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Myosin-interacting guanine exchange factor (MyoGEF) regulates the invasion activity of MDA-MB-231 breast cancer cells through activation of RhoA and RhoC.

Wu D, Asiedu M, Wei Q - Oncogene (2009)

Bottom Line: RNA-interference (RNAi)-mediated depletion of MyoGEF in MDA-MB-231 cells not only suppresses the activation of RhoA and RhoC, but also decreases cell polarity and invasion activity.The dominant-negative mutants of RhoA and RhoC, but not Rac1 and Cdc42, dramatically decrease actin polymerization induced by MyoGEF.Moreover, MyoGEFprotein levels significantly increase in infiltrating ductal carcinomas as well as in invasive breast cancer cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.

ABSTRACT
The small guanine triphosphatase (GTPase) proteins RhoA and RhoC are essential for tumor invasion and/or metastasis in breast carcinomas. However, it is poorly understood how RhoA and RhoC are activated in breast cancer cells. Here we describe the role of myosin-interacting guanine nucleotide exchange factor (Myo-GEF) in regulating RhoA and RhoC activation as well as cell polarity and invasion in an invasive breast cancer cell line MDA-MB-231. RNA-interference (RNAi)-mediated depletion of MyoGEF in MDA-MB-231 cells not only suppresses the activation of RhoA and RhoC, but also decreases cell polarity and invasion activity. The dominant-negative mutants of RhoA and RhoC, but not Rac1 and Cdc42, dramatically decrease actin polymerization induced by MyoGEF. In addition, MyoGEF co-localizes with nonmuscle myosin IIA (NMIIA) to the front of migrating cells, and depletion of NMIIA by RNAi disrupts the polarized localization of MyoGEF at the cell leading edge, suggesting a role for NMIIA in regulating MyoGEF localization and function. Moreover, MyoGEFprotein levels significantly increase in infiltrating ductal carcinomas as well as in invasive breast cancer cell lines. Taken together, our results suggest that MyoGEF cooperates with NMIIA to regulate the polarity and invasion activity of breast cancer cells through activation of RhoA and RhoC.

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NMIIA is required for polarized localization of MyoGEF as well as the formation of MyoGEF-induced actin bundles(A) MDA-MB-231 cells treated with control siRNA (siCont; panel a) or NMIIA siRNA (siIIA; panel b) were subjected to immunofluorescence with anti-MyoGEF antibody. (B) MDA-MB-231 cells treated with siCont or siIIA were subjected to immunoblot analysis with antibodies specific for NMIIA or β-tubulin. (C-D) A plasmid encoding GFP-MyoGEF was cotransfected into HeLa cells with siCont or siIIA. The transfected cells were subjected to immunofluorescence with anti-IIA antibody (C) or phalloidin (D). Bars, 10 μm.
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Figure 7: NMIIA is required for polarized localization of MyoGEF as well as the formation of MyoGEF-induced actin bundles(A) MDA-MB-231 cells treated with control siRNA (siCont; panel a) or NMIIA siRNA (siIIA; panel b) were subjected to immunofluorescence with anti-MyoGEF antibody. (B) MDA-MB-231 cells treated with siCont or siIIA were subjected to immunoblot analysis with antibodies specific for NMIIA or β-tubulin. (C-D) A plasmid encoding GFP-MyoGEF was cotransfected into HeLa cells with siCont or siIIA. The transfected cells were subjected to immunofluorescence with anti-IIA antibody (C) or phalloidin (D). Bars, 10 μm.

Mentions: NMII plays a central role in regulating cell polarity and motility (Conti and Adelstein, 2008; Lo et al., 2004; Meshel et al., 2005; Vicente-Manzanares et al., 2007). In addition, MyoGEF can bind to NMIIA (Figure 5) (Wu et al., 2006). Further, MyoGEF and NMIIA colocalize to the cell leading edge in MDA-MB-231 cells (Figure 4). Therefore, we used RNAi to deplete NMIIA in MDA-MB-231 cells and then carried out immunofluorescence with anti-MyoGEF antibody to examine the effect of NMIIA depletion on MyoGEF localization. As shown in Figure 7A, depletion of NMIIA by RNAi led to cell spreading indiscriminately and the disruption of polarized MyoGEF localization (arrowheads in Figure 7Ab). In contrast, depletion of NMIIB did not impair cell polarity and the polarized distribution of MyoGEF in MDA-MB-231 cells (data not shown), even though it has been demonstrated that depletion of NMIIB also decreases MDA-MB-231 cell migration (Betapudi et al., 2006). These results indicate that NMIIA is required for polarized localization of MyoGEF during cell migration.


Myosin-interacting guanine exchange factor (MyoGEF) regulates the invasion activity of MDA-MB-231 breast cancer cells through activation of RhoA and RhoC.

Wu D, Asiedu M, Wei Q - Oncogene (2009)

NMIIA is required for polarized localization of MyoGEF as well as the formation of MyoGEF-induced actin bundles(A) MDA-MB-231 cells treated with control siRNA (siCont; panel a) or NMIIA siRNA (siIIA; panel b) were subjected to immunofluorescence with anti-MyoGEF antibody. (B) MDA-MB-231 cells treated with siCont or siIIA were subjected to immunoblot analysis with antibodies specific for NMIIA or β-tubulin. (C-D) A plasmid encoding GFP-MyoGEF was cotransfected into HeLa cells with siCont or siIIA. The transfected cells were subjected to immunofluorescence with anti-IIA antibody (C) or phalloidin (D). Bars, 10 μm.
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Figure 7: NMIIA is required for polarized localization of MyoGEF as well as the formation of MyoGEF-induced actin bundles(A) MDA-MB-231 cells treated with control siRNA (siCont; panel a) or NMIIA siRNA (siIIA; panel b) were subjected to immunofluorescence with anti-MyoGEF antibody. (B) MDA-MB-231 cells treated with siCont or siIIA were subjected to immunoblot analysis with antibodies specific for NMIIA or β-tubulin. (C-D) A plasmid encoding GFP-MyoGEF was cotransfected into HeLa cells with siCont or siIIA. The transfected cells were subjected to immunofluorescence with anti-IIA antibody (C) or phalloidin (D). Bars, 10 μm.
Mentions: NMII plays a central role in regulating cell polarity and motility (Conti and Adelstein, 2008; Lo et al., 2004; Meshel et al., 2005; Vicente-Manzanares et al., 2007). In addition, MyoGEF can bind to NMIIA (Figure 5) (Wu et al., 2006). Further, MyoGEF and NMIIA colocalize to the cell leading edge in MDA-MB-231 cells (Figure 4). Therefore, we used RNAi to deplete NMIIA in MDA-MB-231 cells and then carried out immunofluorescence with anti-MyoGEF antibody to examine the effect of NMIIA depletion on MyoGEF localization. As shown in Figure 7A, depletion of NMIIA by RNAi led to cell spreading indiscriminately and the disruption of polarized MyoGEF localization (arrowheads in Figure 7Ab). In contrast, depletion of NMIIB did not impair cell polarity and the polarized distribution of MyoGEF in MDA-MB-231 cells (data not shown), even though it has been demonstrated that depletion of NMIIB also decreases MDA-MB-231 cell migration (Betapudi et al., 2006). These results indicate that NMIIA is required for polarized localization of MyoGEF during cell migration.

Bottom Line: RNA-interference (RNAi)-mediated depletion of MyoGEF in MDA-MB-231 cells not only suppresses the activation of RhoA and RhoC, but also decreases cell polarity and invasion activity.The dominant-negative mutants of RhoA and RhoC, but not Rac1 and Cdc42, dramatically decrease actin polymerization induced by MyoGEF.Moreover, MyoGEFprotein levels significantly increase in infiltrating ductal carcinomas as well as in invasive breast cancer cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.

ABSTRACT
The small guanine triphosphatase (GTPase) proteins RhoA and RhoC are essential for tumor invasion and/or metastasis in breast carcinomas. However, it is poorly understood how RhoA and RhoC are activated in breast cancer cells. Here we describe the role of myosin-interacting guanine nucleotide exchange factor (Myo-GEF) in regulating RhoA and RhoC activation as well as cell polarity and invasion in an invasive breast cancer cell line MDA-MB-231. RNA-interference (RNAi)-mediated depletion of MyoGEF in MDA-MB-231 cells not only suppresses the activation of RhoA and RhoC, but also decreases cell polarity and invasion activity. The dominant-negative mutants of RhoA and RhoC, but not Rac1 and Cdc42, dramatically decrease actin polymerization induced by MyoGEF. In addition, MyoGEF co-localizes with nonmuscle myosin IIA (NMIIA) to the front of migrating cells, and depletion of NMIIA by RNAi disrupts the polarized localization of MyoGEF at the cell leading edge, suggesting a role for NMIIA in regulating MyoGEF localization and function. Moreover, MyoGEFprotein levels significantly increase in infiltrating ductal carcinomas as well as in invasive breast cancer cell lines. Taken together, our results suggest that MyoGEF cooperates with NMIIA to regulate the polarity and invasion activity of breast cancer cells through activation of RhoA and RhoC.

Show MeSH
Related in: MedlinePlus