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Published OnlineFirst November 11, 2016; DOI: 10.1158/1535-7163.MCT-16-0482

Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Pharmacological Inhibition of Myocardin-related Transcription Factor Pathway Blocks Lung Metastases of RhoC-Overexpressing Melanoma Andrew J. Haak1, Kathryn M. Appleton2, Erika M. Lisabeth2, Sean A. Misek2, Yajing Ji2, Susan M. Wade1, Jessica L. Bell3,4, Cheryl E. Rockwell2, Merlin Airik5, Melanie A. Krook5, Scott D. Larsen3,4, Monique Verhaegen6, Elizabeth R. Lawlor5, and Richard R. Neubig2

Abstract

Melanoma is the most dangerous form of skin cancer with concentrations blocks nuclear localization and activity of the majority of deaths arising from metastatic disease. Evi- MRTF-A. In SK-Mel-147 cells, CCG-203971 inhibits cellular dence implicates Rho-activated gene transcription in melano- migration and invasion, and decreases MRTF target gene ma metastasis mediated by the nuclear localization of the expression. In addition, CCG-203971–mediated inhibition of transcriptional coactivator, myocardin-related transcription the Rho/MRTF pathway signiﬁcantly reduces cell growth and factor (MRTF). Here, we highlight a role for Rho and MRTF clonogenicity and causes G1 cell-cycle arrest. In an experimen- signaling and its reversal by pharmacologic inhibition using in tal model of melanoma lung metastasis, the RhoC-overexpres- vitro and in vivo models of human melanoma growth and sing melanoma cells (SK-Mel-147) exhibited pronounced lung metastasis. Using two cellular models of melanoma, we clearly colonization compared with the low RhoC–expressing SK-Mel- show that one cell type, SK-Mel-147, is highly metastatic, has 19. Furthermore, pharmacologic inhibition of the MRTF path- high RhoC expression, and MRTF nuclear localization and way reduced both the number and size of lung metastasis activity. Conversely, SK-Mel-19 melanoma cells have low resulting in a marked reduction of total lung tumor burden. RhoC expression, and decreased levels of MRTF-regulated These data link Rho and MRTF-mediated signaling with aggres- genes. To probe the dependence of melanoma aggressiveness sive phenotypes and support targeting the MRTF transcription- to MRTF transcription, we use a previously developed small- al pathway as a novel approach to melanoma therapeutics. molecule inhibitor, CCG-203971, which at low micromolar Mol Cancer Ther; 16(1); 193–204. 2016 AACR.

Introduction metastasis (4). Rho GTPases are well-known for regulating the actin cytoskeleton (5). RhoA/C activation causes the Rho GTPases play signiﬁcant roles in human cancer (1). formation of myosin-rich F-actin bundles called stress ﬁbers Although RhoA and RhoC share considerable homology, they through their downstream effectors Rho-associated kinase may coordinate different aspects of cancer progression. RhoC (ROCK) and Diaphanous-related formin-1 (mDia1; refs. 6, appears to be most important for cellular invasion and 7). By modulation of the actin cytoskeleton, Rho GTPases also metastasis (2) while RhoA plays a role in transformation and regulate gene expression though myocardin-related transcrip- tumor proliferation (3). In a screen designed to identify tion cofactors (MRTF-A/B) also known as megakaryoblastic genes important for melanoma metastasis, RhoC was found leukemia proteins (MKL1/2). In the nucleus, MRTF cooperates tobeupregulatedanditcontributedtomelanomalung with serum response factor (SRF) to induce transcription of genes involved in proliferation, migration, invasion, and

1 metastasis (8, 9). In the N-terminal region of MRTF are two Department of Pharmacology, University of Michigan Medical Center, Ann – Arbor, Michigan. 2Department of Pharmacology & Toxicology, Michigan State G-actin binding RPEL motifs which sequester MRTF in the University, East Lansing, Michigan. 3Department of Medicinal Chemistry, Uni- cytoplasm. Rho activity reduces cytosolic G-actin, allowing versity of Michigan Medical Center, Ann Arbor, Michigan. 4College of Pharmacy, MRTF to translocate into the nucleus and activate gene tran- University of Michigan Medical Center, Ann Arbor, Michigan. 5Department of scription. Stable knockdown of MRTF or SRF in B16M2 6 Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan. Depart- melanoma or human MDA-MB-231 breast cancer cells with ment of Dermatology, University of Michigan Medical Center, Ann Arbor, high RhoC expression results in a dramatic reduction of in vivo Michigan. lung metastasis and in vitro cellular migration (10). This Note: Supplementary data for this article are available at Molecular Cancer evidence suggests that gene regulation through MRTF med- Therapeutics Online (http://mct.aacrjournals.org/). iates RhoC-induced cancer metastasis. A.J. Haak, K.M. Appleton, and E.M. Lisabeth contributed equally to this article. To assess the potential for targeting MRTF-regulated gene Corresponding Author: Richard R. Neubig, Department of Pharmacology and transcription in cancer metastasis, we used a previously iden- Toxicology, Michigan State University, 1355 Bogue St/B440 Life Sciences, East tiﬁed small-molecule inhibitor of the MRTF pathway (11). Our Lansing, MI 48824. Phone: 517-353-7145; Fax: 517-353-8915; E-mail: initial compound, CCG-1423, selectively blocked Rho GTPase– [email protected]. regulated prostate cancer cell invasion at low micromolar doi: 10.1158/1535-7163.MCT-16-0482 concentrations (11). Further structure–activity relationship 2016 American Association for Cancer Research. optimization of this compound series to reduce toxicity (12)

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and to enhance potency resulted in a new analogue (13), CCG- phalloidin (Cytoskeleton) in 100 mL1 PBS/1% BSA was 203971, that also blocks MRTF-induced gene expression and added to each coverslip for 30 minutes at room temperature. prostate cancer migration. The Rho/MRTF pathway has also Cells were mounted (Prolong Gold antifade-reagent with DAPI, recently been highlighted as a major signaling crossroad in the Invitrogen) and imaged on an upright fluorescence microscope development of pathologic fibrosis in diseases including (Nikon E-800) at 60 magnification. idiopathic pulmonary fibrosis, scleroderma, and Crohn disease fi (14, 15). We have recently shown ef cacy of CCG-203971 in a Real-time PCR fi murine model of skin brosis (16) but a close examination of Cells (1 106) were plated into 60-mm dishes and starved MRTF pathway inhibitors in models of cancer progression has overnight in DMEM containing 0.5% FBS. Cells were lysed and not been performed. RNA was isolated using the RNeasy Kit (Qiagen) following the Here we investigate the Rho/MRTF pathway in two metastatic manufacturer's instructions. DNAse-treated RNA (1 mg) was used melanoma cell lines, SK-Mel-19 and SK-Mel-147. There is a as a template for synthesizing cDNA utilizing the TaqMan clear dichotomy between the two lines where the high level of Reverse-Transcription Reagents Kit (Invitrogen). SYBR green RhoC, constitutive MRTF-A nuclear localization, and function qPCR (SABiosciences) was performed using a Stratagene in SK-Mel-147 is associated with in vivo and in vitro correlates of Mx3000P (Agilent Technologies) and Ct values were analyzed cancer metastasis including clonogenicity, migration, and inva- relative to GAPDH expression. Primer sequences used were as sion. As a novel approach to antimetastasis therapeutics, our follows: GAPDH:50-GGAAGGGCTCATGACCACAG-30,30 ACA- MRTF pathway inhibitor, CCG-203971, blocks cellular migra- GTCTTCTGGGTGGCAGTG-50; CTGF:50-CAGAGTGGAGCGCC- tion and invasion and profoundly suppresses SK-Mel-147 lung TGTT-30,30-CTGCAGGAGGCGTTGTCA-50; CYR61:50-CGCGC- metastasis in vivo. TGCTGTAAGGTCT-30,30–TTTTGCTGCAGTCCTCGTTG-50; MYL9: 50-CATCCATGAGGACCACCTCCG-30,30–CTGGGGTGGCCTAGT- Materials and Methods CGTC-50. Cell culture and proliferation Human cutaneous melanoma cell lines, SK-Mel-19 and SK- Scratch migration assay 5 Mel-147, obtained from Dr. Maria Soengas at the University of Cells (4 10 ) were plated in DMEM containing 10% FBS in Michigan, were cultured in DMEM (Life Technologies) supple- a 12-well plate. After 24 hours, a scratch was made using a 200- m mented with 10% FBS (Life Technologies) including penicillin/ L pipette tip. Medium was replaced with DMEM containing ¼ streptomycin (Life Technologies). Short tandem repeat profiles 2.0% FBS and images of the wound were taken at time 0and fi were conducted on the melanoma cell lines (Genewiz). The 24 hours using a bright eld microscope (Olympus CKX41). fi profiles obtained for the melanoma cell lines do not match any Area quanti cation of the scratch was determined using ImageJ fl established published profiles, and we are unable to identify software as described previously (13, 19). Brie y, the binary published profiles for SK-Mel-19 and SK-Mel-147. Cells were (white and black) threshold for each image was manually expanded and frozen immediately prior to authentication and adjusted to leave only the open area of the scratch black. The then resuscitated only two to three months before experiments. area of this scratch was determined and the percent closure was ¼ We verified the driver mutations in our laboratory using MSU calculated by comparing the difference in area from t 0hour ¼ Sanger sequencing service expected from previous reports (i.e., to t 24 hours. BRAFV600E for SK-Mel-19 and NRASQ61L for SK-Mel-147; refs. 17, 18). For proliferation experiments, 1 104 cells were Transwell migration assay plated into 24-well plates in DMEM containing 10% FBS. Cells Cells (5 104) were added to the top of an 8-mmpore were allowed to attach overnight and then medium was diameter Transwell chamber (Millipore) in DMEM containing replaced with DMEM containing 0.5% FBS. After 72 hours of 0.5% FBS with 10 mmol/L CCG-203971 or 0.1% DMSO con- growth, cells were stained with 0.4% Trypan blue stain (Invi- trol. Medium containing 0.5% FBS and compound or DMSO trogen). Cell counting was performed using the Countess was also added to the bottom chamber. Cells were allowed to Automated Cell Counter (Invitrogen) according to the manu- migrate for 6 hours, and after wiping the remaining cells off the facturer's instructions. top of the Transwell chamber, cells which migrated were fixed and stained in 4% formaldehyde/0.5% crystal violet. The num- Immunocytochemistry and F-actin staining ber of migrated cells for each well was determined by exam- fi Cells (1 105)wereplatedontofibronectin-coated cover- ining four random nonoverlapping elds of view at 20 fi fi slips in DMEM with 10% FBS and allowed to attach overnight. magni cation using a bright eld microscope (Olympus Medium was changed to DMEM with 0.5% FBS for 24 hours CKX41). For knockdown experiments, cells transfected for 48 plus the indicated concentration of CCG-203971 or 0.1% hours with siRNA pools were plated, allowed to migrate, and DMSO. Cells were then fixed in 3.7% formaldehyde for 10 analyzed as stated above. minutes at room temperature and permeabilized with 0.25% Triton X-100 for 10 minutes at room temperature. For immu- siRNA knockdown nocytochemistry studies, primary antibody for MRTF-A (Santa ON-TARGET plus SMART pools (GE Dharmacon) targeting Cruz Biotechnology, cat# SC-21558) was diluted 1:100 and human MRTF-A (GE cat# L-015434-00) or nontargeting pool incubated for 2 hours at room temperature. Fluorophore-con- control (GE cat# D-001810-10-05) were used according to the jugated secondary (Alexa Fluor 594 Donkey Anti-Goat IgG, manufacturer's recommendations. Briefly, 25 nmol/L siRNA Invitrogen) was diluted 1:1,000 and added for 1 hour. For stress smart pools were transfected using DharmaFECT (GE cat# T- fiber assays, a final concentration of 100 nmol/L Acti-Stain 2001-01). Western blot validation of MRTF-A expression was

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conducted using a fraction of the same transfected population PBS) and incubated at room temperature for 30 minutes in the used for the Transwell assays. dark. Fluorescence was detected using a C6 BD Accuri flow cytometer (BD Accuri). Fluorescence was quantified using CFlow Label-free migration and invasion assay software (BD Accuri). Cell-cycle analysis was conducted using Real-time cell analysis (RTCA) of cell invasion was monitored FlowJo v10.0.8 software (Tree Star, Inc.). A total of 1 104 events using a CIM-plate 16 and xCELLigence DP System (Acea Biosci- were detected in experimental triplicate for untreated cells, 0.1% ence, Inc). Matrigel Matrix (BD Biosciences) 5% (v/v) was diluted DMSO, and 10 mmol/L CCG-203971. Three independent experi- 1:20 in basal RPMI media and was added in the top chamber ments were conducted. and allowed to equilibrate for 4 hours in an incubator at 37Cin 5 Western blot analysis 5% CO2. Cells (3 10 /well) were added at t ¼ 5 hours. For compound effects in invasion assays, cells were treated with 10 Cells were starved overnight in DMEM containing 0.5% FBS. mmol/L CCG-203971 upon placement into the top chamber. Cell Cells were lysed [10 mmol/LTris-Cl,1mmol/LEDTA,1% index was measured every 30 minutes. Triton X-100, 0.1% SDS, 140 mmol/L NaCl, protease inhibitor cocktail (Roche cat# 11873580001)], sonicated two times for Luciferase assay 15 seconds, and centrifuged to quantitate soluble proteins Cells (3 104) were seeded into 96-well plates in DMEM using a BCA protein assay as recommended by the manufac- containing 10% FBS overnight. Cells were then cotransfected with turer (Pierce BCA Protein Assay, Thermo Scientific). Protein 56 ng SRE.L- Luciferase reporter selective for Rho/MRTF described lysate (20 mg) were resolved using 15% SDS-PAGE gels, trans- in ref. 20, and 2.3 ng RhoC plasmid diluted with Lipofectamine ferredtoPVDFmembranes,andblockedin5%driedmilk 2000 (Invitrogen) in Opti-MEM (Life Technologies). After 6 in Tween Tris-buffered saline. Membranes were incubated hours, compounds were added for overnight treatment. The next in 1:1,000 diluted primary antibodies RhoC (Cell Signaling day, WST-1 (10 mL/well) reagent (Promega) was added to mea- Technology, cat# 3430), RhoA (Santa Cruz Biotechnology, cat# sure viability. After 1 hour, absorbance was read (450 nm) before SC-418), MRTF-A (Santa Cruz Biotechnology, cat# SC-21558), lysing the cells for luciferase measurement (Promega Luciferase or GAPDH (Cell Signaling Technology, cat# 14C10) overnight Assay System). at 4 C. HRP-conjugated goat anti-mouse (Santa Cruz Biotech- nology, sc-2060), HRP-conjugated goat anti-rabbit (Sigma- Clonogenic assay Aldrich, A0545), or HRP-conjugated bovine anti-goat (Santa Cells were grown for 6 days in DMEM with 2.0% FBS. Two Cruz Biotechnology, cat# SC-2352) antibodies were diluted hundred live cells, determined by Trypan blue exclusion assay, 1:10,000. Blots were developed using SuperSignal Pico chemi- were seeded into a 6-well plate and allowed to form colonies in luminescent substrate (Thermo Scientific). Bands were visual- DMEM containing 2.0% FBS for 10 days. Colonies were fixed ized and quantified using a LI-COR Odyssey Fc or visualized and stained in 3.7% formaldehyde/0.5% crystal violet for 10 using Kodak X-OMAT Film Developer and quantified using minutes at room temperature. Only cell clusters consisting of at ImageJ software. least 50 cells were counted as colonies. For CCG-203971 treatment, cells (2 105) were seeded into 6-well dishes and Mouse metastasis model allowed to adhere overnight. The following day, the medium Ten-week old, female, NOD.CB17-Prkdcscid/J (NOD SCID) was replaced with DMEM containing 0.5% FBS and 0.1% mice were purchased from Jackson Laboratory (stock number: DMSO or 10 mmol/L CCG-203971 was added. Seventy-two 001303). The mice were separated into three groups of 6. One hours later, 200 or 1 103 cells were plated per group in group received tail vein injection of GFP-expressing SK-Mel-19 DMEM with 10% FBS in 6-well dishes and allowed to form (2 106 cells). The other two groups were injected with GFP- colonies for 7 days. Colonies were fixed and stained as stated expressing SK-Mel-147 (2 106 cells). Following tail vein above. Colony counts and mean area quantification were injection, the SK-Mel-147 mice began twice daily intraperito- determined using ImageJ software. Briefly, the image threshold neal injection of 100 mg/kg CCG-203971 or 50-mLDMSO was adjusted to black and white and further processed to (vehicle control). After 18 days, the mice were euthanized and smooth colony pixilation. The images were then analyzed for lungs were collected for histologic analysis. All studies were colony size (pixel2) fixed at 50–infinity and for circularity performed according to protocols approved by the University defined at 0.2–1.0. of Michigan, University Committee on the Use and Care of Animals (UCUCA). Flow cytometry Cells (5 105) were plated in 60-mm dishes in DMEM with IHC 10% FBS and allowed to adhere overnight. Media were replaced For the quantification and imaging of lung tissue from the with DMEM with 0.5% FBS, and 0.1% DMSO or 10 mmol/L CCG- mouse metastasis model, paraffin-embedded sections were 203971 was added. Cells were allowed to grow for 72 hours dewaxed, unmasked, and treated as described previously (21). and were then collected using 1 versene (Gibco), permeabilized, To visualize GFP-expressing melanoma cells in the lung section, and fixed using 70% ethanol for 30 minutes on ice. Cells the Vector Rabbit-on-Mouse basic kit (Vector Laboratories) was were centrifuged (200 g, 5 minutes), and resuspended in DNA utilized according to the manufacturer's instructions. For each extraction buffer (192 mL 0.2 mol/L Na2HPO4, 8 mL 0.1 mol/L experiment, two tissue sections from the same animal were citric acid, pH 7.8, filter sterilized) for 5 minutes. Cells were developed: one with primary antibody [rabbit anti-GFP (1:250 centrifuged (300 g, 5 minutes) to remove DNA extraction buffer Invitrogen A-6455)] included and one with the primary antibody and resuspended in 250 mL of freshly prepared DNA staining left out of the reaction. Visualization of GFP was done using the solution (2 mg/mL propidium iodide, 0.2 mg/mL RNase A in 1 Vectastain Elite ABC kit (Vector Laboratories), used according to

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the manufacturer's instructions. All sections were developed using similar genes as does MRTF-A, such as CYR61,andCTGF (25), 3,30-diaminobenzidine (Vector Laboratories) and counterstained did not show significant differences in patient survival based with hematoxylin. Images were taken at 20 magnification using on high versus low expression levels in cutaneous melanoma a Nikon Eclipse Ti-S microscope with MMI Cell Tools software, (Supplementary Fig. S1). Version 3.47. (MMI). Treatment and control slides were imaged using the same exposure time and LUT settings. The slides were SK-Mel-147 cells exhibit spontaneously nuclear and active fi blindly quanti ed for the number of metastatic nodules on each MRTF-A through F-actin stress fiber formation section followed by the use of ImageJ software to determine the To evaluate MRTF activity, we started by detecting stress m 2 cross-sectional area ( m ) of each nodule. The area was repre- fiber formation in the two melanoma cell lines. Stress fiber sented as an average size for each mouse and the total tumor formation leads to G-actin depletion and MRTF translocation burden was calculated by adding the total cross-sectional areas for into the nucleus (8). SK-Mel-147 cells exhibit strong F-actin all tumors in each mouse then averaged for each group. staining and stress fiber formation in the absence of serum stimulation (Fig. 2A). SK-Mel-19 showed reduced F-actin stain- Statistical analysis ing and very little stress fiber formation. Consistent with the Statistical analysis was performed by ANOVA comparison actin-based mechanism governing MRTF localization and followed by Bonferonni posttest for multiple comparisons where activity, SK-Mel-147 also exhibited spontaneously nuclear P > 0.05, P > 0.01, P > 0.001, and P > 0.0001. Com- MRTF-A under these conditions (Fig. 2A). To show that parison of survival curves was performed by log-rank method MRTF-A localization was dependent on F-actin dynamics, we where P > 0.05 and P > 0.01. stimulated SK-Mel-19 cells with 20% FBS to induce stress fiber formation which induced MRTF-A nuclear localization (Sup- Results plementary Fig. S2). We also treated SK-Mel-147 cells with latrunculin B which inhibits F-actin formation and MRTF-A in vitro SK-Mel-147 exhibits aggressive phenotypes and relocatedtothecytosol(Supplementary Fig. S2). MRTF activity overexpresses RhoC is also regulated by the relative levels of G-actin (26). To assess Previously published results showed that SK-Mel-147 cells MRTF activity, we determined the mRNA expression of three were highly metastatic in immunocompromised mice while known Rho/MRTF target genes, myosin regulatory light chain 9 fi SK-Mel-19 cells were not (22), so to con rm these observa- (MYL9), cysteine-rich angiogenic inducer 61 (CYR61), and tions, we compared phenotypes of SK-Mel-147 and SK-Mel-19 connective tissue growth factor (CTGF). Consistent with the using in vitro models of cancer aggressiveness. SK-Mel-147 predominantly nuclear MRTF-A, SK-Mel-147 shows dramati- readily forms colonies while SK-Mel-19 is unable to form cally increased expression of these three Rho/MRTF-regulated fi quanti able colonies under these conditions (Fig. 1A). We genes compared with SK-Mel-19 (Fig. 2B). Along with being next examined cellular migration and invasion using the MRTF target genes, MYL9, CYR61,andCTGF play important scratch-wound migration assay (Fig. 1B) as well as Matrigel roles in cellular migration and metastasis (10, 27). SK-Mel-147 invasion assay (Fig. 1C), respectively. Consistent with previous cells clearly have increased MRTF activation as compared with results, SK-Mel-147 cells were much more migratory and SK-Mel-19 cells, suggesting that this may contribute to the invasive than SK-Mel-19. Rho GTPase signaling has been metastatic potential of these cells. shown to promote cellular migration and invasion (23). More specifically in melanoma, RhoC expression was elevated in cell lines selected in mice for increased metastatic behavior (5). To MRTF pathway inhibitor CCG-203971 blocks MRTF-A nuclear determine the relative Rho signaling potential within SK-Mel- accumulation and transcription activity 147 and SK-Mel-19, we quantified RhoA and RhoC protein We next assessed the potential for using a novel small- expression (Fig. 1D and E). RhoC protein is overexpressed in molecule inhibitor of the Rho/MRTF pathway to block in vitro the more aggressive melanoma cell line, SK-Mel-147, while and in vivo models of melanoma metastasis. To evaluate tran- RhoA levels are not significantly different between the cell lines scriptional activity of MRTF, we performed luciferase assays fi (Fig. 1E). RhoA plays an important role in proliferation and with a modi ed serum response element promoter (SRE.L) that cytokinesis, whereas RhoC seems to be more involved in is selective for MRTF/SRF but does not respond to TCF/SRF migration, invasion, and metastasis (3, 4). In addition to complexes (11, 28). CCG-203971 inhibits RhoC-induced SRE- increased RhoC expression, SK-Mel-147 also shows modestly Luciferase expression in SK-Mel-147 cells with an IC50 of m increased MRTF-A expression levels compared with SK-Mel-19, approximately 6 mol/L. CCG-203971 does not cause cellular m but it is not statistically significant (Fig. 1D and E). To under- toxicity up to 100 mol/L using the WST-1 viability assay in a stand the potential contribution of RhoC and MRTF in clinical 24-hour time period (Fig. 3A). In addition, CCG-203971 melanoma, we explored survival data for cutaneous melanoma reduces the nuclear localization of MRTF-A in SK-Mel-147 cells datasetsinTheCancerGenomeAtlas(TCGA;ref.24).Strat- in a concentration-dependent manner (Fig. 3B). By inhibiting ification of high top quartile versus low bottom quartile MRTF- the Rho/MRTF pathway, treating SK-Mel-147 cells with CCG- A or RhoC expression levels shows a significant reduction of 203971 also reduces the expression of endogenous MYL9, percent survival in patients with high MRTF-A or RhoC expres- CYR61,andCTGF (Fig. 3C). sion(Fig.1F).Interestingly,stratification of RhoA, RhoB, and MRTF-B high versus low expression does not result in signif- CCG-203971 causes G1 phase cell-cycle arrest and inhibits icant differences in melanoma patient percent survival (Sup- clonogenicity plementary Fig. S1). Of note, the yes-associated protein (YAP) To evaluate the effect of CCG-203971 on cellular growth in and its paralog (TAZ), which have been reported to regulate the aggressive melanoma cell line (SK-Mel-147) compared

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Figure 1. In vitro phenotypes of SK-Mel-19 and SK-Mel-147 and their protein expression levels of RhoA, RhoC, and MRTF-A. A, Clonogenicity was determined by seeding 200 live cells/well into 6-well plates. After 10 days, the colonies were fixed and stained with formaldehyde/crystal violet and the total number of colonies in each well was determined using a >50 cells/colony cutoff. Data are expressed as the mean (SD) of duplicate experiments. B, Cellular migration determined by scratch wound assay. Cells are grown to confluence in 12-well plates then a scratch is made using a 200-mL pipette. Images are taken at 0 and 24 hours in 2.0% serum. Shown are examples of SK-Mel-19 and SK-Mel-147 after 24 hours. Quantification of the open area is determined computationally and the migration of each cell type relative to SK-Mel-19 after 24 hours is shown; Results are expressed as the mean (SEM) of triplicate experiments (, P < 0.001 vs. SK-Mel-19). C, Real-time analysis of melanoma cell invasion. xCELLigence label-free system was used to monitor real-time Matrigel invasion of SK-Mel-147 and SK-Mel-19 in RPMI containing 0.5% FBS. Shown is a representative figure of the change in cell index (SEM) from one experiment performed in triplicate. For each condition, at least two independent experiments were performed. D, Western blot analysis of RhoA, RhoC, MRTF-A, and GAPDH. Each melanoma cell line was starved for 24 hours in 0.5% serum prior to the preparation of total protein lysates. Images are representative blots from three separate experiments. E, Quantitative band density analysis was performed for each experiment comparing the intensity of RhoA, RhoC, and MRTF-A relative to the normalizing protein GAPDH. Results are expressed as the mean (SEM) of triplicate experiments (, P < 0.01 vs. SK-Mel-19). F, The Cancer Genome Atlas (TCGA) cutaneous melanoma dataset was stratified into quartiles based upon expression of RhoC or MRTF-A (n ¼ 107 samples per quartile). Kaplan–Meier plots were generated from the highest (gray) and lowest (black) expressing quartiles. Survival curves were analyzed with the log-rank test with a cutoff of P < 0.05 as statistically significant. with the less aggressive cell line (SK-Mel-19), we determined SK-Mel-147 cells at 72 hours. CCG-203971 is more potent in the effect of Rho/MRTF pathway inhibition on cellular prolif- reducing cell numbers in our cell proliferation assay in the SK- eration. While it had no effect on cell numbers at 24 hours, Mel-147 cell line compared with SK-Mel-19 (Fig. 4A). To CCG-203971 caused a significant reduction in the number of understand whether CCG-203971 affected cell proliferation

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Figure 2. MRTF-A localization and activity is increased with F-actin stress fiber formation in the SK-Mel-147 cells. A, Cellular localization of MRTF-A observed with immunocytochemistry in starved (0.5% FBS, 24 hours) melanoma cells. Images were quantified by scoring an individual cell as predominantly nuclear (N), cytosolic (C), or even distribution (N/C) of MRTF-A. F-actin staining was also performed in the same cells using fluorophore-conjugated phalloidin toxin. Counts are from three independent experiments with at least 100 cells scored blindly for each condition. For stress fiber quantification, only cells expressing prominent stress fiber bundles were determined to be "stress fiber positive". Results are expressed as the mean (SEM) of triplicate experiments (, P < 0.01 vs. SK-Mel-19). Scale bar shown represents 200 mm. B, Expression of MRTF-A target genes known to be regulated by Rho/MRTF activity and involved in cancer migration; myosin regulatory light polypeptide (MYL9), cysteine-rich angiogenic inducer 61 (CYR61), and connective tissue growth factor (CTGF), are quantified by qPCR. Results are expressed as the mean (SEM) of triplicate experiments.

or induced apoptosis, we conducted cell-cycle analysis using city of the SK-Mel-147 cells. Following a 72-hour treatment flow cytometry in SK-Mel-147 cells following 10 mmol/L CCG- with DMSO or CCG-203971, equal numbers of viable cells 203971 treatment for 72 hours. CCG-203971 caused G0–G1 were plated for colony formation in the absence of our Rho/ arrest as evidenced by a significant increase in G0–G1 phase MRTF pathway inhibitor. The CCG-203971–treated cells and a decrease in S-phase compared with DMSO control formed roughly half as many colonies compared with control treatment (Fig. 4B and C). CCG-1423, the precursor com- treatment and the colonies which formed were approximately pound to CCG-203971, has been shown to downregulate 50% reduced in size (Fig. 4D). genes that are involved in cell cycle, specifically G1–Stransi- tion, including targets of the transcription factor E2F (GEO CCG-203971 inhibits cellular migration and invasion Accession number: GSE30188; ref. 29). There was no signifi- To determine whether inhibition of MRTF localization and cant increase in sub-G1 in CCG-203971–treated SK-Mel-147 activity affects phenotypes of the aggressive melanoma line, we cells (Fig. 4B), suggesting that CCG-203971 does not induce investigated the influence of CCG-203971 at the cellular level. apoptosis. Consistent with the effects of CCG-203971 to Using Transwell migration and label-free Matrigel invasion induce G0–G1 cell-cycle arrest, it also inhibited the clonogeni- assays, we found that 10 mmol/L CCG-203971 inhibited cellular

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Figure 3. CCG-203971 regulates cellular localization and blocks target gene expression of MRTF. A, SK-Mel-147 cells were cotransfected with RhoC and the SRE.L reporter plasmid as described in Materials and Methods. After transfection, cells were treated with the indicated concentrations of CCG-203971 overnight before lysis and reading luminescence in the plate reader. Data are graphed as a percentage of the DMSO control. B, Immunocytochemistry was performed on SK-Mel-147 cells which under starved conditions (0.5% FBS, 24 hours) maintained nuclear localization of MRTF-A. Individual cells were assessed for predominately nuclear (N), cytosolic (C), or an even distribution (N/C) of MRTF-A. Data are the averages of three independent experiments with at least 100 cells counted for each condition. Scale bar shown represents 200 mm. C, CCG-203971 effect on gene expression of CTGF, MYL9, and CYR61. SK-Mel-147 cells were starved in 0.5% FBS containing media for 24 hours in the presence of the indicated concentration of CCG-203971 or DMSO before isolation of RNA for qPCR analysis. Results are expressed as the mean (SEM) of triplicate experiments (, P < 0.05, , P < 0.01 vs. DMSO control).

migration (Fig. 5A) and invasion (Fig. 5B) of the RhoC-over- greater by approximately 15 times. To determine the potential expressing melanoma line SK-Mel-147. This migratory phenotype for MRTF pathway inhibitors as metastasis-targeting therapeu- is consistent with an action on MRTF-A as siRNA knockdown of tics, we treated mice with CCG-203971 (100 mg/kg twice MRTF-A expression (Fig. 5C) inhibits SK-Mel-147 Transwell daily) or vehicle control for 18 days following tail vein migration (Fig. 5D). injection of GFP-expressing SK-Mel-147 cells. The CCG- 203971–treated group had about half as many tumors which CCG-203971 blocks melanoma lung metastasis were also about 1/4 the size. This resulted in a ﬁnal tumor To compare the metastatic potential of the high versus low burden around 7 times lower than in the vehicle-treated group RhoC-expressing cell lines, we injected mice with GFP-expres- (Fig. 6B). sing SK-Mel-19 and SK-Mel-147 cells. The RhoC-overexpressing SK-Mel-147 cells readily formed lung metastases following intravenous injection (Fig. 6A). The SK-Mel-147–injected Discussion mice not only displayed more metastatic nodules (3)than Mutations in BRAF and NRAS, leading to constitutive acti- did SK-Mel-19–injected mice, but the tumors were much vation of the MAPK pathway, are found in nearly approximate- larger (5 greater area) than those in the SK-Mel-19– ly 60%–70% of primary human cutaneous melanomas (30). injected mice (Fig. 6B). Thus, the total tumor burden was However, these driver mutations do not appear to govern the

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Figure 4.

CCG-203971 inhibits melanoma cell proliferation through G0–G1 cell-cycle arrest and reduces clonogenicity. A, CCG-203971 inhibits melanoma cell proliferation. Cells were grown in DMEM 0.5% FBS for 72 hours with 0.1% DMSO control or the indicated concentrations of CCG-203971. Cells were harvested and viable cells

counted by Trypan blue exclusion. B and C, CCG-203971 induces G0–G1 cell-cycle arrest. Cells were grown in 0.5% FBS in the presence of 0.1% DMSO or 10 mmol/L CCG-203971 for 72 hours. Cells were stained with prodidium iodide and analyzed using ﬂow cytometry. B, Representative ﬂuorescence intensities versus event counts. C, The results are expressed as the mean (SEM) of triplicates from three independent experiments ( P < 0.05 G0–G1 vs. no treatment and DMSO). D, CCG-203971 reduces clonogenicity of SK-Mel-147. Cells were treated with 10 mmol/L CCG-203971 or 0.1% DMSO for 72 hours. Equal number of viable cells were plated and allowed to form colonies for 7 days. Results are expressed as the mean (SEM) of triplicate experiments (, P < 0.01 vs. no treatment and DMSO).

propensity for metastasis; BRAF vs. NRAS mutation status does activators (GNA12, GNA13, GNAQ, GNA11,orARHGEF11). not predict patient overall survival (31). In addition, current Furthermore, an outlier analysis of melanoma in Oncomine, targeted therapeutics, such as the BRAFV600E inhibitor vemur- (34) showed that 5%–20% of melanomas across several afenib, demonstrate profound initial results in a majority of studies had marked upregulation of RhoC at the transcription- BRAFV600E-expressing melanoma tumors (32). However, these al level. Mechanisms driving this are not known but could responses are limited in duration and resistance typically devel- include regulation by the ETS-1 transcription factor (35, 36). ops within months. Consequently, it is important to better Loss of E-cadherin in melanoma leads to ETS-1 activation and understand factors that drive aggressiveness and metastasis of enhanced RhoC expression (37). RhoC induces c-Jun expres- human melanomas. On the basis of the role of RhoC and sion (likely through MRTF/SRF mechanisms) which contri- MRTF-regulated gene transcription in genetic studies in mouse butes to cell survival (37). This could partially explain the B16F2 melanoma and breast cancer models (2, 4, 11) and in increased clonogenicity observed in SK-Mel-147, the RhoC- the TCGA dataset (24), we wanted to explore the role of those overexpressing melanoma cell line, as well as the increased mechanisms in human metastatic melanoma utilizing our lung colonization. novel Rho/MRTF pathway inhibitor, CCG-203971. In light of the multiple genes and pathways that can lead to While mutations in Rho proteins are rare, review of cancer activation of Rho and MRTF-regulated transcription (38, 39), genomic information through cBioPortal, (33) showed that inhibition at a downstream step in the pathway would be 31% of cutaneous melanomas had ampliﬁcation or mutation beneﬁcial to disrupt signals from the numerous activation of RhoA/C, MKL1/2 (gene names for MRTF-A/B) or upstream mechanisms (40, 37). Interestingly, CCG-203971 treatment

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MRTF Inhibition Blocks Melanoma Metastasis

Figure 5. CCG-203971 inhibits cellular migration and invasion. A, CCG-203971 blocks melanoma migration. Cells were seeded into the top of a Transwell migration chamber with 0.5% FBS on top and bottom to measure basal migration of SK-Mel-19 and SK-Mel-147. CCG-203971 (10 mmol/L) blocked SK-Mel-147 migration. Shown are images at 4 magnification using a bright field microscope after 6 hours of migration. Four random fields of view at 20 magnification were quantified for the number of cells which migrated through the membrane. Results are expressed as the mean (SEM) of triplicate experiments (P < 0.01 vs. SK-Mel-19 with DMSO and vs. SK-Mel-147 with CCG-203971). B, CCG-203971 inhibits invasion of SK-Mel-147 melanoma cells. Real-time analysis of cellular invasion was analyzed using the xCELLigence label-free system. SK-Mel-147 plus 10 mmol/L CCG-203971 or SK-Mel-19 were allowed to invade through Matrigel in RPMI containing 0.5% FBS. Shown is a representative figure of the change in cell index (SEM) from one experiment performed in triplicate. For each condition at least two independent experiments were performed. C, Western blot analysis of MRTF-A in SK-Mel-147 cells knocked down using siRNA for 48 hours. D, MRTF-A knockdown blocks melanoma migration. siRNA-transfected cells were seeded into the top of a Transwell migration chamber with 0.5% FBS on top and bottom. Shown are representative images at 4 magnification using a bright field microscope after 6 hours of migration. Three random fields from experimental duplicates at 20 magnification using bright field microscope are quantified for cell numbers. Results are a mean (SEM) of three independent experiments. (, P < 0.001 vs. no transfection and siNontargeting).

appears to influence SK-Mel-147 cellular morphology in such a using MRTF-A/B double knockdown or silencing SRF (11). We manner that SK-Mel-147 cells more closely resemble SK-Mel-19 also confirm that in SK-Mel-147 cells, siRNA knockdown of cellular morphology. Further studies will need to be conducted MRTF-A alone is sufficient to markedly reduce migration in to better understand this observation. Our compound, CCG- vitro. The majority of recent literature strongly implicates MRTF 203971 prevents the nuclear accumulation of MRTF but the and SRF as being critical factors in the regulation cell motility molecular mechanism remains uncertain. Two proposed mod- (43–45). Despite the commonality between MRTF/SRF- and els include direct binding to MRTF (41) or modulation of the YAP/TEAD–regulated target genes, as well as cell type–specific intranuclear actin regulation by MICAL2 (42). Genetic valida- mutual coactivation of gene expression (46), only MRTF-A tion of our pharmacologic approach to suppress metastasis has expression levels were correlated with patient survival (Fig. 3; already been shown for both melanoma and breast cancer Supplementary Fig. S2).

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Figure 6. Rho/MRTF pathway effect on experimental lung metastasis. A, Immunocompromised mice were separated into three groups. One group received tail vein injection of GFP-expressing SK-Mel-19 (2 106 cells). The other two groups were injected with GFP-expressing SK-Mel-147 (2 106 cells). Following tail vein injection, the mice began twice daily intraperitoneal injection of 100 mg/kg CCG-203971 or 50-mL DMSO (vehicle control). After 18 days, the mice were euthanized and lungs were collected for histologic analysis. Sections from each lung were immunohistologically probed for GFP. Images were taken at 20 magniﬁcation, shown are two representative images of metastatic colonies from each group. Scale bar represents 400 mm. B, Each slide was visually scanned for total number of lung colonies, and the average tumor area was determined using image analysis software. The total tumor burden was calculated by adding together the total cross- sectional area of all colonies identiﬁed within each mouse. Data is represented as the mean (SEM) of n ¼ 6 (SK-Mel-19þDMSO and SK-Mel-147þDMSO) and n ¼ 5 (SK-Mel-147þCCG-203971; , P < 0.05, , P < 0.001 vs. SK-Mel-19þDMSO; þ, P < 0.05, þþþ, P < 0.001 vs. SK-Mel-147þDMSO).

The potential for an antimetastatic small-molecule thera- treatments that would be required for antimetastasis therapies. peutic that targets this gene transcription mechanism is illus- Furthermore, improved bioavailability and pharmacokinetics trated by our result showing marked suppression of SK-Mel- would facilitate both preclinical and potentially clinical 147 lung metastasis by in vivo treatment with CCG-203971. studies. While it has been argued that antimetastatic therapies are not In addition to melanoma, spontaneous nuclear localization of useful or are difﬁcult to test clinically, there is increasing MRTF-A has been reported in MDA-MB-231 breast cancer cells recognition that this approach has value (47). CCG-203971 (11). The MDA-MB-231 cells are often used as a model of also effectively causes G1 cell-cycle arrest which is a phenotype aggressive breast cancer cells. Intriguingly, they also harbor a Ras shared with current MAPK pathway–targeted therapeutics (48, mutation (KRASG13D; ref. 50) similar to the NRASQ61N in SK-Mel- 49). As G1-arrested melanoma cells have increased sensitivity 147. Additional work is needed to assess the generality of these to MAPK pathway inhibitors (49) this suggests the potential observations. beneﬁtofcombinationtreatmentwithRho/MRTFpathway In this report, we provide evidence that Rho-regulated, inhibitors and MAPK pathway inhibitors. Continued efforts MRTF-mediated gene transcription signaling plays an impor- to optimize MRTF transcription inhibitors like CCG-203971 tant role in migration, invasion, and metastasis of human will be needed. In particular, enhancing potency along with cutaneous melanoma. Thus, expression levels of RhoC and reduction of acute toxicity (12, 13) is required for chronic MRTF target genes may provide useful biomarkers to identify

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cancers with propensity for metastasis as well as for targeted Writing, review, and/or revision of the manuscript: A.J. Haak, K.M. Appleton, therapies by this approach. Small-molecule inhibitors of this S.M. Wade, J.L. Bell, C.E. Rockwell, M. Airik, M.A. Krook, S.D. Larsen, pathway, such as enhanced CCG-203971 analogues, represent M.E. Verhaegen, E.R. Lawlor, R.R. Neubig, E.M. Lisabeth Administrative, technical, or material support (i.e., reporting or organizing an exciting class of pharmacologic probes and potential future data, constructing databases): K.M. Appleton, M.A. Krook, M.E. Verhaegen therapeutics. Study supervision: E.R. Lawlor, R.R. Neubig Other (chemical synthesis): J.L. Bell fl Disclosure of Potential Con icts of Interest Acknowledgments A.J. Haak has ownership interest (including patents) in a Patent Appli- We would like to thank Riya Malhotra, Alexandra Turley, and Joseph cation (20160145251). S.D. Larsen has ownership interest (including Zagorski for technical assistance with the flow cytometry experiments. We patents) in a United States Patent Application (20160145251). R.R. Neubig would also like to thank Raelene Van Noord for her assistance with the mouse has ownership interest (including patents) in a United States Patent Appli- metastasis study and Nadia Ayala-Lopez, Humphrey Petersen-Jones, and Dr. fl cation 20160145251. No potential con icts of interest were disclosed by the Stephanie Watts for assistance with the immunohistochemical analysis of other authors. melanoma metastasis.

Authors' Contributions Grant Support Conception and design: A.J. Haak, K.M. Appleton, S.D. Larsen, M.E. Verhaegen, This work was supported in part by a Pharmacological Sciences Training R.R. Neubig, E.M. Lisabeth Program grant GM007767 from NIGMS (to A.J. Haak), NCIT32CA009676 Development of methodology: A.J. Haak, K.M. Appleton, S.M. Wade, (to M.A. Krook), and SPOREU54CA168512 (to E.R. Lawlor). C.E. Rockwell The costs of publication of this article were defrayed in part by the Acquisition of data (provided animals, acquired and managed patients, payment of page charges. This article must therefore be hereby marked provided facilities, etc.): A.J. Haak, K.M. Appleton, S. Misek, Y. Ji, S.M. Wade, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate C.E. Rockwell, M. Airik, M.A. Krook, E.R. Lawlor this fact. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A.J. Haak, K.M. Appleton, S. Misek, Y. Ji, S.M. Wade, Received July 22, 2016; revised October 17, 2016; accepted October 21, 2016; C.E. Rockwell, M. Airik, M.A. Krook, S.D. Larsen, E.R. Lawlor, R.R. Neubig published OnlineFirst November 11, 2016.

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204 Mol Cancer Ther; 16(1) January 2017 Molecular Cancer Therapeutics

Pharmacological Inhibition of Myocardin-related Transcription Factor Pathway Blocks Lung Metastases of RhoC-Overexpressing Melanoma

Andrew J. Haak, Kathryn M. Appleton, Erika M. Lisabeth, et al.

Mol Cancer Ther 2017;16:193-204. Published OnlineFirst November 11, 2016.

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