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Cancer Research 68, 8743, November 1, 2008. doi: 10.1158/0008-5472.CAN-08-0984
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Deleted in Liver Cancer 1 Controls Cell Migration through a Dia1-Dependent Signaling Pathway

Gerlinde Holeiter, Johanna Heering, Patrik Erlmann, Simone Schmid, Ruth Jähne and Monilola A. Olayioye

Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany

Requests for reprints: Monilola A. Olayioye, Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. Phone: 49-711-685-69301; Fax: 49-711-685-67484; E-mail: monilola.olayioye{at}izi.uni-stuttgart.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Deleted in liver cancer (DLC) 1 and 2 are Rho GTPase-activating proteins that are frequently down-regulated in various types of cancer. Ectopic expression in carcinoma cell lines lacking these proteins has been shown to inhibit cell migration and invasion. However, whether the loss of DLC1 or DLC2 is the cause of aberrant Rho signaling in transformed cells has not been investigated. Here, we have down-regulated DLC1 and DLC2 expression in breast cancer cells using a RNA interference approach. Silencing of DLC1 led to the stabilization of stress fibers and focal adhesions and enhanced cell motility in wound-healing as well as chemotactic Transwell assays. We provide evidence that enhanced migration of cells lacking DLC1 is dependent on the Rho effector protein Dia1 but does not require the activity of Rho kinase. By contrast, DLC2 knockdown failed to affect the migratory behavior of cells, suggesting that the two proteins have distinct functions. This is most likely due to their differential subcellular localizations, with DLC1 found in focal adhesions and DLC2 being mainly cytosolic. Collectively, our data show that DLC1 is critically involved in the control of Rho signaling and actin cytoskeleton remodeling and that its cellular loss is sufficient for the acquisition of a more migratory phenotype of breast cancer cells. [Cancer Res 2008;68(21):8743–51]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Cell migration is a biological process involved in development, inflammation, wound healing, and tumor metastasis. The migration of cells is a multistep cycle starting with the formation of membrane protrusions driven by actin polymerization at the cell front. These protrusions are anchored to the extracellular matrix by integrin receptors forming focal adhesions. The migratory cycle is continued by actomyosin-driven contraction of the cell body at the back and is completed by the subsequent detachment of the cell rear. Therefore, cell migration requires coordinated changes in cytoskeletal dynamics (1). The Rho GTPases Rho, Rac, and Cdc42 are key players in the control of cell migration and have defined functions with regard to cytoskeletal remodeling. Active Rho is known to induce the assembly of stress fibers and focal adhesions, whereas Rac and Cdc42 promote the formation of specialized membrane protrusions called lamellipodia and filopodia, respectively (2, 3).

Rho proteins cycle between an inactive GDP-bound state and an active GTP-bound state. When bound to GTP, they interact with effector proteins, modulating their activity and localization. Signaling of growth factor receptors and integrins can induce exchange of GDP for GTP on Rho proteins. This activation of Rho proteins is controlled by the guanine nucleotide exchange factors (GEF), which promote the release of bound GDP and facilitate GTP binding, and the GTPase-activating protein (GAP) proteins, which increase the intrinsic GTPase activity of Rho GTPases to accelerate the return to the inactive state (2). The structurally related proteins deleted in liver cancer (DLC) 1 and 2 belong to the GAP family and display in vitro specificity for Rho and to a lesser extent for Cdc42 (4–6). In addition to their GAP domain, DLC1 and DLC2 further contain a sterile {alpha} motif and a StAR-related lipid transfer (START) domain, which may have regulatory roles that remain to be defined.

The DLC1 gene was originally isolated as a candidate tumor suppressor gene in primary human hepatocellular carcinoma located on chromosome 8p22 (7). Loss of expression due to chromosomal deletion or promoter hypermethylation has subsequently been shown in other tumor types, including breast, colon, prostate, and lung (8). Transfection of the DLC1 cDNA into carcinoma cell lines lacking DLC1 expression inhibited cell growth and tumorigenicity in nude mice (9–12). Microinjection of rat DLC1 suppressed the formation of lysophosphatidic acid–induced stress fibers and focal adhesions (13). Furthermore, stable expression of human DLC1 in hepatocellular and breast carcinoma cell lines was shown to reduce cell motility and invasiveness, consistent with the inhibition of Rho signaling (14, 15). The DLC2 gene whose expression is similarly down-regulated in various tumor types is located on chromosome 13q13 and encodes a protein that is ~60% identical to DLC1 (5). Reminiscent of DLC1, cellular reexpression disrupted the actin cytoskeleton and inhibited cell proliferation and migration (16). It thus seems that DLC1 and DLC2 may be functionally redundant in suppressing Rho signaling and Rho-mediated cellular processes.

Rho is required for Ras-mediated oncogenic transformation and activated mutants were shown to be weakly transforming in murine fibroblasts (17, 18). However, no constitutively active Rho mutants have been identified in human tumors; instead, Rho proteins are rather found to be overexpressed. In mammalian cells, there are three structurally related Rho proteins: RhoA, RhoB, and RhoC. In breast cancer and testicular germ cell tumors, RhoA expression levels correlated positively with the tumor stage (19, 20), and overexpression of RhoC was shown to be causally linked to inflammatory breast cancers (21). In in vivo models of tumor dissemination, RhoC has been identified to enhance the metastatic potential of melanoma cells (22).

An alternative mechanism by which Rho activation can be achieved is the deregulation of GEFs or the loss of its GAPs. In this study, we analyzed the consequences of DLC1 and DLC2 knockdown at the molecular and cellular level. We show that silencing of DLC1 in breast cancer cells augments cellular RhoA levels and enhances cell motility, whereas down-regulation of DLC2 had no effect on the migratory behavior of cells. Our results further shed light onto the underlying molecular mechanisms by identifying Dia1 as the Rho effector involved in DLC1-mediated control of breast cancer cell migration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Antibodies and reagents. Antibodies used were mouse anti-DLC1, mouse anti-Dia1, and mouse anti-paxillin monoclonal antibodies (mAb; Becton Dickinson); mouse anti-RhoA mAb, rabbit anti-RhoA (119) polyclonal antibody (pAb), rabbit anti-Cdc42 pAb, and goat anti-Dia1 pAb (Santa Cruz Biotechnology); and mouse anti-tubulin mAb (Sigma). The anti-DLC2 antiserum was raised by immunizing rabbits with DLC2 peptide (C-373TALPDAGDQSRMHEFH388) coupled to keyhole limpet hemocyanin (Pineda). Antibodies were affinity purified with the SulfoLink Immobilization kit for Peptides (Pierce). Elution was with 100 mmol/L glycine buffer (pH 2.7), and neutralized antibody fractions were pooled and dialyzed against PBS. Horseradish peroxidase (HRP)-labeled secondary anti-mouse and anti-rabbit IgG antibodies were from GE Healthcare; HRP-labeled secondary anti-goat IgG antibody was from Santa Cruz Biotechnology; and Alexa Fluor 488–labeled and Alexa Fluor 546–labeled secondary anti-mouse IgG antibodies and Alexa Fluor 546–labeled phalloidin were from Molecular Probes. The ROCK inhibitors Y27632 and H1152 were from Calbiochem.

DNA cloning. pCS2+MT-DLC1 and pEGFPC1-DLC2{alpha} were kindly provided by Irene Ng (University of Hong Kong, Hong Kong, People's Republic of China). The full-length DLC1 cDNA was amplified by PCR using pCS2+MT-DLC1 as a template with primers containing BamHI restriction sites (DLC1-minusATG-F, 5'-CGCGGATCCTGCAGAAAGAAGCCGGACCC-3'; DLC1-STOP-R, 5'-CGCGGATCCTCACCTAGATTTGGTGTCTTTGG-3'; DLC1-ATG-F, 5'-CGCGGATCCACCATGTGCAGAAAGAAGCCGGACACC-3'; and DLC1-minusSTOP-R, 5'-CGCGGATCCCTAGATTTGGTGTCTTTGGTTTC-3') and cloned into pEGFPC1 and pEGFPN1 vectors, respectively (Clontech). Full-length DLC cDNAs were subcloned by BamHI restriction (for DLC1) into the pmCherryN1 vector and by HindIII restriction (for DLC2) into the pmCherryC1 vector (Clontech). DLC1-K714E was generated by QuikChange Site-Directed PCR Mutagenesis (Stratagene). The forward primer used was 5'-CGTGGCAGACATGCTGGAGCAGTATTTTCGAG-3' (DLC1-K714E-for). All amplified cDNAs were verified by sequencing. Oligonucleotides were purchased from MWG Biotech.

Cell culture and transfection. Cell lines used were cultured in DMEM or RPMI 1640 (Invitrogen) supplemented with 10% FCS (PAA) in a humidified atmosphere of 5% CO2 at 37°C. HEK293T cells were transfected with TransIT (Mirus) and MCF7 cells were transfected with Lipofectamine 2000 and, for RNA interference (RNAi), with Oligofectamine (Invitrogen). The small interfering RNAs (siRNA) used were the following: siDLC1, 5'-GGACACGGUGUUCUACAUCdTdT-3'; siDLC2, 5'-CCAAGGCACUUUCUAUUGAdTdT-3'; siDia1, 5'-GCUGGUCAGAGCCAUGGAUdTdT-3'; and siLacZ, 5'-GCGGCUGCCGGAAUUUACCdTdT-3'. Independent control siRNAs used were the following: siDLC1#2, 5'-UUAAGAACCUGGAGGACUAdTdT-3'; siDLC2#2, 5'-GCUCUCCACGAGUCAUACAdTdT-3'; and siDia1#2, 5'-GAAGUUGUCUGUUGAAGAAdTdT-3'. RhoA-, RhoC-, and Cdc42-specific siRNAs have been described previously (23, 24). All siRNAs were obtained from MWG Biotech.

Semiquantitative reverse transcription-PCR. RNA was extracted using the PureLink Micro-to-Midi Total RNA Purification System (Invitrogen) and reverse transcribed into cDNA using a First Strand cDNA Synthesis kit with random hexamer primers (Fermentas). The cDNA was then used as a template for PCR analysis with REDTaq PCR Master Mix (Sigma). Primers used were the following: DLC1-137F (5'-TGGTCAAGAGAGAGCATGAT-3') and DLC1-643R (5'-TGAAGCTGAAGCTGGACAGT-3'); DLC2-376F (5'-CAAAGGAAAAAGGGTGACGA-3') and DLC2-1282R (5'-TCCTCCAATTAACCCCATTG-3'); DLC2-2021F (5'-AGCCCCTGCCTCAAAGTATT-3') and DLC2-2423R (5'-ATGGGCGTCATCTGATTCTC-3'); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-F (5'-CCCCTTCATTGACCTCAACTA-3') and GAPDH-R (5'-CGCTCCTGGAAGATGGTGAT-3').

Cell lysis, SDS-PAGE, and Western blotting. Cells were lysed in radioimmunoprecipitation assay buffer [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/L sodium orthovanadate, 10 mmol/L sodium fluoride, 20 mmol/L β-glycerophosphate plus Complete protease inhibitors (Roche)]. Lysates were clarified by centrifugation at 16,000 x g for 10 min. Equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Roth). The membrane was blocked with 0.5% blocking reagent (Roche) in PBS containing 0.1% Tween 20 and then incubated with primary antibodies followed by HRP-conjugated secondary antibodies. Visualization was with the enhanced chemiluminescence detection system (Pierce).

RBD pull-downs. BL21 bacteria were transformed with a pGEX vector encoding the RBD of rhotekin and expression was induced with 0.1 mmol/L isopropyl-β-D-1-thiogalactopyranoside for 4 h at 37°C. Bacteria were harvested, resuspended in PBS containing Complete protease inhibitors, and sonicated. Triton X-100 was added (1% final) and the lysate was centrifuged for 10 min at 8,000 x g. GST-RBD was purified with glutathione resin (GE Healthcare). For pull-downs, cells were lysed in RBD extraction buffer [50 mmol/L Tris (pH 7.5), 500 mmol/L NaCl, 10 mmol/L MgCl2, 1% Triton X-100, 1 mmol/L sodium orthovanadate, 10 mmol/L sodium fluoride, 20 mmol/L β-glycerophosphate, 0.5 mmol/L phenylmethylsulfonyl fluoride plus Complete protease inhibitors without EDTA (Roche)]. Equal amounts of cleared lysates were incubated with GST-RBD beads for 45 min at 4°C. Beads were washed with RBD extraction buffer, bound proteins were separated by SDS-PAGE, and RhoA was analyzed by immunoblotting.

Immunofluorescence microscopy. Cells grown on glass coverslips coated with 25 µg/mL collagen (Serva) were fixed with 4% paraformaldehyde for 10 min, permeabilized with PBS containing 0.1% Triton X-100 for 5 min, and blocked with 5% goat serum in PBS containing 0.1% Tween 20 for 30 min. Cells were incubated with primary antibody in blocking buffer for 2 h followed by incubation with secondary antibody in blocking buffer for 1 h. To analyze cytoskeletal structures in cells lacking DLC1 or DLC2 (Fig. 3A), cells were simultaneously fixed and permeabilized with 4% paraformaldehyde in PBS containing 0.1% Triton X-100 for 10 min. Paxillin staining was done as described above. Filamentous actin (F-actin) was stained with Alexa Fluor 546–conjugated phalloidin for 20 min before mounting of coverslips in Fluoromount-G (Southern Biotechnology). Cells were analyzed on a confocal laser scanning microscope (TCS SL, Leica) using 488, 543, and 561 nm excitation and a 40.0/1.25 HCX PL APO oil objective lens. Images were processed with Adobe Photoshop.


Figure 3
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  		Figure 3. Loss of DLC1 enhances stress fiber formation and focal contact assembly. A, MCF7 cells were transiently transfected with siRNAs specific for DLC1 and DLC2 or with LacZ-specific control siRNA and replated onto collagen-coated glass coverslips 3 d after transfection. The next day, cells were fixed and stained with paxillin-specific primary and Alexa Fluor 488–labeled secondary antibody (green). F-actin was visualized by costaining with Alexa Fluor 546–labeled phalloidin (red). B, for subcellular localization studies, MCF7 cells were transiently transfected with expression vectors encoding GFP-DLC1 or GFP-DLC2. The next day, cells were fixed and stained with paxillin-specific primary and Alexa Fluor 546–labeled secondary antibody (red). DLC1-positive focal adhesions are marked with arrowheads in the overlay. The confocal images shown are stacks of three to four sections taken from the bottom of the cell. Scale bars, 20 µm.

 
Cell migration assays. Transwells (8.0 µm; Costar) were coated with 2.5 µg/mL collagen and 105 cells in 100 µL medium with 0.5% FCS were added to the top chamber and allowed to migrate overnight (MCF7) or for 4 h (MDA-MB 436). The bottom chamber was supplemented with 10% FCS. Cells on the upper side of the membranes were removed using a cotton swab and cells on the underside were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. Cells were counted in five independent microscopic fields at a 20-fold magnification. Experiments were performed in duplicate and repeated at least three times. In the case of Rho kinase inhibition, 10 µmol/L Y27632 or 1 µmol/L H1152 was added to cells in the top chamber.

Wound-healing assays. MCF7 cells (6 x 105) were seeded into collagen-coated (25 µg/mL) 12-well dishes. The next day, confluent cell monolayers were wounded with a micropipette tip and plates were returned to the tissue culture incubator. For quantification, images at the beginning and after 14 and 24 h were captured and the wound width of three defined positions was determined.

Rho biosensor assays. HEK293T cells transiently expressing the Raichu-RhoA biosensor and pmCherry-DLC constructs were lysed in 50 mmol/L Tris (pH 7.5), 5 mmol/L β-glycerophosphate, 5 mmol/L sodium fluoride, and 0.5% Triton X-100 and debris was removed by centrifugation at 16,000 x g for 10 min. Emission ratios [fluorescence resonance energy transfer (FRET)/cyan fluorescent protein (CFP)] were determined by measuring CFP and yellow fluorescent protein (YFP) fluorescence after background subtraction at 475 and 530 nm, respectively, using a Tecan Infinite 200M plate reader (excitation, 433 nm). Expression of the DLC proteins was controlled by measuring mCherry emission at 615 nm (excitation, 575 nm).

Luciferase reporter assays. HEK293T cells were grown on collagen-coated 24-well dishes (2.5 µg/mL) and transfected with 50 ng 3DA-Luc firefly luciferase reporter containing three SRF binding elements, 50 ng Renilla luciferase plasmid, and 25 ng of the respective DLC plasmids. After serum starvation overnight, cells were stimulated with 15% serum for 6 h. Cells were lysed with 300 µL passive lysis buffer (Promega) and luciferase activities in 10 µL lysate were measured by addition of 50 µL firefly substrate [470 µmol/L D-luciferin, 530 µmol/L ATP, 270 µmol/L CoA, 33 mmol/L DTT, 20 mmol/L tricine, 2.67 mmol/L MgSO4, 01 mmol/L EDTA (pH 7.8)] followed by addition of 100 µL Renilla substrate [0.7 µmol/L coelenterazine, 2.2 mmol/L Na2EDTA, 0.44 mg/mL bovine serum albumin, 1.1 mol/L NaCl, 1.3 mmol/L NaN3, 0.22 mol/L potassium phosphate buffer (pH 5.0)]. Luminescence was measured with a Tecan Infinite 200M plate reader. DLC protein expression was verified by measuring green fluorescent protein (GFP) fluorescence of the lysates.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
DLC1 and DLC2 inhibit Rho signaling in intact cells. DLC1 and DLC2 have been reported to possess the same substrate selectivity in vitro, with GAP activity being most pronounced for RhoA and lower activity toward Cdc42 (5, 6). In intact cells, substrate specificity of these GAP proteins may depend on additional factors and has not been compared thus far. To do so, we made use of a genetically encoded FRET-based RhoA biosensor, termed Raichu-RhoA (25). This sensor consists of RhoA, the Rho-binding domain (RBD) of the effector PKN, and the fluorescence donor-acceptor pair CFP and YFP. On activation by GTP loading, the RBD binds RhoA, modifying the orientation of the fusion protein and allowing FRET to occur. Because RhoA activation is approximated to be proportional to the ratio of FRET/CFP emission, the activity of GAP proteins expressed along with the biosensor can be measured. As shown in Fig. 1A , cotransfection of Raichu-RhoA with expression plasmids encoding DLC1 and DLC2 into HEK293T cells led to a decrease in the emission ratio, indicating that both proteins increase RhoA-GTP hydrolysis in vivo (Fig. 1A). This can be attributed to the GAP activity of the proteins because an inactive DLC1 variant harboring a point mutation in its GAP domain (DLC1 K714E) only had a minimal effect on the emission ratio of the biosensor (Fig. 1A), as did a DLC2 GAP-inactive mutant (data not shown). Although DLC1 and DLC2 displayed activity for Cdc42 in vitro, we did not observe an effect on the emission ratio of a Raichu-Cdc42 biosensor coexpressed in HEK293T cells (Supplementary Fig. S4C).


Figure 1
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  		Figure 1. DLC1 and DLC2 inhibit Rho signaling in intact cells. A, HEK293T cells were cotransfected with plasmids encoding the Raichu-RhoA biosensor and Cherry-DLC1, Cherry-DLC1-K714E, Cherry-DLC2, or empty vector (control). The next day, the emission ratio of Raichu-RhoA was determined by measuring YFP (FRET) and CFP fluorescence (excitation, 433 nm) in cell lysates. Columns, mean of four independent experiments performed with triplicate samples; bars, SE. Values for DLC1 and DLC2 versus the control were statistically significant (two-tailed unpaired t test, P < 0.0001), whereas those for DLC1 K714E were not significantly different (ns, P = 0.287). B, HEK293T cells were transfected with the SRF-responsive 3DA-Luc firefly luciferase reporter along with plasmids encoding Renilla luciferase and GFP-DLC1, GFP-DLC1-K714E, GFP-DLC2, or empty vector (control), starved overnight, and then either left unstimulated (–) or stimulated with FCS (+) for 6 h. Firefly luciferase activity in cell lysates of triplicate samples was determined and normalized by Renilla luciferase activity. The data correspond to one representative experiment out of three. Columns, mean; bars, SE.

 
To address the question how the two DLC proteins modulate endogenous Rho signaling, we further analyzed their ability to block serum response factor (SRF)-dependent transcription, which is known to require functional Rho (26). HEK293T cells were transfected with a SRF-responsive luciferase reporter along with DLC1 or DLC2 expression plasmids, starved overnight, and then restimulated with serum. As shown in Fig. 1B, both DLC1 and DLC2 wild-type proteins suppressed SRF-dependent transcription in a similar fashion, whereas DLC1 K714E only slightly reduced serum-induced luciferase reporter levels. These results are in accordance with those obtained with the RhoA biosensor and show that both DLC1 and DLC2 target Rho in intact cells.

DLC1 and DLC2 knockdown increases cellular RhoA-GTP levels. DLC1 and DLC2 are thought to act as tumor suppressors by antagonizing Rho signaling because ectopic expression in carcinoma cell lines lacking these proteins reduces Rho-GTP levels and Rho-mediated cellular processes, such as cell migration and invasion (5, 14). However, whether the absence of DLC1 and DLC2 really affects Rho activity and associated cellular events has not been investigated, nor is it clear whether these GAP proteins have redundant functions. To mimic the loss of DLC1 and DLC2, we therefore used a RNAi approach in cancer cells that express both genes and compared the molecular and cellular consequences. To first select appropriate cell lines, we examined expression of the DLC1 and DLC2 genes in a panel of breast cancer cell lines by semiquantitative reverse transcription-PCR (RT-PCR; Fig. 2A ). In most of the cell lines, transcripts specific for both DLC genes could be detected. Whereas DLC1 was absent in a subset of the cell lines, DLC2 was more uniformly expressed. The MCF7 cell line was chosen for further studies because these cells express both genes and can be transfected at high efficiency with siRNAs as tested with a fluorescently labeled control siRNA (data not shown). Specific siRNAs were designed for DLC1 and DLC2 and silencing efficiency was verified by semiquantitative RT-PCR (Fig. 2B). Down-regulation of the individual transcripts was observed by 48 hours after transfection and persisted for at least 96 hours. The siRNAs were found to be selective as knockdown of DLC1 did not affect DLC2 transcript levels and vice versa in comparison with the siLacZ control (Fig. 2B). Specific down-regulation of the DLC1 protein was further verified by immunoblotting (Fig. 2C). Due to the lack of a commercially available antibody for DLC2, we raised a polyclonal DLC2 peptide antibody, which specifically detected the overexpressed protein in Western blots (data not shown). Due to the presence of nonspecific bands, the DLC2 protein could not be visualized in MCF7 cells, but specific siRNA-mediated DLC2 down-regulation could be observed in MDA-MB 436 cells (see Fig. 5D).


Figure 2
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  		Figure 2. DLC1 and DLC2 knockdown increases cellular RhoA-GTP levels. A, semiquantitative RT-PCR analysis of DLC1 and DLC2 genes. cDNA from the breast epithelial cell lines indicated was amplified using specific primers that span introns in the genomic sequence. GAPDH was amplified as a loading control. B and C, MCF7 cells were transiently transfected with DLC1- or DLC2-specific siRNAs, and 3 d after transfection, DLC expression was evaluated by semiquantitative RT-PCR (B) or by Western blotting (C). Cells transfected with a LacZ-specific siRNA were used as a negative control. C, equal amounts of total cell lysates were subjected to SDS-PAGE and transferred to membrane, and DLC1 expression was analyzed by immunoblotting using a DLC1-specific antibody (top). Bottom, equal loading was verified by reprobing the membrane with tubulin-specific antibody. The lanes shown are from the same membrane. D, MCF7 cells were transiently transfected with siRNAs specific for DLC1 and DLC2 or with LacZ-specific control siRNA. Three days after transfection, cells were starved in serum-free medium for 24 h and then either left untreated (–) or restimulated with 20% FCS for 5 min (+). RhoA-GTP was precipitated by incubation of total cell lysates with GST-RBD coupled to glutathione beads. Top, bound proteins were separated by SDS-PAGE and analyzed by Western blotting with RhoA-specific mAb; bottom, total RhoA levels were determined by immunoblotting of cell lysates with RhoA-specific antibody.

 

Figure 5
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  		Figure 5. Down-regulation of DLC1 but not DLC2 stimulates migration of MCF7 and MDA-MB 436 cells. MCF7 (A) and MDA-MB 436 cells (B) were transiently transfected with siRNAs specific for DLC1 and DLC2 or with LacZ-specific control siRNA. Three days after transfection, 105 cells were seeded in medium containing 0.5% FCS into the upper chamber of a Transwell. The lower well contained medium supplemented with 10% FCS. Cells that had migrated across the filter after overnight incubation (MCF7 cells) and after 4 h (MDA-MB 436 cells) were fixed and stained. The number of migrated cells was determined by counting five independent microscopic fields (20-fold magnification). Columns, mean of duplicate wells and representative of at least three independent experiments; bars, SE. C and D, silencing efficiency in siRNA-transfected MDA-MB 436 cells was verified 3 d after transfection by (C) semiquantitative RT-PCR as described in Fig. 2 and (D) immunoblotting of cell lysates using DLC1- and DLC2-specific antibodies (top). Bottom, equal loading was verified by reprobing the membranes with tubulin-specific antibody.

 
We then analyzed RhoA-GTP levels in MCF7 cells lacking DLC1 or DLC2 by GST pull-down assays with the RBD of rhotekin. Cells were transfected with DLC1- and DLC2-specific siRNAs, respectively, starved, and then restimulated with serum for 5 minutes (Fig. 2D). Down-regulation of both DLC1 and DLC2 enhanced serum-induced RhoA-GTP levels compared with the control, suggesting that both proteins indeed reduce the amount of active RhoA. This is consistent with the fact that overexpression of both DLC1 and DLC2 targets RhoA in intact cells (see Fig. 1).

DLC1 depletion enhances stress fiber formation and focal adhesion assembly. Overexpression of DLC1 and DLC2 has been shown to cause cell detachment associated with the disassembly of stress fibers and focal adhesions (13, 14). To investigate whether the loss of endogenous DLC proteins influences the architecture of the actin cytoskeleton, we analyzed the structure of focal adhesions and stress fibers in siRNA-transfected MCF7 cells. To this end, MCF7 cells lacking DLC1 and DLC2 were stained with a paxillin-specific antibody and phalloidin to visualize focal adhesions and F-actin structures, respectively. Analysis by confocal microscopy revealed that silencing of DLC1 stabilized actin stress fibers and promoted an accumulation of focal adhesions located at the tips of these actin-myosin bundles (Fig. 3A ). This was verified with a second independent siRNA for DLC1, proving that the effect was specific (Supplementary Fig. S1). Surprisingly, cells lacking DLC2 showed no obvious morphologic changes and could not be distinguished from siLacZ control cells (Fig. 3A).

DLC1 has been reported to localize to focal adhesions, as shown by colocalization with the focal adhesion protein vinculin (27). Recently, yeast two-hybrid screenings identified DLC1 as a binding partner for members of the tensin family of focal adhesion proteins and this interaction has been proposed to be associated with biological activity (28–30). We thus examined the subcellular localization of DLC1 and DLC2 in MCF7 cells by transiently expressing GFP-tagged variants of the two proteins because low expression levels and/or the quality of specific antibodies precluded visualization of the endogenous proteins. Indirect immunostaining revealed that DLC1 colocalized with paxillin, whereas DLC2 failed to do so (Fig. 3B). These distinct subcellular localizations are likely to provide an explanation for the effect of DLC1 silencing, and not that of DLC2, on stress fiber formation and focal adhesion assembly.

Down-regulation of DLC1 enhances cell migration. Rho proteins are important players in the regulation of cell motility. To study the effect of DLC1 and DLC2 down-regulation on directed cell migration, we performed scratch assays by wounding confluent monolayers of siRNA-transfected MCF7 cells. Compared with the siLacZ control, cells lacking DLC1 closed the wound more rapidly, whereas silencing of DLC2 had a slight inhibition on the speed of wound closure (Fig. 4A ). This is quantified in Fig. 4B: Cells lacking DLC1 closed 53% of the gap after 14 hours and 67% after 24 hours compared with 27% and 38%, respectively, in the case of the siLacZ control. To address the question how DLC proteins affect chemotaxis in the presence of a serum gradient, we measured cell motility in Transwell assays with 0.5% serum in the upper and 10% serum in the lower chamber. In MCF7 cells, the loss of DLC1 typically stimulated migration 3-fold compared with the siLacZ control (Fig. 5A ). A second DLC1-specific siRNA equally enhanced cell migration, confirming that the effect was due to the knockdown of DLC1 (Supplementary Fig. S1). In line with the wounding experiments, silencing of DLC2 had no effect on serum-induced chemotaxis (Fig. 5A). The failure of DLC2 knockdown to enhance cell motility in wounding and Transwell assays was confirmed with an independent siRNA (Supplementary Fig. S2). In the absence of a serum gradient, Transwell migration of MCF7 cells was very poor but also stimulated by the loss of DLC1, indicating that DLC1 is involved in the regulation of both directed and random cell migration (data not shown).


Figure 4
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  		Figure 4. Loss of DLC1 enhances wound closure. A, MCF7 cells were transiently transfected with siRNAs specific for DLC1 and DLC2 or with LacZ-specific control siRNA and replated onto collagen-coated dishes 2 d after transfection. The next day, confluent monolayers were scratched with a white pipette tip. Pictures were taken at a 10-fold magnification to document the scratch at time point zero and after incubation for 24 h. Scale bar, 200 µm. B, quantification of wound closure in MCF7 cells transfected with DLC1- and LacZ-specific siRNAs. Three independent positions of the wounded cell monolayers were photographed at 0, 14, and 24 h and the width of the scratch was determined. Columns, mean of 10 independent measurements per photograph; bars, SE. Results for siDLC1-transfected versus siLacZ-transfected cells at 14 and 24 h were statistically significant (two-tailed unpaired t test, P = 0.0002), whereas the difference of the initial wound width at 0 h was not significant (ns, P = 0.4877).

 
To verify these results with a second cell line, we performed Transwell assays with MDA-MB 436 cells that also express both DLC genes (Fig. 2A). Consistent with the results in MCF7 cells, down-regulation of DLC1 enhanced cell migration, whereas depletion of DLC2 had no effect on the migratory behavior of the cells (Fig. 5B). Efficient silencing of the DLC proteins in MDA-MB 436 cells is shown by semiquantitative RT-PCR and Western blotting (Fig. 5C and D).

DLC1 controls cell migration by modulation of Dia1 signaling. The multiple functions of Rho are mediated by its downstream effectors, the major ones being the Rho kinase (ROCK) and Dia1, the mammalian ortholog of Drosophila diaphanous 1. Rho stimulates actin polymerization through activation of Dia1, which promotes addition of actin monomers to the barbed end of actin filaments. Dia1 acts together with ROCK to mediate Rho-induced stress fiber formation. ROCK phosphorylates and activates LIM kinase, leading to the inhibition of the actin-depolymerizing factor cofilin. In addition, ROCK induces actomyosin-based contractility through phosphorylation-induced inactivation of myosin light chain phosphatase (2, 3).

To identify the molecular pathway underlying DLC1 inhibition of cell migration, we used the pharmacologic inhibitors Y27632 and H1152 to specifically inactivate ROCK. These inhibitors were added to siRNA-transfected MCF7 cells in the upper chamber of the Transwells and the number of migrated cells was then quantified. As shown in Fig. 6A , migration of cells lacking DLC1 was not affected by the presence of either ROCK inhibitor. We next depleted Dia1 using a siRNA that was used previously to show that Dia1 is crucial for stroma cell-derived factor 1{alpha}–induced migration of rat glioma cells (31). Efficient silencing of Dia1 in MCF7 cells was confirmed by Western blot analysis (Fig. 6B, left). Interestingly, simultaneous down-regulation of Dia1 and DLC1 completely abrogated cell migration resulting from DLC1 knockdown, whereas knockdown of Dia1 alone did not affect basal cell migration (Fig. 6B, right). A second Dia1-specific siRNA confirmed these results (Supplementary Fig. S3). We also tested whether silencing of Dia1 prevents the increased migration of cells lacking DLC1 in wounding assays. Dia1 knockdown reduced basal cell motility under these conditions and, in line with the Transwell migration assays, completely blocked increased motility of DLC1-depleted cells (Fig. 6C). Immunostaining revealed that Dia1 accumulated in membrane protrusions of migratory cells at the wound edge, which were especially prominent in cells lacking DLC1 (Fig. 6D). Accordingly, our data indicate a dominant role of Dia1 rather than ROCK in promoting migration of breast carcinoma cells.


Figure 6
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  		Figure 6. Migration of DLC1-depleted cells requires Dia1. A, MCF7 cells were transiently transfected with siRNAs specific for DLC1 or LacZ. Three days after transfection, 105 cells were seeded into the upper chamber of a Transwell in medium containing 0.5% FCS (–) plus 10 µmol/L Y27632 or 1 µmol/L H1152, respectively. The lower well contained medium supplemented with 10% FCS. The number of migrated cells after overnight incubation was determined by counting five independent microscopic fields (20-fold magnification). Columns, mean of duplicate wells; bars, SE. B and C, MCF7 cells were transiently transfected with siRNAs specific for LacZ, DLC1, Dia1, and DLC1 plus Dia1 at a 1:1 ratio. The siRNA amount in each transfection mix was adjusted with LacZ siRNA. B, left, top, silencing efficiency of Dia1 in MCF7 cells was verified 3 d after transfection by immunoblotting of cell lysates using Dia1-specific pAb; left, bottom, equal loading was verified by reprobing the membrane with tubulin-specific antibody; right, MCF7 cells were harvested 3 d after transfection and subjected to migration assays as described in Fig. 5. A representative experiment out of three is shown. C, MCF7 cells were harvested 2 d after transfection and wounding assays were performed and quantified as described in Fig. 4. D, MCF7 cells transfected with siLacZ and siDLC1 were replated onto collagen-coated coverslips and confluent monolayers were wounded with a pipette tip. Cells were fixed 6 h later and stained with a Dia1-specific mAb and Alexa Fluor 488–conjugated secondary antibody. Arrows, Dia1-positive membrane protrusions. Images are stacks of several confocal sections. Scale bar, 20 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
Rho GTPases are proteins with pleiotropic cellular functions that include the regulation of actin cytoskeletal dynamics, proliferation, migration, and invasion, which necessitates precise spatiotemporal control of their activity. Deregulation of Rho activity is involved in oncogenic transformation of cells. DLC1 and DLC2 are structurally related RhoGAP proteins that have been proposed to act as tumor suppressors by antagonizing Rho signaling. Here, we show that both proteins are capable of inactivating Rho when expressed in intact cells based on results obtained with a RhoA biosensor and SRF luciferase assays. To investigate the cellular consequences of DLC1 and DLC2 loss, we knocked down expression in MCF7 breast carcinoma cells. In accordance with their GAP specificity for Rho, RNAi-mediated silencing of DLC1 and DLC2 enhanced cellular RhoA-GTP levels. These observations initially suggested that the two proteins may have redundant cellular functions. However, wound-healing and chemotactic Transwell assays revealed that only cells lacking DLC1, but not DLC2, acquired a more migratory phenotype, providing evidence that this oncosuppressive property is specific to DLC1.

It is becoming apparent that the DLC proteins require precise localization to fulfill their function. In MCF7 cells, GFP-tagged DLC1 was associated with focal adhesions, whereas DLC2 was mainly cytosolic. Recently, DLC1 recruitment to focal adhesions was shown to be mediated by interaction with tensin proteins (28–30). The four tensin members bind to the cytoplasmic tails of β integrins, and tensins 1 to 3 also interact with actin (32). DLC1 mutants deficient in tensin binding were compromised in their ability to inhibit cell growth, suggesting that tensin association is required for biological activity (28, 29). Interaction with tensins has also been shown for the less-characterized DLC3 protein (28), but not for DLC2, making it tempting to speculate that DLC3 may perhaps have functions that overlap with those of DLC1. The exclusion of DLC2 from focal adhesions thus seems to provide an explanation for its inability to regulate actin remodeling in our experimental model. Nevertheless, DLC2 knockdown led to enhanced total RhoA-GTP levels, indicating that it is active as a GAP protein, although its precise biological function still remains to be defined.

In Huh-7 hepatoma cells, DLC2 was reported to target mitochondria via its START domain (33). However, we did not observe mitochondrial localization of DLC2 in MCF7 cells labeled with MitoTracker (data not shown). It is therefore possible that in addition to the START domain cell type–specific cofactors assist in DLC2 mitochondrial targeting. In addition to RhoA, DLC1 was recently shown to possess in vitro GAP activity for RhoB and RhoC (34), which may also be the case for DLC2. Because the Rho proteins present with different subcellular localizations, with RhoA and RhoC found in the cytoplasm and at the plasma membrane and RhoB predominantly at late endosomes (35), DLC2 substrate selection and function will also depend on the spatial distribution of its Rho targets.

The fact that DLC2 depletion did not facilitate migration of MCF7 and MDA-MB 436 cells conflicts with a previous study showing enhanced motility of HepG2 cells in which DLC2 was silenced with a set of four siRNA duplexes (16). It is important to note that in our hands such a commercially available siRNA pool for DLC2 also down-regulated DLC1 transcript levels. It thus cannot be ruled out that the reported enhanced migratory potential of HepG2 cells, which also express DLC1 (7, 12), was in fact due to an off-target effect involving the knockdown of DLC1. Alternatively, the cellular consequences of DLC gene silencing may be cell type specific, possibly depending on Rho expression levels and/or on the balance of GEFs and other GAP proteins that keep Rho in check.

Active Rho is known to stabilize focal adhesions and promote stress fiber formation, which is in line with the morphologic changes observed in MCF7 cells lacking DLC1. We further show that ablation of DLC1 favors directed migration of MCF7 and MDA-MB 436 cells. The contribution of Rho to cell migration has been discussed controversially. Elevated levels of active RhoA were shown to negatively modulate cell migration due to excessive stress fiber formation and adhesion forces (36, 37). In addition, RhoA has always been assumed to act at the back of migrating cells to induce tail retraction via activation of ROCK. More recent studies using biosensors combined with live cell microscopy have provided proof that RhoA activity is detectable throughout cell migration and is not restricted to the rear but also present at the leading edge of cells (38–40). A role for DLC1 at sites of membrane protrusion was proposed by Healy and colleagues (34), who observed local inactivation of a RhoA biosensor in mouse embryonic fibroblasts (MEF) ectopically expressing DLC1.

To investigate the contribution of the different Rho proteins to migration of DLC1-depleted cells, we down-regulated RhoA and RhoC by RNAi. Surprisingly, knockdown of both RhoA and RhoC increased cell migration and did not prevent migration of cells lacking DLC1, suggesting that these Rho isoforms are not the main mediators of cell migration in the absence of DLC1. However, silencing of RhoA/RhoC also reduced DLC1 expression levels, making interpretation of results difficult (Supplementary Fig. S4). Because it is unlikely that both siRNAs have the same nonspecific effect, this observation may indicate that DLC1 protein levels are regulated by RhoA/RhoC expression in a feedback manner. Interestingly, down-regulation of Cdc42 partially inhibited migration of cells lacking DLC1 (Supplementary Fig. S4). Although ectopic DLC1 expression did not lead to measurable GTP hydrolysis of the Raichu-Cdc42 biosensor, negative regulation of endogenous Cdc42 by DLC1 cannot be ruled out. It is also possible that the effect of Cdc42 down-regulation on migration of cells lacking DLC1 is indirect, as Dia1 has been reported to contribute to localization of Cdc42 to the leading edge of migrating cells (31). Unlike Dia2 and Dia3, Dia1 is not a characterized Cdc42 effector, making future studies necessary to address which Rho protein is responsible for promoting cell migration in DLC1-depleted cells.

The Rho effectors ROCK and Dia1 have both been implicated in negative and positive regulation of cell migration depending on the cell type and condition. Elongated protrusive cell movement in three-dimensional matrices, for example, does not require ROCK, as opposed to movement involving a rounded blebbing morphology for which ROCK is essential (41). In MCF7 cells lacking DLC1, pharmacologic suppression of ROCK activity did not impede cell migration. In contrast to this, Dia1 depletion completely abrogated increased migration in the absence of DLC1. This is in accordance with two recent studies, in which Dia1 was identified as a critical component in directed migration of MEFs and glioma cells, respectively (31, 39). Together, our data provide evidence that despite their overlapping substrate specificity toward RhoA, DLC1 and DLC2 have nonredundant cellular functions most likely due to distinct spatial distributions. DLC1 thus seems to be involved in the remodeling of the actin cytoskeleton by local suppression of active Rho proteins and controls directed cell migration through a Dia1-dependent pathway.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: The laboratory of M.A. Olayioye is funded by grants of Deutsche Forschungsgemeinschaft (SFB 495-Junior Research Group) and Deutsche Krebshilfe (OM-106708 and OM-107545). G. Holeiter was supported by a fellowship from Landesgraduiertenförderung.

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.

We thank Cornelius Knabbe (Institute of Clinical Pharmacology, Stuttgart, Germany), Nancy Hynes (Friedrich Miescher Institute, Basel, Switzerland), and Jane Visvader (The Walter and Eliza Hall Institute, Melbourne, Victoria, Australia) for providing breast epithelial cells; Irene Ng for giving pCS2+MT-DLC1 and pEGFPC1-DLC2{alpha}; John Collard (National Cancer Institute, Amsterdam, the Netherlands) for providing pGEX vector encoding RBD-rhotekin; Michiyuki Matsuda (Osaka University, Osaka, Japan) for providing Raichu biosensors; and Angelika Hausser and Klaus Pfizenmaier for helpful comments on the manuscript.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 3/14/08. Revised 8/22/08. Accepted 8/26/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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