Alpha-Smooth Muscle Actin (ACTA2) Is Required for Metastatic Potential of Human Lung Adenocarcinoma

Published OnlineFirst August 30, 2013; DOI: 10.1158/1078-0432.CCR-13-1181

Clinical Cancer Human Cancer Biology Research

Hye Won Lee1,2,3, Young Mi Park3,4, Se Jeong Lee1,4,5, Hyun Jung Cho1,2, Duk-Hwan Kim6,8, Jung-Il Lee2, Myung-Soo Kang3, Ho Jun Seol2, Young Mog Shim7, Do-Hyun Nam1,2,3, Hyeon Ho Kim3,4, and Kyeung Min Joo1,3,4,5

Abstract Purpose: Metastatic relapse of primary lung cancer leads to therapeutic resistance and unfavorable clinical prognosis; therefore, identification of key molecules associated with metastatic conversion has significant clinical implications. We previously identified a link between early brain metastasis of lung adenocarcinoma and amplification of the a-smooth muscle actin (ACTA2) gene. The aim of present study was to investigate the prognostic and functional significance of ACTA2 expression in cancer cells for the metastatic potential of lung adenocarcinomas. Experimental Design: ACTA2 expression was analyzed in tumor cells from 263 patients with primary lung adenocarcinomas by immunohistochemistry, and was correlated with clinicopathologic parameters. The expression of ACTA2 in human lung adenocarcinoma cells was modulated with short hairpin RNAs (shRNA) and siRNAs specifically targeting ACTA2. Results: The patients with lung adenocarcinomas with high ACTA2 expression in tumor cells showed significantly enhanced distant metastasis and unfavorable prognosis. ACTA2 downregulation remarkably impaired in vitro migration, invasion, clonogenicity, and transendothelial penetration of lung adenocarcinoma cells without affecting proliferation. Consistent with the in vitro results, depletion of ACTA2 in human lung adenocarcinoma PC14PE6 cells significantly reduced their metastatic potential without altering their tumorigenic potential. Expression of c-MET and FAK in lung adenocarcinoma cells was also reduced by ACTA2-targeting siRNAs and shRNAs, and was accompanied by a loss of mesenchymal characteristics. Conclusions: These findings indicate that ACTA2 regulates c-MET and FAK expression in lung adenocarcinoma cells, which positively and selectively influence metastatic potential. Therefore, ACTA2 could be a promising prognostic biomarker and/or therapeutic target for metastatic lung adenocarcinoma. Clin Cancer Res; 19(21); 5879–89. 2013 AACR.

Introduction Authors' Affiliations: 1Cancer Stem Cell Research Center and 2Depart- ment of Neurosurgery, Samsung Medical Center; 3Graduate School, Lung cancer is the most common cancer worldwide with Department of Health Sciences and Technology, Samsung Advanced the highest mortality rate (1). The identification of thera- Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University; 4Samsung Biomedical Research Institute, 5Department of Anat- peutic targets in non–small cell lung cancer (NSCLC), such omy and Cell Biology, 6Department of Molecular Cell Biology, 7Center for as EGF receptor (EGFR)-activating mutations, has enabled 8 Genome Research, and Department of Thoracic Surgery, Samsung Med- the development of effective targeted therapies for advanced ical Center, Sungkyunkwan University School of Medicine, Seoul, Korea NSCLC (2). However, the 5-year survival rate for NSCLC Note: Supplementary data for this article are available at Clinical Cancer remains 15% across all stages of the disease. These unfa- Research Online (http://clincancerres.aacrjournals.org/). vorable clinical outcomes originate from its high invasive H.W. Lee and Y.M. Park contributed equally to this work. and metastatic potential (3, 4). In particular, lung adeno- Corresponding Authors: Hyeon Ho Kim, Department of Health Sciences carcinoma is known to establish distant macrometastases in and Technology, Samsung Advanced Institute for Health Sciences and various organs, within months of diagnosis (5). Current Technology (SAIHST), Sungkyunkwan University, 50 Irwon-Dong, Gang- nam-Gu, Seoul 135-710, South Korea. Phone: 82-2-3410-1039; Fax: 82- targeted therapeutics have limited efficacy for the treatment 23410-0534; E-mail: [email protected]; and Kyeung Min Joo, Depart- of distant metastases in lung adenocarcinoma. ment of Anatomy and Cell Biology, Sungkyunkwan University School of The short latency of metastatic relapse in lung adenocar- Medicine, Graduate School for Heath Sciences & Technology Samsung Advanced Institute for Health Science & Technology (SAIHST), Sungkyunk- cinoma implies that the cancer cells in the primary tumor wan University, 50 Irwon-Dong, Gangnam-Gu, Seoul 135-710, South have already acquired numerous multi-organ metastatic Korea. Phone: 82-2-2148-9779; Fax: 82-2148-9829; [email protected] competencies, including cell motility, invasiveness, resis- doi: 10.1158/1078-0432.CCR-13-1181 tance to hypoxia, enhanced angiogenesis, survival after 2013 American Association for Cancer Research. detachment, and evasion of immune surveillance (6, 7).

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November 1994 and August 2004 (11, 12). Written inf- Translational Relevance ormed consent for the use of paraffin-embedded tissues, as The short latency from the initial diagnosis of lung approved by the Institutional Review Board at SMC, was adenocarcinoma, the most common subtype of non– obtained from each patient before surgery. Postoperative small cell lung cancer, to metastatic relapse leads to a follow-up was scheduled at 1 and 2 months, every 3 months high mortality rate. This short latency implies that the during the first 2 years after surgery, and then every 6 cancer cells in a primary lung adenocarcinomas have months thereafter, or more frequently if needed. The already acquired a number of multi-organ metastatic patients consisted of 140 males (53%) and 123 females competencies. In this study, using 263 pathologic sam- (47%), 36–80 years of age. The mean age at surgical resec- ples, we determined that ACTA2 expression in lung tion was 58.9 years. Of the 263 samples, 71, 134, 41, and 7 adenocarcinoma cells at the primary site is significantly were stage I, II, III, and IV, respectively. associated with enhanced distant metastasis. This association was translationally interpreted and validated. Immunohistochemistry and immunofluorescence ACTA2 regulates the expression of the epithelial–mes- staining enchymal transition-associated signaling molecules, c- Tissue microarray sections generated from the above- MET and FAK, and consequently, the in vitro and in vivo mentioned surgical samples were immunostained with metastatic potential of lung adenocarcinoma cells. These mouse monoclonal antibodies against human a-SMA results have high translational and clinical impact (N1584; Dako, clone 1A4 or MA5-15806; Thermo Scientific because ACTA2 could be used for the development of Pierce, clone 4F4). Briefly, endogenous peroxidase activity predictive diagnostics and/or antimetastatic agents for was blocked by incubation in 0.3% hydrogen peroxide. The advanced lung adenocarcinoma. antigen was retrieved by heating sections in 10 mmol/L sodium citrate (pH 6.0) at 95C for 30 minutes. The primary antibodies, biotinylated secondary antibody (Vector Labo- ratories), and then avidin–biotin complex (Vector Labora- Therefore, it might be possible to identify predictive bio- tories) were incubated with the sections at 4C overnight, markers of metastasis and novel therapeutic targets in for 1 hour at room temperature, and for 1 hour at room primary adenocarcinomas with high metastatic potential. temperature, respectively. ACTA2 immunoreactivity was These novel therapeutic targets could then be used to categorized as low (<50% ACTA2-positive tumor cells, overcome the resistance of NSCLC to currently available ACTA2-Low) or high grade (>50% ACTA2-positive tumor targeted treatments. cells, ACTA2-High) in areas with maximal staining. In our previous study, we compared the gene amplifica- To confirm the expression of ACTA2 in lung adenocar- tions and deletions in lung adenocarcinomas with synchro- cinoma tumor cells, immunofluorescence was conducted nous brain metastasis to those in lung adenocarcinomas with the mouse monoclonal anti-ACTA2 (Dako, 1:250) and with metachronous brain metastasis to elucidate the geno- rabbit polyclonal anti-thyroid transcription factor 1 (TTF-1; mic alterations associated with early distant metastasis in Abcam, 1:200) antibody against antigen-retrieved lung lung adenocarcinoma (8). Among several genomic altera- adenocarcinoma paraffin sections. TTF-1 has been reported tions, amplification of a-smooth muscle actin (a-SMA; to be one of several markers differentiating adenocarcinoma hereafter ACTA2) was significantly associated with synchro- from squamous cell carcinoma of the lung (13). The anti- nous brain metastasis. ACTA2 is known to contribute to cell- bodies were visualized by Alexa Fluor 488–labeled goat generated mechanical tension and maintenance of cell anti-mouse IgG and Alexa Fluor 594–labeled goat anti- shape and movement. As cell motility is critically dependent rabbit IgG antibody (Life Technologies), respectively. Sam- on the actin cytoskeleton, the dynamics of cytoskeletal ples were then incubated with DAPI (1:1,000) for 5 minutes structures affected by ACTA2 could be essential to invasion at room temperature to reveal cell nuclei. and metastasis in lung adenocarcinoma (9, 10). Herein, we report that high ACTA2 expression in tumor Cell culture, transfection, and generation of stable cell cells in primary lung adenocarcinoma is closely associated lines with progression of lung adenocarcinoma, and provide the PC14PE6 cells were established from a pleural effusion first evidence of its novel roles in the metastatic potential of developed in a nude mouse injected intravenously with lung adenocarcinoma. To our knowledge, this is the first parental human lung adenocarcinoma PC14 cells (Dr. I. J. report suggesting that ACTA2 is a promising prognostic Fidler, M. D. Anderson Cancer Center, Houston, TX). The biomarker and/or therapeutic target for advanced lung karyotypic analysis of the PC14PE6 cells ruled out contam- adenocarcinoma. ination with murine cells. PC14PE6 cells were maintained at 37 C and 5% CO2 in Dulbecco’s modified Eagle medium Materials and Methods (Hyclone) supplemented with 10% FBS and 1% antibiotic– Study population antimycotic solution (GIBCO). H322, H1299, H460, and Surgical samples were obtained from 263 patients with A549 human NSCLC cell lines were purchased from Amer- lung adenocarcinoma who underwent surgical resection at ican Type Culture Collection and all experiments were the Samsung Medical Center (SMC, Seoul, Korea) between conducted within 6 months of purchase. Authentication

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ACTA2 Confers Metastatic Potential on Lung Adenocarcinoma

of these cell lines was conducted by short tandem repeat 4% paraformaldehyde (PFA). The transmigrated tumor cells (STR) profiling to exclude cross-contamination between the were counted in 10 random fields at 100 magnification. cell lines. H322, H1299, H460, and A549 cells were grown Focus forming assay. Cells were seeded (at 300 cells/ in RPMI (GIBCO) supplemented with 10% FBS and peni- well) in 6-well plates and maintained in complete medium cillin/streptomycin (GIBCO). For transfection, human for 3 weeks. The number of viable colonies per well was NSCLC cells were plated at a density of 5 105 cells per counted after staining with 0.2% crystal violet. dish and transfected with the indicated siRNAs using Lipo- In vivo tumorigenesis and metastasis assay. All animals fectamine 2000 (Invitrogen) according to the manufac- received humane care in compliance with the "Guide for turer’s protocol. siRNAs directed against ACTA2, Focal adhe- the Care and Use of Laboratory Animals" prepared by the sion kinase (FAK), and c-MET were purchased from Santa Institute of Laboratory Animal Resources published by the Cruz Biotechnology. Control siRNA (siCTRL) was synthe- NIH and according to the Animal Experiment Guidelines of sized by Genolution. For stable cell lines, cells were trans- Samsung Biomedical Research Institute. fected with an ACTA2-specific shRNA (shACTA2, Sigma) or Tumorigenicity assay. The effect of ACTA2 silencing on empty pLKO vector (shCTRL) using Lipofectamine 2000, tumorigenic potential of lung adenocarcinoma cells was and then selected by culturing in puromycin (5 mg/mL). analyzed in two different in vivo models. For subcutaneous model, 1 106 cancer cells in 100 mL Hank balanced salt Western blot analysis and quantitative real-time PCR solution (HBSS) were subcutaneously injected into the pos- For Western blot analysis, cells were lysed with radio- terior flank of 6 to 8-week-old female BALB/c nude mice immunoprecipitation buffer containing protease inhibi- (Orient, 7 mice/group). The volume of subcutaneous tumor tors and phosphatase inhibitors (Roche). Equal amounts (0.5 length width2,mm3) was measured on 14, 17, 21, of protein were subjected to SDS-PAGE and transferred to 24, and 28 days after the implantation. At 28 days postim- polyvinylidene difluoride membranes (Millipore). After plantation, subcutaneous tumors were excised, photo- blocking in 5% bovine serum albumin (BSA), membranes graphed, and weighted. For orthotopic model, female 6 to were incubated with the indicated primary antibodies 8-week-old female BALB/c nude mice were anesthetized in overnight, and then with the appropriate secondary anti- the right lateral decubitus position. A 3 mm incision was bodies. For quantitative real-time PCR (qRT-PCR), total made at the lateral dorsal axillary line, 1.5 cmabove the lower RNA was isolated using TRIzol reagent (Invitrogen) rib line, just below the inferior border of the scapula. Cancer according to the manufacturer’s instructions, and then cells (1 106)in40mL HBSS were injected into the left lung used to synthesize cDNA with Superscript III Reverse with a 1 mL insulin syringe and a 30-gauge needle, and then Transcriptase (Invitrogen). Expression of mRNA was the incision was repaired. The body weights of the animals quantified by qRT-PCR (ABI Prism 7600) using power were measured every day and an autopsy was conducted SYBR Green PCR Master Mix (Applied Biosystems). The immediately after a 25% weight loss. Lungs were fixed in primers used in this study were summarized in Supple- formalin, embedded in paraffin, and then stained with H&E. mentary Table S1. Metastasis assay. For experimental metastasis in vivo model, 2 105 cancer cells in 100 mL of HBSS were directly Detection of metastatic activity in vitro injected into the left ventricle of the heart of 6 to 8-week-old Migration and invasion assay. Migratory activity was female BALB/c nude mice with a 1 mL insulin syringe. Four measured by the wound closure assay. Briefly, a confluent weeks later, the animals were sacrificed. Brains, lungs, lower cell surface was scratched with a pipette tip and migration leg bones, adrenal glands, and abnormal organs were har- distance was measured by wound closure. For the invasion vested, fixed in formalin, and embedded in paraffin. Par- assay, Matrigel-coated Transwells (BD) were used. Equal affin-embedded tissues were stained with H&E. numbers of cells in serum-free media were added to the top chamber. The addition of complete media containing 10% Statistical analysis FBS to the bottom compartment stimulated the cells to Overall survival and metastasis-free survival were calcu- invade. After 24-hour incubation, cells were fixed with lated by the Kaplan–Meier method and compared with the 100% methanol and stained with hematoxylin and eosin log-rank test. Group comparisons were analyzed with Stu- (H&E). The number of invading cells on the bottom surface dent two-tailed t test. Tumor metastasis rates were compared of the membrane was counted. by Fisher exact test, two-tailed. Differences with P values less Transendothelial migration assay. Human umbilical than 0.05 were considered statistically significant. vein endothelial cells (HUVEC) were seeded (at 1 105 cells/well) in fibronectin-coated 24-well Transwell inserts Results with a pore size of 8 mm (Corning Costar Corp.), and grown Clinical implications of ACTA2 in lung adenocarcinoma to confluence. Calcein AM-labeled cancer cells (1 105 To confirm the role of ACTA2 in the metastasis of lung cells) were suspended in serum-free medium and added to adenocarcinoma, we assayed ACTA2 expression in 263 lung the endothelial monolayer. After 24-hour incubation, cells adenocarcinomas by immunohistochemistry (Fig. 1A). remaining in the top chamber were completely removed When more than 50% of tumor cells expressed ACTA2, the and the tumor cells that migrated through the endothelial patients were considered as ACTA2-High (n ¼ 14). Specific monolayer to the bottom face of the filter were fixed with detection of ACTA2 protein was verified by same staining

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Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2013 American Association for Cancer Research. 5882 lnCne e;1(1 oebr1 2013 1, November 19(21) Res; Cancer Clin e tal. et Lee Downloaded from BC A DAPI TTF1 ACTA2 DAPI ACTA2-High ACTA2-Low ACTA2 Published OnlineFirstAugust30,2013;DOI:10.1158/1078-0432.CCR-13-1181 TTF1 clincancerres.aacrjournals.org MERGE ACTA2 TTF-1 DAPI 20 µm 20 µm 20 µm 20 µm 20 µm 20 µm on September 24, 2021. © 2013American Association for Cancer D Research. Probability of MFS Probability of OS 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0 0 20 µm 20 µm 50 50 Months Months 100 100 lnclCne Research Cancer Clinical ACTA2-Low ACTA2-High ACTA2-Low ACTA2-High P =0.011 P =0.022 150 150 20 µm 20 µm Published OnlineFirst August 30, 2013; DOI: 10.1158/1078-0432.CCR-13-1181

ACTA2 Confers Metastatic Potential on Lung Adenocarcinoma

patterns of two different antibodies against human ACTA2 metastasis of lung cancer. In contrast, proliferation (Supple- (Supplementary Fig. S1). Moreover, ACTA2 protein in lung mentary Fig. S4A), anoikis resistance (Supplementary Fig. adenocarcinoma cells was confirmed by colocalization of S4B), and adhesion to fibronectin (an extracellular matrix ACTA2 with a lung adenocarcinoma-specific marker, TTF-1 component; Supplementary Fig. S4C) of PC14PE6 cells were (Fig. 1B). The stromal ACTA2 expression in immunohis- not affected by the shRNA-mediated ACTA2 downregula- tochemistry (Fig. 1A) and the presence of ACTA2-positive, tion. Therefore, the inhibitory effects of ACTA2 silencing on TTF-1-negative cells in immunofluorescence staining (Fig. the migration and invasion of PC14PE6 cells did not result 1B) suggested concomitant ACTA2 expression in tumor from decreased proliferation and/or cellular growth. stromal cells (14). ACTA2 staining was also observed in close proximity to the nucleus and/or nuclear domain in Specific implications of ACTA2 in the metastasis of lung TTF-1–positive cancer cells in agreement with previous adenocarcinoma in vivo report (15), although its functional roles need to be further Metastasis is a sequential, multi-step process that includes elucidated. Kaplan–Meier survival analysis was conducted invasion, intravasation, survival in circulation, extravasa- to determine the prognostic significance of ACTA2 expres- tion, and colonization. In vitro downregulation of A-CTA2 sion in patients with lung adenocarcinomas. ACTA2-High significantly affected those processes, which indicated that patients had significantly worse overall survival [OS; medi- ACTA2 could be required for distant metastasis of lung an (95% CI): 28.0 (6.0–50.0) months, P ¼ 0.011; Fig. 1C] adenocarcinoma. To investigate whether ACTA2 has the and metastasis-free survival [MFS; 16.0 (7.6–24.4) months, effect on primary lung tumorigenesis in vivo,1 106 control P ¼ 0.022, Fig. 1D], compared with those of ACTA2-Low (PC14PE6/shCTRL) or ACTA2 knockdown (PC14PE6/ patients [n ¼ 249; OS ¼ 71.0 (55.8–86.2) months; MFS ¼ shACTA2) cells were implanted into the subcutaneous 53.0 (37.4–68.6) months]. These results indicate that not tissue (PC14PE6/shCTRL, n ¼ 7; PC14PE6/shACTA2, only ACTA2 gene amplification (8), but also ACTA2 over- n ¼ 7) or left lung (PC14PE6/shCTRL, n ¼ 7; PC14PE6/ expression in lung adenocarcinoma cells are significantly shACTA2, n ¼ 6) of nude mice. In the subcutaneous model, associated with enhanced metastatic potential and worse both tumor growth rate (P ¼ 0.43) and final tumor weight clinical prognosis of lung adenocarcinoma. (P ¼ 0.49) were not significantly affected by ACTA2 downregulation (Fig. 4A). ACTA2 downregulation also made no In vitro effects of shRNA-mediated downregulation of effects on the tumor formation in the lung cancer orthotopic ACTA2 model, as the survival of mice with intralung injection of We generated 7 PC14PE6 lung adenocarcinoma cell PC14PE6/shACTA2 cells (median survival length ¼ 32 subclones that stably express 6 different ACTA2 shRNAs or days) was comparable with that of the control group (medi- a control vector (pLKO) to assess the effect of ACTA2 an survival length ¼ 25 days; Fig. 4B, P ¼ 0.318). These silencing on the metastatic potential of lung adenocarcino- results are consistent with that of the in vitro cell prolifer- ma cells. Downregulation of ACTA2 mRNA and protein was ation assay, which firmly validates no significant role of confirmed by qRT-PCR (Fig. 2A and Supplementary Fig. S2) ACTA2 in lung tumorigenesis. and Western blot analysis (Fig. 2B), respectively. When To further examine the in vivo biologic role of ACTA2 in ACTA2 expression was downregulated by shRNA, the migra- the metastatic potential of lung adenocarcinoma cells, we tion (Fig. 2C and Supplementary Fig. S2) and invasion (Fig. injected 1 106 PC14PE6/shCTRL or PC14PE6/shACTA2 2D and Supplementary Fig. S2) of PC14PE6 cells were cells into the left ventricle (systemic metastasis animal significantly inhibited. As PC14PE6 cells transfected with model; PC14PE6/shCTRL; n ¼ 9, PC14PE6/shACTA2; n shACTA2#6 showed the lowest expression of ACTA2 mRNA ¼ 7) of nude mice. In contrast with the tumorigenicity and strong suppression of migration and invasion, assays, significantly less metastatic potential was observed PC14PE6/shACTA2#6 cells (hereafter, PC14PE6/shACTA2) in mice that received an intracardiac injection of PC14PE6/ were used for all subsequent experiments. The inhibitory shACTA2 cells (Fig. 4C). These mice had a sole metastatic effects of ACTA2 silencing on the in vitro migration potential tumor in the adrenal gland (14% incidence, 1/7 mice), of lung adenocarcinoma cells were confirmed using various whereas mice that received an intracardiac injection of lung adenocarcinoma cell lines and a specific siRNA target- PC14PE6/shCTRL cells had a 78% incidence (7/9 mice) of ing ACTA2 (Supplementary Fig. S3). metastatic tumors, and the metastatic sites varied (lung, ACTA2 downregulation also significantly decreased trans- adrenal gland, bone, brain, and eyeball; Fig. 4C). Each endothelial migration (Fig. 3A) and clonogenicity (Fig. 3B) metastatic tumor was confirmed pathologically (Fig. 4D). of PC14PE6 cells, properties that are required for distant These in vivo assays indicated that ACTA2 promotes lung

Figure 1. ACTA2 expression is closely associated with early metastasis and poor prognosis of lung adenocarcinoma (ADC). A, representative results of immunohistochemical staining against ACTA2 in 263 lung adenocarcinomas (top, low-expression, ACTA2-Low; bottom, high-expression, ACTA2-High). B, colocalization of ACTA2 (green) with a lung adenocarcinoma–speciﬁc marker, Thyroid transcription factor 1 (TTF-1, red) in lung adenocarcinoma cells. ACTA2þ/TTF-1þ lung adenocarcinoma cells were indicated by arrows. C and D, Kaplan–Meier analysis of overall survival (OS) and metastasis-free survival (MFS) of 263 patients with lung adenocarcinoma. Immunohistochemical staining of ACTA2 was conducted using tissue microarray (TMA) sections from 263 patients. Lung adenocarcinomas were categorized by the frequency of ACTA2-stained cancer cells (>50% tumor cells ¼ ACTA2-High). P values were determined by the log-rank test.

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ABshCTRL shACTA2

1.2

1.0 shCTRL shACTA2 0.8 ACTA2 0.6 *

0.4 GAPDH ACTA2 mRNA ACTA2 0.2

0.0 Figure 2. shRNA-mediated ACTA2 C knockdown inhibited the migratory and invasive activity of PC14PE6 shCTRL shACTA2 shCTRL shACTA2 cells. A and B, knockdown of ACTA2 mRNA and protein was 1.2 verified by qRT-PCR (A) and Western blot analysis (B), 0 h 1.0 respectively. C and D, the effect of ACTA2 silencing on in vitro 0.8 migration and invasion was assessed by the wound closure 0.6 assay (C) and Matrigel-coated * Transwell assay (D), respectively. 0.4 Migration distance was measured 24 h 24 hours (h) after wound formation Migration distance Migration 0.2 on a confluent cell surface and invaded cells were counted under a 0.0 microscope in more than 6 fields for each group. Data shown are the mean plus the SD., P < 0.05. D shCTRL shACTA2 shCTRL shACTA2 1.2

1.0

0.8 *

0.6 Invasion 0.4

0.2

0.0

adenocarcinoma metastasis without affecting primary tion, mRNA and protein of both FAK and c-MET were tumor formation potential, which would be mediated by significantly decreased (Fig. 5A). ACTA2 silencing also increased migration, invasion, and clonogenicity of lung significantly increased E-cadherin expression and decreased adenocarcinoma cells. vimentin expression (Fig. 5B). These results indicated that the expression of FAK and c-MET, which are major signaling Effects of ACTA2 on FAK and c-MET expression molecules for metastasis, is suppressed by ACTA2 down- From the above results, we determined that ACTA2 is a regulation, and that ACTA2 expression is required for main- critical factor for the acquisition of metastatic potential in tenance of the mesenchymal characteristics of PC14PE6 lung adenocarcinoma cells. To address the mechanism cells. To ensure that ACTA2 is associated with FAK and whereby ACTA2 regulates metastatic potential, we analyzed c-MET expression, we checked the downregulation effects the expression of EMT-associated proteins, such as FAK and again with several shRNAs specifically targeting ACTA2.As c-MET, as EMT is a critical process for acquisition of met- shown in Supplementary Fig. S5, shRNA-elicited knock- astatic potential. PC14PE6 cells were transfected with an down of ACTA2 also reduced the expression of FAK and ACTA2-specific siRNA. Forty eight hours after the transfec- c-MET.

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ACTA2 Confers Metastatic Potential on Lung Adenocarcinoma

ABshCTRL shACTA2 shCTRL shACTA2

Figure 3. Effects of shRNA- mediated ACTA2 knockdown on transendothelial migration and clonogenicity of PC14PE6 cells. A, for transendothelial migration, cells were added to a endothelial monolayer. The transmigrated cells shCTRL shACTA2 shCTRL shACTA2 were counted under a ﬂuorescent microscope in more than 10 ﬁelds for each group. B, to assess 1.2 1.2 clonogenicity, cells were seeded in 6-well plates and cultured in 1.0 1.0 complete media for 3 weeks. Colonies were stained with 0.2% 0.8 0.8 crystal violet and counted. Data shown are the mean plus the SD. 0.6 0.6 , P < 0.05. 0.4 TEM activity 0.4 Clonogenicity

0.2 0.2

0.0 0.0

To determine whether FAK and c-MET are required for in vivo. Moreover, our results suggested that the decreased ACTA2 silencing-mediated suppression of migration and metastatic potential induced by ACTA2 silencing is medi- invasion, PC14PE6 cells were transfected with FAK and ated by downregulation of FAK and c-MET expression. c-MET-specific siRNAs (Supplementary Fig. S5). Similarly Although ACTA2 is generally expressed in the smooth to ACTA2 silencing, silencing of either FAK and c-MET muscle cells and activated cancer-associated fibroblasts inhibited migration (Fig. 5C) and invasion (Fig. 5D) of (CAF), tumor cells could also use actin bundles to allow PC14PE6 cells. In accordance with data obtained from them to break away from a primary tumor and invade the PC14PE6, ACTA2 silencing also inhibited migratory and surrounding tissue (9, 10). In many epithelial cancers, TGF- invasive activity of lung adenocarcinoma H322 cells b–elicited EMT induces the expression of ACTA2 (16), through downregulation of FAK and c-MET (Supplemen- increases tumor invasion, and worsens patient survival tary Figs. S6 and S7). These results suggest that decreased (17). The connection between EMT and epithelial stemness expression of FAK and c-MET is involved in ACTA2 down- also suggests that EMT can confer clonogenicity and resis- regulation-mediated suppression of metastatic potential. tance to apoptosis on primary tumor cells (18). Finally, the presence of ACTA2 in the nuclear proximity of some cells Discussion indicated that ACTA2 relate the change of mechanical force Distant metastasis decreases the survival of patients to an altered gene expression via nuclear transcription with NSCLC. While targeted molecular therapies have regulators (15, 19). Therefore, ACTA2 expression and/or greatly improved the management of primary NSCLC, amplification of the ACTA2 gene in lung adenocarcinoma these therapies are ineffective for the treatment of distant cells could be used to predict clinical aggressiveness of lung metastases. Because systemic metastasis is induced by adenocarcinoma, which were shown in this report and our additional metastasis-specific genetic alterations, identify- previous study (8), respectively. ing key molecules that contribute to lung cancer cell However, the possibility that ACTA2 expressing CAFs dissemination is essential for the development of new could synergistically contribute the progression and metas- predictive biomarkers and/or antimetastatic therapeutic tasis of lung adenocarcinomas with adjacent ACTA2-posi- strategies. In this study, we showed that ACTA2 plays a tive cancer cells could not be ruled out. In recent studies, critical role in lung adenocarcinoma metastasis. Clinically, lung CAFs have a significant role in the metastatic potential high expression of ACTA2 in lung adenocarcinoma cells of NSCLC via the direct regulation of gene expression at the was significantly associated with worse prognostic out- invasive front of cancer nests and induction of EMT (20, come and early distant metastasis of patients with lung 21). Although the present study and previous reports high- adenocarcinoma. Experimentally, ACTA2 expression was light the importance of ACTA2 expression of both cancer closely correlated with the metastatic potential of human cells and CAFs in NSCLC progression and metastasis, fur- lung adenocarcinoma cell line, PC14PE6 both in vitro and ther studies are needed to elucidate detail mechanisms how

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A shCTRL 1,800 PC14PE6/shCTRL shACTA2 )

3 PC14PE6/shCTRL 1,500 2.5 PC14PE6/shACTA2 1,200 2.0

900 1.5 PC14PE6/shACTA2 600 1.0

300 0.5 Tumor weight (g) weight Tumor Tumor volume (mm volume Tumor 0 0.0 1 cm 14 16 18 20 22 24 26 28 Day BC shCTRL shACTA2 100 60 1.0 78% (7/9) 44% 50 PC14PE6/shACTA2 80 (4/9) 0.8 (n = 6, median: 32 d) 33% 33% 40 60 (3/9) (3/9) 0.6 22% 30 (2/9) 0.4 40 14% 20 PC14PE6/shCTRL (1/7) 11%

Metastasis (%) 14%

Survival fraction (1/9) 0.2 (n = 7, median: 20 d) 20 (1/7) 10 (%) Metastasis rate P 0.0 Log-rank = 0.318 0% 0% 0% 0% 0 0 0 10 20 30 40 50 60 Lung Adrenal Bone Brain Eye balls gland Days after tumor inoculation shCTRLshACTA2 D Bone Brain Lung Adrenal gland

× 40 × 200 × 40 × 200 × 100 × 100 × 40 × 200

× 200 PC14PE6/shCTRL PC14PE6/shCTRL PC14PE6/shCTRL PC14PE6/shCTRL

× 40 × 200 × 40 × 200 × 40 × 200 × 40 × 200 PC14PE6/shACTA2 PC14PE6/shACTA2 PC14PE6/shACTA2 PC14PE6/shACTA2

Figure 4. Decreased expression of ACTA2 speciﬁcally suppressed systemic metastasis of PC14PE6 cells in vivo. A, downregulation of ACTA2 had no effect on tumorigenic potential of lung adenocarcinoma in subcutaneous model. A total of 1 106 PC14PE6 control (PC14PE6/shCTRL) or ACTA2 knockdown (PC14PE6/shACTA2) cells were implanted into the subcutaneous tissue of nude mice (n ¼ 7/group). The volume of xenograft tumors and relative tumor weight at 28 days postimplantation are shown. Data ¼ mean SEM. Representative photographs of the excised tumors at 28 days postimplantation. B, the effect of ACTA2 on primary tumor growth was evaluated in an orthotopic lung cancer animal model by an intra-lung injection of 1 106 of PC14PE6 control (PC14PE6/shCTRL; n ¼ 7) or ACTA2-knockdown (PC14PE6/shACTA2) cells (n ¼ 6). C, the in vivo effects of ACTA2 on the metastatic potential of lung adenocarcinoma PC14PE6 cells were evaluated by injection of 2 105 PC14PE6/shCTRL (n ¼ 9) or PC14PE6/shACTA2 cells (n ¼ 7) into the left ventricle of female BALB/c nude mice. The incidence of systemic metastases to each organ was compared., P < 0.05. D, representative illustrations of metastatic foci in each organ.

ACTA2 contributes to tumor-promoting effects in CAFs and growth factor (HGF)/c-MET signaling activates a number lung adenocarcinoma cells. of downstream pathways, including mitogen-activated pro- Inhibition of ACTA2 expression in lung adenocarcinoma tein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/ PC14PE6 and H322 cells downregulated the transcription protein kinase B (PKB/Akt), and NF-kB, in epithelial cancer of c-MET and FAK, which decreased the in vitro migration cells (17), and induces proliferation, motility, cell survival, and invasion of PC14PE6 and H322 cells. Hepatocyte and EMT. Recent studies demonstrated that HGF is an

5886 Clin Cancer Res; 19(21) November 1, 2013 Clinical Cancer Research

ACTA2 Confers Metastatic Potential on Lung Adenocarcinoma

A B siCTRL siACTA2 siCTRL siACTA2 c-MET FAK E-cadherin Vimentin 1.2 3.5 siCTRL siACTA2 siCTRL siACTA2 1.2

1.0 3.0 1.0 ACTA2 ACTA2 2.5 0.8 0.8 2.0 c-MET 0.6 E-cadherin 0.6 1.5

mRNA 0.4 mRNA 0.4 FAK Vimentin 1.0 0.2 0.5 0.2 GAPDH GAPDH 0.0 0.0 0.0

C 1.2 siRNA CTRL ACTA2 FAK c-MET 1.0

0.8 0 h 0.6

0.4

Migration distance Migration 0.2 24 h 0.0 CTRL ACTA2 FAK c-MET siRNA D

1.2 siRNA CTRL ACTA2 FAK c-MET 1.0

0.8

0.6

Invasion 0.4

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0.0 CTRL ACTA2 FAK c-MET siRNA

Figure 5. Silencing of ACTA2 suppressed c-MET and FAK expression. A, PC14PE6 cells were transfected with control (siCTRL) or ACTA2 siRNA (siACTA2). At 48 hours after transfection, protein and mRNA levels of ACTA2, c-MET, and FAK were determined by Western blot analysis (left) and qRT-PCR (right), respectively. GAPDH ¼ internal control. B, the protein and mRNA levels of the EMT makers, E-cadherin and vimentin, were determined by Western blot analysis (left) and qRT-PCR (right), respectively. C and D, PC14PE6 cells were transfected with c-MET or FAK siRNA. At 48 hours after transfection, in vitro migration (C) and invasion (D) were determined by wound closure assay and Matrigel Transwell assay, respectively. The data shown are the mean plus SD from 3 separate experiments. , P < 0.05. indicator of poor prognosis in lung adenocarcinoma (22). cantly influence the clinical outcome of lung adenocarci- FAK is activated by integrins that connect the cytoskeleton to noma, this protein could be used as a therapeutic target for the extracellular matrix and act as nucleation sites for the lung adenocarcinomas. assembly of cell adhesions (23, 24). In addition, FAK plays a Maintenance of stemness is critical for lung adenocar- critical role in TGF-b1–induced EMT. Its overexpression was cinoma cells to make distant metastases since establish- significantly associated with positive lymph node metasta- ment of new colonies in unfamiliar microenvironments is sis and worse overall survival of patients with lung adeno- the final step of sequential metastatic processes. Accord- carcinoma (25). Because ACTA2 not only mediates altera- ing to elaborate analysis of genomic features of primary tions in the mechanical properties of cancer cells, but also and metastatic tumors, metastasis is a result from clonal regulates the expression of signaling proteins that signifi- selection of heterogeneous cancer cells in the primary

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Lee et al.

tumor (26, 27). Therefore, clonogenicity is one of key as a possible therapeutic target and a prognostic biomarker phenotypes of stemness, which is required for distant for metastasis. metastasis. Our results indicate that ACTA2 is involved in the clonogenicity of lung adenocarcinoma PC14PE6 cells. Disclosure of Potential Conflicts of Interest Moreover EMT, a transdifferentiating process, was also No potential conflicts of interest were disclosed. affected by ACTA2 expression, in this study. One study postulated that EMT and stem cell properties are com- Authors' Contributions Conception and design: H.W. Lee, Y.M. Park, H.J. Seol, Y.M. Shim, D.-H. bined in invasive cancer cells, which often coexpress EMT Nam, H.H. Kim, K.M. Joo and stem cell markers (28). Moreover, EMTs additionally Development of methodology: H.W. Lee, M.-S. Kang equip more differentiated epithelial cells with stem cell Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H.W. Lee, Y.M. Park, S.J. Lee, H.J. Cho, D.-H. Kim, traits through molecular linking of EMT-inducing tran- J.-I. Lee, M.-S. Kang, H.J. Seol scription factors to self-renewal programs (29, 30). This Analysis and interpretation of data (e.g., statistical analysis, biosta- tistics, computational analysis): H.W. Lee, Y.M. Park, S.J. Lee, H.J. Cho, H. connection between EMT and epithelial stemness indi- J. Seol, H.H. Kim, K.M. Joo cates that by imparting mesenchymal traits to carcinoma Writing, review, and/or revision of the manuscript: H.W. Lee, J.-I. Lee, H. cells, an EMT can confer motility, invasiveness, are resis- H. Kim, K.M. Joo Administrative, technical, or material support (i.e., reporting or orga- tant to apoptosis and metastatic dissemination from nizing data, constructing databases): D.-H. Kim, D.-H. Nam primary tumors (18). Accordingly, stemness of lung ade- Study supervision: H.J. Seol, Y.M. Shim, D.-H. Nam, H.H. Kim, K.M. Joo nocarcinoma cells might be regulated by ACTA2,which could in turn influence the metastatic potential of lung Grant Support adenocarcinoma cells. However, the detailed mechanism This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health & Welfare Affairs, Republic of Korea of how ACTA2 mediates this regulation remains to be (A092255), Samsung Medical Center grant [GFO1130011], and the Basic elucidated further. Science Research Program, National Research Foundation of Korea by the ACTA2 Ministry of Education, Science, and Technology (2011-009329 to H. H. Kim). In this study, we showed for the first time that The costs of publication of this article were defrayed in part by the regulates FAK and c-MET expression in lung adenocarcino- payment of page charges. This article must therefore be hereby marked ma cells, which positively influences in vitro and in vivo advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. metastatic potential. As ACTA2 expression in lung adenocarcinoma cells is significantly associated with poor clinical Received May 2, 2013; revised August 7, 2013; accepted August 12, 2013; outcome and early distant metastasis, ACTA2 could be used published OnlineFirst August 30, 2013.

References 1. Ferlay J, Parkin DM, Steliarova-Foucher E. Estimates of cancer 13. Kim MJ, Shin HC, Shin KC, Ro JY. Best immunohistochemical panel in incidence and mortality in Europe in 2008. Eur J Cancer 2010;46: distinguishing adenocarcinoma from squamous cell carcinoma of 765–81. lung: tissue microarray assay in resected lung cancer specimens. Ann 2. Cufer T, Ovcaricek T, O'Brien ME. Systemic therapy of advanced non- Diagn Pathol 2013;17:85–90. small cell lung cancer: major-developments of the last 5-years. Eur J 14. Li H, Fan X, Houghton J. Tumor microenvironment: the role of the tumor Cancer 2013;49:1216–25. stroma in cancer. J Cell Biochem 2007;101:805–15. 3. Spira A, Ettinger DS. Multidisciplinary management of lung cancer. 15. Valcz G, Sipos F, Krenacs T, Molnar J, Patai AV, Leiszter K, et al. N Engl J Med 2004;350:379–92. Increase of alpha-SMA(þ) and CK (þ) cells as an early sign of epithelial- 4. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, mesenchymal transition during colorectal carcinogenesis. Pathol 2009. CA Cancer J Clin 2009;59:225–49. Oncol Res 2012;18:371–6. 5. Hess KR, Varadhachary GR, Taylor SH, Wei W, Raber MN, Lenzi R, 16. Masszi A, Di Ciano C, Sirokmany G, Arthur WT, Rotstein OD, Wang J, et al. Metastatic patterns in adenocarcinoma. Cancer 2006;106: et al. Central role for Rho in TGF-beta1-induced alpha-smooth muscle 1624–33. actin expression during epithelial-mesenchymal transition. Am J Phy- 6. Bernards R, Weinberg RA. A progression puzzle. Nature 2002; siolo Renal Physiol 2003;284:F911–24. 418:823. 17. Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF. Met, 7. Nguyen DX, Bos PD, Massague J. Metastasis: from dissemination to metastasis, motility and more. Nat Rev Mol Cell Biol 2003;4:915–25. organ-specific colonization. Nat Rev Cancer 2009;9:274–84. 18. Singh A, Settleman J. EMT, cancer stem cells and drug resistance: 8. Lee HW, Seol HJ, Choi YL, Ju HJ, Joo KM, Ko YH, et al. Genomic copy an emerging axis of evil in the war on cancer. Oncogene 2010;29: number alterations associated with the early brain metastasis of non- 4741–51. small cell lung cancer. Int J Oncol 2012;41:2013–20. 19. Thomas CH, Collier JH, Sfeir CS, Healy KE. Engineering gene expres- 9. Fritz G, Kaina B. Rho GTPases: promising cellular targets for novel sion and protein synthesis by modulation of nuclear shape. Proc Natl anticancer drugs. Curr Cancer Drug Targets 2006;6:1–14. Acad Sci U S A 2002;99:1972–7. 10. Lambrechts A, Van Troys M, Ampe C. The actin cytoskeleton in normal 20. Kim SH, Choe C, Shin YS, Jeon MJ, Choi SJ, Lee J, et al. Human lung and pathological cell motility. Int J Biochem Cell Biol 2004;36: cancer-associated fibroblasts enhance motility of non-small cell lung 1890–909. cancer cells in co-culture. Anticancer Res 2013;33:2001–9. 11. Kim JS, Kim JW, Han J, Shim YM, Park J, Kim DH. Cohypermethylation 21. An J, Enomoto A, Weng L, Kato T, Iwakoshi A, Ushida K, et al. of p16 and FHIT promoters as a prognostic factor of recurrence in Significance of cancer-associated fibroblasts in the regulation of gene surgically resected stage I non-small cell lung cancer. Cancer Res expression in the leading cells of invasive lung cancer. J Cancer Res 2006;66:4049–54. Clin Oncol 2013;139:379–88. 12. Kim JS, Han J, Shim YM, Park J, Kim DH. Aberrant methylation of H- 22. Siegfried JM, Weissfeld LA, Luketich JD, Weyant RJ, Gubish CT, cadherin (CDH13) promoter is associated with tumor progression in Landreneau RJ. The clinical significance of hepatocyte growth factor primary nonsmall cell lung carcinoma. Cancer 2005;104:1825–33. for non-small cell lung cancer. Ann Thorac Surg 1998;66:1915–8.

5888 Clin Cancer Res; 19(21) November 1, 2013 Clinical Cancer Research

ACTA2 Confers Metastatic Potential on Lung Adenocarcinoma

23. Guan JL, Shalloway D. Regulation of focal adhesion-associated pro- 27. Gupta GP, Massague J. Cancer metastasis: building a framework. Cell tein tyrosine kinase by both cellular adhesion and oncogenic trans- 2006;127:679–95. formation. Nature 1992;358:690–2. 28. Brabletz S, Brabletz T. The ZEB/miR-200 feedback loop–a motor of 24. Wary KK, Mainiero F, Isakoff SJ, Marcantonio EE, Giancotti FG. The cellular plasticity in development and cancer? EMBO Rep 2010; adaptor protein Shc couples a class of integrins to the control of cell 11:670–7. cycle progression. Cell 1996;87:733–43. 29. Wellner U, Schubert J, Burk UC, Schmalhofer O, Zhu F, Sonntag A, 25. Ji HF, Pang D, Fu SB, Jin Y, Yao L, Qi JP, et al. Overexpression of focal et al. The EMT-activator ZEB1 promotes tumorigenicity by repres- adhesion kinase correlates with increased lymph node metastasis and sing stemness-inhibiting microRNAs. Nat Cell Biol 2009;11:1487– poor prognosis in non-small-cell lung cancer. J Cancer Res Clin Oncol 95. 2013;139:429–35. 30. Shimono Y, Zabala M, Cho RW, Lobo N, Dalerba P, Qian D, et al. 26. Fidler IJ. The pathogenesis of cancer metastasis: the 'seed and soil' Downregulation of miRNA-200c links breast cancer stem cells with hypothesis revisited. Nat Rev Cancer 2003;3:453–8. normal stem cells. Cell 2009;138:592–603.

www.aacrjournals.org Clin Cancer Res; 19(21) November 1, 2013 5889

Alpha-Smooth Muscle Actin (ACTA2) Is Required for Metastatic Potential of Human Lung Adenocarcinoma

Hye Won Lee, Young Mi Park, Se Jeong Lee, et al.

Clin Cancer Res 2013;19:5879-5889. Published OnlineFirst August 30, 2013.

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