Rho family GTPases regulate mammary epithelium cell growth and metastasis through distinguishable pathways

Boumediene Bouzahzah, Chris Albanese, Fayyaz Ahmed, Fiona Pixley, Michael P. Lisanti, Jeffrey E. Segall, John S. Condeelis, David Joyce, Audrey Minden, Channing J. Der, Amanda Chan, Marc Symons, Richard G. Pestell

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Abstract

Background: Relatively few genes have been shown to directly affect the metastatic phenotype of breast cancer epithelial cells in vivo. The Rho family of proteins, including the Rho, Rac and Cdc42 subfamilies, are related to the small GTP binding protein Ras and regulate diverse biological processes including gene transcription, cytoskeletal organization, cell proliferation and transformation. The effects of Cdc42, Rac and Rho on the actin cytoskeleton suggested a possible role for Rho proteins in cellular motility and metastasis, however a formal analysis of the role of Rho proteins in breast cancer cellular growth and metastasis in vivo had not previously been performed. Materials and Methods: We generated a panel of MTLn3 rat mammary adenocarcinoma cells that expressed similar levels of dominant inhibitory mutants of Cdc42-, Rac- and Rho-dependent signaling, to examine the contribution of these GTPases to cell spreading, guided chemotaxis, and metastasis in vivo. The ability of Rho proteins to regulate intravasation into the peripheral blood was determined by implanting MTLn3 cell stable dominant negative lines in nude mice and measuring the formation of breast cancer cell colonies grown from the peripheral blood. Serial sectioning of the lungs was performed to determine the presence of metastasis in mice in which mammary tumors expressing the dominant negative Rho family proteins had grown to a similar size. Results: Cell spreading of MTLn3 cells was selectively abrogated by N17Racl. N19RhoA and N17Cdc42 reduced the number of focal contacts (FCs) and disrupted the colocalization of vinculin with phosphotyrosine at FCs. While N17Racl and N17Cdc42 preferentially inhibited colony formation in soft agar, all three GTPases affected cell growth in vivo. To distinguish effects on tumorigenicity from intravasation into the bloodstream, implanted tumors were grown to the same size in nude mice. Each dominant inhibitory Rho protein reduced intravasation into the peripheral blood. Lung metastasis of MTLn3 cells was also abrogated by the dominant inhibitory Rho proteins, despite the presence of residual CFU. Conclusions: These studies demonstrate for the first time a critical role for the Rho GTPases involving independent signaling pathways to limit mammary tumor cellular growth and metastasis in vivo.

Original languageEnglish (US)
Pages (from-to)816-830
Number of pages15
JournalMolecular Medicine
Volume7
Issue number12
StatePublished - 2001

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rho GTP-Binding Proteins
Breast
Epithelium
Neoplasm Metastasis
Growth
Breast Neoplasms
Proteins
Focal Adhesions
GTP Phosphohydrolases
Nude Mice
Vinculin
Biological Phenomena
Lung
Phosphotyrosine
Aptitude
Chemotaxis
Actin Cytoskeleton
GTP-Binding Proteins
Genes
Agar

ASJC Scopus subject areas

  • Genetics

Cite this

Bouzahzah, B., Albanese, C., Ahmed, F., Pixley, F., Lisanti, M. P., Segall, J. E., ... Pestell, R. G. (2001). Rho family GTPases regulate mammary epithelium cell growth and metastasis through distinguishable pathways. Molecular Medicine, 7(12), 816-830.

Rho family GTPases regulate mammary epithelium cell growth and metastasis through distinguishable pathways. / Bouzahzah, Boumediene; Albanese, Chris; Ahmed, Fayyaz; Pixley, Fiona; Lisanti, Michael P.; Segall, Jeffrey E.; Condeelis, John S.; Joyce, David; Minden, Audrey; Der, Channing J.; Chan, Amanda; Symons, Marc; Pestell, Richard G.

In: Molecular Medicine, Vol. 7, No. 12, 2001, p. 816-830.

Research output: Contribution to journalArticle

Bouzahzah, B, Albanese, C, Ahmed, F, Pixley, F, Lisanti, MP, Segall, JE, Condeelis, JS, Joyce, D, Minden, A, Der, CJ, Chan, A, Symons, M & Pestell, RG 2001, 'Rho family GTPases regulate mammary epithelium cell growth and metastasis through distinguishable pathways', Molecular Medicine, vol. 7, no. 12, pp. 816-830.
Bouzahzah, Boumediene ; Albanese, Chris ; Ahmed, Fayyaz ; Pixley, Fiona ; Lisanti, Michael P. ; Segall, Jeffrey E. ; Condeelis, John S. ; Joyce, David ; Minden, Audrey ; Der, Channing J. ; Chan, Amanda ; Symons, Marc ; Pestell, Richard G. / Rho family GTPases regulate mammary epithelium cell growth and metastasis through distinguishable pathways. In: Molecular Medicine. 2001 ; Vol. 7, No. 12. pp. 816-830.
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abstract = "Background: Relatively few genes have been shown to directly affect the metastatic phenotype of breast cancer epithelial cells in vivo. The Rho family of proteins, including the Rho, Rac and Cdc42 subfamilies, are related to the small GTP binding protein Ras and regulate diverse biological processes including gene transcription, cytoskeletal organization, cell proliferation and transformation. The effects of Cdc42, Rac and Rho on the actin cytoskeleton suggested a possible role for Rho proteins in cellular motility and metastasis, however a formal analysis of the role of Rho proteins in breast cancer cellular growth and metastasis in vivo had not previously been performed. Materials and Methods: We generated a panel of MTLn3 rat mammary adenocarcinoma cells that expressed similar levels of dominant inhibitory mutants of Cdc42-, Rac- and Rho-dependent signaling, to examine the contribution of these GTPases to cell spreading, guided chemotaxis, and metastasis in vivo. The ability of Rho proteins to regulate intravasation into the peripheral blood was determined by implanting MTLn3 cell stable dominant negative lines in nude mice and measuring the formation of breast cancer cell colonies grown from the peripheral blood. Serial sectioning of the lungs was performed to determine the presence of metastasis in mice in which mammary tumors expressing the dominant negative Rho family proteins had grown to a similar size. Results: Cell spreading of MTLn3 cells was selectively abrogated by N17Racl. N19RhoA and N17Cdc42 reduced the number of focal contacts (FCs) and disrupted the colocalization of vinculin with phosphotyrosine at FCs. While N17Racl and N17Cdc42 preferentially inhibited colony formation in soft agar, all three GTPases affected cell growth in vivo. To distinguish effects on tumorigenicity from intravasation into the bloodstream, implanted tumors were grown to the same size in nude mice. Each dominant inhibitory Rho protein reduced intravasation into the peripheral blood. Lung metastasis of MTLn3 cells was also abrogated by the dominant inhibitory Rho proteins, despite the presence of residual CFU. Conclusions: These studies demonstrate for the first time a critical role for the Rho GTPases involving independent signaling pathways to limit mammary tumor cellular growth and metastasis in vivo.",
author = "Boumediene Bouzahzah and Chris Albanese and Fayyaz Ahmed and Fiona Pixley and Lisanti, {Michael P.} and Segall, {Jeffrey E.} and Condeelis, {John S.} and David Joyce and Audrey Minden and Der, {Channing J.} and Amanda Chan and Marc Symons and Pestell, {Richard G.}",
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T1 - Rho family GTPases regulate mammary epithelium cell growth and metastasis through distinguishable pathways

AU - Bouzahzah, Boumediene

AU - Albanese, Chris

AU - Ahmed, Fayyaz

AU - Pixley, Fiona

AU - Lisanti, Michael P.

AU - Segall, Jeffrey E.

AU - Condeelis, John S.

AU - Joyce, David

AU - Minden, Audrey

AU - Der, Channing J.

AU - Chan, Amanda

AU - Symons, Marc

AU - Pestell, Richard G.

PY - 2001

Y1 - 2001

N2 - Background: Relatively few genes have been shown to directly affect the metastatic phenotype of breast cancer epithelial cells in vivo. The Rho family of proteins, including the Rho, Rac and Cdc42 subfamilies, are related to the small GTP binding protein Ras and regulate diverse biological processes including gene transcription, cytoskeletal organization, cell proliferation and transformation. The effects of Cdc42, Rac and Rho on the actin cytoskeleton suggested a possible role for Rho proteins in cellular motility and metastasis, however a formal analysis of the role of Rho proteins in breast cancer cellular growth and metastasis in vivo had not previously been performed. Materials and Methods: We generated a panel of MTLn3 rat mammary adenocarcinoma cells that expressed similar levels of dominant inhibitory mutants of Cdc42-, Rac- and Rho-dependent signaling, to examine the contribution of these GTPases to cell spreading, guided chemotaxis, and metastasis in vivo. The ability of Rho proteins to regulate intravasation into the peripheral blood was determined by implanting MTLn3 cell stable dominant negative lines in nude mice and measuring the formation of breast cancer cell colonies grown from the peripheral blood. Serial sectioning of the lungs was performed to determine the presence of metastasis in mice in which mammary tumors expressing the dominant negative Rho family proteins had grown to a similar size. Results: Cell spreading of MTLn3 cells was selectively abrogated by N17Racl. N19RhoA and N17Cdc42 reduced the number of focal contacts (FCs) and disrupted the colocalization of vinculin with phosphotyrosine at FCs. While N17Racl and N17Cdc42 preferentially inhibited colony formation in soft agar, all three GTPases affected cell growth in vivo. To distinguish effects on tumorigenicity from intravasation into the bloodstream, implanted tumors were grown to the same size in nude mice. Each dominant inhibitory Rho protein reduced intravasation into the peripheral blood. Lung metastasis of MTLn3 cells was also abrogated by the dominant inhibitory Rho proteins, despite the presence of residual CFU. Conclusions: These studies demonstrate for the first time a critical role for the Rho GTPases involving independent signaling pathways to limit mammary tumor cellular growth and metastasis in vivo.

AB - Background: Relatively few genes have been shown to directly affect the metastatic phenotype of breast cancer epithelial cells in vivo. The Rho family of proteins, including the Rho, Rac and Cdc42 subfamilies, are related to the small GTP binding protein Ras and regulate diverse biological processes including gene transcription, cytoskeletal organization, cell proliferation and transformation. The effects of Cdc42, Rac and Rho on the actin cytoskeleton suggested a possible role for Rho proteins in cellular motility and metastasis, however a formal analysis of the role of Rho proteins in breast cancer cellular growth and metastasis in vivo had not previously been performed. Materials and Methods: We generated a panel of MTLn3 rat mammary adenocarcinoma cells that expressed similar levels of dominant inhibitory mutants of Cdc42-, Rac- and Rho-dependent signaling, to examine the contribution of these GTPases to cell spreading, guided chemotaxis, and metastasis in vivo. The ability of Rho proteins to regulate intravasation into the peripheral blood was determined by implanting MTLn3 cell stable dominant negative lines in nude mice and measuring the formation of breast cancer cell colonies grown from the peripheral blood. Serial sectioning of the lungs was performed to determine the presence of metastasis in mice in which mammary tumors expressing the dominant negative Rho family proteins had grown to a similar size. Results: Cell spreading of MTLn3 cells was selectively abrogated by N17Racl. N19RhoA and N17Cdc42 reduced the number of focal contacts (FCs) and disrupted the colocalization of vinculin with phosphotyrosine at FCs. While N17Racl and N17Cdc42 preferentially inhibited colony formation in soft agar, all three GTPases affected cell growth in vivo. To distinguish effects on tumorigenicity from intravasation into the bloodstream, implanted tumors were grown to the same size in nude mice. Each dominant inhibitory Rho protein reduced intravasation into the peripheral blood. Lung metastasis of MTLn3 cells was also abrogated by the dominant inhibitory Rho proteins, despite the presence of residual CFU. Conclusions: These studies demonstrate for the first time a critical role for the Rho GTPases involving independent signaling pathways to limit mammary tumor cellular growth and metastasis in vivo.

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