- Original Articles
- Open Access
Rho Family GTPases Regulate Mammary Epithelium Cell Growth and Metastasis Through Distinguishable Pathways
© Picower Institute Press 2001
- Accepted: 31 October 2001
- Published: 1 December 2001
Relatively few genes have been shown to directly affect the metastatic phenotype of breast cancer epithelial cells in vivo. The Rho family of proteins, including the Rho, Rac and Cdc42 subfamilies, are related to the small GTP binding protein Ras and regulate diverse biological processes including gene transcription, cytoskeletal organization, cell proliferation and transformation. The effects of Cdc42, Rac and Rho on the actin cytoskeleton suggested a possible role for Rho proteins in cellular motility and metastasis, however a formal analysis of the role of Rho proteins in breast cancer cellular growth and metastasis in vivo had not previously been performed.
Materials and Methods
We generated a panel of MTLn3 rat mammary adenocarcinoma cells that expressed similar levels of dominant inhibitory mutants of Cdc42-, Rac- and Rho-dependent signaling, to examine the contribution of these GTPases to cell spreading, guided chemotaxis, and metastasis in vivo. The ability of Rho proteins to regulate intravasation into the peripheral blood was determined by implanting MTLn3 cell stable dominant negative lines in nude mice and measuring the formation of breast cancer cell colonies grown from the peripheral blood. Serial sectioning of the lungs was performed to determine the presence of metastasis in mice in which mammary tumors expressing the dominant negative Rho family proteins had grown to a similar size.
Cell spreading of MTLn3 cells was selectively abrogated by N17Rac1. N19RhoA and N17Cdc42 reduced the number of focal contacts (FCs) and disrupted the colocalization of vinculin with phosphotyrosine at FCs. While N17Rac1 and N17Cdc42 preferentially inhibited colony formation in soft agar, all three GTPases affected cell growth in vivo. To distinguish effects on tumorigenicity from intravasation into the bloodstream, implanted tumors were grown to the same size in nude mice. Each dominant inhibitory Rho protein reduced intravasation into the peripheral blood. Lung metastasis of MTLn3 cells was also abrogated by the dominant inhibitory Rho proteins, despite the presence of residual CFU.
These studies demonstrate for the first time a critical role for the Rho GTPases involving independent signaling pathways to limit mammary tumor cellular growth and metastasis in vivo.
Rho family proteins are closely related to the small GTP binding protein Ras (1). Rho GTPases cycle between an active GTP-bound state and an inactive GDP-bound state to transduce diverse signals from cell surface receptors to intracellular targets (1). Rho proteins regulate a diverse spectrum of biological processes including regulation of gene transcription, superoxide production, changes in cytoskeletal organization, cell proliferation and transformation (1–4). Analyses performed predominantly in fibroblasts, employing microinjection of dominant-active or dominant-negative mutants, demonstrated that specific Rho proteins regulate distinct functions (1). The functional specificity of Rho GTPases is transduced, at least in part, by an interaction with specific effector proteins that coordinate the activation of multiple signaling cascades (1). The regulation of the actin cytoskeleton and specialised cellular adhesion structures is also quite selective for distinct members of the Rho family. Thus, activated RhoA stimulates stress fiber formation (5), Rac1 controls growth factor-induced lamellipod formation in Swiss 3T3 fibroblasts (2) and Cdc42 regulates filopodial protrusion (6,7)
The effects of Cdc42, Rac and Rho on the actin cytoskeleton and cellular adhesion suggested a possible role for Rho proteins in cellular motility. A role for Rho proteins in migration of fibroblasts and neutrophils was demonstrated using the C3 exoenzyme and Rho-GDI (GDP dissociation inhibitor) to block Rho function (8,9). In Bac1.2F5 macrophages, inhibition of Rac and Rho function blocked cell migration in response to CSF-1, while a dominant inhibitory Cdc42 enhanced cell migration (10). Rac activity is also required for PDGF-BB-induced migration of Rat1 cells across a porous membrane (11). In T47D mammary epithelial cells, activated Rac1 and Cdc42, but not RhoA, enhanced cell migration across filters coated with collagen (12), Cellular migration through increasingly complex surfaces such as a three dimensional (3D) collagen gel requires distinct properties of Rho proteins (13). Invasion of 3D collagen matrices by Rat1 cells was inhibited by dominant negative mutants of Cdc42 and Rac1 indicating fibroblast invasion requires optimal level of activity of multiple Rho family members (13). The process of cellular metastasis in vivo is a still more complex process involving several distinct phases including invasion through tissue structures, intravasation and survival in the peripheral blood and the ability to adhere at a distant site. It is predicted that Rho GTPases might regulate mammary epithelial cell invasion and metastasis in vivo. The contribution of the Rho proteins to in vivo mammary tumor bioassays such as tumor blood burden and lung metastasis is therefore of fundamental importance (14,15). As the role of Rac, Rho and Cdc42 to the components governing metastasis in vivo had not previously been formally assessed, we employed a syngeneic model to address this question.
In this study we employed MTLn3 cells that were originally derived from a lung metastasis of the 13762 rat mammary adenocarcinoma (16,17). The effects of Epithelial Growth Factor (EGF) on guided chemotaxis have been extensively studied in the MTLn3 cells. EGF, through binding to its receptor, functions as a chemoattractant for a number of different cell types and is thought to enhance the migration and invasion of tumor cells (18–21). EGF-mediated chemotaxis of MTLn3 cells was shown to include actin polymerization at the leading edge of the lamellipod to enable cellular extension and motility (22–24). In this study, we used MTLn3 cells stably expressing Rho inhibitory proteins to examine the role of the Rho family GTPases in coordinating cytoskeletal reorganization and in the regulation of metastasis. We demonstrate for the first time separable signaling pathways regulated by Rho GTPases that together contribute to in vivo mammary epithelial tumor metastasis.
Cell Culture, Proliferation and Transformation Assays
Cell culture was performed as previously described (24). Cells were grown in a-MEM (Gibco), supplemented with 5% fetal calf serum and antibiotics. For all experiments, unless otherwise mentioned, MTLn3 cells were plated in regular medium for 24 h at low density on tissue culture dishes (Falcon), or MaTek dishes (MaTek Corporation, Ashland, MA), which had been previously coated for 2 h at room temperature with rat tail collagen type 1 (Collaborative Biomedical, Bedford, MA) at 30 µg/ml in DPBS (Gibco). MTLn3 cells were starved for 3 h prior to the experiment in α-MEM medium supplemented with 0.35% bovine serum albumin (BSA) and 12 mM HEPES (starvation medium). Stimulation was done with a final concentration of 5 nM murine EGF (Life Technologies) in starvation medium.
The plasmids pEXV, Myc-N17Rac1, Myc-N19RhoA (25), pCMV5, and Flag-N17Cdc42 (26) have been previously described. MTLn3 cells were stably transfected with the epitope-tagged Rho family GTPase dominant negative mutants and pCNeo, selected using G418 (800 ng/ml) and maintained in G418 (400 ng/ml). Expression of the mutant Rho proteins was confirmed by western blot of the epitope tag. For cellular proliferation assays, the MTLn3 cells encoding Rho family inhibitory proteins, or the parental line containing the empty vector control, (pEXV, pCMV), were plated in 6-well tissue culture plates. After six days growth in regular culture medium (α-MEM, 5% FBS) cells were trypsinized, harvested and counted using a hemocytometer. The soft agar growth assays was performed as previously described (27–29). MTLn3 stable cells (3 × 104 cells) were suspended in 3 ml of α-MEM containing 5% FBS and 0.33% SeaPlaque low-melting-temperature agarose (FMC Bioproducts). The suspension was plated on 60 mm dishes containing a 2 ml layer of solidified α-MEM, 5% FBS and 0.5% SeaPlaque agarose, in quadruplicate. The cells were allowed to settle at the interface between these layers for 30 min at 37°C. Cells were fed every 3 days by overlaying with 2 ml of complete medium containing 0.33% SeaPlaque agarose. After 15 days, the plates were examined and the colonies were counted under a Nikon Phase contrast microscope at 4× or 6× magnification. Experimental values represent the average number of foci in the 60 mm plates for each experimental condition; error bars represent the observed SEM between the 4 plates.
Immunofluorescence and Rhodamine-Phalloidin Staining of Filamentous Actin (F-actin)
Cells were seeded onto 22 mm square glass cover slips (Becton Dickinson, Bedford, MA) coated with rat tail collagen type 1 (27 µg/ml) in 6-well tissue culture plates. When 70–80% confluent, cells were rinsed with 1 × PBS at 37°C, fixed in 3.7% formaldehyde in buffer F (5 mM KCl, 137 mM NaCl, 4 mM NaHCO3, 0.4 mM KH2PO4, 1.1 mM Na2HPO4, 2 mMMgCl2, 5 mM PIPES, pH 7.2, 2 mM EGTA, 5.5 mM glucose) for 5 min at 37°C, extracted in 0.5% Triton X-100 in buffer F for 20 min at room temperature and washed in 0.1 M glycine in buffer F for 10 min at room temperature. The cover slips were washed 5 times for 5 min in 1 × TBS, wicked dry and placed on Parafilm in a humidified chamber and 150 ml of 1% BSA, 10% goat serum in 1 × TBS with rhodamine-phalloidin (0.5 mM) (Molecular Probes, Eugene, OR) added for 20–30 min. Excess rhodamine-phalloidin was washed from the cover slips, 5 times for 5 min with 1% BSA in 1 × TBS and, if no immunofluorescent staining was carried out, the cover slips were mounted in Prolong reagent (Molecular Probes, Eugene, OR). For immunofluorescent staining, the cells were incubated at room temperature for 60 min with primary antibody after blocking. The cover slips were then washed 3 times for 10 min in 1% BSA in 1 × TBS and incubated with secondary antibody for 45–60 min at room temperature before a final series of three 10 min washes in 1% BSA in 1 × TBS. If two primary antibodies were used, the antibodies were added sequentially and each directly followed by incubation with their respective secondary antibodies. The cover slips were mounted as before and examined under an Olympus 1 × 70 inverted microscope with images recorded using a Photometrics CH1 cooled CCD camera.
Antibodies used included an anti-phosphotyrosine (P112300 polyclonal antibody, Transduction Labs), anti-vinculin (V4505, Sigma), anti-Flag antibody (Sigma, (M2)), and anti-Myc (9E10, Santa Cruz, mouse monoclonal).
Chemotactic responses were assessed using the 48-well chemotaxis chamber (Neuroprobe, Cabin, MD, USA) following the manufacturers instructions. A Nucleopore filter with 8 µm pores (Osmonics/Poretics Products, Livermore, CA, USA) was coated with rat tail collagen type 1 at a final concentration of 35.6 µg/ml in DPBS without calcium or magnesium (JRH Biosciences) for 2 hours. MTLn3 cells were cultured in MEM medium with 5% FBS and 0.5% Penicillin/Streptomycin on 10 cm plates. Medium was aspirated and replaced with growth medium containing 12 mM HEPES pH 7.4 and BSA 0.35% in MEM (termed MEMH) without serum or antibiotics for 2 hours. Cells were then harvested with 26.6 mM EDTA in DPBS, resuspended in MEMH and counted. The lower wells of the chamber were filled with 30 µl MEMH containing appropriate concentration of EGF (Life Technologies) or buffer, then the chamber was assembled incorporating the collagen-coated filter. The stock of EGF was prepared in filtered DPBS. The upper wells were then filled with MEMH containing 20,000 cells/well in a total volume of 50 µl. The chamber was incubated at 37°C for 3 h, disassembled and the upper side of the filter scraped to remove cells that had not traveled through the filter. The filters were fixed in 3.7% formaldehyde in DPBS for 30 min, washed twice in water and stained for 12–18 h in hematoxylin. The filters were then rinsed in water and mounted for viewing. Results are means ± SEM from 5- to 8 separate experiments.
Lamellipod Extension and Spreading Assays
Cells were seeded onto 22 mm square collagen-coated glass cover slips in 6-well tissue culture plates and treated or not with 5 nM EGF for 3 min. Cell were fixed in 3.7% formaldehyde, extracted in 0.5% Triton X-100 and mounted. The extension of lamellipodia was assessed as previously described by us (22,28). All samples were examined under an Olympus 1 × 70 inverted microscope with images recorded using a Photometrics CH1 cooled CCD camera. A total of 300 cells were examined.
For the spreading assays, MTLn3 cells were plated (104 cells/well) on a collagen coated (27 µg/ml) 6-well plate. At several time points after plating, the cells were viewed using phase contrast and a 10× objective. The number of spread cells was counted by visual inspection using a 5 × 5 grid. Cells that turned phase-dense during the spreading process and displayed at least one protrusion were counted as spread. A total of 300 cells were counted in five different fields that were selected at random. To observe the F-actin in MTLn3 cells, the cells were fixed and stained with FITC-phalloidin after plating for various times. The data were expressed as mean ± SEM for percentages of positive cells in the five fields.
Immunoprecipitation western blotting was performed as previously described using either the Flag or Myc antibodies (30,31). The abundance of Fak, Talin, and Vinculin proteins was determined by western analysis as previously described (32,33), using anti-Vinculin antibody (Sigma), anti-focal adhesion kinase (FAK) antibody (F15020 polyconal antibody, Transduction Labs), guanine nucleotide dissociation inhibitor (GDI) antibody (Acknowledgments, 25), and anti-Talin monoclonal antibody (8d4, Sigma). Cell homogenates (50 µg) were separated in an SDS-12% polyacrylamide gel and transferred electrophoretically to a nitrocellulose membrane (Micron Separations Inc., Westborough, MA). After transfer, the gel was stained with Coomassie blue as a control for blotting efficiency. The blotting membrane was incubated for 2 h at 25°C in T-PBS (PBS + 0.05% Tween 20) buffer supplemented with 5% (wt/vol) dry milk to block non-specific binding sites. Following 6 h incubation with primary antibody at a 1:5000 dilution (GDI) in T-PBS buffer containing 0.05% (vol/vol) Tween 20, the membrane was washed with the same buffer. The membrane was incubated with goat anti-mouse horseradish peroxidase second antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and washed again. The protein was visualized by the enhanced chemiluminescence system (Kirkegaard and Perry Laboratories, Gaithersburg, MD).
In Vivo Tumor Growth, Blood Burden, and Lung Metastasis Assay
To determine the effect of dominant negative N17Rac, N19Rho and N17Cdc42 on MTLn3 cell metastasis, parental and stably transfected MTLn3 cells were injected into the subcutaneous tissue of 6- to 8-week old nude mice (strain Balb/cAnNCr-nuBR, National Cancer Institute) as previously described (25,34). Experiments were conducted under an approved protocol of the AECOM animal ethics committee. Cells were trypsinized, counted, washed twice and resuspended in ice-cold sterile PBS. Nude mice were anesthetized lightly with Methofane (Schering-Plough Animal Health) and injected with 106 viable cells in 0.1 ml of PBS. Tumors were palpated every 3- to 4 days after the first week and measured with calipers (mean tumor diameter) to quantitate tumor size. Animals were euthanized 5- to 8.5 weeks after cells were injected. Tumors growing at the site of injection were measured and then removed and rinsed in ice-cold PBS. Samples of tumor tissue were rapidly frozen in liquid nitrogen for further analysis.
Tumor cell blood burden was determined as previously described (35) by placing nude mice with a 5 week old tumor under methofane anesthesia and removing 1 ml of blood via heart puncture. The blood was then spun at 5,000 rpm and the serum layer and buffy coat region were plated into α-MEM growth medium. The following day, plates were rinsed 2× with Dulbeco’s PBS (Gibco) to remove red blood cells and regular growth medium (as above) was added. After six days, all clones in the dish were counted. For experiments in which the Rho family dominant inhibitory MTLn3 cells were grown to the same size as vector controls, the subcutaneous tumors were grown for 8.5 weeks.
For measurement of metastasis, excised lungs were placed in 3.7% formaldehyde, mounted in paraffin, sectioned and stained with H&E. Serial slices of 5 µM thickness were methodically viewed throughout the entire lung with sections viewed using a 20× objective and all visible metastases in a section containing more than 5 cells were counted in each section.
Effects of Rho Proteins on EGF-induced Stress Fiber Formation
Tyrosine Phosphorylation of Focal Contacts Involves Rho and Cdc42
Cell Proliferation and Anchorage-independent Growth in MTLn3 Expressing Inhibitory Rho GTPases
Cell Spreading Assays
Effects of Rho GTPases on the Tumorigenicity, Blood Burden and Lung Metastasis of MTLn3 Cells
Tumorigenicity and blood burden in nude mice. The inhibitory Rho family MTLn3 lines were assessed for growth 5 weeks after implantation into nude mice. The mean tumor size ± SEM of at least 3 separate experiments is shown. Colony forming assays were performed on the peripheral blood of nude mice 5 weeks after implantation with MTLn3 lines encoding the dominant inhibitory Rho proteins or control lines.
5 Weeks Growth
Tumor Size Diameter (cm) (Mean ± SEM)
Cells in Blood (range)
Lung Metastasis (Mean ± SEM)
1.36 ± 0.13
26 ± 4.74
1.57 ± 0.23
21 ± 1.56
0.49 ± 0.06
A quantitation of lung metastasis was next performed in animals implanted with the MTLn3 vector control and Rho dominant negative cell lines. At 5 weeks post implantation, all MTLn3 control cells (pEXV, pCDNA3) had formed widespread lung metastatic foci (Fig. 7A). In contrast, none of the cell lines expressing dominant inhibitory Rho GTPases formed detectable macro foci, even though the N19RhoA lines were able to produce detectable tumor burdens in the peripheral blood.
Summary of the role of Rho GTPases derived from analyses of the dominant inhibitory MTLn3 lines. The ability to regulate function completely (>90%, triple arrow), partially (40–70%, double arrow), modestly (10–30% single arrow) or not detected (none).
Focal contacts and
Primary tumor growth
Colony formation (CFU) (cells in blood)
Foci formation (Soft agar)
Several Rho family members have been shown to be essential for Ras transformation (1). Previous studies have identified specific roles for Rho family proteins in cultured cells, although cell type-specific differences have been observed. Different models have been proposed for the relationship between Cdc42, Rac and Rho. Hierarchical interactions have been identified between Rho family members, providing evidence for a “cascade” model (52), regulating the cytoskeleton in Swiss 3T3 fibroblasts, with Cdc42 activating Rac, which in turn activates Rho (7). In contrast with this model, an “antagonistic model” has been proposed in which Cdc42/Rac and Rho function in an antagonistic manner. For example Rac can downregulate Rho activity (53) and inactivation of Rho in neuroblastoma cells leads to neurite extension which is inhibited by either Cdc42-N17 or Rac1N17 (54). A “convergent” pathway model was proposed to define interactions between Rho proteins in several other cell types (49), for example, during integrin-mediated signaling (49,55,56). In fibroblasts, several studies suggested that Ras, Rac, and Rho function in a cascade relationship of interdependency (7), while other studies suggested “convergent” (49,57) or perhaps “independent” pathways to explain the relationships between these proteins in other cell types. Finally, the Rho GTPases have important transcriptional functions in which Rac, Rho and Cdc42 act largely independently of each other (1). Although the current studies suggest that Rac, Rho and Cdc42 each contribute independently to the metastatic phenotype in vivo, further studies are required to distinguish whether the Rho GTPases function in a “cascade” or parallel pathway in mammary epithelial cells.
The role of Rho family members in defining mammary tumor cell morphology was assessed by immunofluorescence of the MTLn3 cytostructural proteins in the stable dominant negative lines. Rho and Cdc42 contributed to the co-localization of pTyr and vinculin without altering the total abundance of vinculin in the cell. pTyr was observed in a more dispersed pattern in cells expressing dominant negative RhoA (Fig. 2D vs. 2A). The loss of pTyr and vinculin co-staining in the N19RhoA MTLn3 cells is consistent with observations that quiescent cells with low Rho activity adhere and spread on ECM, but do not form well developed focal adhesions (58,59). Focal contacts are one form of cell adhesion in cultured cells and contain anchor and cytoskeletal molecules including vinculin, paxillin and talin (36,38), together with signal transduction molecules such as focal adhesion kinase (FAK) (39).
The assembly and tyrosine phosphorylation of FCs depends upon actomyosin contractility and is regulated by cytoplasmic factors, including Rho, or microtubular integrity (37,40,42–45,60). FCs exhibit characteristically high levels of tyrosine phosphorylation (36). Vinculin, a major structural component of FCs, co-localizes at cell-matrix adhesions with pTyr in both primary human fibroblasts (61) and in the MTLn3 cells. Rho-induced FC formation requires a functional cellular contractile apparatus. Inhibiting intracellular contractile forces promotes disassembly and prevents FC formation even though cells remain adherent to the substratum (44,62). Rho is required for complete phosphorylation of FAK induced by matrix adhesion (49), although FAK is phosphorylated at multiple sites and some Rho-independent FAK phosphorylation also occurs upon integrin aggregation (63). The observation that inhibition of Rac signaling did not interfere with pTyr and vinculin co-localization is consistent with findings that FAK phosphorylation and paxillin tyrosine phosphorylation are Rho-dependent and Rac-independent in Rat1 cell fibroblasts (49). The reduction in vinculin staining at FCs in the N17Cdc42 lines suggests that Cdc42 activity contributes to FC formation in MTLn3 cells as previously described in fibroblasts.
The current studies demonstrated a critical role for Rac1 in MTLn3 cell spreading and protrusion. These findings of diminished cell spreading in MTLn3 cells expressing dominant negative Rac1 are consistent with studies of spreading on fibronectin which was shown to be selectively Rac-dependent in T lymphocytes (64) and in fibroblasts (49). The directional migration of Rat1 cells towards PDGF-BB, LPA or fibronectin was blocked by N17Rac1 and not by dominant negative mutants of RhoA or Cdc42 (11,13). Inhibition of spreading by the dominant inhibitory Rac1 was associated with a modest increase in the number of FCs which were PTyr containing. These findings are consistent with observations in NR6 fibroblasts in which FC disassembly was associated with EGF-enhanced migration into an acellular area (46). Rac1 is activated by a variety of tyrosine kinase receptors including platelet-derived growth factor (PDGF), EGF and insulin (65). The adhesion and spreading of T cells provides a more streamlined shape reducing shear imposed upon them by vascular flow and may have analogous functions for mammary epithelial cells. The current studies in MTLn3 mammary adenocarcinoma cells, together with previous studies in Swiss 3T3 and endothelial cells, suggest that the role of Rac1 in cell spreading is well conserved between cell types.
Guided-cell migration in response to a chemotactic gradient involves processes that are distinct from random protrusions and motility (66,67). During guided chemotaxis, focal adhesion disassembly occurs both at the trailing and the leading edge. MTLn3 cells demonstrate classical ameboid chemotaxis on a planar surface in response to EGF (24). Herein, dominant inhibitory Rac1 and Cdc42 reduced EGF-induced guided chemotaxis 50%, and N19RhoA by 90%. Rho function is necessary to establish classical focal adhesion allowing stress fibers to anchor to the exctracellular surface (59). Smaller adhesion complexes at the periphery of the cell are induced by activation of Rac and Cdc42 (7). As the relative abundance of the N19RhoA and N17Rac1 in the stable lines was similar by western blotting, and Rac preferentially regulated MTLn3 cellular spreading, these findings suggest that individual GTPases play distinct roles in spreading compared with guided chemotaxis.
Analysis of Rho family proteins in anchorage-independent growth demonstrated that Cdc42 and Rac function contributes to mammary adenocarcinoma cellular proliferation and growth in soft agar. Although Rho played a dominant role in guided chemotaxis, the contribution of Rho to cellular proliferation and contact-independent growth of the MTLn3 cells assessed in soft agar assays was relatively modest. This is similar to the situation in fibroblasts (47,48,68–71). The induction of anchorage independence by oncogenic Ras however requires Rho, Rac and Cdc42 (47,48,71) and inhibition of Rho as well as Rac and Cdc42 can inhibit G1 phase progression in other cell types (72). The PAK kinases may serve as Rac and Cdc42 effectors involved in integrin-mediated ERK activation and integrin-mediated adhesion is required for efficient coupling of Rac1 to PAK1 (73). Overexpression of activated Rac and Cdc42 can bypass this need and induce anchorage-independent ERK activity (69,74) and expression of the cyclin A gene, a key regulator of S phase progression (68–70). The enhancement of G1 phase progression and cellular growth by Rac appears to be strongly related to the induction of the cyclin D1 gene. Activating mutations of Rac promote DNA synthesis and activate the cyclin D1 promoter directly (28,75) and inhibition of Rac or Cdc42 block cyclin D1 expression and contact independent growth induced by oncogenic Neu (25). The modest effect of blocking Rho on MTLn3 growth in soft agar, compared with Rac1N17 and Cdc42, may reflect the relatively greater importance of Rac/Cdc42 in contact-independent growth of mammary adenocarcinoma cells.
The process of metastasis to the lungs involves several independent processes including migration, invasion of tissues, intravasation and growth. Inhibition of Rac, or Cdc42 blocked growth of MTLn3 cells in nude mice significantly better than N19RhoA at 5 weeks. When tumors were grown to a similar size, each of the dominant inhibitory Rho family members significantly reduced the number of colonies detectable in the peripheral blood by more than 90%. Invasion of tissues may involve induction of proteases through Rho family protein-dependent mechanisms including induction of cytokines such as IL-1α which thereby activates collagenase-1 gene expression (76).
It is clear from a number of studies that Rho family proteins play important but distinguishable roles in cell growth and invasion of cellular matrix (1). Although Rho proteins had been postulated to regulate cellular metastasis, the requirement for Rho proteins in regulating breast cancer metastasis in vivo using a heterologous system had not previously been determined. Dominant negative mutants of Rho family proteins block serum-induced DNA synthesis in Swiss 3T3 cells (72). Rat1 transformation induced by activating mutants of ErbB-2 is also blocked by dominant inhibitory Rho proteins (25). Despite the importance of the EGF receptor and its related family members to mammary epithelial cell growth, the role of Rho proteins in EGF receptor-dependent growth is poorly understood, particularly in the mammary epithelium. MTLn3 cells proliferate in response to EGF or serum (77), and inhibition of either Cdc42 or Rac, but not Rho activity, significantly reduced cellular proliferation (Fig. 5) and growth in soft agar (Fig. 6). An activating mutant of Rac1, but not Cdc42, enhanced cell growth in the absence of serum in Rat1 cells (48) further suggesting that each RhoA protein family member regulates growth in a distinct manner. Our studies using MTLn3 cells suggest an important role for Rac and Cdc42 in MTLn3 cellular proliferation and growth in soft agar.
Taken together, the in vitro studies in MTLn3 cells showed that Rho family GTPases are differentially employed in signaling cell morphology changes, motile functions and aspects of growth that correlate with tumorigenesis and metastasis. Primary mammary tumor size was reduced at 5 weeks by each of the dominant inhibitory Rho family proteins. When tumors were grown to the same size, the reduced intravasation assessed by blood burden, suggested that entry into, and survival in, the peripheral blood is regulated by each of the Rho GTPases examined and is not simply a function of reduced tumor mass. These findings are consistent with recent studies in which RhoC was shown to play an important role in the metastastatic behaviour of melanoma cells (78). The events governing entry into the peripheral blood and survival may include resistance to shear forces, deformability, apoptosis in the absence of substratum and intravasation at the primary site (35). While the role of the Rho family proteins in these events remains to be determined, the N17Rac1 MTLn3 cells were found to have enhanced sensitivity to osmolar stress (RGP, BB, unpublished). In conclusion these experiments establish, for the first time, a critical role for Rho proteins in the regulation of mammary tumor growth and blood burden.
This work was supported in part by RO1CA70897 and RO1CA75503 (to RGP) and awards from the Susan G. Komen Breast Cancer Foundation and Breast Cancer Alliance Inc. RGP is a Monique Weill-Caulier and Irma T. Hirschl Scholar. Work conducted at the Albert Einstein College of Medicine was supported by Cancer Center Core National Institutes of Health grant 5-P30-CA13330-26. MPL is supported by NIH R01-CA-80250. The authors acknowledge the Analytical Imaging Facility and Michael Cammer for skillful help on microscopy and image analysis. We thank Dr. Perry Bickel, Washington University, St Louis, MO, for a generous gift of guanine nucleotide dissociation inhibitor (GDI) antibody.
- Van Aelst L, D’Souza-Schorey C. (1997) Rho GTPases and signaling networks. Genes Dev. 11: 2295–2322.View ArticlePubMedGoogle Scholar
- Ridley AJ, Paterson HF, Johnston CL, et al. (1992) The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70: 401–410.View ArticlePubMedGoogle Scholar
- Ridley AJ. (1994) Membrane ruffling and signal transduction. Bioessays 16: 321–327.View ArticlePubMedGoogle Scholar
- Evers EE, Zondag GCM, Malliri A, et al. (2000) Rho family of proteins in cell adhesion and cell migration. Eur. J. of Cancer 36: 1269–1274.View ArticleGoogle Scholar
- Ridley AJ, Hall A. (1992) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389–399.View ArticlePubMedGoogle Scholar
- Kozma R, Ahmed S, Best A, Lim L. (1995) The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. Mol. Cell. Biol. 15: 1942–1952.View ArticlePubMedPubMed CentralGoogle Scholar
- Nobes CD, Hall A. (1995) Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81: 53–62.View ArticlePubMedGoogle Scholar
- Takaishi K, Kikuchi A, Kuroda S, et al. (1993) Involvement of rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in cell motility. Mol. Cell. Biol. 13(1): 72–79.View ArticlePubMedPubMed CentralGoogle Scholar
- Stasia M-J, Jouan A, Bourmeyster N, et al. (1991) ADP-ribosylation of a small size GTP-binding protein in bovine neutrophils by the C3 exoenzyme of Clostridium botulinum and effect on the cell motility. Biochem. Biophys. Res. Comm. 180: 615–622.View ArticlePubMedGoogle Scholar
- Allen WE, Zicha D, Ridley AJ, Jones GE. (1998) A role for cdc42 in macrophage chemotaxis. J. Cell. Biol. 141: 1147–1157.View ArticlePubMedPubMed CentralGoogle Scholar
- Anand-Apte B, Zetter BR, Viswanathan A, et al. (1997) Platelet-derived growth factor and fibronectin-stimulated migration are differentially regulated by the Rac and extracellular signal-regulated kinase pathways. J. Biol. Chem. 272: 30688–30692.View ArticlePubMedGoogle Scholar
- Keely PJ, Westwick JK, Whitehead IP, et al. (1997) Cdc42 and Rac1 induce integrin-mediated cell motility and invasiveness through PI (3) K. Nature 390: 632–636.View ArticlePubMedGoogle Scholar
- Banyard J, Anand-Apte B, Symons M, Zetter BR. (2000) Motility and invasion are differentially modulated by Rho family GTPases. Oncogene 19: 580–591.View ArticlePubMedGoogle Scholar
- Condeelis JS, Wyckoff JB, Bailly M, et al. (2001) Lamellipodia in Invasion. Seminars in Cancer Biology Apr 11: 119–128.View ArticleGoogle Scholar
- Liotta LA, Steeg PS, Stetler-Stevenson WG. (1991) Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell 64: 327–336.View ArticlePubMedGoogle Scholar
- Neri A, Welch D, Kawaguchi T, Nicolson GL. (1982) Development and biologic properties of malignant cell sublines and clones of a spontaneously metastasizing rat mammary adenocarcinoma. J. Natl. Cancer Inst. 68: 507–517.PubMedGoogle Scholar
- Welch DR, Neri A, Nicolson GL. (1983) Comparison of ‘spontaneous’ and ‘experimental’ metastasis using rat 13762 mammary adenocarcinoma metastatic cell clones. Invasion Metastasis 3(2): 65–80.PubMedGoogle Scholar
- Khazaie K, Schirrmacher V, Lichtner RB. (1993) EGF receptor in neoplasia and metastasis. Cancer Metastasis Rev. 12: 255–74.View ArticlePubMedGoogle Scholar
- Pedersen PH, Ness GO, Engebraaten O, et al. (1994) Heterogeneous response to the growth factors [EGF, PDGF (bb), TGF-alpha, bFGF, IL-2] on glioma spheroid growth, migration and invasion. Int. J. Cancer 56(2): 255–261.View ArticlePubMedGoogle Scholar
- Grotendorst GR, Soma Y, Takehara K, Charette M. (1989) EGF and TGF-alpha are potent chemoattractants for endothelial cells and EGF-like peptides are present at sites of tissue regeneration. J. Cell Physiol. 139(3): 617–623.View ArticlePubMedGoogle Scholar
- Blay J, Brown KD. (1985) Epidermal growth factor promotes the chemotactic migration of cultured rat intestinal epithelial cells. J. Cell Physiol. 124: 107–112.View ArticlePubMedGoogle Scholar
- Segall JE, Tyerich S, Boselli L, et al. (1996) EGF stimulates lamellipod extension in metastatic mammary adenocarcinoma cells by an actin-dependent mechanism. Clin. Exp. Metastasis 14: 61–72.View ArticlePubMedGoogle Scholar
- Chan A, Raft S, Bailly M, et al. (1998) EGF stimulates actin nucleation at the tip of the lamellipod in mammary adenocarcinoma cells. J. Cell. Science 111: 199–211.PubMedGoogle Scholar
- Bailly M, Yan L, Whitesides GM, et al. (1998) Regulation of protrusion shape and adhesion to the substratum during chemotactic responses of mammalian carcinoma cells. Exp. Cell Res. 241: 285–299.View ArticlePubMedGoogle Scholar
- Lee RJ, Albanese C, Fu M, et al. (2000) Cyclin D1 is required for transformation by activated Neu and is induced through an E2F-dependent signaling pathway. Mol. Cell. Biol. 20: 672–683.View ArticlePubMedPubMed CentralGoogle Scholar
- Clarke N, Arenzana N, Hai T, et al. (1998) Epidermal growth factor induction of the c-jun promoter by a Rac pathway. Mol. Cell. Biol. 18(2): 1065–1073.View ArticlePubMedPubMed CentralGoogle Scholar
- Westwick JK, Lee RJ, Lambert QT, et al. (1998) Transforming potential of Dbl family proteins correlates with transcription from the cyclin D1 promoter but not with activation of Jun NH2-terminal kinase, p38/Mpk2, serum response factor, or c-Jun. J. Biol. Chem. 273: 16739–16747.View ArticlePubMedGoogle Scholar
- Westwick JK, Lambert QT, Clark GJ, et al. (1997) Rac regulation of transformation, gene expression and actin organisation by multiple, PAK-independent pathways. Mol. Cell. Biol. 17: 1324–1335.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang W, Razani B, Altschuler Y, et al. (2000) Caveolin-1 inhibits EGF-stimulated lamellipod extension and cell migration in metastatic mammary adenocarcinoma cells (MTLn3).Transformation suppressor effects of adenoviral-mediated gene delivery of caveolin-1. J. Biol. Chem. 275: 20717–20725.View ArticlePubMedGoogle Scholar
- Fu M, Wang C, Reutens AT, et al. (2000) p300 and P/CAF acetylate the androgen receptor at sites governing hormone-dependent transactivation. J. Biol. Chem. 275: 20853–20860.View ArticlePubMedGoogle Scholar
- Bouzahzah B, Fu M, Iaavarone A, et al. (2000) Transforming growth factor β1 recruits histone deacetylase 1 to a p130 repressor complex in trangenic mice in vivo. Cancer Res. 60: 4531–537.PubMedGoogle Scholar
- Albanese C, Johnson J, Watanabe G, et al. (1995) Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J. Biol. Chem. 270: 23589–23597.View ArticlePubMedGoogle Scholar
- Watanabe G, Lee RJ, Albanese C, et al. (1996) Angiotensin II (AII) activation of cyclin D1-dependent kinase activity. J. Biol. Chem. 271: 22570–22577.View ArticlePubMedGoogle Scholar
- Galbiati F, Volonte D, Engelman JA, et al. (1998) Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and activate the p42/p44 MAP kinase cascade. EMBO J. 17: 6633–6648.View ArticlePubMedPubMed CentralGoogle Scholar
- Wyckoff JB, Jones JG, Condeelis JC, Segall JE. (2000) A critical step in metastasis: in vivo analysis of intravasation at the primary tumor. Cancer Res. 60: 2504–2511.PubMedGoogle Scholar
- Burridge K, Turner CE, Romer LH. (1992) Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J. Cell Biol. 119: 893–903.View ArticlePubMedGoogle Scholar
- Jockusch BM, Bubeck P, Giehl K, et al. (1995) The molecular architecture of focal adhesions. Annu. Rev. Cell Dev. Biol. 11: 379–416.View ArticlePubMedGoogle Scholar
- Yamada KM, Geiger B. (1997) Molecular interactions in cell adhesion complexes. Curr. Opin. Cell Biol. 9: 76–85.View ArticlePubMedGoogle Scholar
- Yamada KM, Miyamoto S. (1995) Integrin transmembrane signaling and cytoskeletal control. Curr. Opin. Cell Biol. 7: 681–689.View ArticlePubMedGoogle Scholar
- Zamir E, Katz B-Z, Aota S-I, et al. (1999) Molecular diversity of cell-matrix adhesions. J. Cell Sci. 1129: 1655–1669.Google Scholar
- Helfman DM, Levy ET, Berthier C, et al. (1999) Caldesmon inhibits nonmuscle cell contractility and interferes with the formation of focal adhesions. Mol. Biol. Cell. 10: 3097–3112.View ArticlePubMedPubMed CentralGoogle Scholar
- Pelham RJ, Wang YL. (1997) Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl. Acad. Sci. USA 94: 13661–13665.View ArticlePubMedGoogle Scholar
- Craig SW, Johnson RP. (1996) Assembly of focal adhesions: progress, paradigms, and portents. Curr. Opin. Cell Biol. 8: 74–85.View ArticlePubMedGoogle Scholar
- Chrzanowska-Wodnicka M, Burridge K. (1996) Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J. Cell Biol. 133: 1403–1415.View ArticlePubMedGoogle Scholar
- Bershadsky A, Chausovsky A, Becker E, et al. (1996) Involvement of microtubules in the control of adhesion-dependent signal transduction. Curr. Biol. 6: 1279–1289.View ArticlePubMedGoogle Scholar
- Xie H, Pallero MA, Gupta K, et al. (1998) EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of the motility-associated PLC (gamma) signaling pathway. J. Cell Science 111(5): 615–624.PubMedGoogle Scholar
- Qiu R-G, Chen J, McCormick F, Symons M. (1995) A role for Rho in Ras transformation. Proc. Natl. Acad. Sci. USA 92: 11781–11785.View ArticlePubMedGoogle Scholar
- Qiu R-G, Abo A, McCormick F, Symons M. (1997) Cdc42 regulates anchorage-independent growth and is necessary for Ras transformation. Mol. Cell. Biol. 17: 3449–3458.View ArticlePubMedPubMed CentralGoogle Scholar
- Clark EA, King WG, Brugge JS, et al. (1998) Integrin-mediated Signals Regulated by Members of the Rho Family of GTPases. J. Cell Biol. 142: 573–586.View ArticlePubMedPubMed CentralGoogle Scholar
- Lichtner RB, Kaufmann AM, Kittmann A, et al. (1995) Ligand mediated activation of ectopic EGF receptor promotes matrix protein adhesion and lung colonization of rat mammary adenocarcinoma cells. Oncogene 10: 1823–1832.PubMedGoogle Scholar
- Kaufmann AM, Lichtner RB, Schirrmacher V, Khazaie K. (1996) Induction of apoptosis by EGF receptor in rat mammary adenocarcinoma cells coincides with enhanced spontaneous tumour metastasis. Oncogene 13: 2349–2358.PubMedGoogle Scholar
- Chant J, Stowers L. (1995) GTPase cascades choreographing cellular behavior: movement, morphogenesis, and more. Cell 81(1): 1–4.View ArticlePubMedGoogle Scholar
- Sander EE, ten Klooster JP, van Delft S, et al. (1999) Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior. J. Cell Biol. 147: 1009–1022.View ArticlePubMedPubMed CentralGoogle Scholar
- Kozma R, Sarner S, Ahmed S, Lim L. (1997) Rho family GT-Pases and neuronal growth cone remodeling: relationship between increased complexity induced by Cdc42Hs, Rac1 and acetylcholine and collapse induced by RhoA and lysophosphatidic acid. Mol. Cell. Biol. 17(3): 1201–1211.View ArticlePubMedPubMed CentralGoogle Scholar
- King WG, Mattaliano MD, Chan TO, et al. (1997) Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation. Mol. Cell Biol. 17: 4406–4418.View ArticlePubMedPubMed CentralGoogle Scholar
- Clark EA, Hynes RO. (1996) Ras activation is necessary for integrin-mediated activation of extracellular signal-regulated kinase 2 and cytosolic phospholipase A2 but not for cytoskeletal organization. J. Biol. Chem. 271: 14814–14818.View ArticlePubMedGoogle Scholar
- Rodriguez-Viciana P, Warne PH, Khwaja A, et al. (1997) Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by ras. Cell 89: 457–467.View ArticlePubMedGoogle Scholar
- Ben-Ze’ev A, Geiger B. (1998) Differential molecular interactions of beta-catenin and plakoglobin in adhesion, signaling and cancer. Curr Opin Cell Biol. 10: 629–639.View ArticlePubMedGoogle Scholar
- Hotchin NA, Hall A. (1995) The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases. J. Cell. Biol. 131: 1857–1865.View ArticlePubMedGoogle Scholar
- Burridge K, Chrzanowska-Wodnicka M, Zhong CL. (1997) Focal adhesion assembly. Trends Cell Biol. 7: 342–347.View ArticlePubMedGoogle Scholar
- Katz BZ, Zamir E, Bershadsky A, et al. (2000) Physical State of the Extracellular Matrix Regulates the Structure and Molecular Composition of Cell-Matrix Adhesions. Mol. Bio. Cell 11(3): 1047–1060.View ArticleGoogle Scholar
- Volberg T, Geiger B, Citi S, Bershadsky AD. (1994) Effects of protein-kinase inhibitor H-7 on the contractility, integrity, and membrane anchorage of the microfilament system. Cell Motil. Cytoskel. 29: 321–338.View ArticleGoogle Scholar
- Miyamoto S, Akiyama SK, Yamada KM. (1995) Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science 267: 883–885.View ArticlePubMedGoogle Scholar
- D’Souza-Schorey C, Boettner B, Van Aelst L. (1998) Rac Regulates Integrin-Mediated Spreading and Increased Adhesion of T Lymphocytes. Mol. Cell Biol. 18: 3936–3946.View ArticlePubMedPubMed CentralGoogle Scholar
- Kjoller L, Hall A. (1999) Signaling to Rho GTPases. Exp. Cell Research 253: 166–179.View ArticleGoogle Scholar
- Aznavoorian S, Stracke ML, Parsons J, et al. (1996) Integrin alphav-beta3 mediates chemotactic and haptotactic motility in human melanoma cells through different signaling pathways. J. Biol. Chem. 271: 3247–3254.View ArticlePubMedGoogle Scholar
- Huttenlocher A, Ginsberg MH, Horwitz AF. (1996) Modulation of cell migration by integrin-mediated cytoskeletal linkages and ligand-binding affinity. J. Cell Biol. 134: 1551–1562.View ArticlePubMedGoogle Scholar
- Gjoerup O, Lukas J, Bartek J, Willumsen BM. (1998) Rac and Cdc42 are potent stimulators of E2F-dependent transcription capable of promoting retinoblastoma susceptibility gene product hyperphosphorylation. J. Biol. Chem. 273: 18812–18818.View ArticlePubMedGoogle Scholar
- Aplin AE, Juliano RL. (1999) Integrin and cytoskeletal regulation of growth factor signaling to the MAP kinase pathway. J. Cell Sci. 112(5): 695–706.PubMedGoogle Scholar
- Philips A, Roux P, Coulon V, et al. (2000) Differential effect of rac and cdc42 on p38 kinase activity and cell cycle progression of nonadherent primary mouse fibroblasts. J. Biol Chem. 275(8): 5911–5917.View ArticlePubMedGoogle Scholar
- Qiu R-G, Chen J, Kirn D, et al. (1995) An essential role for Rac in Ras transformation. Nature 374: 457–459.View ArticlePubMedGoogle Scholar
- Olson MF, Ashworth A, Hall A. (1995) An essential role for Rho, Rac and Cdc42 GTPases in cell cycle progression through G1. Science 269: 1270–1272.View ArticlePubMedGoogle Scholar
- Coniglio SJ, Jou T-S, Symons M. (2001) Rac1 protects epithelial cells against anoikis. J. Biol. Chem. 276: 28113–28120.View ArticlePubMedGoogle Scholar
- del Pozo MA, Price LS, Alderson NB, et al. (2000) Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK. EMBO J. 19: 2008–2014.View ArticlePubMedPubMed CentralGoogle Scholar
- Joyce D, Bouzahzah B, Fu M, et al. (1999) Integration of Rac-dependent regulation of cyclin D1 transcription through an NF-κB-dependent pathway. J. Biol. Chem. 274: 25245–25249.View ArticlePubMedGoogle Scholar
- Kheradmand F, Werner E, Tremble P, et al. (1998) Role of Rac1 and oxygen radicals in collagenase-1 expression induced by cell shape change. Science 280: 898–902.View ArticlePubMedGoogle Scholar
- Lichtner RB, Wiedemuth M, Kittmann A, et al. (1992) Lig-and-induced activation of epidermal growth factor receptor in intact rat mammary adenocarcinoma cells without detectable receptor phosphorylation. J Biol Chem. 267: 11872–11880.PubMedGoogle Scholar
- Clark EA, Golub TR, Lander ES, Hynes RO. (2000) Genomic analysis of metastasis reveals an essential role for RhoC. Nature 406: 532–535.View ArticlePubMedGoogle Scholar