that the RhoA/ROCK signaling pathway mediated EMT in rat PMCs in response to TGF-β1. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background Statins have recently been highlighted for their pleiotropic actions distinct from cholesterol-lowering effects. Despite this interest, it is currently unknown whether statin therapy inhibits peritoneal dialysis (PD)-related epithelial-mesenchymal transition (EMT). Methods In vitro, human peritoneal mesothelial cells (HPMCs) were exposed to 5.6 mM glucose (NG) or 100 mM glucose (HG) with or without simvastatin (1 µM). In vivo, PD catheters were inserted into 32 Sprague-Dawley rats, and saline (C, n = 16) or 4.25% peritoneal dialysis fluid (PDF) (PD, n = 16) was infused for 4 weeks. Eight rats from each group were treated with 5 mg/kg/day of simvastatin intraperitoneally. Changes in the protein expression of EMT markers such as E-cadherin, α-SMA, Snail, and fibronectin in HPMCs and the peritoneum were evaluated by Western blot analysis and immunofluorescence or immunohistochemical staining. We also explored whether activation of the mevalonate pathway and its downstream small GTPases were involved in dialysis-related peritoneal EMT and could be inhibited by statin treatment. Results Compared to NG cells, E-cadherin expression was significantly decreased, while α-SMA, Snail, and fibronectin expression were significantly increased in HPMCs exposed to HG, and these changes were abrogated by simvastatin (p<0.05). In addition, the cobblestone-like appearance of normal HPMCs was converted into a fibroblast-like morphology after HG treatment, which was reversed by simvastatin. These EMT-like changes were also observed in HPMCs treated with geranyl-geranyl pyrophosphate (5 µM). HG significantly increased the protein expression of RhoA and Rac1 in the membrane fractions, and these increases were ameliorated by simvastatin (p<0.05). In PD rats, E-cadherin in the peritoneum was significantly decreased, whereas α-SMA, Snail, and fibronectin expression were significantly increased (p<0.05) compared to C rats. The thickness of the mesothelial layer in the peritoneum were also significantly greater in PD rats than in C rats (p<0.05). These changes of the peritoneum in PD rats were significantly attenuated by simvastatin. Conclusion This study demonstrated that PD-related EMT was mediated via the mevalonate pathway, and statin treatment inhibited the EMT changes in HG-treated HPMCs and PDF-stimulated PD rats. These findings suggest that statins may be a promising therapeutic strategy for preservation of peritoneal membrane integrity in long-term PD patients.
    PLoS ONE 10/2014; 9(10):e109628. DOI:10.1371/journal.pone.0109628 · 3.23 Impact Factor
  • Source
    • "IQGAP1 may be involved in mediating the strength of cell–cell adhesions on the effect of Cdk5 on Rac. Rac1 effector IQGAP1 negatively regulates E-cadherin-mediated cell adhesion by interacting with β-catenin [9,32,40-44]. This is supported by the findings that Rac1 activity decreases after E-cadherin-mediated adhesion in the Cdk5-deficient adherent confluent epithelium. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Although cyclin-dependent kinase 5 (Cdk5) inhibits the formation of junctions containing N-cadherin, the effect of Cdk5 on junctions containing E-cadherin is less clear. The present study investigates the functional significance of Cdk5 in forming and maintaining cell-cell stability in corneal epithelial cells. A Cdk5-deficient human corneal limbal epithelial cell line was generated by lentiviral transduction of small hairpin RNA specific for Cdk5 (shCdk5-HCLE cells). A blasticidin-inducible vector for expression of Cdk5-specific short hairpin RNA (ShCdk5) was generated by recombination and packaged into non-replicative lentiviral particles for transduction of human corneal limbal epithelial (HCLE) cells. Blasticidin-resistant cells were isolated for analysis. Cell aggregations were performed using HCLE, Cdk5 inhibitor olomoucine, ShCdk5, and MDA-MB 231 cells in the presence and absence of calcium, and particle size was measured using image analysis software. Relative protein concentrations were measured with immunoblotting and quantitative densitometry. Total internal reflection fluorescence (TIRF) microscopy was performed on cells transfected with green fluorescent protein (GFP)-E-cadherin or GFP-p120, and internalization of boundary-localized proteins was analyzed with particle tracking software. The stability of surface-exposed proteins was determined by measuring the recovery of biotin-labeled proteins with affinity chromatography. Rho and Rac activity was measured with affinity chromatography and immunoblotting. Examining the effect of Cdk5 on E-cadherin containing epithelial cell-cell adhesions using a corneal epithelial cell line (HCLE), we found that Cdk5 and Cdk5 (pY15) coimmunoprecipitate with E-cadherin and Cdk5 (pY15) colocalizes with E-cadherin at cell-cell junctions. Inhibiting Cdk5 activity in HCLE or suppressing Cdk5 expression in a stable HCLE-derived cell line (ShHCLE) decreased calcium-dependent cell adhesion, promoted the cytoplasmic localization of E-cadherin, and accelerated the loss of surface-biotinylated E-cadherin. TIRF microscopy of GFP-E-cadherin in transfected HCLE cells showed an actively internalized sub-population of E-cadherin, which was not bound to p120 as it was trafficked away from the cell-cell boundary. This population increased in the absence of Cdk5 activity, suggesting that Cdk5 inhibition promotes dissociation of p120/E-cadherin junctional complexes. These effects of Cdk5 inhibition or suppression were accompanied by decreased Rac activity, increased Rho activity, and enhanced binding of E-cadherin to the Rac effector Ras GTPase-activating-like protein (IQGAP1). Cdk5 inhibition also reduced adhesion in a cadherin-deficient cell line (MDA-MB-231) expressing exogenous E-cadherin, although Cdk5 inhibition promoted adhesion when these cells were transfected with N-cadherin, as previous studies of Cdk5 and N-cadherin predicted. Moreover, Cdk5 inhibition induced N-cadherin expression and formation of N-cadherin/p120 complexes in HCLE cells. These results indicate that loss of Cdk5 activity destabilizes junctional complexes containing E-cadherin, leading to internalization of E-cadherin and upregulation of N-cadherin. Thus, Cdk5 activity promotes stability of E-cadherin-based cell-cell junctions and inhibits the E-cadherin-to-N-cadherin switch typical of epithelial-mesenchymal transitions.
    Molecular vision 02/2013; 19:319-32. · 1.99 Impact Factor
Show more