2 Mediate the Effects of Loss of the Tumor Suppressor Deleted in Liver Cancer 1

Oncogene (2012) 31, 3913–3923 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE The transcriptional coactivators megakaryoblastic leukemia 1/2 mediate the effects of loss of the tumor suppressor deleted in liver cancer 1

S Muehlich1, V Hampl1, S Khalid1, S Singer2, N Frank1, K Breuhahn2, T Gudermann1,4 and R Prywes3,4

1Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-University, Munich, Germany; 2Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany and 3Department of Biological Sciences, Columbia University, New York, NY, USA

Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor accumulates in the nucleus and activates gene transcrip- whose allele is lost in 50% of liver, breast, lung and 70% tion through SRF (Miralles et al., 2003). Nuclear export of colon cancers. Here, we show that the transcrip- of MKL1 is facilitated by binding of G-actin to the N- tional coactivators Megakaryoblastic Leukemia 1 and 2 terminus of MKL1 (Vartiainen et al., 2007). Work from (MKL1/2) are constitutively localized to the nucleus our laboratory revealed that phosphorylation of MKL1 in hepatocellular and mammary carcinoma cells that by ERK1/2 enhances G-actin binding and nuclear lack DLC1. Moreover, DLC1 loss and MKL1 nuclear export of MKL1 (Muehlich et al., 2008). Although localization correlate in primary human hepatocellular the regulation of MKL1 nuclear entry and export is carcinoma. Nuclear accumulation of MKL1 in DLC1- well established in fibroblasts, the pathophysiologic deficient cancer cells is accomplished by activation of the relevance, however, remains to be investigated. RhoA/actin signaling pathway and concomitant impair- Both the RhoA and ERK 1/2 MAP kinase pathways ment of MKL1 phosphorylation, which results in con- that regulate MKL1 nuclear export have been impli- stitutive activation of MKL1/2 target genes. We provide cated in cancer. Activation of ERK1/2 MAP kinase evidence that MKL1/2 mediates cancerous transforma- signaling by the oncogenes Ras and Raf and their role in tion in DLC1-deficient hepatocellular and mammary tumor progression are well characterized (Wilhelm et al., carcinoma cells. Depletion of MKL1/2 suppresses cell 2004; Giehl, 2005; Gollob et al., 2006). RhoA over- migration, cell proliferation and anchorage-independent expression has been found in various cancers (Gomez cell growth induced by DLC1 loss. del Pulgar et al., 2005), but the mechanisms for RhoA Oncogene (2012) 31, 3913–3923; doi:10.1038/onc.2011.560; activation, the downstream mediators and transcription published online 5 December 2011 factors involved remain largely elusive. The closely related RhoC gene has particularly been shown to have Keywords: DLC1; MKL1; MKL2; MRTF; Rho; SRF a role in metastasis. High RhoC expression was found to be required for the metastatic potential of melanoma cells in mice and loss of RhoC reduced metastases in Polyoma middle T transgenic mice (Clark et al., 2000; Introduction Hakem et al., 2005). Further evidence to support a role for Rho pathways Megakaryoblastic Leukemia 1 and 2 proteins (MKL1/2) in cancer has come from the identification of the are coactivators of the transcription factor serum tumor suppressor Deleted in Liver Cancer 1 (DLC1) response factor (SRF). SRF controls many fundamental whose loss potentiates RhoA activity (Yuan et al., 1998; biological processes such as cell growth, cell migration, Xue et al., 2008). DLC1 is a RhoGAP protein, which differentiation and organization of the cytoskeleton increases the intrinsic GTPase activity of Rho GTPases via approximately 160 different target genes (Pipes to accelerate their return to the inactive state (Jaffe and et al., 2006). The association of SRF with the coacti- Hall, 2005). The DLC1 gene was originally identified as vator MKL1 (MRTF-A, MAL, BSAC) and MKL2 a candidate tumor suppressor gene in human hepato- (MRTF-B) links gene transcription to changes in actin cellular carcinoma (HCC) (Yuan et al., 1998). HCC is dynamics. In response to RhoA-induced actin polymer- among the most lethal and common human cancers in ization and concomitant G-actin depletion, MKL1/2 the human population (Farazi and DePinho, 2006). Despite its significance, the molecular mechanisms that Correspondence: Dr S Muehlich, Walther-Straub-Institute of drive liver cell transformation upon DLC1 loss remain Pharmacology and Toxicology, Ludwig-Maximilians-University unclear. A representative oligonucleotide microarray Munich, Goethestr. 33, Munich 80336, Germany. analysis showed heterozygous deletion of DLC1 in E-mail: [email protected] B50% of liver, breast, lung and 70% of colon cancers, 4These authors contributed equally to this work. Received 22 August 2011; revised 26 October 2011; accepted 3 almost as frequently as p53 mutations in these cancers November 2011; published online 5 December 2011 (Lahoz and Hall, 2008; Xue et al., 2008). MKL1/2 mediate tumorigenesis upon DLC1 loss S Muehlich et al 3914 In this study, we report that DLC1 loss causes nuclear for altered expression of the tumor suppressor DLC1, localization of MKL1/2 in hepatocellular and mammary which has RhoGAP activity. HuH7, HCC1143, MDA- carcinoma cell lines. A correlation between MKL1 MB-453 and MDA-MB-468 cells express low levels of nuclear localization and DLC1 loss was also observed in the tumor suppressor DLC1 protein (Figure 1b). This HCC. Nuclear accumulation of MKL1/2 was accompa- distinguishes them from Hep3B, HepG2, MCF10F and nied by constitutive activation of tumor-relevant MKL/ MCF7 cells that did express DLC1. SRF-dependent target genes. In addition, we demonstrate To investigate whether DLC1 loss leads to nuclear a requirement of MKL1/2 for cancer cell specific features localization of MKL1 and MKL2, we used hepato- caused by DLC1 loss, such as anchorage-independent cell cytes that were depleted of DLC1 with a specific shRNA growth, cell migration and cell proliferation. (Xue et al., 2008). The knockdown (KD) efficiency of DLC1 is shown in Figure 6a and Supplementary Figure S1. Compared with control shRNA, DLC1 Results shRNA resulted in nuclear accumulation of MKL1 and MKL2 (Figure 1c). We also reintroduced Flag-tagged Nuclear localization of MKL1/2 in cancer cell lines with DLC1 in HuH7 cells. HuH7 cells transfected with empty DLC1 loss vector displayed nuclear localization of MKL1 and 2. We first analyzed the localization of MKL1 in several In contrast, HuH7 cells expressing recombinant DLC1 hepatocellular and breast carcinoma cell lines (HuH7, showed a redistribution of MKL1 and 2 to the cyto- HCC1143, MDA-MB-453, MDA-MB-468, MCF7), as plasm (Figure 1d and Supplementary Figure S2). Taken well as in breast epithelial cells (MCF10F) and human together, these results implicate an effect of DLC1 hepatoma cells (Hep3B, HepG2). MKL1 exhibited expression on MKL1/2 subcellular distribution. predominantly nuclear localization in HuH7, HCC1143, MDA-MB-453 and MDA-MB-468 cells, whereas cytoplasmic MKL1 localization was observed in Hep3B, Correlation between MKL1 nuclear localization and HepG2, MCF7 and MCF10F cells (Figure 1a). MKL2 DLC1 loss in human HCC showed the same subcellular distribution (Figure 1a). As DLC1 was originally identified as a tumor suppressor MKL1/2 localization is regulated by RhoA, we screened frequently deleted in human HCC (Yuan et al., 1998).

MKL1 MKL2 100 Control shRNA DLC1 shRNA

80 MKL1 MKL1

40 MKL2 MKL2

20 % cells with nuclear MKL1/2

0 Hep Hep MCF MCF HuH HCC MDA-MDA- Hep MCF HuH MDA- 3B G2 10F 7 7 1143 MB- MB- G2 7 7 MB- 453 468 468

Control vector Flag-DLC1 vector DLC1 MKL1 MKL1

Tubulin MCF MDA- MCF7 MDA- 10F MB- MB- 453 468

DLC1 Flag Flag

Tubulin HepG2 HuH7 Hep3B HCC 1143

Figure 1 Nuclear localization of MKL1 in cancer cell lines with DLC1 loss. (a) The indicated cells were stained with anti-MKL1 and anti-MKL2 antibody for immunfluorescence analysis. In each cell line, subcellular localization was scored as predominantly cytoplasmic, evenly distributed or predominantly nuclear. The percentage of cells exhibiting MKL1 nuclear localization is shown for each cell line. Statistical analysis was carried out for three independent experiments with 100 cells per condition; error bars, s.d. of the mean. (b) DLC1 expression in indicated cell lines was analyzed by immunoblotting with DLC1 and Tubulin antibody as a loading control. (c) Hepatocytes expressing DLC1 shRNA or control shRNA were subjected to immunofluorescence analysis with anti-MKL1 and anti-MKL2 antibody. (d) HuH7 cells were transfected with a Flag-DLC1 or control vector and processed for immunofluorescence analysis using anti-Flag and anti-MKL1 antibodies.

Oncogene MKL1/2 mediate tumorigenesis upon DLC1 loss S Muehlich et al 3915 * HCC Control 12.00 9.00 8.00 6.00 5.00 4.00 MKL1 3.50 3.00 2.00

HCC Control MKL1 nuclear *** 3.00 2.50 2.00 DLC1 1.50 1.00

Ki-67 IHC score0.50 DLC1 IHC score 0.00

HCC Control HCC Control MKL1 nuclear Figure 2 Correlation between MKL1 nuclear localization and decreased DLC1 expression in HCC. (a) Immunostaining of MKL1 in HCC tissue. (b) Immunostaining of MKL1 in normal liver tissue (control). (c) DLC1 immunostaining in the same HCC specimen as shown in A. (d) DLC1 immunostaining in the same normal liver tissue specimen (control) as shown in B. Scale bar represents 50 mm. (e) Scatter plot shows DLC1 expression in HCCs with nuclear accumulation of MKL1 compared with normal liver tissue. Horizontal lines indicate the median (*Po0.05). (f) Scatter plot shows Ki-67 expression in HCCs with nuclear accumulation of MKL1 compared with normal liver tissue. Horizontal lines indicate the median (***Po0.001).

Along with the finding that MKL1 resides in the nucleus that specifically recognizes GTP-bound RhoA. The of HuH7 cells, we analyzed MKL1 expression and amount of GTP-bound RhoA was strongly increased subcellular localization in primary human HCC versus in DLC1-deficient MDA-MB-468 cells, whereas total normal liver tissue to determine the correlation between RhoA levels were similar (Figure 3a). To generate a DLC1 loss and MKL1 nuclear localization in vivo. quantitative result, we performed a Rho G-LISA assay On the basis of immunohistochemical analyses, demonstrating a significant increase of active, GTP- normal liver samples did not show significant levels of bound RhoA in MDA-MB-468 cells as compared with MKL1 expression, whereas MKL1 was detectable MCF7 cells (Figure 3a, right). in 26% of HCCs (30/118). In these samples, MKL1 In agreement with these observations, the DLC1- was nuclear in 53% of cases (16/30). We analyzed deficient HCC cell line HuH7 also showed higher DLC1 expression by immunohistochemistry in the same RhoA-GTP levels than the hepatoma cell line HepG2 tumor samples. Consistent with our results in hepato- (Figure 3b). RhoA activation was readily detectable in cellular and mammary carcinoma cells, HCCs positive HepG2 cells upon stimulation with lysophosphatidic for nuclear MKL1 exhibited significantly decreased acid, whereas there was no further RhoA activation in DLC1 expression compared with normal liver tissue HuH7 cells. These data indicate that RhoA is constitu- (Figures 2a–e). Nuclear accumulation of MKL1 was tively activated in carcinoma cells with DLC1 loss. also associated with a significantly higher expression of In order to analyze whether MKL1 nuclear accumu- the proliferation marker Ki-67 (Figure 2f). These data lation is due to RhoA activation upon DLC1 loss, we suggest that loss of DLC1 and subsequent MKL1 accu- tested the effect of the Rho Kinase inhibitor Y27632 in mulation in the nucleus are likely to be pro-tumorigenic MDA-MB-468 and HuH7 cells. This inhibitor partially events in human hepatocarcinogenesis. blocks the effects of RhoA on actin polymerization (Maekawa et al., 1999). We observed that treatment with Y27632 resulted in cytoplasmic localization of Activation of the RhoA/actin signaling pathway upon MKL1 in HuH7 and MDA-MB-468 cells suggesting DLC1 loss leads to nuclear localization of MKL1 that the RhoA pathway is required for MKL1 nuclear On the basis of the nuclear localization of MKL1 in localization (Figure 3c and Supplementary Figure S3). DLC1-deficient cells, we hypothesized that activation To further delineate the importance of the RhoA/ of the RhoA/actin signaling pathway upon DLC1 loss actin signaling pathway for MKL1 nuclear accumula- may cause MKL1 nuclear accumulation. To test this tion, we analyzed the actin cytoskeleton of HepG2, hypothesis, we assessed the RhoA activation status in HuH7, MCF7 and MDA-MB-468 cells. Immuno- DLC1-deficient MDA-MB-468 versus DLC1-expressing fluorescence analysis using a fluorescent phalloidin MCF7 cells by immunoprecipitation with an antibody conjugate revealed increased stress fiber formation in

Oncogene MKL1/2 mediate tumorigenesis upon DLC1 loss S Muehlich et al 3916 1.5 *** RhoA-GTP 1 HepG2 HuH7 total RhoA

MCF7 MDA- MDA- 0.5 MB- MB- RhoA activity 468 468/ beads 0 only MCF7 MDA- MB- MCF7 MDA- 468 MB- 468 2.5

RhoA-GTP 2.0

1.5

HepG2 total RhoA 1.0 RhoA-GTP Jasplakinolide - + - + 0.5 HuH7 total RhoA SS PP

- + LPA ratio RhoA-GTP/total RhoA 0 -+-+LPA Actin HepG2 HuH7

C 120 0.9 * C/N 0.8 ** 100 N 0.7 80 0.6 0.5 60 0.4 % cells 40 0.3 0.2

20 ratio F-actin/G-actin 0.1 0 0 CoY23762 LatB HepG2MCF7 HuH7 MDA- MB-468 Figure 3 Activation of the RhoA/actin signaling pathway upon DLC1 loss leads to nuclear localization of MKL1. (a) MCF7 and MDA-MB-468 cells were immunoprecipitated with anti-active RhoA antibody and then immunoblotted with anti-RhoA antibody. A portion of the cell lysate (1/30) was also directly immunoblotted with anti-RhoA antibody. For the beads only point, no antibody was added in the immunoprecipitation. Right: Active RhoA levels were assessed by using an ELISA assay. Data are represented as the means±s.d. of three independent experiments (***Po0.001, Student’s t-test). (b) HepG2 and HuH7 cells were serum-starved for 16 h, stimulated with LPA (10 mM) for 2 min and lysed. The lysates were analyzed for RhoA expression as in (a). The blot is representative of three independent experiments. Right: Quantitation of the ratio of active RhoA versus total RhoA. (c) MDA-MB-468 cells were treated with latrunculin B (1 mM) or Y27632 (5 mM) for 45 min. Cells were ﬁxed, permeabilized and stained with anti-MKL1 antibody for immunoﬂuorescence analysis. Subcellular localization was scored as predominantly cytoplasmic (c), evenly distributed (C/N) or predominantly nuclear (N). Statistical analysis was carried out for three independent experiments with 100 cells per condition; error bars, s.e.m. (d) F-actin staining in the indicated cell lines was visualized by Alexa Fluor 488 phalloidin binding. (e) HuH7 cells were treated with jasplakinolide (0.3 mM) for 90 min before lysis and separation into supernatant (S) and pellet (P) fractions by ultracentrifugation. Equal aliquots of these fractions were detected after SDS–PAGE using anti-actin antibody. (f) The indicated cell lines were fractionated by ultracentrifugation as described above. The intensity of the bands representing the supernatant and pellet was measured with the NIH ImageJ 1.42 program. The F-/G-actin ratio was obtained by dividing the values of pellet and supernatant. Data are mean s.d. from triplicate experiments (*Po0.05; **Po0.01).

DLC1-deficient HuH7 and MDA-MB-468 cells. In localization of MKL1, we treated these cells with contrast, barely any stress fibers were detected in HepG2 the actin polymerization inhibitor latrunculin B and and MCF7 cells (Figure 3d). For quantitative assess- examined MKL1 localization by immunofluorescence. ment, F- and G-actin was separated by ultracentrifuga- As a result, MKL1 was redistributed to the cytoplasm tion into 100 000 g supernatant and pellet fractions. (Figure 3c and Supplementary Figure S3). Taken The cytoskeletal drug jasplakinolide, which stabilizes together, these data indicate that potentiation of the F-actin, served as a control to ensure correct partition- RhoA/actin signaling pathway upon DLC1 loss affects ing between the supernatant containing G-actin and the MKL1 localization. pellet fractions with F-actin. Treatment with jasplakinolide resulted in quantitative recovery of cellular actin in the pellet fraction (Figure 3e). Consistent with the MKL1 phosphorylation status contributes to nuclear immunofluorescence analysis, the DLC1-deficient cell localization in DLC1-deficient cells lines HuH7 and MDA-MB-468 were characterized by Our previous studies demonstrated that phosphoryla- an approximately 1.7-fold increase in mean cellular tion of MKL1 affects MKL1 nuclear export (Muehlich F-actin content compared with the other cell lines et al., 2008). Besides changes in the actin cytoskeleton, (Figure 3f). impairment of MKL1 phosphorylation could also To investigate whether the cytoskeletal alterations in contribute to nuclear localization of MKL1 in DLC1- DLC1-deficient cancer cells account for the nuclear deficient cells. To investigate this issue, we used a

Oncogene MKL1/2 mediate tumorigenesis upon DLC1 loss S Muehlich et al 3917 p-MKL1

MKL1 HepG2 HuH7 GAPDH

-+ -+ - + - + serum ERKpT202/pY204

MCF7 MDA-MB-468 HepG2 HuH7 ERK

pT202/pY204 HepG2 ERK p-MKL1

HepG2 ERK MKL1 HuH7 ERKpT202/pY204 - + - + Simvastatin HuH7 ERK

0 7.5 15 30 60 120 min serum

10 10 HepG2 HuH7 8 8

pT202/pY204 6 6 pT202/pY204

4 4

2 2 Relative ERK Relative ERK 0 0 0 7.5 15 30 60 120 min serum 0 7.5 15 30 60 120 min serum Figure 4 The MKL1 phosphorylation status contributes to MKL1 nuclear localization in DLC1-deficient cells. (a) Phosphorylation of MKL1 was determined in cell lysates stimulated with 20% serum for 30 min by immunoblotting using phospho-MKL1 antibody. (b) Serum-starved HepG2 and HuH7 cells were serum-induced for the indicated time points. After immunoblotting with ERKpT202/pY204 and total ERK antibodies, the relative ratio of p-ERK versus total ERK was quantitated and depicted graphically (±s.d.). The blot is representative of three independent experiments. (c) ERK, ERKpT202/pY204 and phospho-MKL1 expression was detected by immunoblotting in HepG2 and HuH7 cells treated with 10 mM simvastatin for 18 h. p-MKL1 antibody that specifically recognizes MKL1 tumor-relevant SRF target genes. Thus, we generated phosphorylated at serine 454. Phosphorylation of HuH7 cells transformed with a lentiviral shRNA vector MKL1 occurred in HepG2 and MCF7 cells subsequent targeting both MKL1 and MKL2, here referred to as to serum induction (Figure 4a). In contrast, hardly any MKL1/2 KD cells. This ‘double-KD’ strategy was used phosphorylation was detectable in MDA-MB-468 and because our previous results in fibroblasts demonstrated HuH7 cells. As ERK1/2 is the kinase responsible that a single KD of MKL1 is not sufficient to suppress for MKL1 phosphorylation (Muehlich et al., 2008), we immediate early gene expression (Cen et al., 2003). tested whether the lack of MKL1 phosphorylation There is only one shRNA sequence targeting MKL1 reflects the absence of activated, phosphorylated and MKL2, which could be used to generate stable ERK1/2 in HuH7 cells. Treatment with serum resulted MKL1/2 KD cells (Medjkane et al., 2009; Lee et al., in 6-fold activation of ERK phosphorylation in HepG2 2010). Control cells contained a lentiviral vector with an cells, whereas ERK activation in HuH7 cells remained shRNA sequence that does not target known genes. nearly unchanged (Figure 4b). Of note, Morin et al. MKL1 expression was downregulated by 85% at have reported reduced ERK activation in cells expres- protein and RNA levels (Figure 5a). sing constitutively active RhoA (Morin et al., 2009). Two immediate early genes known to be SRF target Consistent with this notion, inhibition of RhoA by genes (Selvaraj and Prywes, 2004; Miano et al., 2007; simvastatin in HuH7 cells led to a derepression of ERK Muehlich et al., 2007), namely connective tissue growth and enabled phosphorylation of MKL1 (Figure 4c) factor (CTGF) and integrin alpha 5 (Itga5), showed (Porter et al., 2004). It is thus likely that RhoA acti- preferential expression in HuH7 versus HepG2 cells, as vation caused by DLC1 loss limits ERK activation, well as MDA-MB-468 versus MCF7 cells (Figure 5b thereby reducing phosphorylation and nuclear export and Supplementary Figure S4). Both have been reported of MKL1. to have an important role in tumorigenesis (Friedl and Wolf, 2003; Mazzocca et al., 2010). However, MKL dependence has not been determined yet. We found that Activation of MKL/SRF target genes in DLC1-deficient MKL1/2 shRNAs resulted in a marked reduction in cells CTGF and Itga5 expression, indicating that both genes Previous studies have shown that DLC1 is a potent are MKL-dependent (Figure 5b). Next, we performed tumor suppressor and its loss promotes HCC (Xue quantitative RT–PCR for HuH7 and HepG2 cells et al., 2008). We therefore hypothesized that MKL1 treated with and without serum. We reasoned that may mediate the effects on tumorigenesis by affecting if MKL1/2 is constitutively nuclear in HuH7 cells, it

Oncogene MKL1/2 mediate tumorigenesis upon DLC1 loss S Muehlich et al 3918 HepG2 HuH7

10 *

1.2 5 MKL1 1.0 expression CTGF mRNA 0.8 0 - + - + serum MKL2 0.6 0.4 ** HepG2 HuH7 HSP90 0.2 40 MKL1 mRNA expression 0 HuH7 HuH7 HuH7 HuH7 * MKL1/2 KD 30 MKL1/2 KD 20 expression Itga5 mRNA 10

8 40 0 - + - + serum 7 35

6 30 1

5 25 CTGF 0.8 4 20 DLC1 0.6 3 15 * 2 * 10 0.4 Itga5 mRNA expression CTGF mRNA expression Tubulin * 1 5 Itga5 mRNA expression 0.2 Co DLC1 0 0 HepG2 Huh7 Huh7 HepG2 Huh7 Huh7 0 MKL1/2 KD MKL1/2 KD Co DLC1 Figure 5 Activation of MKL/SRF target genes in DLC1-deficient cells. (a) The KD efficiency of MKL1 and MKL2 in HuH7 and HuH7 MKL1/2 KD cells was determined by immunoblotting and quantitative real time RT–PCR using 18S rRNA levels for normalization. Data are mean s.d. from triplicate experiments. (**Po0.01). (b) Total RNA was isolated from HepG2, HuH7 and HuH7 MKL1/2 KD cells. CTGF and Itga5 mRNA levels were measured as in (a). Means of three independent experiments are shown with s.d. (*Po0.05). (c) Serum-starved HepG2 and HuH7 cells were treated with serum for 2 h. The abundance of CTGF and Itga5 mRNA was analyzed as in (a) and represented as the mean±s.d. of three independent experiments (*Po0.05). (d) HuH7 cells were transfected with Flag-DLC1 and processed for western blotting using CTGF and Flag antibodies or RT–PCR-analysis as described in (a). The blot is representative of three independent experiments. *Po0.05, significant repression.

should constitutively activate target genes even in monolayers expressing DLC1 shRNA and/or MKL1/2 serum-starved cells. This is exactly what was found shRNA. A DLC1 KD efficiency of about 90% was when transcript levels of CTGF and Itga5 were tested by achieved, as determined by RT–PCR. MKL1 expression quantitative RT–PCR in HuH7 cells, whereas serum- was downregulated by 85% at the protein level (Figure 6a). induced expression was observed in HepG2 cells The wound-healing assays revealed a strong increase (Figure 5c and Supplementary Figure S4). The same in cell motility in the absence of DLC1 (Figure 6b). expression pattern was observed for Cyr61, a known MKL1/2 shRNA was able to revert this effect, suggest- MKL/SRF-dependent immediate early gene that pro- ing that MKL1 exerts the effect on cell motility caused motes tumor growth (Supplementary Figure S5) by DLC1 loss. To corroborate the anti-migratory effect To confirm that enhanced CTGF and Itga5 expres- of MKL1 in tumor cells with DLC1 loss, we used the sion was caused by loss of DLC1 expression, we established HuH7 MKL1/2 KD cell line for the scratch reconstituted DLC1 expression by transient transfection assays. While wound closure was observed in HuH7 with Flag-DLC1-coding vectors. DLC1 strongly sup- control cells within 18 h, it was significantly delayed in pressed CTGF and Itga5 expression (Figure 5d). We HuH7 MKL1/2 KD cells (Figure 6c). conclude that DLC1 loss leads to constitutive activation We also stably introduced the lentiviral MKL1/2 of the MKL-dependent, tumor-relevant target genes shRNA into the DLC1-deficient mammary carcinoma CTGF, Itga5 and Cyr61. cell line MDA-MB-468 to exclude a liver cell-specific effect. We also observed a significant reduction in cell motility of about 4-fold (Figure 6d). Moreover, MKL1/2 is required for cell motility upon DLC1 loss upon MKL1/2 depletion, phalloidin staining revealed a Itga5 has a key role in tumor-cell motility and metastasis significant decrease in the number of protrusive struc- (Friedl and Wolf, 2003). This fact and the role of RhoA tures, named filopodia, as well as a reduction in actin in regulating actin stress fibers led us to investigate stress fibers that cross the cell (Figure 6e). These data whether MKL1/2 and DLC1 affect cell migration. provide compelling evidence that MKL1/2 is required Therefore, we performed scratch assays in hepatocyte for cell motility upon DLC1 loss.

Oncogene MKL1/2 mediate tumorigenesis upon DLC1 loss S Muehlich et al 3919 0 h 18 h HuH7 MKL1 100 HSP90

Co 80 HuH7 1.0 60 closure 0.8 40 ** 0.6 20 HuH7

MKL1/2 KD 0 0.4 Co MKL1/2KD

DLC1 mRNA expression MDA-MB-468 0.2 100 0.0 - - + + DLC1 shRNA 80 - + -+ MKL1/2 shRNA MKL1 60 HSP90 closure 100 40 468 *** 20

80 MDA-MB- MDA-MB-

0 60 468 MKL1/2 KD Co MKL1/2KD *

closure 40 stress HuH7 HuH7 MKL1/2 KD ﬁbers

0 - - + + DLC1 shRNA filopodia - + -+ MKL1/2 shRNA Figure 6 MKL1/2 is required for cell motility upon DLC1 loss. (a) Hepatocytes stably expressing control shRNA, DLC1 shRNA or MKL1/2 shRNA were subjected to immunoblotting using anti-MKL1 and anti-hsp90 antibodies. DLC1 mRNA expression was measured by RT–PCR. (b) Scratch-wound assay in the above-mentioned hepatocytes. Cell migration into the wound after 18 h was quantified in three independent experiments. *Po0.05 significant repression. (c) Scratch-wound assay in HuH7 control and HuH7 MKL1/2 KD cells as described in (b). The difference in wound closure was statistically significant, with a **P value of o0.01. (d) MKL1 KD efficiency in MDA-MB-468 cells determined as in (a). The scratch-wound assay was performed as described in (b); ***Po0.001. (e) The actin cytoskeleton in HuH7 and HuH7 MKL1/2 KD cells was visualized by Alexa Fluor 488 phalloidin binding. Arrows point at filopodia and stress fibers.

MKL1/2 is required for cell growth upon DLC1 loss Discussion Besides enhanced cell migration, another DLC1-dependent hallmark of HCC cells is anchorage-independent Nuclear localization of MKL1/2 is a prerequisite for its cell growth (Wong et al., 2005). To further substantiate function as a transcriptional activator. In fibroblasts, that MKL1/2 confer cancer cell-specific features caused inactive MKL1 shuttles continuously between cyto- by DLC1 loss, we compared anchorage-independent plasm and nucleus; nuclear accumulation of MKL1 growth of HuH7 control cells and HuH7 MKL1/2 KD occurs only upon stimulation with serum due to an inhi- cells in soft agar culture. Intriguingly, MKL1/2 KD cells bition of nuclear export (Miralles et al., 2003; Vartiainen did not grow on soft agar at all (Figure 7a). This finding et al., 2007). In this report, we provide evidence that suggests that MKL1/2 depletion is sufficient to abolish MKL1/2 exhibits nuclear localization in various hepa- anchorage-independent cell growth in HuH7 cells. tocellular and mammary carcinoma cell lines and Next, we asked whether MKL1/2 is required for cell human HCC tissue samples that lack the tumor proliferation in HCC cells that lack DLC1. We observed suppressor DLC1. We propose a model in which that HuH7 MKL1/2 KD cells exhibited a growth rate of DLC1 loss activates RhoA, which controls F-actin about 50% compared with HuH7 cells (Figure 7b). The formation leading to nuclear accumulation of MKL1/2 effect of MKL1/2 KD on HuH7 cell proliferation and activation of tumor-relevant SRF target genes resembled that of DLC1 reconstitution (Figure 7b). (Figure 7d). Besides activation of RhoA/actin signaling, The notion that depletion of MKL1 is able to revert impairment of MKL1 phosphorylation in tumor cells enhanced cell proliferation caused by DLC1 loss was lacking DLC1 contributes to nuclear localization of further supported by a 50% reduction of cell prolifera- MKL1. Previous studies by others and us have tion in MDA-MB-468 MKL1/2 KD cells (Figure 7c). suggested that nuclear export is facilitated by ERK 1/ Collectively, the findings presented here reveal that 2-mediated MKL1 phosphorylation and increa- MKL1/2 is required for anchorage-independent cell sed actin binding (Vartiainen et al., 2007; Muehlich growth and enhanced cell proliferation. et al., 2008). Impairment of MKL1 phosphorylation in

Oncogene MKL1/2 mediate tumorigenesis upon DLC1 loss S Muehlich et al 3920 HuH7 HuH7 MKL1/2 KD

30 ] HuH7 5 25 HuH7 MKL1/2 KD

x10 HuH7 DLC1 [ 20 15 10 5 Cell number 0 12345 Time (days)

12 MDA-MB-468

] MDA-MB-468 5 10 MKL1/2 KD

x10 8 [ 6 4

Cell number 2 0 1234567 Time (days) Figure 7 MKL1/2 is required for cell proliferation upon DLC1 loss. (a) HuH7 control cells and HuH7 MKL1/2 KD cells were grown in soft agar for 2 weeks and colony growth were examined. The image is representative of two experiments with 10 plates each of HuH7 and HuH7 MKL1/2 KD cells. (b) The indicated cells were counted daily for 5 days. Points, mean of three independent experiments; bars, s.d. (c) Cell proliferation was monitored as described in (b). (d) Model for MKL1/2 as mediators of tumorigenesis upon DLC1 loss: Loss of DLC1 activates RhoA, which controls F-actin formation leading to nuclear accumulation of MKL1/2 and activation of SRF target genes. This process is required for enhanced cell migration, cell proliferation and anchorage-independent cell growth induced by DLC1 loss.

DLC1-deficient cancer cells is due to a failure of of CTGF expression and strong serum inducibility. ERK activation caused by constitutively active RhoA, Activation of CTGF expression was due to DLC1 loss, because ERK activation as well as MKL1 phosphory- because rescue of DLC1 suppressed CTGF expression. lation were reconstituted when RhoA was inhibited. Hepatocellular and breast cancers contain high levels of Nuclear localization of MKL1 is required, but not CTGF, which has been shown to have a role in tumor sufficient for activation of target genes. For maximal progression (Xie et al., 2001; Mazzocca et al., 2010). transcriptional activity, MKL1 has to dissociate from These data provided the basis for our hypothesis that actin (Vartiainen et al., 2007). In addition, a group of MKL1/2 might mediate the effects of DLC1 loss on SRF-transcriptional co-regulators named ternary com- tumorigenesis via MKL1/2 target genes. Consistent with plex factors has been shown to function as repressors this, the known MKL/SRF-dependent immediate early of some MKL1/2 target genes (Lee et al.,2010).We gene Cyr61, which promotes tumor growth was strongly therefore sought to determine whether the nuclear induced in DLC1-deficient HuH7 cells. We identified a localization of MKL1/2 in DLC1-deficient hepato- novel target gene, Itga5, that exhibited a DLC1- and cellular and mammary carcinoma cells results in acti- MKL-dependent regulation. Itga5 has a key role in cell vation of its target genes. We first tested the expression of motility and metastasis (Friedl and Wolf, 2003). Altera- CTGF, which has been classified as an SRF-dependent tions in the expression profile of Itga5 have been shown target gene (Muehlich et al., 2007; Medjkane et al., 2009). to have dramatic consequences for cell-matrix adhesion CTGF also appeared in a recent microarray study by dynamics and cell motility (Truong and Danen, 2009). Medjkane et al. as a MKL-dependent target gene We analyzed whether MKL1/2 might therefore be (Medjkane et al., 2009). Indeed, CTGF expression was responsible for the enhanced cell motility that was constitutively activated in DLC1-deficient HuH7 cells, observed in response to DLC1 loss (Goodison et al., whereas HepG2 hepatoma cells displayed low basal levels 2005; Heering et al., 2009). To test this hypothesis, we

Oncogene MKL1/2 mediate tumorigenesis upon DLC1 loss S Muehlich et al 3921 generated DLC1/MKL1/2 KD hepatocytes and subjected cells were cultivated in RPMI medium, Hep3B cells in MEM them to wound-healing assays. We observed a strong medium and MDA-MB-453 cells in Leibovitz medium. For enhancement of cell motility in DLC1 KD hepatocytes. MCF10F, DMEM:F12 (1:1) medium containing 5% horse Consistent with this result, Wong et al. also found serum, 20 ng/ml EGF, 0.01 mg/ml insulin and 500 mg/ml increased cell motility in DLC1-deficient HCC cells. They hydrocortison was used. went on to show that this increase was due to DLC1 loss, HuH7 and MDA-MB-468 cells stably expressing MKL1/2 shRNA or control shRNA (non-target shRNA control because reconstitution with DLC1 significantly inhibited vector, SHC002, Sigma, Taufkirchen, Germany) were gener- cell motility of HCC cells (Wong et al., 2005). ated by lentiviral infection followed by puromycin (1 mg/ml) We found that MKL1/2 depletion abrogated the selection. A sequence (50-CATGGAGCTGGTGGAGAAGAA- increase in cell motility upon DLC1 KD, indicating that 30) targeting both MKL1 and MKL2 was used for shRNA MKL1/2 is required for cell motility upon DLC1 loss. expression as described (Lee et al., 2010). A similar reduction in cell motility was achieved upon For transient transfections, Lipofectamine 2000 reagent MKL1/2 KD in the DLC1-deficient mammary carcino- (Invitrogen, Karlsruhe, Germany) was used. RhoA activity ma cell line MDA-MB-468 and HCC cell line HuH7. was measured with RhoA Activation Assay Kits according to Depletion of MKL1/2 in these cells resulted in a the manufacturer’s instructions (Cytoskeleton, Denver, CO, significant reduction in filopodia. These protrusive USA and NewEast Biosciences, King of Prussia, PA, USA). structures of a motile cell are considered a characteristic of invasive cancer cells (Mattila and Lappalainen, 2008). Actin fractionation The phenotype of MKL1/2 KD HuH7 cells resembles Cells were lysed in actin lysis buffer (50 mM NaCl, 1 mM SRFÀ/À phenotypes that display an almost complete EDTA, 0.5% Triton X-100, 20 mM 4-(2-hydroxyethyl)-1- lack of filopodia (Knoll et al., 2006; Knoll, 2010). These piperazineethanesulfonic acid). After ultracentrifugation of results strongly argue that DLC1 exerts its effects on cell the lysate for 1 h at 100 000 g, the supernatant was separated from the pellet. The pellet was resuspended in actin lysis buffer migration through MKL1 and SRF. and sonicated. 5 ml of each fraction was subjected to One of the most intriguing pathogenetic insights immunoblotting using anti-actin antibody (Sigma). gleaned from our study is that anchorage-independent cell growth is completely abrogated in MKL1/2 KD HCC cells. The capability of anchorage-independent cell Immunofluorescence growth is a hallmark of DLC1-deficient HCC cells Cells were fixed with 4% paraformaldehyde in phosphate- (Wong et al., 2005). This finding provides compelling saline buffer for 10 min. Latrunculin B (1 mM, Calbiochem, Darmstadt, Germany) and Y27632 (5 mM, Biozol) were added evidence that depletion of MKL1/2 is able to suppress to the cells 45 min before fixation. Cells were then permeabi- cancer cell growth caused by DLC1 loss. lized with 0.2% Triton X-100 in phosphate-saline buffer for DLC1-deficient HCC cells are also characterized by 7 min and blocked with 1% bovine serum albumin in enhanced cell proliferation. This pathology of tumors phosphate-saline buffer for 30 min at 37 1C. Incubations with appears to be derived from DLC1 KD as seen by the high goat anti-MRTF-A (C-19) antibody (Santa Cruz Biotechnol- proliferative index measured by Ki-67 immunohisto- ogy, Santa Cruz, CA, USA), rabbit anti-MKL2 antibody chemistry (Xue et al., 2008). We found that depletion of (Hanna et al., 2009) and mouse anti-FLAG M2 anti- MKL1/2 in HCC cells inhibits cell proliferation, thereby body (Sigma) were carried out for 1 h. The secondary anti- resembling the effect of DLC1 reintroduction into these bodies, labeled with the Alexa Fluor 488/555 dye (Invitrogen), cells (Wong et al., 2005). Further support for a function were added to the cells for 30 min. Cells were washed four times in phosphate-saline buffer after antibody incu- of MKL1/2 as mediators of cancer cell growth upon bations and before mounting. Actin filaments were stained DLC1 loss comes from the notion that nuclear staining of with phalloidin coupled to Alexa Fluor 488 (Invitrogen). MKL1 in HCCs correlated with Ki-67 expression. Images were obtained using a confocal microscope (Zeiss, Medjkane et al. reported that depletion of MKL1/2 did Oberkochen, Germany). not affect cell proliferation of MDA-MB-231 and B16F2 cells. This discrepancy may be attributable to the presence of DLC1 in MDA-MB-231 and B16F2 cells. Consistent Immunoblotting Cells were lysed in 100 ml radioimmunoprecipitation assay with our results, they showed that MKL1/2 are required (RIPA) buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.5% for motility and experimental metastasis in these two cell Sodium deoxycholate, 2 mM EDTA, 1% Nonidet P-40, 0.1% lines (Medjkane et al., 2009). SDS) containing a protease inhibitor mixture (Calbiochem). Our study provides novel insight into the mechanism The lysates were kept on ice for 15 min and were then cleared by which loss of DLC1 initiates tumorigenesis. As by centrifugation at 13 000 g for 10 min at 4 1C. The super- MKL1/2 has a key role in this process, this pathway natant was boiled with Laemmli sample buffer, and the may provide promising pharmacological targets for proteins subsequently resolved on SDS–polyacrylamide gel cancer therapy. electrophoresis (SDS–PAGE). The proteins were then trans- ferred to polyvinylidene fluoride (PVDF) membranes and immunoblotted with the respective antibody. The primary antibodies used were goat anti-MRTF-A (C-19), goat anti- Materials and methods CTGF (L-20) (Santa Cruz) and mouse anti-DLC1 (BD Biosciences, Heidelberg, Germany). The phospho-specific Cell culture, transfections and reagents MKL antibody and the MKL2 antibody were described HuH7, MDA-MB-468, MCF7 cells and DLC1 KD hepato- previously (Cen et al., 2003; Muehlich et al., 2008). Rat anti- cytes were grown in DMEM medium. HepG2 and HCC1143 tubulin (Millipore, Schwalbach, Germany), mouse monoclonal

Oncogene MKL1/2 mediate tumorigenesis upon DLC1 loss S Muehlich et al 3922 anti-hsp90 (Santa Cruz) and rabbit anti-GAPDH (Sigma) were of association. The non-parametric Mann–Whitney U-test was used for loading controls. Blots were detected at a luminescent used for statistical comparison. imager (Peqlab, Erlangen, Germany). Soft agar assay RNA extraction, cDNA synthesis and quantitative real time The feeder layer consisted of 0.6% agar (Difco, Lawrence, KS, PCR analysis USA). 4 Â 104 cells were mixed to form a top layer of 0.3% Total RNA was extracted from hepatocellular and mammary agar. Following 2 weeks of incubation, images were taken after carcinoma cell lines using TRIzol reagent according to the staining with 1 mg/ml Iodonitrotetrazoliumchlorid (Serva, manufacturer’s directions (Invitrogen). RNA (1 mg) was Heidelberg, Germany) in Hank’s BSS (Sigma). primed with random hexamers and reverse-transcribed into cDNA with SuperScript II Reverse Transcriptase (Invitrogen). Amounts of cDNA corresponding to 100 ng of RNA were Cell proliferation assay used in SYBR Green based real-time PCR. PCR reactions Cells were harvested at 24-hour intervals for 7 days and cell were carried out using the LightCycler 480 Real-Time PCR numbers were counted with hematocytometer. System (Roche, Mannheim, Germany). Specificity of amplified products was monitored by performing melting curves at the Scratch-wound assay end of each amplification. The relative mRNA level was Cells were allowed to grow to a confluent monolayer before calculated using the determination of cDNA levels of 18S scratching a wound using a pipette tip. The mobilization of rRNA as a control. cells behind the wound edge was monitored using a Zeiss microscope and the AxioVision software. Tissues and immunohistochemistry Formalin-fixed and paraffin-embedded tissue specimens were provided by the tissue bank of the National Center for Tumor Diseases (ethics proposal 206/2005, University of Heidelberg). Conflict of interest The tissue micro-array contained two representative areas of 120 HCCs and 20 normal livers without significant patholo- The authors declare no conflict of interest. gical alterations. MKL1 and DLC1 expression was assessed by a scoring system, as previously described (Singer et al., 2007). An IHC score of >2 was considered as positive for nuclear MKL1 staining. The evaluation was done independently by Acknowledgements two experienced investigators (SS and KB). Immunohisto- chemistry was performed, as previously described, with citrate We thank Dr Scott Lowe for the DLC1 knockdown buffer pretreatment at pH 6 (Prange et al., 2003). The primary hepatocytes and Dr Monilola Olayioye for DLC1 vectors. antibodies were as follows: rabbit anti-human MKL1, rabbit Breast carcinoma cell lines were kindly provided by Dr Ramon anti-human DLC1 (Atlas Antibodies, Stockholm, Sweden) Parsons. This work was funded by grant MU 2737/2-1 from and rabbit anti-human Ki-67 (DAKO, Hamburg, Germany). the German Research Foundation (DFG) to SM and grant The Spearman rank coefficient served as a statistical measure CA050329 from the National Cancer Institute to RP.

References

Cen B, Selvaraj A, Burgess RC, Hitzler JK, Ma Z, Morris SW Hakem A, Sanchez-Sweatman O, You-Ten A, Duncan G, Wakeham A, et al. (2003). Megakaryoblastic leukemia 1, a potent transcrip- Khokha R et al. (2005). RhoC is dispensable for embryogenesis and tional coactivator for serum response factor (SRF), is required tumor initiation but essential for metastasis. Genes Dev 19: 1974–1979. for serum induction of SRF target genes. Mol Cell Biol 23: Hanna M, Liu H, Amir J, Sun Y, Morris SW, Siddiqui MA et al. 6597–6608. (2009). Mechanical regulation of the proangiogenic factor CCN1/ Clark EA, Golub TR, Lander ES, Hynes RO. (2000). Genomic CYR61 gene requires the combined activities of MRTF-A and analysis of metastasis reveals an essential role for RhoC. Nature CREB-binding protein histone acetyltransferase. J Biol Chem 284: 406: 532–535. 23125–23136. Farazi PA, DePinho RA. (2006). Hepatocellular carcinoma pathogen- Heering J, Erlmann P, Olayioye MA. (2009). Simultaneous loss of the esis: from genes to environment. Nat Rev Cancer 6: 674–687. DLC1 and PTEN tumor suppressors enhances breast cancer cell Friedl P, Wolf K. (2003). Tumour-cell invasion and migration: migration. Exp Cell Res 315: 2505–2514. diversity and escape mechanisms. Nat Rev Cancer 3: 362–374. Jaffe AB, Hall A. (2005). Rho GTPases: biochemistry and biology. Giehl K. (2005). Oncogenic Ras in tumour progression and metastasis. Annu Rev Cell Dev Biol 21: 247–269. Biol Chem 386: 193–205. Knoll B. (2010). Actin-mediated gene expression in neurons: the Gollob JA, Wilhelm S, Carter C, Kelley SL. (2006). Role of Raf kinase MRTF-SRF connection. Biol Chem 391: 591–597. in cancer: therapeutic potential of targeting the Raf/MEK/ERK Knoll B, Kretz O, Fiedler C, Alberti S, Schutz G, Frotscher M et al. signal transduction pathway. Semin Oncol 33: 392–406. (2006). Serum response factor controls neuronal circuit assembly in Gomez del Pulgar T, Benitah SA, Valeron PF, Espina C, Lacal JC. the hippocampus. Nat Neurosci 9: 195–204. (2005). Rho GTPase expression in tumourigenesis: evidence for a Lahoz A, Hall A. (2008). DLC1: a signiﬁcant GAP in the cancer signiﬁcant link. Bioessays 27: 602–613. genome. Genes Dev 22: 1724–1730. Goodison S, Yuan J, Sloan D, Kim R, Li C, Popescu NC et al. (2005). Lee SM, Vasishtha M, Prywes R. (2010). Activation and repression of The RhoGAP protein DLC-1 functions as a metastasis suppressor cellular immediate early genes by serum response factor cofactors. in breast cancer cells. Cancer Res 65: 6042–6053. J Biol Chem 285: 22036–22049.

Oncogene MKL1/2 mediate tumorigenesis upon DLC1 loss S Muehlich et al 3923 Maekawa M, Ishizaki T, Boku S, Watanabe N, Fujita A, Iwamatsu A Prange W, Breuhahn K, Fischer F, Zilkens C, Pietsch T, Petmecky K et al. (1999). Signaling from Rho to the actin cytoskeleton through et al. (2003). Beta-catenin accumulation in the progression of protein kinases ROCK and LIM-kinase. Science 285: 895–898. human hepatocarcinogenesis correlates with loss of E-cadherin and Mattila PK, Lappalainen P. (2008). Filopodia: molecular architecture accumulation of p53, but not with expression of conventional and cellular functions. Nat Rev Mol Cell Biol 9: 446–454. WNT-1 target genes. J Pathol 201: 250–259. Mazzocca A, Fransvea E, Dituri F, Lupo L, Antonaci S, Giannelli G. Selvaraj A, Prywes R. (2004). Expression profiling of serum inducible (2010). Down-regulation of connective tissue growth factor by genes identifies a subset of SRF target genes that are MKL inhibition of transforming growth factor beta blocks the tumor- dependent. BMC Mol Biol 5: 13. stroma cross-talk and tumor progression in hepatocellular carcino- Singer S, Ehemann V, Brauckhoff A, Keith M, Vreden S, Schirmacher ma. Hepatology 51: 523–534. P et al. (2007). Protumorigenic overexpression of stathmin/Op18 by Medjkane S, Perez-Sanchez C, Gaggioli C, Sahai E, Treisman R. gain-of-function mutation in p53 in human hepatocarcinogenesis. (2009). Myocardin-related transcription factors and SRF are Hepatology 46: 759–768. required for cytoskeletal dynamics and experimental metastasis. Truong H, Danen EH. (2009). Integrin switching modulates adhesion Nat Cell Biol 11: 257–268. dynamics and cell migration. Cell Adh Migr 3: 179–181. Miano JM, Long X, Fujiwara K. (2007). Serum response factor: Vartiainen MK, Guettler S, Larijani B, Treisman R. (2007). Nuclear master regulator of the actin cytoskeleton and contractile apparatus. actin regulates dynamic subcellular localization and activity of the Am J Physiol Cell Physiol 292: C70–C81. SRF cofactor MAL. Science 316: 1749–1752. Miralles F, Posern G, Zaromytidou AI, Treisman R. (2003). Actin Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H et al. dynamics control SRF activity by regulation of its coactivator (2004). BAY 43-9006 exhibits broad spectrum oral antitumor MAL. Cell 113: 329–342. activity and targets the RAF/MEK/ERK pathway and receptor Morin P, Flors C, Olson MF. (2009). Constitutively active RhoA tyrosine kinases involved in tumor progression and angiogenesis. inhibits proliferation by retarding G(1) to S phase cell cycle Cancer Res 64: 7099–7109. progression and impairing cytokinesis. Eur J Cell Biol 88: 495–507. Wong CM, Yam JW, Ching YP, Yau TO, Leung TH, Jin DY et al. Muehlich S, Cicha I, Garlichs CD, Krueger B, Posern G, Goppelt- (2005). Rho GTPase-activating protein deleted in liver cancer Struebe M. (2007). Actin-dependent regulation of connective tissue suppresses cell proliferation and invasion in hepatocellular carcino- growth factor. Am J Physiol Cell Physiol 292: C1732–C1738. ma. Cancer Res 65: 8861–8868. Muehlich S, Wang R, Lee SM, Lewis TC, Dai C, Prywes R. (2008). Xie D, Nakachi K, Wang H, Elashoff R, Koeffler HP. (2001). Elevated Serum-induced phosphorylation of the serum response factor levels of connective tissue growth factor, WISP-1, and CYR61 in coactivator MKL1 by the extracellular signal-regulated kinase primary breast cancers associated with more advanced features. 1/2 pathway inhibits its nuclear localization. Mol Cell Biol 28: Cancer Res 61: 8917–8923. 6302–6313. Xue W, Krasnitz A, Lucito R, Sordella R, Vanaelst L, Cordon- Pipes GC, Creemers EE, Olson EN. (2006). The myocardin family of Cardo C et al. (2008). DLC1 is a chromosome 8p tumor suppressor transcriptional coactivators: versatile regulators of cell growth, whose loss promotes hepatocellular carcinoma. Genes Dev 22: migration, and myogenesis. Genes Dev 20: 1545–1556. 1439–1444. Porter KE, Turner NA, O’Regan DJ, Balmforth AJ, Ball SG. (2004). Yuan BZ, Miller MJ, Keck CL, Zimonjic DB, Thorgeirsson SS, Simvastatin reduces human atrial myofibroblast proliferation Popescu NC. (1998). Cloning, characterization, and chromosomal independently of cholesterol lowering via inhibition of RhoA. localization of a gene frequently deleted in human liver cancer Cardiovasc Res 61: 745–755. (DLC-1) homologous to rat RhoGAP. Cancer Res 58: 2196–2199.

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