Rho Gtpase-Activating Protein Deleted in Liver Cancer Suppresses Cell Proliferation and Invasion in Hepatocellular Carcinoma

Research Article

Rho GTPase-Activating Protein Deleted in Liver Cancer Suppresses Cell Proliferation and Invasion in Hepatocellular Carcinoma

Chun-Ming Wong,1 Judy Wai-Ping Yam,1 Yick-Pang Ching,1,2 Tai-On Yau,1 Thomas Ho-Yin Leung,1 Dong-Yan Jin,2 and Irene Oi-Lin Ng1

1Department of Pathology, S.H. Ho Foundation Research Laboratories and Jockey Club Clinical Research Centre, and 2Department of Biochemistry, Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong

Abstract tumor suppressor genes may be located in these chromosome Deleted in liver cancer (DLC1) is a candidate tumor sup- arms (2–7). pressor gene recently isolated from human hepatocellular Recently, a candidate tumor suppressor gene—frequently deleted carcinoma. Structurally, DLC1 protein contains a conserved in liver cancer (DLC1)—was identified in primary hepatocellular GTPase-activating protein for Rho family protein (RhoGAP) carcinoma sample (8) and mapped to 8p21.3-22, where chromo- domain, which has been thought to regulate the activity of somal deletion is particularly common in human cancers (2, 9–11). Rho family proteins. Previous studies indicated that DLC1 was DLC1 mRNA encodes a protein with 1,091 amino acid residues and frequently inactivated in cancer cells. In the present study, we is ubiquitously expressed in human tissues (8). The amino acid aimed to characterize the tumor suppressor roles of DLC1 in sequence of DLC1 shares 86% homology with rat p122RhoG and hepatocellular carcinoma. We showed that DLC1 significantly this strongly suggests that DLC1 is a p122RhoGAP human inhibited cell proliferation, anchorage-independent growth, orthologue. p122RhoGAP was first identified as a phospholipase y and in vivo tumorigenicity when stably expressed in hepato- C- 1 binding protein in a rat brain expression library screening cellular carcinoma cells. Moreover, DLC1 expression greatly (12). Structurally, both p122RhoGAP and DLC1 contain three reduced the motility and invasiveness of hepatocellular conserved functional domains—sterile a motif (SAM), RhoGAP, carcinoma cells. With RhoGAP-deficient DLC1 mutant and steroidogenic acute regulatory related lipid transfer (START) (DLC1-K714E), we showed that the RhoGAP activity was domains (13, 14). Proteins containing a RhoGAP domain usually essential for DLC1-mediated tumor suppressor function. function to catalyze the hydrolysis of GTP that is bound to Rho Furthermore, the 292– to 648–amino acid region and the family proteins. When the bound GTP is hydrolyzed to GDP, Rho steroidogenic acute regulatory related lipid transfer domain proteins return to the inactive basal state. Thus, RhoGAPs played an auxiliary role to RhoGAP and tumor suppressor negatively regulate Rho-mediated cellular processes, such as actin function of DLC1. Taken together, our findings showed that cytoskeleton organization, gene expression, and cell cycle progres- DLC1 functions as a tumor suppressor in hepatocellular sion (15, 16). carcinoma and provide the first evidence to support the Evidence has shown that hyperactivation of Rho proteins is hypothesis that DLC1 suppresses cancer cell growth by oncogenic (17–20). Clinically, Rho proteins were found to be negatively regulating the activity of Rho proteins. (Cancer Res overexpressed in different human cancers and overexpression of 2005; 65(19): 8861-8) Rho proteins was associated with more aggressive tumor behavior (21, 22). Several members of the RhoGAP family have been suggested Introduction to possess potential tumor suppressor function. For instance, antitumor function of p190RhoGAP has been shown by in vitro Hepatocellular carcinoma is the fifth commonest cancer studies. Overexpression of antisense p190RhoGAP RNA is able to worldwide and is the second leading fatal cancer in Southeast transform normal NIH3T3 fibroblasts. In contrast, overexpression of Asia and Hong Kong. Etiologically, hepatocellular carcinoma is the wild-type p190RhoGAP can suppress v-Ha-Ras–induced trans- closely associated with chronic hepatitis B and C virus infection, formation (23). Therefore, it is logical to hypothesize that DLC1 cirrhosis, and aflatoxin B1 intake (1). However, the molecular might have tumor suppressor functions by negatively regulating mechanisms leading to the development and progression of tumor cell growth or tumor progression through down-regulation of hepatocellular carcinoma remain unclear. In hepatocellular oncogenic activity of Rho family proteins. carcinoma, deletions of chromosomal materials are common and In this study, we showed that DLC1 reduced the proliferation of a nonrandom pattern, with recurrent deletions on chromosomes rate, anchorage-independent growth ability, and in vivo tumorige- 1p, 4q, 8p, 13q, 16q, and 17p. These strongly suggest that putative nicity of hepatocellular carcinoma cells. Stable expression of DLC1 also significantly suppressed the motility and invasiveness of hepatocellular carcinoma cells. Furthermore, DLC1 inhibited Rho- dependent stress fiber formation in fibroblasts and hepatocellular Note: C-M. Wong and J.W-P. Yam contributed equally to this work. carcinoma cells, and this Rho inhibitory function was essential for Supplementary data for this article are available at Cancer Research Online (http:// the DLC1-mediated growth inhibition in hepatocellular carcinoma cancerres.aacrjournals.org/). Requests for reprints: Irene O.L. Ng, Department of Pathology, University of Hong cells. Thus, our findings support the hypothesis that DLC1 is a Kong, Queen Mary Hospital, Room 127B, University Pathology Building, Pokfulam, RhoGAP antagonizing the oncogenic activity of Rho proteins and Hong Kong. Phone: 852-2855-4197; Fax: 852-2872-5197; E-mail: [email protected]. I2005 American Association for Cancer Research. serves as a tumor suppressor gene in human hepatocellular doi:10.1158/0008-5472.CAN-05-1318 carcinoma. www.aacrjournals.org 8861 Cancer Res 2005; 65: (19). October 1, 2005

j z Materials and Methods the 37 CCO2 incubator. Colonies ( 10 cells) formed in the soft agarose culture were counted and photographed 2 weeks after inoculation. Cell lines. 5 Human hepatocellular carcinoma cell lines, SMMC-7721 and Cell motility assays. For cell motility assay, 8 Â 10 cells were seeded onto BEL7402, which lack DLC1 expression, were obtained from Shanghai 60-mm dishes and grown for 24 hours. Cells were treated with 10 Ag/mL Institute of Cell Biology (14, 24). The murine fibroblast cell line Swiss-3T3 mitomycin C (Sigma, St. Louis, MO) for 3 hours before the wounding. A linear was obtained from American Type Culture Collection (Manassas, VA). The wound was made by scraping a pipette tip across the confluent cell rodent fibroblast cell line Rat 6 was kindly provided by Dr. W.L. Hsiao (Hong monolayer. Cells were rinsed with PBS and grown in DMEM supplemented Kong Baptist University, Hong Kong; ref. 25). SMMC-7721 and BEL7402 were with 10% FBS for additional 48 hours. The cell motility in terms of wound maintained in DMEM supplemented with 10% (v/v) fetal bovine serum closure was measured by photographing at three random fields at the time of (FBS) and antibiotics. Swiss-3T3 and Rat 6 cells were cultured in DMEM wounding (time 0) and at 24 and 48 hours after wounding. j supplemented with 10% (v/v) calf serum and antibiotics at 37 Cina In vitro cell invasion assay. For in vitro cell invasion assay, 3 Â 105 cells humidified incubator with 5% CO2. were suspended in 300 AL serum-free DMEM and loaded onto the upper Plasmid construction. A 3.5-kb fragment of full-length coding sequence compartment of invasion chamber that contained a polycarbonate of the DLC1 gene was amplified from normal liver tissue and cloned into membrane with an 8-Am pore size and coated with a layer of extracellular pcDNA3.1(À) vector (26). Truncated fragments were subcloned into matrix (ECM; Chemicon International, Temecula, CA). After 60 hours of eukaryotic expression vector, pCS2+MT, of Myc-tagged protein as follows: incubation, the invasive cells that had migrated through the ECM layer to DLC1-FL (1-1,091 amino acids), DLC1-SAM (1-292 amino acids), DLC1-GAP the complete medium in the lower compartment were stained, and the (648-839 amino acids), DLC1-START (879-1,091 amino acids), DLC1-NDC numbers of invasive cells were photographed and counted under the (1-595 amino acids), DLC1-CDN (648-1,091 amino acids), DLC1-DSAM (292- microscope. 1,091 amino acids), and DLC1-DSAMDSTART (292-839 amino acids). The In vivo tumorigenicity assay. SMMC-7721 and SMMC-7721/DLC1 cells DNA sequences and the reading frame of the recombinant plasmids were were harvested and resuspended in PBS. Cells (1 Â 106) were inoculated confirmed by DNA sequencing. s.c. into the right flank of 6-week-old female BALB/c nude mice using a Mutagenesis. A DLC1 fragment (1-804 amino acids) was released from 25-gauge needle (5 mice for each group). Tumor size was monitored weekly pcDNA3.1/DLC1 plasmid by EcoRI digestion and subcloned into pBlue- by measure the largest and smallest diameters of tumor and estimated script II SK(+) vector. The site-specific mutation of K714E was introduced according to the formula: volume = 1 / 2 Â (largest diameter) Â (smallest into DLC1 cDNA by PCR mutagenesis with two consecutive PCR diameter)2. This experiment was preformed following the Animals (Control amplifications. For the first PCR amplification, PCR fragments contain of Experiments) Ordinance (H.K.) and Institute’s guidance on animal DLC1-K714E mutation was generated from pSK+/DLC1 (1-804 amino experiments. acids) with two set of PCR primers: 5V-GGATGGATGAGGAGAAGCTG-3V Immunofluorescence microscopy. Cells (5 Â 104) were seeded onto (set 1, forward), 5V-GATCTCGAAAATACTGCTCCA-3V(set 1, reverse), coverslips 16 hours before transfection. At 24 hours after transfection, 5V-TGCTGGAGCAGTATTTTCGAG-3V (set 2, forward), and 5V-TAATAC- cells were serum starved for an additional 24 hours and treated with 200 GACTCACTATAGGG-3V (set 2, reverse). Subsequently, an aliquot of the ng/mL lysophosphatidic acid (LPA; Sigma) for 30 minutes. Cells were first PCR products were used as templates for the second PCR, which was fixed in 4% paraformaldehyde in PBS and permeabilized with 0.2% Triton designed to amplify the full-length insert with the desired mutation in the X-100 in PBS for 10 minutes. Fixed cells were blocked with 3% bovine DLC1 cDNA with the set 1 forward primer and set 2 reverse primer that serum albumin and incubated with 1:1,000 rabbit polyclonal anti-Myc have been used in the first PCR amplification. The final amplified product antibody (A14; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours was excised and gel purified. The NocI/BamHI fragment of DLC1 cDNA in followed by 1:100 FITC-conjugated goat anti-rabbit antibody (Amersham, pSK+/DLC1 (1-804 amino acids) plasmid was then replaced by this PCR- Piscataway, NJ) and 1:1,000 rhodamine-conjugated phalloidin (Sigma) for 1 amplified fragment. The full-length DLC1-K714E mutant was generated by hour with extensive washing in between. Cells were counterstained with releasing the DLC1-K714E fragment (1-804 amino acids) from pSK+/DLC1 4V,6-diamidino-2-phenylindole (Calbiochem, San Diego, CA) and mounted K714E plasmids by EcoRI digestion and subcloned into pCS2+MT/DLC1 in Vectashield antifade mountant (Vector Laboratories, Burlingame, CA). (805-1,091 amino acids) plasmid, which was then verified by DNA Images were captured under magnification of Â1,000 by a fluorescence sequencing. microscope equipped with a charge-coupled device camera (Leica, Establishment of DLC1 stably expressing cells. pcDNA3.1(À)/DLC1 Wetzlar, Germany). plasmid (2 Ag) was transfected into SMMC-7721 cells with FuGene 6 Colony suppression assay. Cells (2 Â 105) were seeded onto 35-mm Transfection Reagent as described by the manufacturer (Roche Molecular dish 1 day before transfection. Each of the various pCS2+MT/DLC1 Biochemicals, Indianapolis, IN). After 24 hours, the transfected cells were constructs (2 Ag) or the pCS2+MT empty vector was cotransfected with trypsinized and split onto 100-mm culture dish at a density of 1 Â 104 per 0.2 Ag pBABE-puromycin selection vector into the cells using FuGene 6 dish. Transfected cells were selected for 2 weeks in culture medium Transfection Reagent. After 24 hours, cells were seeded onto 10-cm culture containing 0.75 mg/mL G418 (Invitrogen, Carlsbad, CA). Single G418- dishes in 1:5 dilution and grown in culture medium containing 0.6 Ag/mL resistant clones were isolated from the culture dish using a Cloning cylinder puromycin (Sigma) for 2 weeks. The puromycin-resistant colonies were (Bellco Biotechnology, Vineland, NJ). The expression of DLC1 was fixed with 3.7% formaldehyde, and the colony formation efficiency was determined by reverse transcription-PCR (RT-PCR) and Northern blotting examined by Giemsa staining (Sigma). as described previously (14). Cell proliferation assay. Cells (2.5 Â 104) were plated in each well of 12-well culture plates containing 1 mL DMEM supplemented with 10% or Results 0.5% FBS. Cells were harvested at 24-hour intervals for 7 days. Cell numbers DLC1 inhibited cell proliferation, anchorage-independent were counted by trypan blue exclusion assay with hematocytometer. The growth, and tumorigenicity of hepatocellular carcinoma cells. doubling time of proliferating cells was determined by nonlinear regression We have shown previously that ectopic overexpression of DLC1 (exponential growth) model using GraphPad Prism version 3.00 (GraphPad cDNA can suppress cancer cell growth in colony suppression assay Software, San Diego, CA). (26). To further characterize the tumor suppressor function of Anchorage-independent growth assay in soft agar. Agarose culture medium (0.5%, 5 mL) containing 10% FBS was used to coat the bottom of DLC1 in hepatocellular carcinoma, in this study, we established a each of the wells of the six-well culture plates. After hardening, 1 Â 104 cells cellular model in which DLC1 cDNA was stably expressed in were suspended in agarose culture medium (0.3%, 2 mL) containing 10% SMMC-7721 cells that lack endogenous DLC1 expression. The FBS warmed at 40jC and plated onto the bottom layer. The cell solution ectopic expression of DLC1 in stably transfected cells was was allowed to set for 30 minutes at room temperature before moving into confirmed by semiquantitative RT-PCR and Northern blot analysis

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DLC1 suppressed cell motility and invasiveness of hepatocellular carcinoma cells. To test whether DLC1 affects the motility of hepatocellular carcinoma cells, wound healing assay was done on SMMC-7721 and SMMC-7721/DLC1 cells in the absence or presence of a cell division inhibitor, mitomycin C. Our results showed that the motility of DLC1 stably expressing cells was significantly reduced. Similar results were also obtained when the cells were pretreated with mitomycin C, which indicated that the reduction of cell motility in DLC1-expressing cells was not due to the difference in proliferation rate (Fig. 3A). Consistently, the cell motility of SMMC-7721/DLC1 was significantly reduced in cell motility assay using invasion chamber without ECM coating, which confirmed that expression of DLC1 inhibited cell motility (Supplementary Fig. S1). Furthermore, we asked whether DLC1 could suppress the invasiveness of hepatocellular carcinoma cells. To this end, we did in vitro cell invasion assay using invasion chamber coated with an ECM layer (27). In sharp contrast with the parental SMMC-7721 cells, the number of invasive cells was Figure 1. Stable expression of DLC1 inhibited cell proliferation and dramatically reduced in DLC1 stably expressing cells (Fig. 3B). anchorage-independent growth. A, stable expression of DLC1 in SMMC-7721. Thus, our results clearly showed that DLC1 suppressed the cell Left, RT-PCR; right, Northern blotting. B, DLC1 inhibited cell proliferation in SMMC-7721. Cells were harvested and counted at 24-hour intervals for motility and invasiveness of hepatocellular carcinoma cells. 7 days. Points, mean of three independent experiments; bars, SD. C, DLC1 Expression of DLC1 inhibited Rho-mediated stress fiber expression suppressed anchorage-independent growth of hepatocellular formation. The existence of RhoGAP domain on DLC1 protein carcinoma cells (P = 0.011, t test). Cells were grown in soft agar culture for 2 weeks and the efficiency of colony growth was examined under the strongly suggests that DLC1 may function as a negative regulator microscope. Columns, mean of three independent experiments; bars, SD. toward the activity of Rho family proteins. Previously, we have shown that bacterially expressed DLC1 protein significantly enhanced the intrinsic GTP hydrolysis activity of two well- (Fig. 1A). To determine the effect of DLC1 on cell proliferation, we characterized Rho family proteins, RhoA and Cdc42 (14). This monitored the proliferation rate of DLC1 stably expressing SMMC- observation implied that DLC1 facilitated the conversion of the 7721 cells (SMMC-7721/DLC1) for 7 days in a mitogen-dependent active GTP-bound Rho proteins to the GDP-bound inactive form. (10% FBS) and a mitogen-independent (0.5% FBS) manner. In both It is well known that Rho proteins play a critical role in regulating culture conditions, we observed that SMMC 7721/DLC1 exhibited cytoskeleton organization and mediate actin stress fiber a significantly slower growing phenotype. The doubling time of formation upon extracellular stimulations (28, 29). Actin stress SMMC-7721/DLC1 cells in mitogen-dependent and mitogen- fibers are dissembled in serum-starved cells but restored by LPA independent conditions were 37.8 hours [95% confidence interval treatment through activation of Rho proteins. In this study, we (95% CI), 34.4-41.8 hours] and 55.0 hours (95% CI, 46.3-67.8 hours), further tested the in vivo RhoGAP activity of DLC1 by transiently respectively. In contrast, the doubling time of parental SMMC-7721 expressing Myc-tagged DLC1 cDNA in hepatocellular carcinoma cells were 29.5 hours (95% CI, 27.5-31.8 hours) and 41.1 hours (95% cell lines and fibroblast cell lines. The effects of DLC1 on the CI, 37.6-45.4 hours), respectively (Fig. 1B). These results indicated activity of Rho proteins were examined under fluorescence that DLC1 exerts growth inhibitory effects on hepatocellular microscope with regard to morphologic changes and actin stress carcinoma cells. fiber formation. As shown in Fig. 4B, LPA induced the formation The capability of anchorage-independent growth is an important of actin stress fibers in serum-starved SMMC-7721 cells. However, indicator of malignant transformation. To substantiate the tumor suppression function of DLC1, we compared the anchorage- independent growth ability of SMMC-7721/DLC1 and SMMC- 7721 cells in soft agar culture. As expected, the DLC1 stably transfected cells exhibited a dramatically reduced ability to form colonies on soft agar. Both the number and the size of colonies formed by DLC1 stably expressing cells were significantly reduced compared with those of the parental SMMC-7721 cells (P = 0.011; Fig. 1C). Our findings suggest that ectopic expression of DLC1 is sufficient to suppress anchorage-independent growth in SMMC- 7721 cells. Next, we determine the effects of DLC1 expression on the in vivo tumorigenicity in nude mice xenograft model. Cells (1 Â 106) were inoculated into the right flank of nude mice and the formation of solid tumor was monitored for 63 days. We observed that growth rate of DLC1-expressing SMMC-7721 cells was significantly suppressed in nude mice (Fig. 2). The above finding strongly suggests Figure 2. DLC1 suppressed tumorigenicity in nude mice. Cells (1 Â 106) were inoculated s.c. into the right flank of 6-week-old female BALB/c nude mice that DLC1 gene plays a tumor suppression role in human (5 mice for each group). Tumor size was estimated according to the formula: carcinogenesis. volume = 1 / 2 Â (largest diameter) Â (smallest diameter)2. www.aacrjournals.org 8863 Cancer Res 2005; 65: (19). October 1, 2005

Figure 3. DLC1 inhibited cell motility and invasiveness in hepatocellular carcinoma. A, ability of SMMC-7721 and SMMC-7721/DLC1 grown in a confluent monolayer to migrate into a linear wound created by a pipette tip in the absence (left) or presence (right) of the cell division inhibitor, mitomycin C (10 Ag/mL), was monitored for 48 hours. B, invasiveness of the hepatocellular carcinoma cells was assayed with ECM-coated invasion chamber. Invasive cells were stained, photographed, and counted under the microscope. Columns, mean of three independent experiments; bars, SD. Expression of DLC1 significantly inhibited cell invasion of SMMC-7721 (P < 0.001, t test).

in DLC1-overexpressing cells, the formation of actin stress fibers the empty vector control (Fig. 4B). Similar findings were also was significantly suppressed. Furthermore, overexpression of reproduced in another DLC1-null hepatocellular carcinoma cell line DLC1 resulted in extensive cell rounding and cortical retraction. (BEL7402) as well as in fibroblast cell lines (Swiss-3T3 and Rat 6; data It is of note that these morphologic changes were remarkably not shown). Taken together, our results strongly suggest that DLC1 similar to that caused by the Rho inhibitor, C3 exoenzyme, in functions as a RhoGAP and negatively regulates the activity of Rho HeLa cells (30), suggesting that these morphologic changes were proteins in vivo. due to inhibition of Rho proteins. Consistent observation was RhoGAP activity was essential for DLC1-mediated tumor obtained from both hepatocellular carcinoma cell lines SMMC- suppressor function. We hypothesized that DLC1 suppressed 7721 and BEL7402 and fibroblast cell lines Swiss-3T3 and Rat 6, cancer cell growth through negatively regulating the activity of Rho suggesting that the effects of DLC1 on actin stress fiber formation proteins. To test this hypothesis, colony suppression assays were and cell morphologic changes were not cell line–specific response. done on SMMC-7721 cells transfected with wild-type and RhoGAP- In silico analysis had revealed that several lysine and arginine deficient mutant DLC1 cDNA. We found that ectopic overexpression residues are highly conserved in various mammalian RhoGAPs. A of wild-type DLC1 significantly suppressed the colony formation in previous study on the rat p122RhoGAP (DLC1 orthologue) has SMMC-7721 cells (Fig. 5A). Indeed, this finding was highly consistent provided evidence that Lys706 residue is involved in stimulating the with that found in cancer cell lines of different tissue types as GTP hydrolysis of active GTP-bound Rho proteins (13). To further reported by us and others (26, 31, 32). In contrast to wild-type DLC1, confirm the RhoGAP function of DLC1, we constructed a RhoGAP- expression of the RhoGAP-deficient mutant had no effect on the deficient mutant by converting the Lys714 into glutamate (K714E), colony formation ability of SMMC-7721 cells. The number of colony which corresponds to the Lys706 in rat p122RhoGAP. Expression of formed by DLC1-K714E transfected cells was comparable with that DLC1-K714E mutant abolished the DLC1-induced cell rounding and of the empty vector (Fig. 5A). Thus, our data suggest that the cortical retraction. In addition, on LPA induction, DLC1-K714E RhoGAP activity is essential for DLC1-mediated tumor suppressor mutant-expressing cells formed actin stress fibers as efficiently as function and lend further support to the notion that DLC1 exerts

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. Functional Characterization of DLC1 Gene tumor suppressor function by negatively regulating the activity of tocellular carcinoma cells, respectively. Because RhoGAP is Rho proteins. essential for DLC1-mediated tumor suppressor function, we first Cooperation among the multifunctional domains of DLC1 asked whether the RhoGAP domain alone was sufficient to inhibit was required for its growth inhibition of hepatocellular the activity of Rho proteins and suppress colony formation in carcinoma cells. DLC1 contains three conserved functional hepatocellular carcinoma cells as efficiently as the wild-type DLC1. domains. Apart from the RhoGAP domain, SAM and START In this regard, we transiently expressed the DLC1 fragment domains are located at the NH2 and COOH termini of the DLC1 containing the RhoGAP domain (DLC1-GAP) in SMMC-7721 cells. protein, respectively (Fig. 4A). We have shown in this study that the Surprisingly, the DLC1-GAP failed to induce morphologic change RhoGAP domain of DLC1 is essential for negative regulation of Rho and inhibit actin stress fiber formation when overexpressed in activity and suppression of cancer cell growth. However, the roles hepatocellular carcinoma cells (Fig. 4B). Moreover, we did not find of the SAM and START domains in DLC1 functions are largely any significant difference in the colony formation ability between unclear. To address this question, we expressed different truncated hepatocellular carcinoma cells expressing DLC1-GAP and those DLC1 cDNAs in SMMC-7721 cells (Fig. 4A). The expression of the transfected with empty vector control (Fig. 5A). Similarly, the truncated DLC1 proteins was confirmed by Western blotting using colony formation ability of hepatocellular carcinoma cells express- anti-Myc antibody (Fig. 5B). The RhoGAP activity and tumor ing DLC1 fragment containing either the SAM domain (DLC1-SAM) suppressor function of these truncated constructs were determined or the START domain (DLC1-START) alone was also comparable by their ability to inhibit Rho-mediated actin stress fiber formation with that of the empty vector control (Fig. 5A). DLC1-SAM and (Fig. 4B) and to suppress colony formation (Fig. 5A) in hepa- DLC1-START also had no effect on hepatocellular carcinoma cell

Figure 4. Effect of DLC1 on cell morphology and Rho-dependent stress fiber formation. A, schematic representation of the various constructs of DLC1. The full-length DLC1, DLC1-FL (1-1,091 amino acids), contains three major functional domains—SAM, RhoGAP, and START. Various fragments of DLC1 cDNA were subcloned into Myc-tagged expression vector, pCS2+MT. +, significant inhibition of actin stress fiber formation or colony formation in hepatocellular carcinoma cells; À, no significant inhibition of actin stress fiber formation or colony formation in hepatocellular carcinoma cells. B, morphologic change and actin stress fiber formation of SMMC-7721 cells transfected with various DLC1 mutants. SMMC-7721 cells grown on glass coverslips were transfected with Myc-tagged DLC1 mutants or empty vector. Cells were serum starved for 18 hours and the formation of stress fibers was induced by LPA. The expression of DLC1 was determined by polyclonal anti-Myc antibody (A14) followed by FITC-conjugated goat anti-rabbit antibody. The actin stress fibers were stained with rhodamine-conjugated phalloidin. Arrowheads, cell expression of DLC1-FL and DLC1-DSAM significantly inhibited stress fiber formation and induced cell rounding and cortical retraction.

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domain–deleted fragment (DLC1-DSAMDSTART), respectively, in hepatocellular carcinoma cells. Like the COOH-terminal fragment, expression of the NH2-terminal fragment of DLC1 also had no effects on the cell morphology and colony formation efficiency of hepatocellular carcinoma cells. However, compared with DLC1- DSAM, further deletion of the START domain resulted in loss of the inhibitory effect on actin stress fiber formation and colony formation in hepatocellular carcinoma cells (Figs. 4B and 5A). These findings indicate that the START domain is indispensable for DLC1-mediated RhoGAP and growth inhibitory function. The concurrence of RhoGAP activity and growth inhibitory functions among these various truncated DLC1 constructs provides further evidence to support the notion that the RhoGAP activity is essential for DLC1-mediated tumor suppression (Fig. 4A). Similar results were also obtained when these experiments were done in BEL7402 and Rat 6 cell lines, indicating that these findings were not merely due to a cell line–specific phenotype (data not shown).

Discussion DLC1 is a candidate tumor suppressor gene recently isolated from PCR-based subtractive hybridization in human hepatocellular carcinoma (8). Follow-up studies indicate that inactivation of DLC1 is implicated in hepatocarcinogenesis (14, 26, 33, 34). Although somatic mutations in DLC1 gene are rare (14, 35, 36), loss of heterozygosity is present in approximately half of primary hepatocellular carcinomas (8, 14, 35). DLC1 mRNA expression is significantly down-regulated in primary hepatocellular carcinoma samples (14, 26). Earlier in vitro studies using hepatoma cell lines not expressing DLC1 have revealed that epigenetic alterations, including promoter methylation, are also responsible for DLC1 silencing in hepatocellular carcinoma (14, 34). In addition, Figure 5. Tumor suppressor function of DLC1 mutants. A, colony methylation of the DLC1 promoter was detected in f24% of suppression assays of SMMC-7721 cells transfected with DLC1 mutants. primary hepatocellular carcinoma samples (14). These findings Columns, mean of three independent experiments; bars, SD. Similar findings were also obtained from another hepatocellular carcinoma cell line BEL7402 suggest that epigenetic silencing of DLC1 is not merely an artifact lacking endogenous DLC1 expression (data not shown). B, expression of introduced by in vitro tissue culture but may substantially DLC1 mutants in SMMC-7721 cells. DLC1 expression were detected by contribute to human carcinogenesis (14). The frequent inactivation Western blotting and confirmed that all DLC1 mutants were at expected size and were expressed at similar level. NS, nonspecific bands. of the DLC1 gene in hepatocellular carcinoma prompted us to further investigate its tumor suppressor functions. We showed previously that transfection of DLC1 cDNA into hepatocellular morphology as well as stress fiber formation (data not shown). carcinoma cell lines resulted in inhibition of colony formation (26). These observations suggest that SAM, RhoGAP, or START domain Recently, inactivation of the DLC1 gene has also been observed in on their own is insufficient to inhibit the activity of Rho proteins or human breast, gastric, and lung cancers (31, 32, 37, 38), suggesting suppress colony formation in hepatocellular carcinoma cells. Thus, that DLC1 functions as a bona fide tumor suppressor gene. cooperation among multifunctional domains in DLC1 is required Although several lines of evidence support the tumor suppression for RhoGAP and tumor suppressor functions. role of DLC1, little is known about the functions of the DLC1 and Next, we tested the RhoGAP and tumor suppressor functions of how it is implicated in human carcinogenesis. COOH-terminal (DLC1-CDN) and SAM domain–deleted (DLC1- To further substantiate the tumor suppressor function of DLC1, DSAM) fragments of DLC1 in SMMC-7721 cells. COOH terminus of we stably expressed DLC1 cDNA in SMMC-7721 cells. Expression of DLC1 showed no suppressive effects on the stress fibers and colony DLC1 cDNA significantly suppressed cell proliferation in both formation, whereas expression of DLC1-DSAM resulted in extensive mitogen-dependent and mitogen-independent cultures. In addition, cell rounding and cortical retraction accompanied with loss of stable expression of DLC1 resulted in reduction of anchorage- actin stress fiber formation (Fig. 4B). In addition, DLC1-DSAM also independent growth capability. DLC1 expression also suppressed significantly suppressed colony formation in hepatocellular carci- the tumorigenicity of SMMC-7721 cells in nude mice xenograft noma cells (Fig. 5A). These findings indicate that SAM domain is model. Consistent with our findings, Yuan et al. recently showed not necessary for the RhoGAP and tumor suppressor function of that introduction of the DLC1 cDNA into other cancer cell lines also DLC1, whereas an unidentified functional domain located between abolished the in vivo tumorigenicity in nude mice (32, 33). The SAM and RhoGAP (i.e., amino acid residues 292-639) may play above findings, when taken together, strongly suggest that the DLC1 an important role in regulating DLC1 functions. To test the role gene functions as a tumor suppressor in human carcinogenesis. of START domain in DLC1-mediated functions, we further ex- Our previous in vitro study indicated that DLC1 negatively pressed the NH2 terminus (DLC1-NDC) and the SAM and START regulated the activity of Rho family proteins by promoting the GTP

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. Functional Characterization of DLC1 Gene hydrolysis (14). In the present study, we further showed that DLC1 indicate that, apart from the RhoGAP domain, other functional expression could significantly suppress Rho-dependent actin stress domains of the DLC1 protein may also play some cooperative role fiber formation in hepatocellular carcinoma and fibroblast cell in regulating the DLC1 function in vivo. Indeed, deletion of START lines. Thus, our findings strongly imply that overexpression of domain of DLC1 resulted in impaired inhibition on actin stress DLC1 can inhibit the activity of Rho proteins and further support fiber and colony formation, suggesting that the START domain may the notion that DLC1 functions as RhoGAP in vivo. play an auxiliary role to DLC1 in inhibiting Rho activity and tumor Cell motility is tightly regulated by the activity of Rho proteins cell growth. The exact function of START domain is largely through actin cytoskeletal rearrangements (29). Recently, a growing unknown, but a previous study on the other START domain– body of evidence has suggested that deregulation of Rho proteins containing proteins indicated that this domain might be involved are implicated in cancer cell invasion and metastasis. For instance, in membrane targeting and lipid binding (46). We speculate that overexpression of wild-type RhoA in rat hepatoma cells induced cell START domain in DLC1 may be responsible for targeting DLC1 to invasion in both in vitro and in vivo conditions (39). On the other particular subcellular localization where its specific substrates hand, dominant-negative RhoA (RhoA N19) was sufficient to inhibit locate. We also noticed that the expression of COOH terminus of the invasiveness of human prostate cancer cells (40). It has also DLC1 (648-1,091 amino acids), which contains intact RhoGAP and been documented that the RhoA-specific inhibitor C3 exoenzyme START domain, did not affect the actin stress fiber formation and greatly suppressed the cell motility of hepatocellular carcinoma cell was unable to suppress colony formation in hepatocellular lines, Li7 and KYN-2. In sharp contrast, the cell motility of these cell carcinoma cells. However, DLC1-DSAM (292-1,091 amino acids) lines was strongly induced by LPA treatment (41). Based on the significantly inhibited the activity of Rho proteins and colony above evidence, we hypothesize that DLC1, a negative regulator of formation ability of hepatocellular carcinoma cells. This result Rho proteins, will reduce cell motility. Indeed, our data clearly strongly suggests that an unidentified functional domain located at showed that expression of DLC1 significantly inhibited cell motility 292 to 648 may be important for the DLC1-mediated RhoGAP and and invasiveness of hepatocellular carcinoma cells. tumor suppressor functions. Thus, we propose that both RhoGAP More than 30 potential downstream effectors of Rho family and START domains and an unidentified domain located within proteins have been identified (42). These effectors may be the region of 292– to 648–amino acid residues are required for responsible for the different specific cellular functions mediated DLC1-mediated growth and stress fiber inhibition, and further by the Rho proteins. ROCK is one of the best characterized investigations are warranted in this regard. Furthermore, similar downstream effectors, which selectively binds to active GTP- with the RhoGAP-deficient mutant, we noticed a close association bound Rho proteins and regulates cell cytoskeletal reorganization between RhoGAP activity and tumor suppressor function of various through phosphorylating a wide range of cytoskeletal proteins truncated DLC1 cDNAs. Thus, our results suggest that the tumor (43, 44). Recent studies indicated that deregulation of Rho/ROCK suppressor function of DLC1 is related to its Rho protein inhibitory signaling pathway is responsible for cancer invasion and metas- activity. tasis (22, 41, 45). It is tempting to speculate that DLC1 suppresses In conclusion, our findings thus far have indicated that DLC1 cell motility and invasiveness through down-regulation of the significantly inhibited Rho-mediated actin stress fiber formation, Rho/ROCK signaling pathway. Further experiments are warrant to and the RhoGAP activity of DLC1 was essential for tumor clarify the functional relationship between DLC1 and Rho/ROCK suppressor function. Expression of DLC1 suppressed cell prolifera- signaling pathway. tion, anchorage-independent growth, tumorigenicity, and invasive- To further characterize the Rho inhibitory and tumor suppressor ness of hepatocellular carcinoma cells. activity of DLC1, various DLC1 truncated mutants was generated. RhoGAP-deficient mutant abolished DLC1-mediated growth inhi- Acknowledgments bition when expressed in hepatocellular carcinoma cells. Thus, our Received 4/19/2005; revised 6/16/2005; accepted 7/18/2005. results have shown for the first time that the RhoGAP activity of Grant support: Association for International Cancer Research grant 03-216 and DLC1 is essential for its tumor suppressor function. Surprisingly, Hong Kong Research Grants Council Project HKU 7436/04M. The costs of publication of this article were defrayed in part by the payment of page ectopic expression of the DLC1 RhoGAP domain alone did not charges. This article must therefore be hereby marked advertisement in accordance inhibit stress fiber and colony formation. These observations with 18 U.S.C. Section 1734 solely to indicate this fact.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. Rho GTPase-Activating Protein Deleted in Liver Cancer Suppresses Cell Proliferation and Invasion in Hepatocellular Carcinoma

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