Oncogene (2009) 28, 2219–2230 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE -interacting guanine exchange factor (MyoGEF) regulates the invasion activity of MDA-MB-231 breast cancer cells through activation of RhoA and RhoC

D Wu, M Asiedu and Q Wei

Department of Biochemistry, Kansas State University, Manhattan, KS, USA

The small guanine triphosphatase (GTPase) proteins 2005). Cell migration involves lamellipodia formation RhoA and RhoC are essential for tumor invasion and/or and membrane protrusion at the front and retraction at metastasis in breast carcinomas. However, it is poorly the rear part of migrating cells. Actin polymerization at understood how RhoA and RhoC are activated in breast the front of migrating cells is critically important for cancer cells. Here we describe the role of myosin- membrane protrusion, whereas myosin-based contrac- interacting guanine nucleotide exchange factor (Myo- tility is required for the retraction of the rear of GEF) in regulating RhoA and RhoC activation as well as migrating cells (Lauffenburger and Horwitz, 1996; cell polarity and invasion in an invasive breast cancer cell Webb et al., 2002; Ridley et al., 2003; Raftopoulou line MDA-MB-231. RNA-interference (RNAi)-mediated and Hall, 2004). depletion of MyoGEF in MDA-MB-231 cells not only The small guanine triphosphatase (GTPase) proteins, suppresses the activation of RhoA and RhoC, but also such as Rac1, Cdc42 and RhoA, are key regulators of decreases cell polarity and invasion activity. The domi- the actin cytoskeleton. RhoA stimulates the assembly of nant-negative mutants of RhoA and RhoC, but not Rac1 contractile actomyosin filaments and associated focal and Cdc42, dramatically decrease actin polymerization adhesion complexes (Ridley and Hall, 1992). Rac1 induced by MyoGEF. In addition, MyoGEF colocalizes induces the formation of lamellipodia and membrane with nonmuscle myosin IIA (NMIIA) to the front of ruffles (Ridley et al., 1992), whereas Cdc42 induces migrating cells, and depletion of NMIIA by RNAi filopodia (Kozma et al., 1995). Both lamellipodia and disrupts the polarized localization of MyoGEF at the cell filopodia are actin-rich membrane protrusions, which leading edge, suggesting a role for NMIIA in regulating define the leading edge of a migrating cell. Earlier MyoGEF localization and function. Moreover, MyoGEF studies suggest that Rac1 and Cdc42 are restricted to the protein levels significantly increase in infiltrating ductal front of migrating cells and are responsible for carcinomas as well as in invasive breast cancer cell lines. membrane protrusion, whereas RhoA is localized to Taken together, our results suggest that MyoGEF the rear of migrating cells and is responsible for tail cooperates with NMIIA to regulate the polarity and retraction (Kraynov et al., 2000; Nalbant et al., 2004; invasion activity of breast cancer cells through activation Raftopoulou and Hall, 2004; Sastry et al., 2006). of RhoA and RhoC. However, studies using fluorescence resonance energy Oncogene (2009) 28, 2219–2230; doi:10.1038/onc.2009.96; transfer (FRET) to monitor the distribution of active published online 4 May 2009 RhoA in randomly migrating cells concluded that active RhoA also localizes to the front of migrating cells Keywords: MyoGEF; RhoA; RhoC; MDA-MB-231; (Kurokawa and Matsuda, 2005; Pertz et al., 2006), cell motility; nonmuscle myosin II consistent with findings that RhoA can induce mem- brane protrusion and ruffling in epithelial cells (Kawano et al., 1999; O’Connor et al., 2000). It is now believed that active RhoA can localize to the front of migrating Introduction cells in a cell type- and/or signal-specific manner. In randomly migrating cells, high levels of active RhoA are Cell migration plays a critical role in normal physio- found at the cell leading edge. In contrast, low levels of logical processes, such as embryogenesis, immune active RhoA are found in platelet-derived growth factor surveillance and angiogenesis, as well as in pathological (PDGF)-induced membrane protrusions (Pertz et al., processes, such as tumor invasion and metastasis 2006). (Burridge and Wennerberg, 2004; Jaffe and Hall, Tumor invasion and metastasis involve uncontrolled cell migration. Accumulating evidence suggests that activation of small GTPase proteins—RhoA and Correspondence: Dr Q Wei, Department of Biochemistry, Kansas RhoC—is criticalfor tumor invasion and/or metastasis State University, 141 Chalmers Hall, Manhattan, KS 66506, USA. E-mail: [email protected] in breast carcinoma (van Golen et al., 1999, 2000; Clark Received 3 November 2008; revised 23 March 2009; accepted 28 March et al., 2000; Kleer et al., 2002, 2004, 2005; Simpson et al., 2009; published online 4 May 2009 2004; Hakem et al., 2005; Pille et al., 2005; Kusama Regulation of cell migration by MyoGEF DWuet al 2220 et al., 2006). RhoA and RhoC are activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). However, the GEFs that are responsible for RhoA/RhoC activation in breast cancer cells remain to be identified. In this article, we have demonstrated that MyoGEF can activate RhoA and RhoC in invasive breast cancer cell line MDA-MB-231 cells. MyoGEF colocalizes with nonmuscle myosin IIA (NMIIA) to the front of migrating cells. In addition, depletion of myosin- interacting guanine exchange factor (MyoGEF) in MDA-MB-231 cells by RNAi impairs cell polarity and invasion. Further, MyoGEF is highly expressed in invasive breast cancer cell lines as well as in infiltrating ductalcarcinomas. Our resultsindicate that MyoGEF plays an important role in regulating the polarity and invasion activity of invasive breast cancer cells by activating RhoA/RhoC.

Results

MyoGEF is required for the invasion activity of MDA-MB-231 cells To correlate the expression level of MyoGEF with the invasive potential of breast cancer cells, immunoblot analysis with an antibody specific for MyoGEF was carried out to examine MyoGEF protein levels in breast cancer cell lines. As shown in Figure 1A, MyoGEF is highly expressed in invasive breast cancer cells MDA- Figure 1 Myosin-interacting guanine exchange factor (MyoGEF) MB-231 and MDA-MB-435S, but is not detectable in is required for the invasion activity of MDA-MB-231 cells. noninvasive (MDA-MB-361 and MCF-7) or poorly (A) Immunoblot analysis with anti-MyoGEF antibody shows that invasive (MDA-MB-468) breast cancer cells. This MyoGEF is expressed in MDA-MB-231 and MDA-MB-435S cells, but not in MDA-MB-361, MDA-MB-468 and MCF-7 cells. finding indicates that MyoGEF may play a critical role (B) Immunoblot analysis confirms the depletion of MyoGEF in in regulating the invasion activity of breast cancer cells. MDA-MB-231 cells by RNA interference (RNAi). (C) MDA-MB- Therefore, we used RNA interference (RNAi) to deplete 231 cells depleted of MyoGEF and/or nonmuscle myosin IIA MyoGEF in MDA-MD-231 cells, and then examined (NMIIA) were subjected to Matrigelinvasion assays. ( D) Images in (C) were quantitated by using the NationalInstitutes of Health the effect of MyoGEF depletion on the invasion activity (NIH) ImageJ program. (E) Immunohistochemicalanalysisof a of MDA-MB-231 cells. At 48 h after transfection with breast cancer-tissue array with MyoGEF antibody. Three arrays controlor MyoGEF smallinterference RNA (siRNA), were analyzed independently and similar results were obtained. transfected MDA-MB-231 cells were subjected to Immunohistochemistry with pre-immune serum shows light, back- Matrigelinvasion assays. As shown in Figures 1C and ground straining (data not shown). Images in (C) and (E) were taken by using a  20 objective (Leica DMI 6000 B microscope). D, depletion of MyoGEF dramatically inhibited MDA- MB-231 cell invasion activity. Nonmuscle myosin II (NMII) has also been implicated as an important Rockville, MD, USA). This tissue array contains 21 regulator of cell invasion and migration (Lo et al., cases of infiltrating ductal carcinomas with normal and 2004; Meshel et al., 2005; Vicente-Manzanares et al., adjacent tissues. As shown in Figure 1E, MyoGEF 2007; Conti and Adelstein, 2008). In addition, MyoGEF protein levels significantly increase in infiltrating ductal can interact with NMIIA (Wu et al., 2006). Therefore, carcinomas as compared with normalor adjacent breast we also depleted both NMIIA and MyoGEF to examine tissues (compare panels a and c with panel b). We found whether NMIIA and MyoGEF synergistically regulate that 17 out of the 21 cases of infiltrating ductal cell invasion. As shown in Figures 1C and D, RNAi- carcinomas (n ¼ 17/21 cases) show a dramatic increase mediated depletion of both NMIIA and MyoGEF in MyoGEF protein levels. further decreased the invasion activity of MDA-MB- 231 cells, suggesting that MyoGEF and NMIIA may Activation of RhoA and RhoC by MyoGEF cooperate in regulating breast cancer-cell invasion. To determine whether MyoGEF can activate RhoA and We then asked whether MyoGEF protein levels RhoC in breast cancer cells, MDA-MB-231 cells treated increase in invasive breast carcinomas. To this end, we with controlor MyoGEF siRNAs were subjected to carried out immunohistochemicalanalysiswith Myo- rhotekin Rho-binding domain (RBD) or p21-activated GEF antibody to examine MyoGEF protein levels in a kinase 1 (PAK1)/p21 binding domain (PBD) pull-down human breast cancer-tissue array (US Biomax Inc., assays to estimate the activity of the small GTPase

Oncogene Regulation of cell migration by MyoGEF DWuet al 2221 proteins, including RhoA, RhoC, Rac1 and Cdc42. or GDP (Figures 3e and f), indicating that MyoGEF RBD binds to activated (guanine triphosphate (GTP)- could bind to GTP- and GDP-bound RhoA and RhoC. bound) RhoA and RhoC, whereas PBD binds to In contrast, MyoGEF only bound to Rac1 that was activated (GTP-bound) Rac1 and Cdc42 (Abe et al., preloaded with GTP (Figure 3g), suggesting that 2000; Liu and Burridge, 2000; Arthur et al., 2002). As MyoGEF differentially bound to the active form of shown in Figures 2c and d, RBD-conjugated agarose Rac1 (GTP-Rac1). However, RNAi-mediated depletion beads could precipitate significant amounts of of MyoGEF in HeLa and MDA-MB-231 cells decreased GTP-RhoA and RhoC from MDA-MB-231 cells treated the amount of activated RhoA/RhoC (but not Rac1and with controlsiRNA (lane1 in Figures 2c and d), Cdc42) (Figure 2) (Wu et al., 2006). Therefore, our whereas depletion of MyoGEF by RNAi dramatically results indicate that RhoA and RhoC are most likely the decreased the amount of active RhoA and RhoC physiological effectors of MyoGEF. precipitated by RBD beads (lane 2 in Figures 2c and d). In contrast, MyoGEF depletion did not dramatically affect the amount of active Rac1 and Cdc42 pulled MyoGEF colocalizes with actin-myosin filaments at the down by PBD-conjugated agarose beads (Figures 2e cell leading edge and f). These results suggest that MyoGEF can activate To determine the localization of MyoGEF in migrating RhoA and RhoC, but not Rac1 and Cdc42, in MDA- MDA-MB-231 cells, immunofluorescence with anti- MB-231 cells. MyoGEF antibody was carried out in fixed MDA- To further characterize the in vitro activity of MB-231 cells after B6 h of culture on fibronectin-coated MyoGEF towards RhoA, RhoC, Rac1 and Cdc42, coverslips. As shown in Figure 4A, endogenous Myo- HeLa cells expressing Myc-MyoGEF were subjected to GEF colocalized with actin filaments at the front of immunoprecipitation with anti-Myc antibody, and then migrating cells (arrowheads in panels a–c and a0–c0). the immunoprecipitated Myc-MyoGEF was used for the Consistent with these observations, exogenously ex- analysis of MyoGEF activity towards RhoA, RhoC, pressed green fluorescent protein (GFP)-MyoGEF also Rac1 or Cdc42 with a fluorescence-based GTPase colocalized with actin filaments at the cell leading edge in vitro assay. As shown in Figure 3, Myc-MyoGEF (Figure 4C, panels a–c and a0–c0). It should be noted that could activate RhoA (3a), RhoC (3b) and Rac1 (3c), but expression of exogenous GFP-MyoGEF could induce not Cdc42 (3d). Consistent with these findings, in vitro the formation of thick actin bundles (panels b0 and c0). pull-down assays demonstrated that full-length Myo- In addition, GFP-MyoGEF formed filament-like struc- GEF (ThioHis-MyoGEF) or a truncated MyoGEF tures that overlap with these thick actin bundles fragment 71-388 (glutathione S- (GST)-71- (arrowheads in panelc 0). These results indicate that 388 containing the Dbl homology (DH) domain) could MyoGEF can localize to the cell leading edge, where it bind to RhoA and RhoC that were preloaded with GTP induces actin filament formation.

Figure 2 Depletion of myosin-interacting guanine exchange factor (MyoGEF) represses RhoA and RhoC activation in MDA-MB- 231 cells. (a) Immunoblot analysis confirms the depletion of MyoGEF in MDA-MB-231 cells by RNA interference (RNAi). (b) The image in (a) was quantitated by using the NationalInstitutes of Health(NIH) ImageJ program to estimate the efficiency of MyoGEF depletion in MDA-MB-231 cells by RNAi. (c–f) Depletion of MyoGEF decreases the amount of active RhoA (c) and RhoC (d), but not Rac1 (e) and CDc42 (f), in MDA-MB-231 cells. Almost 6% of transfected-cell lysates were used as control to estimate the amount of totalRhoA, RhoC, Rac1 and Cdc42. ( g) The images in (c), (d), (e) and (f) were quantitated by using the NIH ImageJ program.

Oncogene Regulation of cell migration by MyoGEF DWuet al 2222

Figure 3 In vitro activation of RhoA, RhoC and Rac1, but not Cdc42, by Myosin-interacting guanine exchange factor (MyoGEF). (a–d) The immunoprecipitated Myc-MyoGEF from transfected HeLa cells could activate RhoA (a), RhoC (b) and Rac1 (c), but not Cdc42 (d) in a fluorescence-based guanine exchange factor (GEF) assay. (e) ThioHis-MyoGEF (full-length) could bind both guanine diphosphate (GDP)-RhoA (lane 4) and guanine triphosphate (GTP)-RhoA (lane 5). (f) A MyoGEF fragment (amino acids 71–388) that contain the Dbl homology (DH) domain could bind both GDP-RhoC (lane 4) and GTP-RhoC (lane 5). (g) ThioHis-MyoGEF could bind GTP-Rac1 (lane 5), but not GDP-Rac1 (lane 4). D, preloaded with GDP; T, preloaded with GTP.

NMII has been shown to localize to the cell leading MDA-MB-231 cells as indicated in Figure 4E (arrow- edge, and it plays a critical role in regulating cell polarity heads in panels a0–d0). and motility (Kolega, 2006; Sandquist et al., 2006; Vicente-Manzanares et al., 2007; Conti and Adelstein, 2008). We also reported earlier that MyoGEF interacts MyoGEF preferentially interacts with NMIIA with NMIIA (Wu et al., 2006). Therefore, we also in MDA-MB-231 cells examined whether MyoGEF and NMIIA colocalized in We earlier showed that MyoGEF interacts with NMIIA randomly migrating MDA-MB-231 cells. MDA-MB- in vitro (Wu et al., 2006). Three isoforms of the 231 cells expressing GFP-tagged nonmuscle myosin nonmuscle myosin heavy chain (NMHC), IIA, IIB and heavy chain IIA (GFP-IIA) were subjected to immuno- IIC, have been identified in humans and mice (Bresnick, fluorescence with anti-MyoGEF antibody. As shown in 1999; Sellers, 2000; Golomb et al., 2004; Krendeland Figure 4D, MyoGEF colocalized with GFP-IIA at the Mooseker, 2005; Conti and Adelstein, 2008). MDA- cell leading edge (arrowheads in panels a0–d0). Consis- MB-231 cells express IIA and IIB, but not IIC (Betapudi tently, exogenously expressed GFP-MyoGEF also et al., 2006). To confirm the interaction between colocalized with endogenous NMIIA in transfected MyoGEF and NMII, MDA-MB-231 cells expressing

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Figure 4 Myosin-interacting guanine exchange factor (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 rhodamine-phalloidin (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 green fluorescent protein (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 nonmuscle myosin IIA (NMIIA) (red) at the cell leading edge of transfected MDA-MB-231 cells. Bars, 10 mm.

Myc-MyoGEF were subjected to immunoprecipitation Depletion of MyoGEF by RNAi impairs cell polarity with anti-Myc antibody followed by immunoblot Rho-GTPase signaling is essential for cell polarity and analysis with anti-IIA or anti-IIB antibodies. As shown cell migration (Ridley et al., 2003). Localization of in Figure 5a, endogenous NMIIA, but not NMIIB, MyoGEF to the cell leading edge (Figure 4) suggests could be co-immunoprecipitated with Myc-MyoGEF that MyoGEF may play a role in regulating cell from total-cell lysates of transfected MDA-MB-231 polarity. Therefore, we examined whether depletion of cells, indicating that MyoGEF can preferentially inter- MyoGEF had an effect on the polarity of MDA-MB- act with NMIIA in MDA-MB-231 cells. To further 231 cells. As shown in Figures 6A and B, only B25% of characterize the interaction between MyoGEF and MDA-MB-231 cells treated with control siRNA NMIIA, we generated different truncated versions of (n ¼ 200 cells) remain unpolarized after 6 h of culture MyoGEF (Figure 5b). Plasmids encoding these Myc- on fibronectin-coated coverslips. However, B60% of tagged MyoGEF fragments were transfected into a MDA-MB-231 cells (n ¼ 250 cells) did not polarize HeLa cell line that only expresses NMIIA, but not following MyoGEF depletion (Figures 6A and B). NMIIB and NMIIC (Wei and Adelstein, 2000). As Consistent with these findings, MyoGEF localized to shown in Figure 5c, Myc-MyoGEF full-length, Myc- the cell leading edge in MDA-MB-231 cells transfected pleckstrin homology (PH), Myc-1-409, and Myc-1-500 with control siRNA, and these cells showed polarized could immunoprecipitate a significant amount of actin polymerization (Figure 6C; arrowheads in panels NMIIA, suggesting that the pleckstrin homology (PH) a–c). In contrast, depletion of MyoGEF by RNAi domain as well as the 351–409 region,C-terminal to the decreased MyoGEF protein levels (Figure 6C; compare Dblhomology(DH) domain, are required for interac- panel b with panel e), and MyoGEF-depleted cells did tion with NMIIA. not show polarized actin organization (panels d and f).

Oncogene Regulation of cell migration by MyoGEF DWuet al 2224 Figures 6De and Ee). Consistently, p-mrlc staining was also found predominantly at the cell periphery (arrow- heads in Figures 6Dd and Ed). However, we did not observe a dramatic decrease in NMIIA, NMIIB and p- mrlc staining. These results suggest that MyoGEF depletion impairs cell polarity as well as polarized actin–myosin organization, without having an obvious effect on overall actin–myosin filament formation.

NMII filaments are required for polarized distribution of MyoGEF NMII plays a central role in regulating cell polarity and motility (Lo et al., 2004; Meshel et al., 2005; Vicente- Manzanares et al., 2007; Conti and Adelstein, 2008). 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 immuno- fluorescence with anti-MyoGEF antibody to examine the effect of NMIIA depletion on MyoGEF localiza- tion. As shown in Figure 7A, depletion of NMIIA by RNAi led to indiscriminate cell spreading and the disruption of polarized MyoGEF localization (arrow- heads in Figure 7Ab). In contrast, depletion of NMIIB did not impair cell polarity and the polarized distribu- tion 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 Figure 5 Myosin-interacting guanine exchange factor (MyoGEF) interacts with nonmuscle myosin IIA (NMIIA). (a) MDA-MB-231 that NMIIA is required for polarized localization of cells expressing Myc-MyoGEF were subjected to immunoprecipi- MyoGEF during cell migration. tation with anti-Myc antibody followed by immunoblot analysis To further confirm that NMIIA is required for with anti-IIA or anti-IIB antibodies. Note that Myc-MyoGEF MyoGEF function in other epithelial cells, we transfected binds to NMIIA but not NMIIB. (b) Schematic diagram of MyoGEF fragments that were used in (c). (c) Interactions between NMIIA siRNA and a plasmid encoding GFP-MyoGEF Myc-tagged MyoGEF fragments and endogenous NMIIA. Full- into a HeLa cell line that expresses only NMIIA (Wei and length MyoGEF (lane 3) as well as MyoGEF fragments Myc- Adelstein, 2000; Golomb et al., 2004), and then examined pleckstrin homology (PH) (lane 5), Myc-1-409 (lane 8) and Myc-1- the formation of actin–myosin bundles induced by 500 (lane 9) could pull down a significant amount of endogenous exogenously expressed GFP-MyoGEF. As shown in NMIIA. Note that cell lysate from lane 3 was also used for immunoprecipitation with normal IgG (lane 2). Almost 5% of total Figure 7C, GFP-MyoGEF induced the formation of cell lysates were loaded. massive myosin bundles in the presence of control siRNA (arrowheads in panels a–c), and GFP-MyoGEF coloca- lized with these NMIIA bundles (arrowheads in panel c). Notably, actin filaments were predominantly assembled In contrast, RNAi-mediated depletion of NMIIA com- at the periphery of MyoGEF-depleted cells (Figure 6C; pletely abrogated the formation of massive myosin panels d and f). These results suggest that MyoGEF is bundles induced by GFP-MyoGEF (Figure 7C, panels required for cell polarization in MDA-MB-231 cells, d–f). We also examined the effect of NMIIA depletion on even though MyoGEF-depletion did not dramatically the formation of actin bundles induced by GFP- decrease actin filament formation (based on the intensity MyoGEF. As shown in Figure 7D, depletion of NMIIA of actin filament staining; data not shown). by siRNA dramatically reduced the formation of massive We then examined the effect of MyoGEF-depletion actin bundles induced by GFP-MyoGEF (compare panels on myosin filament organization. MDA-MB-231 cells a–c with panels d–f). These results suggest that the treated with controlsiRNA (siCont) were ableto presence of NMIIA and MyoGEF–myosin II interaction polarize, and a significant amount of phosphorylated may be critical for MyoGEF localization and function. myosin regulatory light chain (p-mrlc), NMIIA, and NMIIB localized at the cell leading edge of randomly migrating cells (arrowheads in Figures 6Da–c and Expression of dominant-negative mutants N19RhoA Ea–c). In contrast, cells treated with MyoGEF siRNA or N19RhoC interferes with MyoGEF-induced actin did not polarize (Figures 6Dd–f and Ed–f). NMIIA and polymerization NMIIB filaments were predominantly assembled at the To determine whether RhoA and/or RhoC are required periphery of MyoGEF-depleted cells (arrowheads in for MyoGEF-induced actin bundles, a plasmid encoding

Oncogene Regulation of cell migration by MyoGEF DWuet al 2225

Figure 6 Depletion of myosin-interacting guanine exchange factor (MyoGEF) by RNA interference (RNAi) impairs MDA-MB-231 cell polarity. (A) MDA-MB-231 cells treated with control small interference RNA (siRNA) (siCont) or MyoGEF siRNA (siMyoGEF) for 48 h were trypsinized, replated on fibronectin-coated coverslips and cultured for an additional 6 h. Note that cells depleted of MyoGEF did not polarize. (B) Quantitation of nonpolarized MDA-MB-231 cells treated with control or MyoGEF siRNAs. (C) MDA-MB-231 cells treated with siCont or siMyoGEF were subjected to immunofluorescence with MyoGEF antibody (red) and FITC-phalloidin (green). (D) MDA-MB-231 cells treated with siCont or siMyoGEF were stained with antibodies specific for phosphorylated myosin regulatory light chain (p-mrlc) (green) and nonmuscle myosin IIA (NMIIA) (red). (E) MDA-MB-231 cells treated with siCont or siMyoGEF were stained with antibodies specific for p-mrlc (green) and NMIIB (red). Bar in (a), 80 mm; Bars in (c), (d) and (e), 10 mm.

GFP-MyoGEF was co-transfected into HeLa cells with of GTP-Rac1 to potentialtarget sites. Interestingly,it plasmids encoding the dominant-negative mutants of has been reported earlier that a Rho-specific GEF, the small GTPase proteins RhoA, RhoC, Rac1 or DBL’s big sister (DBS), can bind to GTP-Rac1 and act Cdc42. Actin filaments were visualized by staining with as a downstream effector of GTP-Rac1 (Cheng et al., rhodamine-conjugated phalloidin. Co-transfection of an 2004). However, it is yet to be determined whether empty vector did not affect the formation of massive MyoGEF activity towards RhoA and RhoC can be actin bundles induced by GFP-MyoGEF (Figure 8; regulated by Rac signaling. arrowheads in panelb). 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 Discussion and h with panelb). These findings indicate that RhoA or RhoC is required for MyoGEF-induced actin In this article, we have demonstrated that MyoGEF can bundles. However, expression of dominant-negative activate RhoA and RhoC in a breast cancer cell line mutant N17Rac1 or N17Cdc42 did not significantly MDA-MB-231. We also show that MyoGEF plays a decrease the formation of actin bundles induced by role in regulating the polarity and invasion activity of GFP-MyoGEF (Figure 8, panels j–o), even though MDA-MB-231 cells. In addition, polarized distribution expression of N17Rac1 induced the formation of of MyoGEF at the cell leading edge is dependent on the filopodia in GFP-MyoGEF-expressing cells, and it integrity of the actin–myosin cytoskeleton. Importantly, appeared that GFP-MyoGEF predominantly localized MyoGEF is highly expressed in invasive (MDA-MB-231 to the cell periphery in the presence of N17Rac1 and MDA-MB-435s) breast cancer cells, but is not (compare panels a–c with panels j–l). In addition, detectable in noninvasive (MDA-MB-361 and MCF-7) MyoGEF can specifically bind to GTP-Rac1 (see or poorly invasive (MDA-MB-468) breast cancer cells. Figure 3g). Therefore, our results suggest that MyoGEF Expression of dominant-negative mutants N19RhoA or may act as a downstream effector of Rac1 or as a carrier N19RhoC interferes with the formation of MyoGEF-

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Figure 7 Nonmuscle myosin IIA (NMIIA) is required for polarized localization of myosin-interacting guanine exchange factor (MyoGEF) as well as the formation of MyoGEF-induced actin bundles. (A) MDA-MB-231 cells treated with control small interfering RNA (siRNA) (siCont; panela) or NMIIA siRNA (siIIA; panelb) 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 b-. (C, D) A plasmid encoding green fluorescent protein (GFP)-MyoGEF was co-transfected into HeLa cells with siCont or siIIA. The transfected cells were subjected to immunofluorescence with anti-IIA antibody (C) or phalloidin (D). Bars, 10 mm.

induced actin–myosin bundles. Our results suggest that and invasion activity of MDA-MB-231 cells through the MyoGEF and NMII cooperate to regulate cell polarity activation of RhoA and/or RhoC. Although a number and motility in invasive breast cancer cells. of studies suggest an essentialrolefor RhoC in breast Rho GTPase signaling has been implicated in cancer metastasis, our results show that MyoGEF can regulating cell polarity and motility (Arthur and activate both RhoA and RhoC in MDA-MB-231 cells. Burridge, 2001; Worthylake and Burridge, 2003; Kur- It remains to be determined whether both RhoA and okawa et al., 2005; Yamana et al., 2006). Rac1 is widely RhoC are required for MyoGEF-mediated regulation of considered as a key regulator of cell migration in cell migration. Nonetheless, a line of evidence suggests different cell lines and organisms (Ridley, 2001; Ridley that RhoA also plays a critical role in regulating the et al., 2003). However, accumulating evidence also motility and invasion of MDA-MB-231 cells (Pille et al., suggests that Rac1 activity is not required for 2005, 2006; Kusama et al., 2006; Sahai et al., 2007). cell migration in a number of cell lines, such as Therefore, it is likely that both RhoA and RhoC are colon carcinoma cells, rat fibroblasts and macrophages important for MyoGEF-mediated regulation of MDA- (Ahram et al., 2000; O’Connor et al., 2000; Wells et al., MB-231 cell invasion. Another possibility is that 2004). Instead, it has been demonstrated that RhoC is a MyoGEF-RhoA and MyoGEF-RhoC may differen- key pro-metastatic protein that is essentialfor cell tially function in different breast cancer cells. migration and invasion in breast cancer cells (Clark RhoA and RhoC can induce actin–myosin filament et al., 2000; van Golen et al., 2000; Simpson et al., 2004; formation through the activation of ROCK and mDia. Hakem et al., 2005; Kleer et al., 2005). ROCK inhibits myosin phosphatase and directly phos- However, it is poorly understood whether there are phorylates myosin regulatory light chains, resulting in specific GEFs that are responsible for RhoA and/or an increase in myosin contractile activity (Kimura et al., RhoC activation in breast cancer cells. We have shown 1996; Matsui et al., 1996). mDia can induce actin that MyoGEF can activate both RhoA and RhoC in polymerization through association with the barbed end MDA-MB-231 cells (Figures 2 and 3). Depletion of of growing actin filaments (Watanabe et al., 1999; MyoGEF by RNAi impairs cell polarity and invasion Tominaga et al., 2000; Chang and Peter, 2002; Li and activity (Figures 1 and 6). Thus, our findings point to a Higgs, 2003; Higashida et al., 2004; Zigmond, 2004). mechanism by which MyoGEF regulates the polarity We observed the reorganization of the actin–myosin

Oncogene Regulation of cell migration by MyoGEF DWuet al 2227

Figure 8 Expression of dominant-negative mutants N19RhoA and N19RhoC inhibits the formation of myosin-interacting guanine exchange factor (MyoGEF)-induced actin bundles. A plasmid encoding green fluorescent protein (GFP)-MyoGEF was co-transfected 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 mm. cytoskeleton following MyoGEF depletion, even though that other actin–myosin promoting pathways can depletion of MyoGEF does not dramatically reduce compensate for the loss of RhoA/RhoC activity result- actin–myosin filament formation (Figure 6). It is likely ing from MyoGEF depletion. For instance, in addition

Oncogene Regulation of cell migration by MyoGEF DWuet al 2228 to ROCK, myosin light chain kinase (MLCK) can also (Manassas, VA, USA). MDA-MB-231, MDA-MB-435S, phosphorylate mrlc, thus increasing the phosphoryla- MDA-MB-361 and MDA-MB-468 were grown in Leibovitz’s tion of mrlc as well as myosin contractile activity. L-15 medium (ATCC) supplemented with 10% fetal bovine Importantly, it has been reported that myosin light serum. HeLa cells were purchased from Clontech (Mountain chain kinase (MLCK) predominantly phosphorylates View, CA, USA). HeLa and MCF-7 cells were grown in mrlc at the cell periphery, whereas ROCK is responsible Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetalbovine serum. Transfection was done with for mrlc phosphorylation at the cell center (Totsukawa Lipofectamine 2000 according to the manufacturer’s instruc- et al., 2004). Consistent with this conclusion, we found tions (Invitrogen, Carlsbad, CA, USA). MyoGEF siRNA has that actin–myosin filaments are predominantly as- been described earlier (Wu et al., 2006). sembled at the cell periphery in MyoGEF-depleted cells (Figure 6). Although Rho–ROCK signaling has been implicated Immunoprecipitation and immunoblotting in cell motility in a number of cell lines as well as in Immunoprecipitation was carried out as described earlier (Wei, animal models, our results indicated that treatment with 2005; Asiedu et al., 2008, 2009), except that 2 mM adenosine Y27632 does not affect cell polarity and polarized triphosphate (ATP) and 2 mM MgCl2 were included in the lysis buffer. The following primary antibodies were used: mouse distribution of MyoGEF in MDA-MB-231 cells (data anti-Myc (9E10, 1:2000; Santa Cruz Biotechnology, Santa not shown). Instead, MDA-MB-231 cells often show an Cruz, CA, USA), rabbit anti-b-tubulin (1:2000; Santa Cruz elongated morphology, indicating that the tail retraction Biotechnology), mouse anti-RhoA (1:200; Santa Cruz Bio- is not completed in the presence of Y27632 (data not technology), goat anti-RhoC (1:100; Santa Cruz Biotechno- shown), whereas depletion of MyoGEF impairs cell logy), mouse anti-NMIIB (1:1000; Developmental Studies polarity, leading to rounded cell morphology (Figure 6). Hybridoma Bank, Iowa City, IA, USA) and rabbit anti- These results suggest that Rho–ROCK signaling may be MyoGEF (1:200) (Wu et al., 2006). dispensable for MyoGEF function in regulating cell polarity and invasion activity. These observations are Immunofluorescence consistent with the finding that RhoA–ROCK is Immunofluorescence was carried out as described earlier required for the invasion activity of MDA-MB-435S, (Asiedu et al., 2009). MDA-MB-231 cells transfected with but not MDA-MB-231 cells (Demou et al., 2005). plasmids or siRNA were trypsinized, cultured on coverslips for Furthermore, a correlation between the invasion activity an additional6–12 h, and then fixed with 4% paraformalde- of different tumor cells and the requirement of Rho– hyde. The primary antibodies used for immunofluorescence ROCK signaling has been suggested (Sahai and were as follows: rabbit polyclonal NMIIA antibody (1:1000), Marshall, 2003). One of the future challenges is to mouse monoclonal p-mrlc antibody (1:200; Cell Signaling, determine whether Rho–mDia signaling is required for Beverly, MA, USA) and rabbit polyclonal MyoGEF antibody MyoGEF function in the regulation of cell polarity and (1:100). The secondary antibodies rhodamine goat anti-mouse invasion. IgG (1:500) and rhodamine goat anti-rabbit IgG (1:500) were purchased from Invitrogen. The nuclei were visualized by NMII plays an essential role in the regulation of cell DAPI (Sigma, St Louis, MO, USA). Actin filaments were polarity and cell migration. MyoGEF interacts with stained with rhodamine-phalloidin (Invitrogen). Images were NMIIA and both proteins colocalize at the cell leading taken using a Leica DMI 6000 B microscope (Leica, Deerfield, edge (Figure 4). Disruption of NMIIA by RNAi impairs IL, USA) and processed by blind deconvolution. To determine cell polarity as well as polarized distribution of the cell polarity, long (L) and short (S) axes of individual cells MyoGEF (Figure 7). In addition, exogenously expressed were measured using the NationalInstitutes of Health(NIH) GFP-MyoGEF forms filament-like structures that over- ImageJ program. Cells were counted as polarized (L/S ratio lap with actin–myosin bundles (Figure 4). Depletion of >2) or nonpolarized (L/S o2). NMIIA completely abrogates the formation of Myo- GEF-induced massive actin bundles (Figure 7C). These findings indicate that NMIIA may act as a scaffold to Immunohistochemistry The breast cancer tissue arrays were purchased from US anchor MyoGEF to the cell leading edge. In turn, Biomax (Rockville, MD, USA; Cat # BC08032). After MyoGEF locally activates RhoA and/or RhoC at the incubation at 60 1C for 2 h, the paraffin-embedded sections front of migrating cells, thereby forming a positive were deparaffinized in three changes of xylene and then MyoGEF-NMIIA loop and promoting cell polarization rehydrated using graded alcohols. Antigen retrieval was done and invasion. with 10 mM citrate buffer, pH 6, in the microwave for 30 min and allowed to cool to room temperature, followed by a wash with phosphate buffer solution (PBS). The slides were then blocked with normal goat serum and incubated with anti- Materials and methods MyoGEF antibody (1:100) at room temperature for 1 h. Pre- immune serum was used on a duplicate slide in place of Plasmids and cell culture MyoGEF antibody as a negative control. After washing three pEGFP-MyoGEF, pCS3-MyoGEF and pEGFP-NMHC-IIA times with PBS, the slides were incubated with biotinylated were described earlier (Wei and Adelstein, 2000; Wu et al., secondary goat anti-rabbit antibody at room temperature for 2006). MyoGEF fragments were cloned into XhoI/XbaI sites of 30 min. After washing three times with PBS, the slides were pCS3 þ MT vector. Breast cancer cell lines MDA-MB-231, stained with the ABC/DAB Elite kit (goat IgG type; MDA-MB-435S, MDA-MB-361, MDA-MB-468 and MCF-7 Vectorlabs, Burlingame, CA, USA). Finally, the slides were were purchased from the American Type Cell Culture (ATCC) counterstained with hematoxylin, dehydrated, cleared and

Oncogene Regulation of cell migration by MyoGEF DWuet al 2229 then mounted with Permount mounting medium (Fisher In vitro guanine nucleotide exchange analysis Scientific, Pittsburgh, PA, USA). The GEF exchange assay was described earlier (Asiedu et al., 2008).

Matrigel invasion assays Transfected MDA-MB-231 cells were trypsinized and approxi- Conflict of interest mately 1 Â 105 cells (in Leibovitz’s L-15 medium containing 3% of bovine serum albumin (BSA)) were seeded on the upper The authors declare no conflict of interest. wells of Biocoat Matrigel chambers (BD Biosciences, Bedford, MA, USA). The lower wells were filled with Leibovitz’s L-15 medium containing 10% fetalbovine serum (FBS). The Acknowledgements transfected cells then underwent chemoattracting across the matrigeland filter(pore size: 8 mm) to the lower surface of the We thank Dr Robert S Adelstein and Dr Mary Anne Conti for trans-wells for 22 h. The non-migrating cells on the upper their criticalreading and comments on the manuscript. We chambers were removed by a cotton swab. The migrating cells thank Dr Keith Burridge (University of North Carolina at on the lower surface were fixed in 4% paraformaldehyde, Chapel Hill, Chapel Hill, NC, USA) and Dr Rick Cerione stained with 1% crystalvioletand then photographed with an (Cornell University, Ithaca, NY, USA) for providing GST- Â 20 objective. Data were collected from three independent RBD and GST-PBD plasmids. This publication was made experiments, each done in triplicate. possible by National Institutes of Health (NIH) Grant no. P20 RR015563 from the NationalCenter for Research Resources. This work was also supported by NIHGrant k22 HL071542 RhoA, RhoC, Rac1 and Cdc42 activation assays (to QW), as well as a fund from Terry C. Johnson Center for RhoA, RhoC, Rac1 and Cdc42 activation assays were Basic Cancer Research (to QW). This is contribution 09-136-J described earlier (Glaven et al., 1999; Liu and Burridge, from the Kansas Agricultural Experiment Station, Manhattan, 2000; Wu et al., 2006; Asiedu et al., 2008). KS, USA.

References

Abe K, Rossman KL, Liu B, Ritola KD, Chiang D, Campbell SL et al. Demou ZN, Awad M, McKee T, Perentes JY, Wang X, Munn LL (2000). Vav2 is an activator of Cdc42, Rac1, and RhoA. J Biol et al. (2005). Lack of telopeptides in fibrillar collagen I promotes Chem 275: 10141–10149. the invasion of a metastatic breast tumor cell line. Cancer Res 65: Ahram M, Sameni M, Qiu RG, Linebaugh B, Kirn D, Sloane BF. 5674–5682. (2000). Rac1-induced endocytosis is associated with intracellular Glaven JA, Whitehead I, Bagrodia S, Kay R, Cerione RA. (1999). The proteolysis during migration through a three-dimensional matrix. Dbl-related protein, Lfc, localizes to microtubules and mediates the Exp Cell Res 260: 292–303. activation of Rac signaling pathways in cells. J Biol Chem 274: Arthur WT, Burridge K. (2001). RhoA inactivation by p190RhoGAP 2279–2285. regulates cell spreading and migration by promoting membrane Golomb E, Ma X, Jana SS, Preston YA, Kawamoto S, Shoham NG protrusion and polarity. Mol Biol Cell 12: 2711–2720. et al. (2004). Identification and characterization of nonmuscle Arthur WT, Ellerbroek SM, Der CJ, Burridge K, Wennerberg K. myosin II-C, a new member of the myosin II family. J Biol Chem (2002). XPLN, a guanine nucleotide exchange factor for RhoA and 279: 2800–2808. RhoB, but not RhoC. J Biol Chem 277: 42964–42972. Hakem A, Sanchez-Sweatman O, You-Ten A, Duncan G, Wakeham Asiedu M, Wu D, Matsumura F, Wei Q. (2008). Phosphorylation of A, Khokha R et al. (2005). RhoC is dispensable for embryogenesis MyoGEF on Thr-574 by Plk1 promotes MyoGEF localization to and tumor initiation but essentialfor metastasis. Genes Dev 19: the centralspindle. J Biol Chem 283: 28392–28400. 1974–1979. Asiedu M, Wu D, Matsumura F, Wei Q. (2009). Centrosome/spindle Higashida C, Miyoshi T, Fujita A, Oceguera-Yanez F, Monypenny J, pole-associated protein regulates cytokinesis via promoting the Andou Y et al. (2004). Actin polymerization-driven molecular recruitment of MyoGEF to the centralspindle. Mol Biol Cell 20: movement of mDia1 in living cells. Science 303: 2007–2010. 1428–1440. Jaffe AB, Hall A. (2005). Rho : biochemistry and biology. Betapudi V, Licate LS, Egelhoff TT. (2006). Distinct roles of Annu Rev Cell Dev Biol 21: 247–269. nonmuscle myosin II isoforms in the regulation of MDA-MB-231 Kawano Y, Fukata Y, Oshiro N, Amano M, Nakamura T, Ito M et al. breast cancer cell spreading and migration. Cancer Res 66: (1999). Phosphorylation of myosin-binding subunit (MBS) 4725–4733. of myosin phosphatase by Rho-kinase in vivo. J Cell Biol 147: Bresnick AR. (1999). Molecular mechanisms of nonmuscle myosin-II 1023–1038. regulation. Curr Opin Cell Biol 11: 26–33. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M Burridge K, Wennerberg K. (2004). Rho and Rac take center stage. et al. (1996). Regulation of myosin phosphatase by Rho and Cell 116: 167–179. Rho-associated kinase (Rho-kinase). Science 273: 245–248. Chang F, Peter M. (2002). Cell biology. Formins set the record Kleer CG, Griffith KA, Sabel MS, Gallagher G, van Golen KL, Wu straight. Science 297: 531–532. ZF et al. (2005). RhoC-GTPase is a noveltissue biomarker Cheng L, Mahon GM, Kostenko EV, Whitehead IP. (2004). associated with biologically aggressive carcinomas of the breast. Pleckstrin homology domain-mediated activation of the rho-specific Breast Cancer Res Treat 93: 101–110. guanine nucleotide exchange factor Dbs by Rac1. J Biol Chem 279: Kleer CG, van Golen KL, Zhang Y, Wu ZF, Rubin MA, 12786–12793. Merajver SD. (2002). Characterization of RhoC expression in Clark EA, Golub TR, Lander ES, Hynes RO. (2000). Genomic benign and malignant breast disease: a potential new marker for analysis of metastasis reveals an essential role for RhoC. Nature small breast carcinomas with metastatic ability. Am J Pathol 160: 406: 532–535. 579–584. Conti MA, Adelstein RS. (2008). Nonmuscle myosin II moves in new Kleer CG, Zhang Y, Pan Q, Gallagher G, Wu M, Wu ZF et al. (2004). directions. J Cell Sci 121: 11–18. WISP3 and RhoC guanosine triphosphatase cooperate in the

Oncogene Regulation of cell migration by MyoGEF DWuet al 2230 development of inflammatory breast cancer. Breast Cancer Res 6: Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, R110–R115. Borisy G et al. (2003). Cell migration: integrating signals from front Kolega J. (2006). The role of myosin II motor activity in distributing to back. Science 302: 1704–1709. myosin asymmetrically and coupling protrusive activity to cell Sahai E, Garcia-Medina R, Pouyssegur J, VialE. (2007). Smurf1 translocation. Mol Biol Cell 17: 4435–4445. regulates tumor cell plasticity and motility through degradation of Kozma R, Ahmed S, Best A, Lim L. (1995). The Ras-related protein RhoA leading to localized inhibition of contractility. J Cell Biol 176: Cdc42Hs and bradykinin promote formation of peripheralactin 35–42. microspikes and filopodia in Swiss 3T3 fibroblasts. Mol Cell Biol 15: Sahai E, Marshall CJ. (2003). Differing modes of tumour cell invasion 1942–1952. have distinct requirements for Rho/ROCK signalling and extra- Kraynov VS, Chamberlain C, Bokoch GM, Schwartz MA, Slabaugh cellular proteolysis. Nat Cell Biol 5: 711–719. S, Hahn KM. (2000). Localized Rac activation dynamics visualized Sandquist JC, Swenson KI, Demali KA, Burridge K, Means AR. in living cells. Science 290: 333–337. (2006). Rho kinase differentially regulates phosphorylation of KrendelM, Mooseker MS. (2005). : tails(and heads) of nonmuscle myosin II isoforms A and B during cell rounding and functionaldiversity. Physiology (Bethesda) 20: 239–251. migration. J Biol Chem 281: 35873–35883. Kurokawa K, Matsuda M. (2005). Localized RhoA activation as a Sastry SK, Rajfur Z, Liu BP, Cote JF, Tremblay ML, Burridge K. requirement for the induction of membrane ruffling. Mol Biol Cell (2006). PTP-PEST couples membrane protrusion and tail retraction 16: 4294–4303. via VAV2 and p190RhoGAP. J Biol Chem 281: 11627–11636. Kurokawa K, Nakamura T, Aoki K, Matsuda M. (2005). Mechanism Sellers JR. (2000). Myosins: a diverse superfamily. Biochim Biophys and role of localized activation of Rho-family GTPases in growth Acta 1496: 3–22. factor-stimulated fibroblasts and neuronal cells. Biochem Soc Trans Simpson KJ, Dugan AS, Mercurio AM. (2004). Functionalanalysisof 33: 631–634. the contribution of RhoA and RhoC GTPases to invasive breast Kusama T, Mukai M, Tatsuta M, Nakamura H, Inoue M. (2006). carcinoma. Cancer Res 64: 8694–8701. Inhibition of transendothelial migration and invasion of human Tominaga T, Sahai E, Chardin P, McCormick F, Courtneidge SA, breast cancer cells by preventing geranylgeranylation of Rho. Int J Alberts AS. (2000). Diaphanous-related formins bridge Rho Oncol 29: 217–223. GTPase and Src tyrosine kinase signaling. Mol Cell 5: 13–25. Lauffenburger DA, Horwitz AF. (1996). Cell migration: a physically Totsukawa G, Wu Y, Sasaki Y, Hartshorne DJ, Yamakita Y, integrated molecular process. Cell 84: 359–369. Yamashiro S et al. (2004). Distinct roles of MLCK and ROCK in Li F, Higgs HN. (2003). The mouse Formin mDia1 is a potent the regulation of membrane protrusions and focal adhesion actin nucleation factor regulated by autoinhibition. Curr Biol 13: dynamics during cell migration of fibroblasts. J Cell Biol 164: 1335–1340. 427–439. Liu BP, Burridge K. (2000). Vav2 activates Rac1, Cdc42, and RhoA van Golen KL, Davies S, Wu ZF, Wang Y, Bucana CD, Root H et al. downstream from growth factor receptors but not beta1 integrins. (1999). A novel putative low-affinity insulin-like growth factor- Mol Cell Biol 20: 7160–7169. binding protein, LIBC (lost in inflammatory breast cancer), and Lo CM, Buxton DB, Chua GC, Dembo M, Adelstein RS, Wang YL. RhoC GTPase correlate with the inflammatory breast cancer (2004). Nonmuscle myosin IIb is involved in the guidance of phenotype. Clin Cancer Res 5: 2511–2519. fibroblast migration. Mol Biol Cell 15: 982–989. van Golen KL, Wu ZF, Qiao XT, Bao LW, Merajver SD. (2000). Matsui T, Amano M, Yamamoto T, Chihara K, Nakafuku M, Ito M RhoC GTPase, a noveltransforming oncogene for human et al. (1996). Rho-associated kinase, a novelserine/threonine kinase, mammary epithelial cells that partially recapitulates the inflamma- as a putative target for small GTP binding protein Rho. EMBO J tory breast cancer phenotype. Cancer Res 60: 5832–5838. 15: 2208–2216. Vicente-Manzanares M, Zareno J, Whitmore L, Choi CK, Horwitz MeshelAS, Wei Q, Adelstein RS, Sheetz MP. (2005). Basic mechanism AF. (2007). Regulation of protrusion, adhesion dynamics, and of three-dimensional collagen fibre transport by fibroblasts. Nat Cell polarity by myosins IIA and IIB in migrating cells. J Cell Biol 176: Biol 7: 157–164. 573–580. Nalbant P, Hodgson L, Kraynov V, Toutchkine A, Hahn KM. (2004). Watanabe N, Kato T, Fujita A, Ishizaki T, Narumiya S. (1999). Activation of endogenous Cdc42 visualized in living cells. Science Cooperation between mDia1 and ROCK in Rho-induced actin 305: 1615–1619. reorganization. Nat Cell Biol 1: 136–143. O’Connor KL, Nguyen BK, Mercurio AM. (2000). RhoA function in Webb DJ, Parsons JT, Horwitz AF. (2002). Adhesion assembly, lamellae formation and migration is regulated by the alpha6beta4 disassembly and turnover in migrating cells—over and over and integrin and cAMP metabolism. J Cell Biol 148: 253–258. over again. Nat Cell Biol 4: E97–E100. Pertz O, Hodgson L, Klemke RL, Hahn KM. (2006). Spatiotemporal Wei Q. (2005). Pitx2a binds to human papillomavirus type 18 E6 dynamics of RhoA activity in migrating cells. Nature 440: protein and inhibits E6-mediated P53 degradation in HeLa cells. 1069–1072. J Biol Chem 280: 37790–37797. Pille JY, Denoyelle C, Varet J, Bertrand JR, Soria J, Opolon P et al. Wei Q, Adelstein RS. (2000). Conditional expression of a truncated (2005). Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation fragment of nonmuscle myosin II-A alters cell shape but not and invasiveness of MDA-MB-231 breast cancer cells in vitro and cytokinesis in HeLa cells. Mol Biol Cell 11: 3617–3627. in vivo. Mol Ther 11: 267–274. Wells CM, Walmsley M, Ooi S, Tybulewicz V, Ridley AJ. (2004). Pille JY, Li H, Blot E, Bertrand JR, Pritchard LL, Opolon P et al. Rac1-deficient macrophages exhibit defects in cell spreading and (2006). Intravenous delivery of anti-RhoA small interfering RNA membrane ruffling but not migration. J Cell Sci 117: 1259–1268. loaded in nanoparticles of chitosan in mice: safety and efficacy in Worthylake RA, Burridge K. (2003). RhoA and ROCK promote xenografted aggressive breast cancer. Hum Gene Ther 17: 1019–1026. migration by limiting membrane protrusions. J Biol Chem 278: Raftopoulou M, Hall A. (2004). Cell migration: Rho GTPases lead the 13578–13584. way. Dev Biol 265: 23–32. Wu D, Asiedu M, Adelstein RS, Wei Q. (2006). A novel guanine Ridley AJ. (2001). Rho GTPases and cell migration. J Cell Sci 114: nucleotide exchange factor MyoGEF is required for cytokinesis. 2713–2722. Cell Cycle 5: 1234–1239. Ridley AJ, Hall A. (1992). The small GTP-binding protein rho Yamana N, Arakawa Y, Nishino T, Kurokawa K, Tanji M, Itoh RE regulates the assembly of focal adhesions and actin stress fibers in et al. (2006). The Rho-mDia1 pathway regulates cell polarity and response to growth factors. Cell 70: 389–399. focal adhesion turnover in migrating cells through mobilizing Apc Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A. (1992). and c-Src. Mol Cell Biol 26: 6844–6858. The small GTP-binding protein regulates growth factor-induced Zigmond SH. (2004). Formin-induced nucleation of actin filaments. membrane ruffling. Cell 70: 401–410. Curr Opin Cell Biol 16: 99–105.

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