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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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Expression of dominant negative mutants N19RhoA and N19RhoC inhibits the formation of MyoGEF-induced actin bundlesA plasmid encoding GFP-MyoGEF was cotransfected into HeLa cells with an empty vector (a-c) or plasmids encoding N19RhoA (d-f), N19RhoC (g-i), N17Rac1 (j-l), or N17Cdc42 (m-o). Note that co-transfection of N19RhoA or N19RhoC decreases the formation of massive actin bundles induced by GFP-MyoGEF. Bars, 60 μm.
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Figure 8: Expression of dominant negative mutants N19RhoA and N19RhoC inhibits the formation of MyoGEF-induced actin bundlesA plasmid encoding GFP-MyoGEF was cotransfected into HeLa cells with an empty vector (a-c) or plasmids encoding N19RhoA (d-f), N19RhoC (g-i), N17Rac1 (j-l), or N17Cdc42 (m-o). Note that co-transfection of N19RhoA or N19RhoC decreases the formation of massive actin bundles induced by GFP-MyoGEF. Bars, 60 μm.

Mentions: To determine whether RhoA and/or RhoC are required for MyoGEF-induced actin bundles, a plasmid encoding GFP-MyoGEF was co-transfected into HeLa cells with plasmids encoding the dominant negative mutants of the small GTPase proteins RhoA, RhoC, Rac1, or Cdc42. Actin filaments were visualized by staining with rhodamine-conjugated phalloidin. Co-transfection of an empty vector did not affect the formation of massive actin bundles induced by GFP-MyoGEF (Figure 8; arrowheads in panel b). In contrast, transfection with plasmids encoding N19RhoA (d-f) or N19RhoC (g-i) significantly decreased the formation of massive actin bundles induced by GFP-MyoGEF (compare panels e and h with panel b). These findings indicate that RhoA or RhoC is required for MyoGEF-induced actin bundles. However, expression of dominant negative mutants N17Rac1 or N17Cdc42 did not significantly decrease the formation of actin bundles induced by GFP-MyoGEF (Figure 8; panels j-o), even though expression of N17Rac1 induced the formation of filopodia in GFP-MyoGEF-expressing cells, and it appeared that GFP-MyoGEF predominantly localized to cell periphery in the presence of N17Rac1 (compare panels a-c with panels j-i). In addition, MyoGEF can specifically bind to GTP-Rac1 (see Figure 3G). Therefore, our results suggest that MyoGEF may act as a downstream effector of Rac1 or as a carrier of GTP-Rac1 to potential target sites. Interestingly, it has been reported previously that a Rho-specific GEF, DBL's big sister (DBS), can bind to GTP-Rac1 and act as a downstream effector of GTP-Rac1 (Cheng et al., 2004). However, it is yet to be determined whether MyoGEF activity towards RhoA and RhoC can be regulated by Rac signaling.


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)

Expression of dominant negative mutants N19RhoA and N19RhoC inhibits the formation of MyoGEF-induced actin bundlesA plasmid encoding GFP-MyoGEF was cotransfected into HeLa cells with an empty vector (a-c) or plasmids encoding N19RhoA (d-f), N19RhoC (g-i), N17Rac1 (j-l), or N17Cdc42 (m-o). Note that co-transfection of N19RhoA or N19RhoC decreases the formation of massive actin bundles induced by GFP-MyoGEF. Bars, 60 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2692373&req=5

Figure 8: Expression of dominant negative mutants N19RhoA and N19RhoC inhibits the formation of MyoGEF-induced actin bundlesA plasmid encoding GFP-MyoGEF was cotransfected into HeLa cells with an empty vector (a-c) or plasmids encoding N19RhoA (d-f), N19RhoC (g-i), N17Rac1 (j-l), or N17Cdc42 (m-o). Note that co-transfection of N19RhoA or N19RhoC decreases the formation of massive actin bundles induced by GFP-MyoGEF. Bars, 60 μm.
Mentions: To determine whether RhoA and/or RhoC are required for MyoGEF-induced actin bundles, a plasmid encoding GFP-MyoGEF was co-transfected into HeLa cells with plasmids encoding the dominant negative mutants of the small GTPase proteins RhoA, RhoC, Rac1, or Cdc42. Actin filaments were visualized by staining with rhodamine-conjugated phalloidin. Co-transfection of an empty vector did not affect the formation of massive actin bundles induced by GFP-MyoGEF (Figure 8; arrowheads in panel b). In contrast, transfection with plasmids encoding N19RhoA (d-f) or N19RhoC (g-i) significantly decreased the formation of massive actin bundles induced by GFP-MyoGEF (compare panels e and h with panel b). These findings indicate that RhoA or RhoC is required for MyoGEF-induced actin bundles. However, expression of dominant negative mutants N17Rac1 or N17Cdc42 did not significantly decrease the formation of actin bundles induced by GFP-MyoGEF (Figure 8; panels j-o), even though expression of N17Rac1 induced the formation of filopodia in GFP-MyoGEF-expressing cells, and it appeared that GFP-MyoGEF predominantly localized to cell periphery in the presence of N17Rac1 (compare panels a-c with panels j-i). In addition, MyoGEF can specifically bind to GTP-Rac1 (see Figure 3G). Therefore, our results suggest that MyoGEF may act as a downstream effector of Rac1 or as a carrier of GTP-Rac1 to potential target sites. Interestingly, it has been reported previously that a Rho-specific GEF, DBL's big sister (DBS), can bind to GTP-Rac1 and act as a downstream effector of GTP-Rac1 (Cheng et al., 2004). However, it is yet to be determined whether MyoGEF activity towards RhoA and RhoC can be regulated by Rac signaling.

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