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. Ó2016 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 Ó2016 American Association for Cancer Research. optimization of this compound series to reduce toxicity (12) www.aacrjournals.org 193 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst November 11, 2016; DOI: 10.1158/1535-7163.MCT-16-0482 Haak et al. 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