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

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.

[1]  G. Nolan,et al.  Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. , 1996, Human gene therapy.

[2]  T. Reid,et al.  Identification and Characterization of hPEM-2, a Guanine Nucleotide Exchange Factor Specific for Cdc42* , 1999, The Journal of Biological Chemistry.

[3]  John G. Collard,et al.  The Guanine Nucleotide Exchange Factor Tiam1 Affects Neuronal Morphology; Opposing Roles for the Small GTPases Rac and Rho , 1997, The Journal of cell biology.

[4]  M. Kinch,et al.  Rho-stimulated contractility contributes to the fibroblastic phenotype of Ras-transformed epithelial cells. , 1997, Molecular biology of the cell.

[5]  D. Bar-Sagi,et al.  Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins. , 1986, Science.

[6]  John G. Collard,et al.  A role for Rac in Tiaml-induced membrane ruffling and invasion , 1995, Nature.

[7]  John G. Collard,et al.  Inhibition of invasion of epithelial cells by Tiam1-Rac signaling. , 1997, Science.

[8]  W. Kiosses,et al.  Regulation of the small GTP‐binding protein Rho by cell adhesion and the cytoskeleton , 1999, The EMBO journal.

[9]  C. Der,et al.  Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation , 1995, Molecular and cellular biology.

[10]  C. Marshall.  Ras effectors. , 1996, Current opinion in cell biology.

[11]  W. Fiers,et al.  Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role , 1991, Cell.

[12]  G. Bokoch,et al.  Inhibition of myosin light chain kinase by p21-activated kinase. , 1999, Science.

[13]  David A. Cheresh,et al.  Regulation of Cell Motility by Mitogen-activated Protein Kinase , 1997, The Journal of cell biology.

[14]  N. Nomura,et al.  Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherin- mediated cell-cell adhesion. , 1998, Science.

[15]  L. Van Aelst,et al.  Rac Regulates Integrin-Mediated Spreading and Increased Adhesion of T Lymphocytes , 1998, Molecular and Cellular Biology.

[16]  C. Marshall,et al.  Signals from Ras and Rho GTPases interact to regulate expression of p21Waf1/Cip1 , 1998, Nature.

[17]  C. Betsholtz,et al.  EPS8 and E3B1 transduce signals from Ras to Rac , 1999, Nature.

[18]  John G. Collard,et al.  Identification of an invasion-inducing gene, Tiam-1, that encodes a protein with homology to GDP-GTP exchangers for Rho-like proteins , 1994, Cell.

[19]  H. Kotani,et al.  Regulation of Cell–Cell Adhesion by Rac and Rho Small G Proteins in MDCK Cells , 1997, The Journal of cell biology.

[20]  M. Sone,et al.  Identification of the stef Gene That Encodes a Novel Guanine Nucleotide Exchange Factor Specific for Rac1* , 1999, The Journal of Biological Chemistry.

[21]  A. Hall,et al.  The Small GTPases Rho and Rac Are Required for the Establishment of Cadherin-dependent Cell–Cell Contacts , 1997, The Journal of cell biology.

[22]  F. McCormick,et al.  A role for Rho in Ras transformation. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[23]  C. Der,et al.  Tyrosine phosphorylation regulates the adhesions of ras-transformed breast epithelia , 1995, The Journal of cell biology.

[24]  John G. Collard,et al.  Rac Downregulates Rho Activity: Reciprocal Balance between Both Gtpases Determines Cellular Morphology and Migratory Behavior , 1999 .

[25]  C. L. Adams,et al.  Cytomechanics of cadherin-mediated cell-cell adhesion. , 1998, Current opinion in cell biology.

[26]  M. White,et al.  Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. , 1998, Science.

[27]  L. Lim,et al.  Rho family GTPases and neuronal growth cone remodelling: relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid , 1997, Molecular and cellular biology.

[28]  P. Comoglio,et al.  Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells , 1995, Molecular and cellular biology.

[29]  R Kemler,et al.  Cadherins and tissue formation: integrating adhesion and signaling , 1999, BioEssays : news and reviews in molecular, cellular and developmental biology.

[30]  F. McCormick,et al.  An essential role for Rac in Ras transformation , 1995, Nature.

[31]  W. Birchmeier,et al.  Dissecting tumor cell invasion: epithelial cells acquire invasive properties after the loss of uvomorulin-mediated cell-cell adhesion , 1989, The Journal of cell biology.

[32]  G. Nolan,et al.  Expression of Rho GTPases using retroviral vectors. , 2000, Methods in enzymology.

[33]  Anne J. Ridley,et al.  The small GTP-binding protein rac regulates growth factor-induced membrane ruffling , 1992, Cell.

[34]  A. Hall,et al.  Rho GTPases and the actin cytoskeleton. , 1998, Science.

[35]  Frits Michiels,et al.  Matrix-dependent Tiam1/Rac Signaling in Epithelial Cells Promotes Either Cell–Cell Adhesion or Cell Migration and Is Regulated by Phosphatidylinositol 3-Kinase , 1998, The Journal of cell biology.

[36]  John G. Collard,et al.  Rac regulates phosphorylation of the myosin-II heavy chain, actinomyosin disassembly and cell spreading , 1999, Nature Cell Biology.

[37]  D. Bar-Sagi,et al.  Coupling of Ras and Rac guanosine triphosphatases through the Ras exchanger Sos. , 1998, Science.

[38]  C. Der,et al.  Cdc42 and Rac1 induce integrin-mediated cell motility and invasiveness through PI(3)K , 1997, Nature.

[39]  L. Shaw,et al.  Activation of phosphoinositide 3-OH kinase by the alpha6beta4 integrin promotes carcinoma invasion. , 1997, Cell.

[40]  Leslie M Shaw,et al.  Activation of Phosphoinositide 3-OH Kinase by the α6β4 Integrin Promotes Carcinoma Invasion , 1997, Cell.

[41]  P. Warne,et al.  Role of Phosphoinositide 3-OH Kinase in Cell Transformation and Control of the Actin Cytoskeleton by Ras , 1997, Cell.