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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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MyoGEF colocalizes with actin-myosin filaments at the cell leading edge(A) MDA-MB-231 cells were subjected to immunofluorescence with anti-MyoGEF antibody (green) and rhodaminephalloidin (red). (B) Immunoblot analysis of total cell lysates from MDA-MB-231 with anti-MyoGEF antibody. Note that a single band was recognized by MyoGEF antibody in MDA-MB-231 cell lysates. (C) Exogenously expressed GFP-MyoGEF (green) colocalizes with actin filaments (red) in transfected MDA-MB-231 cells. (D) Exogenously expressed GFP-IIA (green) colocalizes with endogenous MyoGEF (red) at the cell leading edge of transfected MDA-MB-231 cells. (E) Exogenously expressed GFP-MyoGEF (green) colocalizes with endogenous NMIIA (red) at the cell leading edge of transfected MDA-MB-231 cells. Bars, 10 μm.
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Figure 4: MyoGEF colocalizes with actin-myosin filaments at the cell leading edge(A) MDA-MB-231 cells were subjected to immunofluorescence with anti-MyoGEF antibody (green) and rhodaminephalloidin (red). (B) Immunoblot analysis of total cell lysates from MDA-MB-231 with anti-MyoGEF antibody. Note that a single band was recognized by MyoGEF antibody in MDA-MB-231 cell lysates. (C) Exogenously expressed GFP-MyoGEF (green) colocalizes with actin filaments (red) in transfected MDA-MB-231 cells. (D) Exogenously expressed GFP-IIA (green) colocalizes with endogenous MyoGEF (red) at the cell leading edge of transfected MDA-MB-231 cells. (E) Exogenously expressed GFP-MyoGEF (green) colocalizes with endogenous NMIIA (red) at the cell leading edge of transfected MDA-MB-231 cells. Bars, 10 μm.

Mentions: To determine the localization of MyoGEF in migrating MDA-MB-231 cells, immunofluorescence with anti-MyoGEF antibody was carried out in fixed MDA-MB-231 cells after ~6 h of culture on fibronectin-coated coverslips. As shown in Figure 4A, endogenous MyoGEF colocalized with actin filaments at the front of migrating cells (arrowheads in panels a-c and a'-c'). Consistent with these observations, exogenously expressed GFP-MyoGEF also colocalized with actin filaments at the cell leading edge (Figure 4C, panels a-c and a'-c'). It should be noted that expression of exogenous GFP-MyoGEF could induce the formation of thick actin bundles (panels b' and c'). In addition, GFP-MyoGEF formed filament-like structures that overlap with these thick actin bundles (arrowheads in panel c'). These results indicate that MyoGEF can localize to the cell leading edge, where it induces actin filament formation.


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)

MyoGEF colocalizes with actin-myosin filaments at the cell leading edge(A) MDA-MB-231 cells were subjected to immunofluorescence with anti-MyoGEF antibody (green) and rhodaminephalloidin (red). (B) Immunoblot analysis of total cell lysates from MDA-MB-231 with anti-MyoGEF antibody. Note that a single band was recognized by MyoGEF antibody in MDA-MB-231 cell lysates. (C) Exogenously expressed GFP-MyoGEF (green) colocalizes with actin filaments (red) in transfected MDA-MB-231 cells. (D) Exogenously expressed GFP-IIA (green) colocalizes with endogenous MyoGEF (red) at the cell leading edge of transfected MDA-MB-231 cells. (E) Exogenously expressed GFP-MyoGEF (green) colocalizes with endogenous NMIIA (red) at the cell leading edge of transfected MDA-MB-231 cells. Bars, 10 μm.
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Figure 4: MyoGEF colocalizes with actin-myosin filaments at the cell leading edge(A) MDA-MB-231 cells were subjected to immunofluorescence with anti-MyoGEF antibody (green) and rhodaminephalloidin (red). (B) Immunoblot analysis of total cell lysates from MDA-MB-231 with anti-MyoGEF antibody. Note that a single band was recognized by MyoGEF antibody in MDA-MB-231 cell lysates. (C) Exogenously expressed GFP-MyoGEF (green) colocalizes with actin filaments (red) in transfected MDA-MB-231 cells. (D) Exogenously expressed GFP-IIA (green) colocalizes with endogenous MyoGEF (red) at the cell leading edge of transfected MDA-MB-231 cells. (E) Exogenously expressed GFP-MyoGEF (green) colocalizes with endogenous NMIIA (red) at the cell leading edge of transfected MDA-MB-231 cells. Bars, 10 μm.
Mentions: To determine the localization of MyoGEF in migrating MDA-MB-231 cells, immunofluorescence with anti-MyoGEF antibody was carried out in fixed MDA-MB-231 cells after ~6 h of culture on fibronectin-coated coverslips. As shown in Figure 4A, endogenous MyoGEF colocalized with actin filaments at the front of migrating cells (arrowheads in panels a-c and a'-c'). Consistent with these observations, exogenously expressed GFP-MyoGEF also colocalized with actin filaments at the cell leading edge (Figure 4C, panels a-c and a'-c'). It should be noted that expression of exogenous GFP-MyoGEF could induce the formation of thick actin bundles (panels b' and c'). In addition, GFP-MyoGEF formed filament-like structures that overlap with these thick actin bundles (arrowheads in panel c'). These results indicate that MyoGEF can localize to the cell leading edge, where it induces actin filament formation.

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