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Originally published In Press as doi:10.1074/jbc.M411625200 on January 27, 2005

J. Biol. Chem., Vol. 280, Issue 15, 14675-14683, April 15, 2005
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H-Ras-specific Activation of Rac-MKK3/6-p38 Pathway

ITS CRITICAL ROLE IN INVASION AND MIGRATION OF BREAST EPITHELIAL CELLS*

Ilchung Shin{ddagger}§, Seonhoe Kim{ddagger}§, Hyun Song{ddagger}, Hyeong-Reh Choi Kim¶, and Aree Moon{ddagger}||

From the {ddagger}College of Pharmacy, Duksung Women's University, Seoul 132-714, Korea and the Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201

Received for publication, October 12, 2004 , and in revised form, January 10, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human tumors frequently exhibit constitutively activated Ras signaling, which contributes to the malignant phenotype. Mounting evidence suggests unique roles of the Ras family members, H-Ras, N-Ras and K-Ras, in normal and pathological conditions. In an effort to dissect distinct Ras isoform-specific functions in malignant phenotypic changes, we previously established H-Ras- and N-Ras-activated MCF10A human breast epithelial cell lines. Using these, we showed that p38 kinase is a key signaling molecule differentially regulated between H-Ras and N-Ras, leading to H-Ras-specific induction of invasive and migrative phenotypes. The present study is to further investigate H-Ras- and N-Ras-mediated signaling pathways and to unveil how these pathways are integrated for regulation of invasive/migrative phenotypic conversion of human breast epithelial cells. Here we report that the Rac-MAPK kinase (MKK)3/6-p38 pathway is a unique signaling pathway activated by H-Ras, leading to the invasive/migrative phenotype. In contrast, Raf-MEK-ERK and phosphatidylinositol 3-kinase-Akt pathways, which are fundamental to proliferation and differentiation, are activated by both H-Ras and N-Ras. A significant role for p38 in cell invasion is further supported by the observation that p38 activation by MKK6 transfection is sufficient to induce invasive and migrative phenotypes in MCF10A cells. Activation of the MKK6-p38 pathway results in a marked induction of matrix metalloproteinase (MMP)-2, whereas it had little effect on MMP-9, suggesting MMP-2 up-regulation by MKK6-p38 pathway as a key step for H-Ras-induced invasion and migration. We also provide evidence for cross-talk among the Rac, Raf, and phosphatidylinositol 3-kinase pathways critical for regulation of MMP-2 and MMP-9 expression and invasive phenotype. Taken together, the present study elucidated the role of the Rac-MKK3/6-p38 pathway leading to H-Ras-specific induction of malignant progression in breast epithelial cells, providing implications for developing therapeutic strategies for mammary carcinoma to target Ras downstream signaling molecules required for malignant cancer cell behavior but less critical for normal cell functions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ras proteins are small GTPases that link cell surface receptors to intracellular effector pathways regulating cell proliferation, differentiation, and survival. Human tumors frequently express Ras proteins activated by point mutation; about 20% of all tumors have undergone an activating mutation in one of the ras genes (1). In these tumors, the activated Ras protein contributes to malignant phenotype, including invasiveness and angiogenesis (2). The role of Ras and Ras-dependent signaling pathways in cell invasion and migration, which are crucial events for metastatic process, has been intensively investigated (3–5).

Three members of the Ras family, Harvey-Ras (H-Ras), Kirsten-Ras (K-Ras), and N-Ras, are found to be activated by mutation in human tumors (6). The amino-terminal 85 amino acids are identical, and the middle 80 amino acids contain 85% homology between Ras proteins, whereas the carboxyl-terminal sequence is highly divergent (7–9). Although these Ras proteins share many common signaling pathways leading to similar cellular responses, studies clearly demonstrate unique roles of the Ras family members in normal physiological conditions and pathological conditions. Whereas H-Ras is more transforming than N- or K-Ras in murine fibroblasts, N-Ras is more transforming in human hemopoietic cells (10). Unique functions of the Ras family members at the molecular level are supported by the demonstration that there are differences in the signal transduction pathways induced by Ras proteins (11–13).

The biological effects of Ras proteins are exerted through the activation of several downstream effectors, including Raf, Rac, phosphatidylinositol 3-kinase (PI3K),1 and Ral (14). Ras stimulates serine/threonine kinase Raf, followed by activation of the downstream kinase MAPK/ERK kinase (MEK), which in turn phosphorylates ERKs (15, 16). In addition to the Ras/Raf/ERK pathway, the small GTPase Rac and PI3K are shown to be critical for the mitogenic and oncogenic effect of Ras (17, 18). PI3K is activated either by G-protein-coupled receptors in response to extracellular stimuli (19) or by a direct interaction with Ras (20, 21). Interestingly, studies have demonstrated that the three Ras isoforms can differentially activate effector molecules. K-Ras activates Raf-1 more effectively than H-Ras and N-Ras (11, 22). H-Ras is a more potent activator of PI3K than K-Ras (22), whereas K-Ras activates Rac more efficiently than H-Ras (13).

In an effort to dissect distinct ras isoform-specific pathways critical for malignant phenotypic changes, we previously established a MCF10A human breast epithelial cell system with which the roles of H-Ras and N-Ras can be investigated. Using this, we showed that H-Ras, but not N-Ras, induced invasive and migrative phenotypes, whereas both induced transformed phenotype in human breast epithelial cells, through up-regulation of matrix metalloproteinase (MMP)-2 rather than MMP-9 (23, 24). H-Ras activation of both p38 and ERKs is essential for cell invasion and motility, whereas N-Ras activation of ERKs alone is insufficient, revealing p38 kinase as a key signaling molecule differentially regulated by H-Ras and N-Ras (24). The present study is aimed to further investigate H-Ras- and N-Ras-mediated signal transduction pathways and to unveil how these signaling pathways are integrated for the regulation of invasive/migrative phenotypic conversion of human breast epithelial cells. Here we report that whereas the Raf-MEK-ERK and PI3K-Akt (protein kinase B) pathways are activated by both H-Ras and N-Ras, the Rac-MAP kinase kinase (MKK)3/6-p38 pathway is a unique signaling pathway activated by H-Ras, leading to the invasive/migrative phenotype. We provide evidence that although activation of Raf and PI3K pathways is insufficient to mediate invasive phenotype, these pathways interact with the Rac-MKK3/6-p38 pathway to further induce these phenotypic changes. Thus, our study suggests that the Rac-MKK3/6-p38 pathway is critical for the H-Ras-mediated malignant phenotypic changes in breast epithelial cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines—Development and characterization of MCF10A, H-Ras MCF10A, and N-Ras MCF10A cells have been described previously (23). The cells were cultured in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 5% horse serum, 0.5 µg/ml hydrocortisone, 10 µg/ml insulin, 20 ng/ml epidermal growth factor, 0.1 µg/ml cholera enterotoxin, 100 units/ml penicillin/streptomycin, 2 mM L-glutamine, and 0.5 µg/ml amphotericin B. Stable transfectants of MCF10A expressing the constitutively activated mutant of MKK6 were cultured in MCF10A media containing 400 µg/ml of G418 (Invitrogen).

Transfection—Transfection was performed using Lipofectamine reagent (Invitrogen) following the manufacturer's instructions. A dominant negative mutant of Rac1 (DN Rac1), N17Rac1, was provided by Dr. H. Kim (Seoul National University, Seoul, Korea).

Immunoblot Analysis—Equal amounts of protein extracts in SDS-lysis buffer were subjected to 12% SDS-PAGE analysis and electrophoretically transferred to nitrocellulose membrane. Anti-Akt, anti-phosphorylated Akt, anti-JNK, anti-phosphorylated JNK, anti-ERK-1/2, anti-phosphorylated ERK-1/2, anti-p38, anti-phosphorylated p38, anti-MKK3, anti-phosphorylated MKK3/MKK6, anti-MEK1/2, anti-phosphorylated MEK1/2, and anti-phosphorylated Raf-1 antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-Rac1 and anti-phosphotyrosine antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-PI3K and anti-MKK6 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The ECL system (Amersham Biosciences) was used for detection. Relative band intensities were determined by quantitation of each band with an Image Analyzer (Vilber Lourmet, France).

In Vitro Invasion Assay—An in vitro invasion assay was performed using a 24-well Transwell as previously described (23). The lower side of the filter was coated with type I collagen, and the upper side was coated with Matrigel (Collaborative Research, Lexington, KY). The lower compartment was filled with serum-free media containing 0.1% bovine serum albumin. Cells were placed in the upper part of the Transwell plate, incubated for 17 h, fixed with methanol, and stained with hematoxylin for 10 min followed briefly by eosin. The invasive phenotypes were determined by counting the cells that migrated to the lower side of the filter with microscopy at x400. Thirteen fields were counted for each filter, and each sample was assayed in triplicate.

Wound Migration Assay—Wound migration assay was performed as previously described (24). Briefly, cells were pretreated with mitomycin C (25 µg/ml) for 30 min before the injury line was made. The injury line was made on the cells plated in culture dishes at 90% confluence. After they were rinsed with phosphate-buffered saline, cells were allowed to migrate in complete media, and photographs were taken (x40) at the indicated time points.

In Vitro Migration Assay Using Transwell—An in vitro migration assay was performed using a 24-well Transwell unit with polycarbonate filters. Experimental procedures were the same as for the in vitro invasion assay described above except that the filter was not coated with Matrigel for the migration assay.

Gelatin Zymography—A gelatin zymogram assay was performed as previously described (23). Briefly, cells were cultured in serum-free Dulbecco's modified Eagle's medium/F-12 medium for 48 h. Equal amounts of protein and conditioned media were mixed with 2x Laemmli nonreducing sample buffer, incubated for 15 min at room temperature, and then electrophoresed on 10% SDS-polyacrylamide gels containing 1 mg/ml gelatin. The gels were washed, rinsed and incubated overnight at 37 °C. After the gels were stained and destained, areas of gelatinase activity were detected as clear bands against the blue-stained gelatin background.

Rac1 Activity Assay—The levels of Rac1-GTP were measured by affinity precipitation using PAK-1 p21-binding domain Rac assay reagent (Upstate Biotechnology, Inc., Lake Placid, NY) following the manufacturer's instructions as previously described (25). Briefly, cells were lysed in MLB buffer, and PAK-1-agarose was immediately added to the cell lysate and incubated for 1 h at 4°C. The bead pellet was washed three times with MLB buffer. The bead pellet was finally suspended in Laemmli sample buffer. Proteins were separated by 12% SDS-PAGE, transferred to nitrocellulose membrane, and blotted with anti-Rac1 antibody.

Raf-1 Activity Assay—Cells were lysed for 15 min at 4 °C in a radioimmune precipitation buffer containing 20 mM Tris (pH 8.0), 137 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Nonidet P-40 (NP-40), 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM aprotinin, 20 µM leupeptin, and 5 mM sodium vanadate. The lysate was preincubated with protein A-Sepharose suspension (Roche Applied Science). The supernatant was incubated with anti-Raf-1 antibody. Protein A-Sepharose suspension was added, and incubation was extended overnight on ice with shaking. The antibody-antigen complex was washed twice in lysis buffer and twice in high salt buffer, and then the SDS-PAGE loading buffer was added. Immunoblot analysis was performed to assess the activity of immunoprecipitated Raf-1 with anti-phosphorylated Raf-1 antibody.

PI3K Activity Assay—The PI3K activity was determined as previously described (26). Briefly, cells were lysed in a radioimmune precipitation assay buffer at 4 °C for 30 min. The lysates were centrifuged for 15 min at 12,000 x g to remove debris, immunoprecipitated using anti-phosphotyrosine antibody and immobilized protein G-agarose beads (Pierce) to the antigen-antibody complex, and incubated for 2 h at 4 °C. Immunoprecipitates were washed three times with radioimmune precipitation buffer and denatured in Laemmli sample buffer. The supernatants were resolved by 8% reducing SDS-PAGE. Tyrosine-phosphorylated PI3K proteins were detected by immunoblotting using anti-PI3K antibody.

Immunofluorescence Assay—Cells grown on a coverslip were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.2% Triton X-100. After incubation with SuperBlock (Scy Tek Laboratories, Logan, UT) and avidin for 20 min, the cells were incubated with anti-phosphorylated p38 antibody containing 2% bovine serum albumin and biotin for 1 h at room temperature, followed by incubation with biotinylated conjugated anti-mouse IgG antibody (Molecular Probes, Inc., Eugene, OR). Cells were incubated with fluorescence avidin DCS for 30 min at room temperature. The staining was analyzed by a confocal microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Rac-MKK3/6-p38 Pathway Is Activated by H-Ras but Not by N-Ras—To dissect the signaling pathway from H-Ras to p38, leading to H-Ras-specific invasive and migrative phenotypes in MCF10A cells, we determined whether the H-Ras and N-Ras isoforms differentially regulate Rac activity, since mounting evidence indicates that p38 lies downstream of Rac (27, 28). To directly assess the abilities of H-Ras and N-Ras to activate Rac1, we performed a Rac1 activity assay that specifically recognizes the activated GTP-bound form of Rac1 (29). As shown in Fig. 1A, a prominent activation of Rac1 was induced by H-Ras but not by N-Ras, which is consistent with H-Ras being a strong inducer of MCF10A cell motility. The immunoblots shown are representative of several blots from independent experiments.



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  		FIG. 1.
Rac-MKK3/6-p38 pathway is activated by H-Ras but not by N-Ras. A, Rac1 activity was determined in MCF10A (10A), H-Ras MCF10A (H), and N-Ras MCF10A (N) cell lines. Cell lysates were incubated with GST-p21-bindig domain fusion protein, and the bound active Rac1-GTP molecules were analyzed by immunoblotting using anti-Rac1 antibody. The levels of activated MKK3/6 and p38 were determined by immunoblot analysis using phosphospecific antiMKK3/6 and p38 antibodies (pMKK3/6 and pp38, respectively) and total anti-MKK3/6 and p38 antibodies. WB, Western blotting. B, H-Ras MCF10A cells were transiently transfected with either the control vector or DN Rac1 construct.

 
We next examined the activation of direct upstream kinases of p38, MKK3 and MKK6 (30, 31), in these cells. A marked activation of MKK3 and MKK6 was observed in H-Ras MCF10A cells but not in N-Ras MCF10A cells (Fig. 1A). The phospho-MKK3/6 antibody recognizes both phosphorylated MKK3 and phosphorylated MKK6 with indistinguishable mobilities (32). To investigate whether H-Ras activation of MKK3/6 is Rac-dependent, we inhibited the Rac1 pathway using a DN Rac1 construct (33). As shown in Fig. 1B, transfection of DN Rac1 effectively inhibited Rac1 activation in H-Ras MCF10A cells. In order to determine the transient transfection efficiency, we cotransfected H-Ras MCF10A cells with control or DN Rac1 expression vector together with green fluorescent protein plasmid, followed by observation under a fluorescent microscope (data not shown). The number of total cells and the fluorescent cells were counted, and transfection efficiency was determined as the ratio of the fluorescent cell number to the total cell number. The transfection efficiencies were 71.7 and 73.8% for vector transfection and DN Rac1 transfection, respectively. Importantly, DN Rac1-mediated Rac1 inactivation markedly inhibited the activation of MKK3/6 in H-Ras MCF10A cells, demonstrating that Rac1 activity is crucial for H-Ras-induced MKK3/6 activation in MCF10A cells. These data show that the Rac-MKK3/6-p38 signaling pathway is uniquely activated by H-Ras.

The Raf-MEK-ERK Pathway Is Shared by H-Ras and N-Ras—Our previous and the present studies have shown that ERKs are activated by both H-Ras and N-Ras, whereas p38 activation is H-Ras-specific, showing that MAPK members are differentially activated between H-Ras and N-Ras. Since Raf-1 connects the Ras signal to the MAPK pathway, which regulates fundamental cellular processes including proliferation, transformation, and differentiation (34, 35), we asked whether H-Ras and N-Ras differentially regulate Raf-1 and its downstream effector molecule, MEK, and if the Raf-1 pathway interacts with the Rac1 pathway. As shown in Fig. 2A, Raf-1 and MEK were activated in both H-Ras and N-Ras MCF10A cells, showing that the Raf-MEK-ERK pathway is shared by H-Ras and N-Ras in MCF10A human breast epithelial cells. We then investigated the regulation of MEK by Rac1 in H-Ras MCF10A cells. As shown in Fig. 2B, phosphorylation of MEK was not altered by DN Rac1 transfection in H-Ras MCF10A cells. The result demonstrates that the Rac1 pathway is not involved in H-Ras activation of MEK in MCF10A cells.



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  		FIG. 2.
Raf-MEK-ERK pathway is activated by H-Ras and NRas. A, Raf-1 activity was examined in the parental (10A), H-Ras(H), and N-Ras (N) MCF10A cells. Cell lysates were immunoprecipitated (IP) with anti-Raf-1 antibody and probed with phosphorylated Raf-1 antibody. WB, Western blotting. Activation of MEK and ERKs was determined by immunoblot analysis using phosphospecific MEK and ERK-1/2 antibodies. B, Rac1 activity and the active form of MEK were detected in H-Ras MCF10A cells transfected with the control vector or DN Rac1 construct.

 
Both H-Ras and N-Ras Activate the PI3K Pathway in MCF10A Cells—It has been reported that Ras activates PI3K by direct binding, which in turn stimulates Akt (36, 37). To examine whether the PI3K pathway is differentially activated between H-Ras and N-Ras, we examined the activation of PI3K and its primary signal mediator, Akt (38, 39), in H-Ras and N-Ras MCF10A cells. PI3K activity and the phosphorylated Akt level were increased in both of these cell lines (Fig. 3A), indicating that the PI3K pathway is effectively activated by both H-Ras and N-Ras in MCF10A cells.



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  		FIG. 3.
Both H-Ras and N-Ras activate PI3K pathway in MCF10A cells. A, PI3K activity was detected in the parental (10A), H-Ras (H), and N-Ras (N) MCF10A cells. Cell lysates were immunoprecipitated (IP) with anti-phosphotyrosine antibody, and the immunoprecipitates were subjected to immunoblot analysis with anti-PI3K antibody. WB, Western blotting. Activation of Akt was determined by immunoblot analysis of whole cell lysates using antibodies against phosphospecific Akt (pAkt) and total Akt. B, immunoblot analysis for detecting the activation of PI3K, Akt, MKK3/6, and MEK was performed on whole cell lysates from H-Ras MCF10A cells treated with 10 µM LY294002 (LY) for 30 min. Control cells were treated with Me2SO.

 
To examine the role of the PI3K pathway in the activation of MKK3/6 and MEK in H-Ras MCF10A cells, we detected the levels of phosphorylated forms of these molecules upon inhibition of PI3K pathway by a chemical inhibitor of PI3K, LY294002. When the H-Ras MCF10A cells were treated with 10 µM LY294002 for 30 min, the levels of active PI3K and Akt were significantly decreased, confirming that the treatment effectively inhibited the PI3K pathway in H-Ras MCF10A cells (Fig. 3B). Phosphorylated forms of neither MKK3/6 nor MEK were affected when the PI3K pathway was abolished by LY294002, indicating that the activation of MKK3/6 and MEK was independent of the PI3K pathway.

Rac1 Lies Upstream of PI3K—The signaling order between Rac and PI3K has been reported to be controversial in different cell systems. To evaluate the significance of the signal cross-talk between H-Ras-activated Rac1 and PI3K in H-Ras MCF10A cells, we examined the effect of Rac1 inhibition on PI3K activation and vice versa. As shown in Fig. 4A, inhibition of the Rac pathway by DN Rac1 markedly reduced the levels of active PI3K as well as active Akt in H-Ras MCF10A cells. In contrast, the level of the active Rac1 (Rac1-GTP) was not affected when the PI3K pathway was inhibited by treatment with 10 µM LY294002 for 30 min (Fig. 4B). The data reveal that Rac1 lies upstream of PI3K in the H-Ras signaling pathway of human breast epithelial cells.



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  		FIG. 4.
Rac1 lies upstream of PI3K in H-Ras MCF10A cells. A, H-Ras MCF10A cells were transiently transfected with either the control vector or DN Rac1 construct. Activation of PI3K and Akt in the transfectants was examined as described in the legend to Fig. 3. B, H-Ras MCF10A cells were treated with 10 µM LY294002 (LY) for 30 min. PI3K and Rac1 activities were determined as described in the legends for Fig. 3 and Fig. 1, respectively. C, N-Ras MCF10A cells were transiently transfected with either the control vector or DN Rac1 construct. Rac1 activity and activation of Akt were examined as described in the legend to Fig. 1 and 3, respectively.

 
In order to compare the role of the Rac1 pathway in Akt activity between H-Ras- and N-Ras MCF10A cells, we also transfected the N-Ras MCF10A cells with a DN Rac1 mutant. As shown in Fig. 4C, the basal level of Rac1 activity was further inhibited by DN Rac1 in N-Ras MCF10A cells. Interestingly, however, DN Rac1 had little effect on activation of Akt in N-Ras MCF10A cells, whereas it markedly down-regulated Akt activity in H-Ras MCF10A cells (Fig. 4A). These results suggest that whereas the activation of Akt by H-Ras signaling heavily depends on Rac1 activity, N-Ras-induced Akt activation is likely to be mediated through other signaling molecule(s) rather than Rac1.

Rac1 Is Critical for H-Ras Activation of p38 and ERKs—To evaluate the significance of the Rac pathway in H-Ras-activated MAPK family members, we examined the effects of Rac1 inhibition in the H-Ras-induced MAPKs. As shown in Fig. 5A, phosphorylated forms of p38 and ERKs were significantly decreased when the Rac1 pathway was blocked by DN Rac1 transfection, whereas activation of JNK was not significantly affected by DN Rac1. These data suggest that Rac1 is a critical component for the maximum activation of ERKs and p38 in H-Ras MCF10A cells.



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  		FIG. 5.
Rac1 activity is critical for H-Ras-induced activation of ERKs and p38, invasion, migration, and MMP-2/9 up-regulation. H-Ras MCF10A cells were transiently transfected with either the control vector or DN Rac1 construct. A, activated JNK, ERK-1/2 and p38 in transfectant cells were detected by immunoblot analysis of whole cell lysates using antibodies for phosphospecific MAPKs and total MAPKs. B, the transfected cells were subjected to in vitro invasion assay. The number of invaded cells per field was counted (x400) in 13 fields. The results represent means ± S.E. of triplicates. **, statistically different from control at p < 0.01. C, migratory ability of the H-Ras MCF10A cell transfectants was determined by wound migration assay. Cellular migration was observed with light microscope (x40) at the indicated time points. The width of the injury line from three independent experiments was measured and plotted as a percentage of the width at 0 h. The results presented were means ± S.E. of triplicates. D, conditioned media of the transfectants were subjected to immunoblot analysis for detecting secreted levels of MMP-2 (72 kDa) and MMP-9 (92 kDa).

 
H-Ras-induced Invasion, Motility, and MMP-2/9 Up-regulation Are Rac1-dependent—To evaluate the functional significance of the Rac pathway in the H-Ras-mediated invasive phenotype of MCF10A cells, we performed an in vitro invasion assay. Transfection with DN Rac1 significantly reduced the number of invaded cells by 49.5% (Fig. 5B). We also evaluated the role of the Rac pathway in the migratory property of H-Ras MCF10A cells by wound migration assay. H-Ras MCF10A cell migration was significantly inhibited (0 versus 51.4%) by DN Rac1 transfection after 12 h (Fig. 5C). DN Rac1 transfection did not lead to a significant reduction in cell viability compared with the transfection of control vector (data not shown), indicating that DN Rac1-mediated inhibition of H-Ras MCF10A cell invasion/motility was not due to a cytotoxic effect. The results demonstrate that activation of the Rac pathway by H-Ras is critical for H-Ras-induced breast epithelial cell migration and invasion.

Invasive phenotype of cancer cells is often associated with increased expression of MMP-2 and/or MMP-9, which can degrade type IV collagen, the major structural collagen of the basement membrane (40). We previously showed that H-Ras activation of both p38 and ERKs induced MMP-2 and MMP-9 expression, resulting in the invasive phenotype of MCF10A cells (24). Since the present study showed that the Rac pathway is critical for H-Ras-mediated invasion, we asked if Rac1 mediates up-regulation of MMP-2 and/or MMP-9 expression by H-Ras. To this end, conditioned medium of H-Ras MCF10A cells transfected with DN Rac1 was subjected to immunoblot analysis. Blocking the Rac pathway by DN Rac1 markedly decreased the protein levels of MMP-2 and MMP-9 in H-Ras MCF10A cells (Fig. 5D). The data suggest the requirement of the Rac pathway in H-Ras-induced MMP-2 and MMP-9 up-regulation in MCF10A cells.

PI3K Is Required for H-Ras-mediated p38 and ERK Activation, Resulting in Invasive and Migrative Phenotypic Changes via Increased Expression of MMP-2/9—Since PI3K and Akt have been reported to promote the invasive phenotype in many cell systems (41, 42), we evaluated whether the PI3K pathway plays a role in H-Ras-induced human breast epithelial cell invasion and migration. Inhibition of the PI3K pathway by 10 µM LY294002 for 30 min down-regulated ERKs and p38 without affecting JNK activation (Fig. 6A). Treatment with LY294002 significantly decreased invasion and migration of H-Ras MCF10A cells, as shown in Fig. 6, B and C, respectively. This treatment was not cytotoxic (data not shown), indicating that the inhibition of invasion and motility was not due to the cytotoxic effect of LY294002. Given that PI3K and Akt are activated not only in H-Ras MCF10A cells but also in noninvasive/nonmigrative N-Ras MCF10A cells (Fig. 3A), these results suggest that the activation of the PI3K pathway is required but may not be sufficient for H-Ras-induced invasion and motility.



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  		FIG. 6.
PI3K is required for H-Rasinduced activation of ERKs and p38, invasion, migration, and MMP-2/9 upregulation. A, immunoblot analysis was performed on whole cell lysates from HRas MCF10A cells treated with 10 µM LY294002 (LY) for 30 min. The levels of activated JNK, ERK-1/2, and p38 were determined by immunoblot analysis. B, H-Ras MCF10A cells pretreated with 10 µM of LY294002 for 30 min were subjected to in vitro invasion assay as described in the legend to Fig. 5B. *, statistically different from control at p < 0.05. C, migratory ability of the H-Ras MCF10A cells treated with 10 µM of LY294002 was determined by a wound migration assay as described in the legend to Fig. 5C. D, conditioned media of the H-Ras MCF10A cells treated with various concentrations of LY294002 for 48 h were subjected to immunoblot analysis for detecting secreted levels of MMP-2 (72 kDa) and MMP-9 (92 kDa).

 
To determine the functional role of PI3K activation in H-Ras-induced up-regulation of MMP-2 and MMP-9, a dose-response study of immunoblot analysis was conducted on the conditioned medium of H-Ras MCF10A cells treated with various concentrations of LY294002 for 48 h. The treatment resulted in a significant down-regulation of MMP-2 and MMP-9 in a dose-dependent manner (Fig. 6D), demonstrating the critical role for the PI3K pathway in the regulation of MMP-2 and MMP-9 expression in H-Ras MCF10A cells.

MKK6 Renders MCF10A Cells Invasive and Motile—Our data (Fig. 5), along with the previous results (24), suggest that the Rac-MKK3/6-p38 signaling pathway, activated by only H-Ras, may be responsible for H-Ras-specific induction of invasive and migrative phenotypes in MCF10A cells. To validate these results obtained by using a chemical inhibitor and DN mutant constructs of Rac1 and p38, we performed a gain-of-function experiment. We tested whether the activation of p38, the end signaling molecule of the Rac-MKK3/6-p38 pathway, can confer invasive and migrative abilities in MCF10A cells, which are originally noninvasive and nonmigrative. By transfecting MCF10A cells with a constitutively active mutant of MKK6, a direct upstream activator of p38, we established two stable transfectant MCF10A cell lines, MKK6-MCF10A1 (M1) and MKK6-MCF10A2 (M2).

MKK6 induced changes in the MCF10A cell morphology from cuboidal to an elongated spindle-like shape, a typical morphology of mesenchymal-like cells that is similar to the morphology of H-Ras MCF10A cells (Fig. 7A). A prominent activation of MKK6 and p38 in M1 and M2 cells was confirmed (Fig. 7B). The levels of phosphorylated MKK6 and p38 in M1 cells were lower than those in M2 or H-Ras MCF10A cells. Phosphorylated p38 in M2 cells was comparable with that in H-Ras MCF10A cells, demonstrating that both H-Ras and MKK6 are equally potent activators of p38. Other MAPKs, ERKs and JNKs, were not significantly induced in these cells, indicating the selective activation of p38 by MKK6 in MCF10A cells. We then performed an immunofluorescence assay to examine the activation and subcellular localization of phosphorylated p38 in these transfectant cells. As shown in Fig. 7C, activated p38 was detected in M1 and M2 cells as well as in H-Ras MCF10A cells but neither in the control vector-transfected MCF10A cells nor in the N-Ras MCF10A cells. Phosphorylated p38 localized along the lateral edges and leading edges (arrows in Fig. 7C), suggesting the potential involvement of activated p38 in cell migration. Indeed, MKK6-activated p38 resulted in the induction of invasive and migrative phenotypes in MCF10A cells (Fig. 7D). Of note, H-Ras exerted a stronger induction of invasion and migration compared with MKK6 in MCF10A cells. Invasive and migrative abilities of M1 cells were lower than those of M2 cells (29 versus 58% for invasion and 65 versus 82% for migration in M1 and M2 cells, respectively).



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  		FIG. 7.
MKK6 renders MCF10A cells invasive and motile. A, cell morphology was observed under light microscopy (x100). B, MKK6, p38, ERK-1/2, and JNK-1/2 were detected in MCF10A cells transfected with control vector (Vector), MKK6 (M1 and M2), and H-Ras MCF10A cells (H-ras) by immunoblot analysis of whole cell lysates using antibodies for phosphospecific MAPKs and total MAPKs. C, immunofluorescence assay was performed on the cells for activated p38 with anti-phosphorylated p38 antibody. The immunostained cells were observed under a confocal microscope. The arrows indicate subcellular localization of phosphorylated p38. D, left, in vitro invasion assay. The number of invaded cells per field was counted (x400) in 13 fields. The results represent means ± S.E. of triplicates. Right, Transwell migration assay. The number of migrated cells was counted in 13 microscopic fields (x400). The results presented were means ± S.E. of triplicates. * and **, statistically different from control at p < 0.05 and at p < 0.01, respectively. E, a gelatin zymogram assay was performed to analyze the gelatinolytic activity of secreted MMP-2 (72 kDa) and MMP-9 (92 kDa). F, activation of MKK6 and p38 in N-Ras MCF10A cells transfected with either control vector or MKK6 was examined by immunoblot analysis. In vitro invasion assay and in vitro migration assay were performed on the N-Ras MCF10A cells transfected with control vector or MKK6. **, statistically different from control at p < 0.01.

 
We then examined the effect of MKK6 activation on MMP-2 and MMP-9 expression by gelatin zymogram assay. As shown in Fig. 7E, a marked induction of MMP-2 was detected in both M1 and M2 cells with a higher expression in M2 cells than in M1 cells. The level of MMP-2 expression in M2 cells was comparable with that detected in H-Ras MCF10A cells, indicating that MKK6-p38 is an efficient signaling pathway for MMP-2 up-regulation in MCF10A cells. Interestingly, MKK6-activated p38 had little effect on MMP-9, whereas H-Ras enhanced expression of both MMP-2 and MMP-9. These results indicate that MKK6-p38 up-regulation of MMP-2 alone can induce invasion and migration in MCF10A cells, but less effectively than H-Ras activation, suggesting that besides MKK6-p38, other pathway(s) activated by H-Ras, such as ERKs and PI3K, are required for the maximum induction of invasive/migrative phenotypes in human breast epithelial cells. This is consistent with our previous finding that p38 and MMP-2 are critical for the H-Ras-induced invasive/migrative phenotype, whereas H-Ras-induced activation of ERKs involving MMP-9 up-regulation is necessary for the completion of H-Ras-induced invasion and migration of human breast epithelial cells.

In order to investigate whether the activation of MKK6 induces the invasive/migrative phenotypes in N-Ras MCF10A cells, we transiently transfected the N-Ras MCF10A cells with the active mutant of MKK6 and examined invasive and migrative properties. Activation of MKK6 and p38 in N-Ras MCF10A cells transfected with the active MKK6 construct was confirmed (Fig. 7F). Invasion and migration were significantly induced by MKK6 transfection in N-Ras MCF10A cells, demonstrating that the activation of MKK6 signaling pathway can confer invasive and migrative phenotypes in the N-Ras MCF10A cell line.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The Ras proteins play central roles in controlling signal pathways that regulate normal cellular proliferation and malignant transformation. Thus, inhibition of general Ras activities can be detrimental not only to cancer cells but also to normal cells. Rational therapies that target Ras downstream signaling molecules essential for malignant cancer cell behavior, but less critical for normal cell functions, would therefore have a potential impact (43). In an effort to identify distinct signaling molecules required for H-Ras-specific invasion and migration in MCF10A human breast epithelial cells, we have investigated the signal pathways selectively activated by H-Ras and those commonly regulated by H- and N-Ras. Here we show that oncogenic H-Ras signaling is mediated by the Rac-MKK3/6-p38 pathway in breast epithelial cells, whereas the Raf-MEK-ERK and PI3K-Akt pathways are common signaling pathways activated by H-Ras and N-Ras.

The small GTP-binding protein Rac promotes actin cytoskeletal reorganization, leading to membrane ruffling, lamellipodia formation, cell migration, and invasion (44, 45). Our previous finding of enhanced motility induced by H-Ras (24) suggested that H-Ras might be a more effective activator of the Rac pathway compared with N-Ras in MCF10A cells. Consistently, a marked activation of Rac1 was exerted by H-Ras but not by N-Ras in MCF10A cells (Fig. 1A). Activation of p38 has been shown to activate MAPK-activated protein kinase-2 and -3 and consequently phosphorylate heat shock protein 27 (HSP27), which modulates F-actin polymerization and cytoskeletal remodeling (46). Besides its effect on the actin cytoskeleton, p38 has also been reported to modulate microtubule organization by activating stathmin, a microtubule destabilizer protein (47, 48). Paxillin, a focal adhesion-associated adaptor protein involved in adhesion and cell migration (49), is phosphorylated by p38 in the neurite extension of PC-12 cells (50). Our study clearly revealed the subcellular localization of the phosphorylated p38 in the lateral edges and leading edges of MKK6activated MCF10A cells (Fig. 7C). Further studies need to be performed to identify the active substrate(s) for p38, leading to focal adhesion and migration of MCF10A cells.

The first mammalian effector of Ras to be characterized is Raf (43, 51). Raf-MEK-ERK pathway can promote cell cycle progression by mediating Ras-induced cell survival and proliferation (35). PI3K has emerged as a crucial facilitator of Ras function (52) to regulate cell survival, cell cycle entry, and the structure of the actin cytoskeleton (53). Promotion of cell survival by Ras proceeds through both the Raf-MEK-ERK and PI3K-Akt pathways (54, 55), and the cross-talk between these pathways was demonstrated in the MCF-7 human breast cancer cell line (56, 57). The present study showed that these two pathways were activated not only in H-Ras MCF10A cells but also in N-Ras MCF10A cells, unlike the Rac-MKK3/6-p38 pathway, which was activated solely by H-Ras. These findings suggest that the Raf-MEK-ERK and PI3K-Akt pathways, which are fundamental to general Ras activities, including cell survival and proliferation, are insufficient for mediating malignant cell progression of breast epithelial cells.

Rac has been demonstrated to lie both upstream and downstream of PI3K in different systems. Several studies suggest that PI3K acts upstream of Rac1 in a pathway for membrane ruffling and chemotaxis (53, 58). Rac1 has been reported to mediate signaling from PI3K to p38, and the resulting PI3KRac-p38 pathway is involved in inducing the malignant phenotype of signet-ring cell carcinoma (59). Conversely, the activated form of Rac1 was shown to act upstream of PI3K to increase cellular motility and invasiveness (60, 61). Activation of Rac1 disrupted the normal polarization of mammary epithelial cells and promoted motility and invasion through PI3K (60). Our data showed that the PI3K pathway was dependent on Rac1 activity (Fig. 4A), whereas Rac1 activity was not affected by PI3K inhibition (Fig. 4B), suggesting that Rac1 may lie upstream of PI3K in H-Ras-activated MCF10A cells. Given that Ras has been shown to interact directly with the catalytic subunit of PI3K (20, 62), it remains to be investigated how Rac1 and PI3K signaling pathways are integrated in H-Ras MCF10A cells.

Ras is thought to be localized to the inner surface of the plasma membrane to be biologically active. Interestingly, however, recent studies demonstrate that Ras is also activated on and transmits signals from the endoplasmic reticulum and Golgi apparatus, suggesting that the plasma membrane may not be the exclusive platform from which Ras regulates signaling (63, 64). The interaction of Ras with its effectors is mediated by the effector binding loop, which spans residues 32–40. Despite the fact that the amino acid sequence corresponding to the effector binding loop is identical among Ras proteins, recent studies have demonstrated that the three Ras isoforms can differentially activate the effector molecules. These hierarchies appear to result, at least in part, from differences in the mechanisms of membrane attachment of the three Ras isoforms (22, 65). The hypervariable domain is highly divergent with the exception of the last four amino acids (the CAAX motif), which are required for post-translational processing and plasma membrane association (11, 66). Recently, a considerable body of evidence has suggested that functional differences among the Ras isoforms could be due to variations in plasma membrane microlocalization. Differential activation of Raf-1 and PI3K by K-Ras and H-Ras (22) may be explained by the differences in the microdomain localization of these Ras proteins (22, 67, 68). Mutation of the H-Ras C terminus changed effector pathway utilization (65), suggesting a role of the lipidated C terminus in the biological functions of Ras proteins. These studies demonstrate that the localization of Ras proteins to different microdomains of the plasma membrane may be critical for signaling specificity. Here, we observed that H-Ras and N-Ras differentially regulated Rac1 activity, whereas they did not vary in their abilities to activate Raf-1 and PI3K in MCF10A cells. Detailed comparison of microlocalization of H-Ras and N-Ras has not been elucidated thus far. It would be of importance to investigate whether differential microlocalization in plasma membrane accounts for the distinct regulation of the Rac1 signaling by the highly homologous H-Ras and N-Ras proteins.

Based on our previous findings (23, 24) and the observations shown in this study, a working model is proposed for H-Rasunique and H-Ras/N-Ras overlapping pathways leading to the invasive phenotype of MCF10A human breast epithelial cells (Fig. 8). We reveal the Rac-MKK3/6-p38 pathway as a unique signaling pathway activated only by H-Ras, resulting in phenotypic conversion of noninvasive MCF10A cells to an invasive phenotype. Raf-MEK-ERK and PI3K-Akt pathways, which are fundamental to proliferation, differentiation, and transformation, are shared signaling pathways activated by both H-Ras and N-Ras. Interestingly, our data showed that DN Rac1 transfection inhibited the activation of ERK (Fig. 5A) but not that of its upstream activator, MEK (Fig. 2B). Similarly, treatment with LY294002, an inhibitor of PI3K, resulted in the inhibition of ERK (Fig. 6A) but not MEK (Fig. 3B). These findings suggest that MEK may not be the sole activator of ERK in the H-Ras MCF10A cell system, and PI3K may also be critical for maximum activation of ERK as proposed in Fig. 8.



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  		FIG. 8.
A proposed model for the H-Ras-unique and H-Ras/NRas overlapping pathways in MCF10A cells.

 
It should be noted that Rac1 is required for both MMP-2 and MMP-9 induction (Fig. 5D), whereas MKK6 and p38 activate MMP-2 only (Fig. 7E). A plausible explanation is that the role of Rac1 in H-Ras-induced MMP-9 up-regulation may be mediated by PI3K and ERK pathway, which are shown to be downstream signaling molecules of Rac1 (Figs. 4A and 5A). Tumor cell invasion is a complex process involving cell motility and extracellular matrix-degrading proteinase activity. Undoubtedly, multiple gene products are required for invasive activity. The present study showed that MMP-2 up-regulation by MKK6-p38 pathway is a key step for H-Ras-induced invasion and migration (Fig. 7, D and E), whereas H-Ras-induced MMP-9 is necessary for the completion of H-Ras-induced invasion of human breast epithelial cells.

Induction of MMP-9 expression has been reported to be mediated by Ras-ERK and PI3K-Akt pathways, in which the transcription factors NF-{kappa}B and AP-1 are involved (69, 70). The signaling pathways regulating MMP-2 expression have not been fully elucidated. Recently, it has been reported that insulin-like growth factor-I up-regulates MMP-2 expression via PI3K-Akt signaling while concomitantly transmitting a negative regulatory signal via the Raf-ERK pathway in lung carcinoma cells (71). Given that up-regulation of MMP-2 rather than MMP-9 plays a key role in the H-Ras-induced invasive phenotype of MCF10A breast epithelial cells (24), our findings that reveal MKK6-p38 as the signaling pathway responsible for an efficient induction of MMP-2 would be of importance in understanding the malignant phenotypic conversion of breast epithelial cells at the molecular level. Delineation of MMP-2 regulation may have implications for development of therapeutic strategies to arrest breast cancer cell invasion. MMP-2 activity can be regulated by gene expression, proenzyme activation, and inhibition of enzyme activity by tissue inhibitor of metalloproteinase-2. Using 5'-deletional MMP-2 reporter constructs, it has been revealed that the transcriptional factors Sp1, Sp3, and AP-2 are required for MMP-2 gene expression in astroglioma cells (72). We are currently investigating the sequence requirements and transcription factors for transcriptional activation of the MMP-2 gene by the Rac-MKK3/6-p38 pathway in H-Ras MCF10A cells and MKK6-activated MCF10A (M1 and M2) cells.

Taken together, we show that Rac and MKK3/6 mediate H-Ras activation of p38, leading to H-Ras-specific cell invasive and migrative phenotypes of MCF10A human breast epithelial cells. Our findings may contribute to the development of therapeutic interventions that target the Ras signaling molecules required for the malignant cell behavior but less critical for normal cell functions.


   FOOTNOTES
 
* This work was supported by Basic Research Program of the Korea Science and Engineering Foundation Grant R04-2003-000-10063-0 and Korea Research Foundation Grant for Leading Researchers 041E00036 (to A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These two authors contributed equally to this work. Back

|| To whom correspondence should be addressed. Tel.: 82-2-901-8394; Fax: 82-2-901-8386; E-mail: armoon{at}duksung.ac.kr.

1 The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase kinase; MEK, MAPK/ERK kinase; MMP, matrix metalloproteinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal protein kinase; DN, dominant negative. Back



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