, kathryn m. appleton , erika m. lisabeth , sean misek ... · 1 pharmacological inhibition of...

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Pharmacological inhibition of Myocardin-related transcription factor pathway blocks lung metastases

of RhoC overexpressing melanoma

Andrew J. Haak1*, Kathryn M. Appleton5*, Erika M. Lisabeth5*, Sean Misek5, Yajing Ji5, Susan M. Wade1,

Jessica L. Bell2, Cheryl E. Rockwell5, Merlin Airik4, Melanie A. Krook4, Scott D. Larsen2, Monique Verhaegen3,

Elizabeth R. Lawlor4, Richard R. Neubig5.

Departments of 1Pharmacology and 2Medicinal Chemistry, and College of Pharmacy, and 3Dermatology, and

4Pediatrics, University of Michigan Medical Center, Ann Arbor, MI. 5Department of Pharmacology & Toxicology,

Michigan State University, East Lansing, MI.

Running Title: MRTF inhibition blocks melanoma metastasis

Keywords: Rho, MKL1, ROCK, MRTF-A, SRF

Financial Support: This work was supported in part by a Pharmacological Sciences Training Program grant

GM007767 from NIGMS (A.J. Haak); NCI T32CA009676 (M.A. Krook) and SPORE U54CA168512 (E.R.

Lawlor).

Corresponding Author: Richard R. Neubig, Michigan State University, Department of Pharmacology and

Toxicology. 1355 Bogue St / B440 Life Sciences East Lansing, MI 48824. Phone: 517 353-7145. Fax: 517-353-

8915. E-mail: [email protected].

Disclosure of potential conflicts of interest: Drs. Larsen, Neubig, and Haak filed a United States Patent

(Application 20160145251) for analogs related to CCG-203971.

Manuscript notes: 4,452 total words, 6 figures

*Authors made equal contributions

on January 9, 2020. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 11, 2016; DOI: 10.1158/1535-7163.MCT-16-0482

http://mct.aacrjournals.org/

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Abstract

Melanoma is the most dangerous form of skin cancer with the majority of deaths arising from metastatic

disease. Evidence implicates Rho-activated gene transcription in melanoma metastasis mediated by the

nuclear localization of the transcriptional coactivator, myocardin-related transcription factor (MRTF). Here, we

highlight a role for Rho and MRTF signaling and its reversal by pharmacologic inhibition using in vitro and in

vivo models of human melanoma growth and metastasis. Using two cellular models of melanoma, we clearly

show that one cell type, SK-Mel-147, is highly metastatic, has high RhoC expression, and MRTF nuclear

localization and activity. Conversely, SK-Mel-19 melanoma cells have low RhoC expression, and decreased

levels of MRTF-regulated genes. To probe the dependence of melanoma aggressiveness to MRTF

transcription, we use a previously developed small molecule inhibitor, CCG-203971, which at low micro-molar

concentrations blocks nuclear localization and activity of MRTF-A. In SK-Mel-147 cells, CCG-203971 inhibits

cellular migration and invasion, and decreases MRTF target gene expression. In addition, CCG-203971-

mediated inhibition of the Rho/MRTF pathway significantly reduces cell growth and clonogenicity and causes

G1 cell cycle arrest. In an experimental model of melanoma lung metastasis, the RhoC-overexpressing

melanoma cells (SK-Mel-147) exhibited pronounced lung colonization compared to the low RhoC expressing

SK-Mel-19. Furthermore, pharmacological inhibition of the MRTF pathway reduced both the number and size

of lung metastasis resulting in a marked reduction of total lung tumor burden. These data link Rho and MRTF-

mediated signaling with aggressive phenotypes and support targeting the MRTF transcriptional pathway as a

novel approach to melanoma therapeutics.




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Introduction

Rho GTPases play significant roles in human cancer (1). Although RhoA and RhoC share considerable

homology, they may coordinate different aspects of cancer progression. RhoC appears to be most important

for cellular invasion and metastasis (2) while RhoA plays a role in transformation and tumor proliferation (3). In

a screen designed to identify genes important for melanoma metastasis, RhoC was found to be up-regulated

and it contributed to melanoma lung metastasis (4). Rho GTPases are well-known for regulating the actin

cytoskeleton (5). RhoA/C activation causes the formation of myosin-rich F-actin bundles called stress fibers

through their downstream effectors Rho associated kinase (ROCK) and Diaphanous-related formin-1 (mDia1)

(6, 7). By modulation of the actin cytoskeleton, Rho GTPases also regulate gene expression though

myocardin-related transcription cofactors (MRTF-A/B) also known as megakaryoblastic leukemia proteins

(MKL1/2). In the nucleus, MRTF cooperates with serum response factor (SRF) to induce transcription of genes

involved in proliferation, migration, invasion, and metastasis (8, 9). In the N-terminal region of MRTF are two

G-actin binding RPEL motifs which sequester MRTF in the cytoplasm. Rho activity reduces cytosolic G-actin,

allowing MRTF to translocate into the nucleus and activate gene transcription. Stable knockdown of MRTF or

SRF in B16M2 melanoma or human MDA-MB-231 breast cancer cells with high RhoC expression results in a

dramatic reduction of in vivo lung metastasis and in vitro cellular migration (10). This evidence suggests that

gene regulation through MRTF mediates RhoC-induced cancer metastasis.

To assess the potential for targeting MRTF-regulated gene transcription in cancer metastasis, we used

a previously identified small-molecule inhibitor of the MRTF pathway (11). Our initial compound, CCG-1423,

selectively blocked Rho GTPase regulated prostate cancer cell invasion at low micro-molar concentrations

(11). Further structure-activity relationship optimization of this compound series to reduce toxicity (12) and to

enhance potency resulted in a new analog (13), CCG-203971, that also blocks MRTF-induced gene

expression and prostate cancer migration. The Rho/MRTF pathway has also recently been highlighted as a

major signaling crossroad in the development of pathological fibrosis in diseases including idiopathic

pulmonary fibrosis, scleroderma, and Crohn’s disease (14, 15). We have recently shown efficacy of CCG-




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203971 in a murine model of skin fibrosis (16) but a close examination of MRTF pathway inhibitors in models

of cancer progression has not been performed.

Here we investigate the Rho/MRTF pathway in two metastatic melanoma cell lines; SK-Mel-19, and

SK-Mel-147. There is a clear dichotomy between the two lines where the high level of RhoC, constitutive

MRTF-A nuclear localization and function in SK-Mel-147 is associated with in vivo and in vitro correlates of

cancer metastasis including clonogenicity, migration, and invasion. As a novel approach to anti-metastasis

therapeutics, our MRTF pathway inhibitor, CCG-203971, blocks cellular migration and invasion and profoundly

suppresses SK-Mel-147 lung metastasis in vivo.




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MATERIALS AND METHODS

Cell Culture and Proliferation

Human cutaneous melanoma cell lines, SK-Mel-19 and SK-Mel-147, obtained from Dr. Maria Soengas at the

University of Michigan, were cultured in DMEM (Life Technologies) supplemented with 10% fetal bovine serum

(Life Technologies) including penicillin/streptomycin (Life Technologies). Short tandem repeat profiles were

conducted on the melanoma cell lines (Genewiz, South Plainfield, NJ). The profiles obtained for the melanoma

cell lines do not match any established published profiles, and we are unable to identify published profiles for

SK-Mel-19 and SK-Mel-147. Cells were expanded and frozen immediately prior to authentication and then

resuscitated only two to three months before experiments. We verified the driver mutations in our lab using

MSU Sanger sequencing service expected from previous reports (i.e. BRAFV600E for SK-Mel-19 and NRASQ61L

for SK-Mel-147 (17, 18)). For proliferation experiments 1x104 cells were plated into 24-well plates in DMEM

containing 10% FBS. Cells were allowed to attach overnight and then medium was replaced with DMEM

containing 0.5% FBS. After 72 hours of growth, cells were stained with 0.4% trypan blue stain (Invitrogen). Cell

counting was performed using the Countess® Automated Cell Counter (Invitrogen, Carlsbad, CA) according to

manufacturer’s instructions.

Immunocytochemistry and F-actin staining

Cells (1.0x105) were plated onto fibronectin-coated cover slips in DMEM with 10% FBS and allowed to attach

overnight. Medium was changed to DMEM with 0.5% FBS for 24 hours plus the indicated concentration of

CCG-203971 or 0.1% DMSO. Cells were then fixed in 3.7% formaldehyde for 10 minutes at room temperature

(RT) and permeabilized with 0.25% Triton X-100 for 10 minutes at RT. For immunocytochemistry studies,

primary antibody for MRTF-A (Santa Cruz, cat# SC-21558) was diluted 1:100 and incubated for 2 hours at RT.

Fluorophore-conjugated secondary (Alexa Fluor® 594 Donkey Anti-Goat IgG, Invitrogen) was diluted 1:1,000

and added for 1 hour. For stress fiber assays, a final concentration of 100 nM Acti-Stain® phalloidin

(Cytoskeleton) in 100 µL 1X PBS/1% BSA was added to each cover slip for 30 minutes at RT. Cells were

mounted (Prolong Gold ® antifade-reagent with DAPI, Invitrogen) and imaged on an upright fluorescence

microscope (Nikon E-800, Melville, NY) at 60X magnification.




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Real-time PCR

Cells (1.0x106) were plated into 60 mm dishes and starved overnight in DMEM containing 0.5% FBS. Cells

were lysed and RNA was isolated using the RNeasy® kit (Qiagen) following the manufacturer’s directions.

DNAse-treated RNA (1 µg) was used as a template for synthesizing cDNA utilizing the Taqman® Reverse-

Transcription Reagents kit (Invitrogen). SYBR green qPCR (SABiosciences) was performed using a

Stratagene Mx3000P (Agilent Technologies, Santa Clara, CA) and cT values were analyzed relative to GAPDH

expression. Primer sequences: GAPDH; 5’ GGAAGGGCTCATGACCACAG. 3’

ACAGTCTTCTGGGTGGCAGTG. CTGF; 5’ CAGAGTGGAGCGCCTGTT. 3’ CTGCAGGAGGCGTTGTCA.

CYR61; 5’ CGCGCTGCTGTAAGGTCT. 3’ TTTTGCTGCAGTCCTCGTTG. MYL9; 5’

CATCCATGAGGACCACCTCCG. 3’ CTGGGGTGGCCTAGTCGTC.

Scratch Migration Assay

Cells (4.0x105) were plated in DMEM containing 10% FBS in a 12-well plate. After 24 hours, a scratch was

made using a 200 µL pipette tip. Medium was replaced with DMEM containing 2.0% FBS and images of the

wound were taken at time=0 and 24 hours using a bright field microscope (Olympus CKX41, Center Valley,

PA). Area quantification of the scratch was determined using ImageJ software as previously described (13,

19). Briefly, the binary (white and black) threshold for each image was manually adjusted to leave only the

open area of the scratch black. The area of this scratch was determined and the percent closure was

calculated by comparing the difference in area from t=0 hour to t=24 hour.

Transwell Migration Assay

Cells (5.0x104) were added to the top of an 8 µm pore diameter transwell chamber (Millipore) in DMEM

containing 0.5% FBS with 10 µM CCG-203971 or 0.1% DMSO control. Medium containing 0.5% FBS and

compound or DMSO was also added to the bottom chamber. Cells were allowed to migrate for 6 hours, and

after wiping the remaining cells off the top of the transwell chamber, cells which migrated were fixed and

stained in 4% formaldehyde/0.5% crystal violet. The number of migrated cells for each well was determined by

examining four random non-overlapping fields of view at 20X magnification using a bright field microscope




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(Olympus CKX41, Center Valley, PA). For knockdown experiments, cells transfected for 48 hours with siRNA

pools were plated, allowed to migrate, and analyzed as stated above.

Small Interfering RNA knockdown

ON-TARGET plus SMART pools (GE Dharmacon, Lafayette, CO) targeting human MRTF-A (GE cat# L-

015434-00) or non-targeting pool control (GE cat# D-001810-10-05) were used according to manufacturer’s

recommendations. Briefly, 25 nM siRNA smart pools were transfected using DharmaFECT (GE cat# T-2001-

01). Western blot validation of MRTF-A expression was conducted using a fraction of the same transfected

population used for transwell assays.

Label-Free Migration and Invasion Assay

Real-Time Cell Analysis (RTCA) of cell invasion was monitored using a CIM-plate 16 and xCELLigence DP

System (Acea Bioscience, Inc, San Diego, CA.). Matrigel™ Matrix (BD BioSciences) 5% (v/v) was diluted 1:20

in basal RPMI media and was added in the upper chamber and allowed to equilibrate for 4 hours in an

incubator at 37°C in 5% CO2. Cells (3x105/well) were added at t=5 hours. For compound effects in invasion

assays, cells were treated with 10 μM CCG-203971 upon placement into the upper chamber. Cell index was

measured every 30 minutes.

Luciferase Assay

Cells (3.0x104) were seeded into 96-well plates in DMEM containing 10% FBS overnight. Cells were then

cotransfected with 56 ng SRE.L- Luciferase reporter selective for Rho/MRTF described in ref. (20), and 2.3 ng

RhoC plasmid diluted with Lipofectamine 2000 (Invitrogen) in Opti-MEM (Life Technologies). After 6 hours,

compounds were added for overnight treatment. The next day WST-1 (10 µL/well) reagent (Promega) was

added to measure viability. After 1 hour, absorbance was read (450 nm) before lysing the cells for luciferase

measurement (Promega Luciferase Assay System).

Clonogenic Assay

Cells were grown for 6 days in DMEM with 2.0% FBS. Two hundred live cells, determined by trypan blue

exclusion assay, were seeded into a 6-well plate and allowed to form colonies in DMEM containing 2.0% FBS

for 10 days. Colonies were fixed and stained in 3.7% formaldehyde/0.5% crystal violet for 10 minutes at RT.




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Only cell clusters consisting of at least 50 cells were counted as colonies. For CCG-203971 treatment, cells

(2.0x105) were seeded into 6-well dishes and allowed to adhere overnight. The following day, the medium was

replaced with DMEM containing 0.5% FBS and 0.1% DMSO or 10 µM CCG-203971 was added. 72 hours

later, two hundred or 1x103 cells were plated per group in DMEM with 10% FBS in 6-well dishes and allowed to

form colonies for seven days. Colonies were fixed and stained as stated above. Colony counts and mean area

quantification were determined using ImageJ software. Briefly, the image threshold was adjusted to black and

white and further processed to smooth colony pixilation. The images were then analyzed for colony size

(pixel^2) fixed at 50-infinity and for circularity defined at 0.2-1.0.

Flow Cytometry

Cells (5.0x105) were plated in 60mm dishes in DMEM with 10% FBS and allowed to adhere overnight. Media

was replaced with DMEM with 0.5% FBS, and 0.1% DMSO or 10 µM CCG-203971 was added. Cells were

allowed to grow for 72 hours and were then collected using 1X versene (Gibco), permeabilized, and fixed using

70% ethanol for 30 minutes on ice. Cells were centrifuged (200 x g, 5 minutes), and resuspended in DNA

extraction buffer (192 mL 0.2 M Na2HPO4, 8mL 0.1 M citric acid, pH 7.8, filter sterilized) for five minutes. Cells

were centrifuged (300 x g, 5 minutes) to remove DNA extraction buffer and resuspended in 250 µl of freshly

prepared DNA staining solution (2 µg/mL propidium iodide, 0.2 mg/mL RNase A in 1X PBS) and incubated at

RT for 30 minutes in the dark. Fluorescence was detected using a C6 BD Accuri flow cytometer (BD Accuri,

San Jose, CA). Fluorescence was quantified using CFlow software (BD Accuri). Cell cycle analysis was

conducted using FlowJo v10.0.8 software (Tree Star, Inc., Ashland, OR). 10,000 events were detected in

experimental triplicate for untreated cells, 0.1% DMSO, and 10 µM CCG-203971. Three independent

experiments were conducted.

Western Blot Analysis

Cells were starved overnight in DMEM containing 0.5% FBS. Cells were lysed (10mM Tris-Cl, 1mM EDTA, 1%

Triton X-100, 0.1% SDS, 140mM NaCl, protease inhibitor cocktail (Roche cat# 11873580001)), sonicated 2x

for 15 seconds, and centrifuged to quantitate soluble proteins using a BCA protein assay as recommended by

the manufacturer (Pierce BCA Protein Assay, Thermo Scientific). Protein lysate (20 µg) were resolved using




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15% SDS-PAGE gels, transferred to PVDF membranes, and blocked in 5% dried milk in tween tris-buffered

saline. Membranes were incubated in 1:1000 diluted primary antibodies RhoC (Cell Signaling, cat# 3430),

RhoA (Santa Cruz, cat# SC-418), MRTF-A (Santa Cruz, cat# SC-21558) or GAPDH (Cell Signaling, cat#

14C10) overnight at 4º C. HRP-conjugated goat anti-mouse (Santa Cruz Biotechnology, sc-2060), HRP-

conjugated goat anti-rabbit (Sigma-Aldrich, A0545) or HRP-conjugated bovine anti-goat (Santa Cruz, cat# SC-

2352) antibodies were diluted 1:10,000. Blots were developed using SuperSignal® Pico chemiluminescent

substrate (Thermo Scientific, Waltham, MA). Bands were visualized and quantified using a Li-Cor Odyssey Fc

or visualized using Kodak X-OMAT Film Developer (Rochester, NY) and quantified using ImageJ software.

Mouse Metastasis Model

Ten-week old, female, NOD.CB17-Prkdcscid/J (NOD SCID) mice were purchased from Jackson Laboratory (Bar

Harbor, ME, stock number: 001303). The mice were separated into three groups of six. One group received tail

vein injection of GFP-expressing SK-Mel-19 (2.0x106 cells). The other two groups were injected with GFP-

expressing SK-Mel-147 (2.0x106 cells). Following tail vein injection, the SK-Mel-147 mice began twice daily I.P.

injection of 100 mg/kg CCG-203971 or 50μL DMSO (vehicle control). After 18 days the mice were euthanized

and lungs were collected for histological analysis. All studies were performed according to protocols approved

by the University of Michigan, University Committee on the Use and Care of Animals (UCUCA).

Immunohistochemistry

For the quantification and imaging of lung tissue from the mouse metastasis model, paraffin-embedded

sections were dewaxed, unmasked, and treated as previously described (21). To visualize GFP-expressing

melanoma cells in the lung section, the Vector Rabbit-on-Mouse basic kit (Vector Laboratories) was utilized

according to the manufacturer's instructions. For each experiment, two tissue sections from the same animal

were developed: one with primary antibody (rabbit anti-GFP (1:250 Invitrogen A-6455)) included and one with

the primary antibody left out of the reaction. Visualization of GFP was done using the Vectastain Elite ABC kit

(Vector Laboratories), used according to the manufacturer's instructions. All sections were developed using

3,3′-diaminobenzidine (Vector Laboratories) and counterstained with haematoxylin. Images were taken at 20X

magnification using a Nikon Eclipse Ti-S microscope with mmi Cell Tools software, Version 3.47. (MMI,




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Eching, Germany). Treatment and control slides were imaged using the same exposure time and LUT settings.

The slides were blindly quantified for the number of metastatic nodules on each section followed by the use of

ImageJ software to determine the cross sectional area (µm2) of each nodule. The area was represented as an

average size for each mouse and the total tumor burden was calculated by adding the total cross sectional

areas for all tumors in each mouse then averaged for each group.

Statistics

Statistical analysis was performed by ANOVA comparison followed by Bonferonni post-test for multiple

comparisons where * p> 0.05, ** P>0.01, *** P>0.001, and **** P>0.0001. Comparison of survival curves were

performed by log-rank method where * p> 0.05, ** P>0.01.




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Results

SK-Mel-147 exhibits aggressive in vitro phenotypes and overexpresses RhoC

Previously published results showed that SK-Mel-147 cells were highly metastatic in immunocompromised

mice while SK-Mel-19 cells were not (22), so to confirm these observations, we compared phenotypes of SK-

Mel-147 and SK-Mel-19 using in vitro models of cancer aggressiveness. SK-Mel-147 readily forms colonies

while SK-Mel-19 is unable to form quantifiable colonies under these conditions (Fig. 1A). We next examined

cellular migration and invasion using the scratch-wound migration assay (Fig. 1B) as well as Matrigel®

invasion assay (Fig. 1C), respectively. Consistent with previous results, SK-Mel-147 cells were much more

migratory and invasive than SK-Mel-19. Rho GTPase signaling has been 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 determine the relative Rho signaling potential within SK-Mel-147 and

SK-Mel-19, we quantified RhoA and RhoC protein expression (Fig. 1D,E). RhoC protein is overexpressed in

the more aggressive melanoma cell line, SK-Mel-147, while RhoA levels are not significantly different between

the cell lines (Fig. 1E). RhoA plays an important role in proliferation and cytokinesis whereas RhoC seems to

be more involved in migration, invasion, and metastasis (3, 4). In addition to increased RhoC expression, SK-

Mel-147 also shows modestly increased MRTF-A expression levels compared to SK-Mel-19, but it is not

statistically significant (Fig. 1D,E). To understand the potential contribution of RhoC and MRTF in clinical

melanoma, we explored survival data for cutaneous melanoma datasets in The Cancer Genome Atlas (TCGA)

(24). Stratification of high top quartile versus low bottom quartile MRTF-A or RhoC expression levels shows a

significant reduction of percent survival in patients with high MRTF-A or RhoC expression (Fig. 1F).

Interestingly, stratification of RhoA, RhoB and MRTF-B high versus low expression does not result in

significant differences in melanoma patient percent survival (Fig. S1). Of note, the yes-associated protein

(YAP) and its paralog (TAZ), which have been reported to regulate similar genes as does MRTF-A, such as

CYR61, and CTGF (25), did not show significant differences in patient survival based on high versus low

expression levels in cutaneous melanoma (Fig. S1).




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SK-Mel-147 cells exhibit spontaneously nuclear and active MRTF-A through F-actin stress fiber

formation

To evaluate MRTF activity, we started by detecting stress fiber formation in the two melanoma cell lines. Stress

fiber formation leads to G-actin depletion and MRTF translocation into the nucleus (8). SK-Mel-147 cells exhibit

strong F-actin staining and stress fiber formation in the absence of serum stimulation (Fig. 2A). SK-Mel-19

showed reduced F-actin staining and very little stress fiber formation. Consistent with the actin-based

mechanism governing MRTF localization and activity, SK-Mel-147 also exhibited spontaneously nuclear

MRTF-A under these conditions (Fig. 2A). To show that MRTF-A localization was dependent on F-actin

dynamics, we stimulated SK-Mel-19 cells with 20% FBS to induce stress fiber formation which induced MRTF-

A nuclear localization (Fig. S2). We also treated SK-Mel-147 cells with latrunculin B which inhibits F-actin

formation and MRTF-A relocated to the cytosol (Fig. S2). MRTF activity is also regulated by the relative levels

of G-actin (26). To assess MRTF activity, we determined the mRNA expression of three known Rho/MRTF

target genes, myosin regulatory light chain 9 (MYL9), cysteine-rich angiogenic inducer 61 (CYR61), and

connective tissue growth factor (CTGF). Consistent with the predominantly nuclear MRTF-A, SK-Mel-147

shows dramatically increased expression of these three Rho/MRTF-regulated genes compared to SK-Mel-19

(Fig. 2B). Along with being MRTF target genes, MYL9, CYR61, and CTGF play important roles in cellular

migration and metastasis (10, 27). SK-Mel-147 cells clearly have increased MRTF activation as compared to

SK-Mel-19 cells, suggesting that this may contribute to the metastatic potential of these cells.

MRTF pathway inhibitor CCG-203971 blocks MRTF-A nuclear accumulation and transcription activity

We next assessed the potential for using a novel small-molecule inhibitor of the Rho/MRTF pathway to block in

vitro and in vivo models of melanoma metastasis. In order to evaluate transcriptional activity of MRTF, we

performed luciferase assays with a modified serum response element promoter (SRE.L) that is selective for

MRTF/SRF but does not respond to TCF/SRF complexes (11, 28). CCG-203971 inhibits RhoC-induced SRE-

Luciferase expression in SK-Mel-147 cells with an IC50 of ~6 µM. CCG-203971 does not cause cellular toxicity

up to 100 µM using the WST-1 viability assay in a 24 hour time period (Fig. 3A). In addition, CCG-203971




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reduces the nuclear localization of MRTF-A in SK-Mel-147 cells in a concentration dependent manner (Fig.

3B). By inhibiting the Rho/MRTF pathway, treating SK-Mel-147 cells with CCG-203971 also reduces the

expression of endogenous MYL9, CYR61, and CTGF (Fig. 3C).

CCG-203971 causes G1 phase cell cycle arrest and inhibits clonogenicity

To evaluate the effect of CCG-203971 on cellular growth in the aggressive melanoma cell line (SK-Mel-147)

compared to the less aggressive cell line (SK-Mel-19), we determined the effect of Rho/MRTF pathway

inhibition on cellular proliferation. While it had no effect on cell numbers at 24 hours, CCG-203971 caused a

significant reduction in the number of SK-Mel-147 cells at 72 hours. CCG-203971 is more potent in reducing

cell numbers in our cell proliferation assay in the SK-Mel-147 cell line compared to SK-Mel-19 (Fig. 4A). To

understand whether CCG-203971 affected cell proliferation or induced apoptosis, we conducted cell cycle

analysis using flow cytometry in SK-Mel-147 cells following 10 µM CCG-203971 treatment for 72 hours. CCG-

203971 caused G0/G1 arrest as evidenced by a significant increase in G0/G1 phase and a decrease in S-

phase compared to DMSO control treatment (Fig. 4B,C). CCG-1423, the precursor compound to CCG-

203971, has been shown to down-regulate genes that are involved in cell cycle, specifically G1/S transition,

including targets of the transcription factor E2F (GEO Accession number: GSE30188) (29). There was no

significant increase in Sub-G1 in CCG-203971 treated SK-Mel-147 cells (Fig 4B), suggesting that CCG-203971

does not induced apoptosis. Consistent with the effects of CCG-203971 to induce G0/G1 cell cycle arrest, it

also inhibited the clonogenicity of the SK-Mel-147 cells. Following a 72-hour treatment with DMSO or CCG-

203971, equal numbers of viable cells were plated for colony formation in the absence of our Rho/MRTF

pathway inhibitor. The CCG-203971 treated cells formed roughly half as many colonies compared to control

treatment and the colonies which formed were ~50% reduced in size (Fig 4D).

CCG-203971 inhibits cellular migration and invasion

To determine whether inhibition of MRTF localization and activity affects phenotypes of the aggressive

melanoma line, we investigated the influence of CCG-203971 at the cellular level. Using transwell migration




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and label-free Matrigel invasion assays, we found that 10 µM CCG-203971 inhibited cellular migration (Fig. 5A)

and invasion (Fig. 5B) of the RhoC-overexpressing melanoma line SK-Mel-147. This migratory phenotype is

consistent with an action on MRTF-A since siRNA knockdown of MRTF-A expression (Fig. 5C) inhibits SK-Mel-

147 transwell migration (Fig. 5D).

CCG-203971 blocks melanoma lung metastasis

To compare the metastatic potential of the high vs. low RhoC-expressing cell lines, we injected mice with GFP-

expressing SK-Mel-19 and SK-Mel-147 cells. The RhoC-overexpressing SK-Mel-147 cells readily formed lung

metastases following I.V. injection (Fig. 6A). The SK-Mel-147 injected mice not only displayed more metastatic

nodules (~3X) than did SK-Mel-19 injected mice, but the tumors were much larger (~5X greater area) than

those in the SK-Mel-19 injected mice (Fig. 6B). Thus the total tumor burden was greater by ~15X. To

determine the potential for MRTF pathway inhibitors as metastasis-targeting therapeutics, we treated mice with

CCG-203971 (100 mg/kg BID) or vehicle control for 18 days following tail vein injection of GFP-expressing SK-

Mel-147 cells. The CCG-203971 treated group had about half as many tumors which were also about 1/4 the

size. This resulted in a final tumor burden around 7X lower than in the vehicle treated group (Fig. 6B).




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Discussion

Mutations in BRAF and NRAS, leading to constitutive activation of the mitogen-activated protein kinase

(MAPK) pathway, are found in nearly ~60-70% of primary human cutaneous melanomas (30). However, these

driver mutations do not appear to govern the propensity for metastasis; BRAF vs. NRAS mutation status does

not predict patient overall survival (31). In addition, current targeted therapeutics, such as the BRAFV600E

inhibitor Vemurafenib, demonstrate profound initial results in a majority of BRAFV600E expressing melanoma

tumors (32). However, these responses are limited in duration and resistance typically develops within months

. Consequently, it is important to better understand factors that drive aggressiveness and metastasis of human

melanomas. Based on the role of RhoC and MRTF-regulated gene transcription in genetic studies in mouse

B16F2 melanoma and breast cancer models (2, 4, 11) and in the TCGA data set (24), we wanted to explore

the role of those mechanisms in human metastatic melanoma utilizing our novel Rho/MRTF pathway inhibitor,

CCG-203971.

While mutations in Rho proteins are rare, review of cancer genomic information through cBioPortal,

(33) showed that 31% of cutaneous melanomas had amplification or mutation of RhoA/C, MKL1/2 (gene

names for MRTF-A/B) or upstream activators (GNA12, GNA13, GNAQ, GNA11 or ARHGEF11). Furthermore,

an outlier analysis of melanoma in Oncomine, (34) showed that 5-20% of melanomas across several studies

had marked up-regulation of RhoC at the transcriptional level. Mechanisms driving this are not known but could

include regulation by the ETS-1 transcription factor (35, 36). Loss of E-cadherin in melanoma leads to ETS-1

activation and enhanced RhoC expression (40). RhoC induces c-Jun expression (likely through MRTF/SRF

mechanisms) which contributes to cell survival (40). This could partially explain the increased clonogenicity

observed in SK-Mel-147, the RhoC-overexpressing melanoma cell line, as well as the increased lung

colonization.

In light of the multiple genes and pathways that can lead to activation of Rho and MRTF-regulated

transcription (37, 38), inhibition at a downstream step in the pathway would be beneficial to disrupt signals from

the numerous activation mechanisms (39, 40). Interestingly, CCG-203971 treatment appears to influence SK-

Mel-147 cellular morphology in such a manner that SK-Mel-147 cells more closely resemble SK-Mel-19 cellular




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morphology. Further studies will need to be conducted to better understand this observation. Our compound,

CCG-203971 prevents the nuclear accumulation of MRTF but the molecular mechanism remains uncertain.

Two proposed models include direct binding to MRTF (41) or modulation of the intranuclear actin regulation by

MICAL2 (42). Genetic validation of our pharmacologic approach to suppress metastasis has already been

shown for both melanoma and breast cancer using MRTF-A/B double knock-down or silencing SRF (11). We

also confirm that in SK-Mel-147 cells, siRNA knockdown of MRTF-A alone is sufficient to markedly reduce

migration in vitro. The majority of recent literature strongly implicates MRTF and SRF as being critical factors

in the regulation cell motility (43-45). Despite the commonality between MRTF/SRF and YAP/TEAD regulated

target genes, as well as cell type specific mutual co-activation of gene expression (46), only MRTF-A

expression levels were correlated with patient survival (Fig. 3 and Fig. S2).

The potential for an anti-metastatic small molecule therapeutic that targets this gene transcription

mechanism is illustrated by our result showing marked suppression of SK-Mel-147 lung metastasis by in vivo

treatment with CCG-203971. While it has been argued that antimetastatic therapies are not useful or are

difficult to test clinically, there is increasing recognition that this approach has value (47). CCG-203971 also

effectively causes G1 cell cycle arrest which is a phenotype shared with current MAPK pathway targeted

therapeutics (48, 49). Since G1-arrested melanoma cells have increased sensitivity to MAPK pathway

inhibitors (49) this suggests the potential benefit of combination treatment with Rho/MRTF pathway inhibitors

and MAPK pathway inhibitors. Continued efforts to optimize MRTF-transcription inhibitors like CCG-203971 will

be needed. In particular enhancing potency along with reduction of acute toxicity (12, 13) is required for

chronic treatments that would be required for anti-metastasis therapies. Furthermore, improved bioavailability

and pharmacokinetics would facilitate both preclinical and potentially clinical studies.

In addition to melanoma, spontaneous nuclear localization of MRTF-A has been reported in MDA-MB-

231 breast cancer cells (11). The MDA-MB-231 cells are often used as a model of aggressive breast cancer

cells. Intriguingly they also harbor a Ras mutation (KRASG13D) (50) similar to the NRASQ61N in SK-Mel-147.

Additional work is needed to assess the generality of these observations.




17

In this report, we provide evidence that Rho-regulated, MRTF-mediated gene transcription signaling

plays an important role in migration, invasion, and metastasis of human cutaneous melanoma. Thus,

expression levels of RhoC and MRTF target genes may provide useful biomarkers to identify cancers with

propensity for metastasis as well as for targeted therapies by this approach. Small molecule inhibitors of this

pathway, such as enhanced CCG-203971 analogs, represent an exciting class of pharmacological probes and

potential future therapeutics.




18

Acknowledgements

We would like to thank Riya Malhotra, Alexandra Turley, and Joseph Zagorski for technical assistance with the

flow cytometry experiments. We would also like to thank Raelene Van Noord for her assistance with the

mouse metastasis study and Nadia Ayala-Lopez, Humphrey Petersen-Jones, and Dr. Stephanie Watts for

assistance with the immunohistochemistry analysis of melanoma metastasis.




19

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24

Figure Legends

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 cut off. Data are expressed as the mean (±SD) of duplicate

experiments. B. Cellular migration determined by wound assay. Cells are grown to confluence in 12-well plates

then a scratch is made using a 200 µL 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 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 = 107 samples per quartile). Kaplan-Meier plots were generated from the

highest (grey) and lowest (black) expressing quartiles. Survival curves were analyzed with the log-rank test

with a cutoff of P < 0.05 as statistically significant.

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




25

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 200µm 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.

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

200µm. 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).

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 µM CCG-203971 for 72 hours. Cells were

stained with prodidium iodide and analyzed using flow cytometry. B. Shows representative fluorescence

intensities versus event counts. C. Shows the results expressed as the mean (±SEM) of triplicates from three




26

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 µM CCG-203971 or 0.1% DMSO for 72 hours. Equal

number of viable cells were plated and allowed to form colonies for seven days. Results are expressed as the

mean (±SEM) of triplicate experiments (** P< 0.01 vs. No Treatment and DMSO).

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 µM) blocked SK-Mel-147 migration. Shown

are images at 4X magnification using a bright field microscope after six hours of migration. Four random fields

of view at 20X 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 10μM 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 of

MRTF-A in SK-Mel-147 cells knock 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 4X magnification using a bright field microscope after

six hours of migration. Three random fields from experimental duplicates at 20X 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 siNon-Targeting).

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 (2x106 cells).

The other two groups were injected with GFP-expressing SK-Mel-147 (2x106 cells). Following tail vein injection

the mice began twice daily I.P. injection of 100mg/kg CCG-203971 or 50μL DMSO (vehicle control). After 18

days the mice were euthanized and lungs were collected for histological analysis. Sections from each lung




27

were immunohistologically probed for GFP. Images were taken at 20X magnification, shown are two

representative images of metastatic colonies from each group. Scale bar represents 400µm. 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=6 (SK-Mel-19+DMSO

and SK-Mel-147+DMSO) and n=5 (SK-Mel-147+CCG-203971). (* P< 0.05, *** P< 0.001 vs. SK-Mel-

19+DMSO). (+ P< 0.05, +++ P< 0.001 vs. SK-Mel-147+DMSO).




Published OnlineFirst November 11, 2016.Mol Cancer Ther Andrew J. Haak, Kathryn M Appleton, Erika M. Lisabeth, et al. melanoma

overexpressingfactor pathway blocks lung metastases of RhoC Pharmacological inhibition of Myocardin-related transcription

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