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1 1 Alpha-Smooth Muscle Actin (ACTA2) Is Required for Metastatic Potential of Human 1 Lung Adenocarcinoma 2 3 Hye Won Lee*1,2,3

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1 1 Alpha-Smooth Muscle Actin (ACTA2) Is Required for Metastatic Potential of Human 1 Lung Adenocarcinoma 2 3 Hye Won Lee*1,2,3 Author Manuscript Published OnlineFirst on August 30, 2013; DOI: 10.1158/1078-0432.CCR-13-1181 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 1 Alpha-smooth muscle actin (ACTA2) is required for metastatic potential of human 2 lung adenocarcinoma 3 4 Hye Won Lee*1,2,3, Young Mi Park*3,4, Se Jeong Lee1,4,5, Hyun Jung Cho1,2, Duk-Hwan Kim6,7, 5 Jung-Il Lee2, Myung-Soo Kang3, Ho Jun Seol2, Young Mog Shim8, Do-Hyun Nam1,2,3, Hyeon 6 Ho Kim3,4, Kyeung Min Joo1,3,4,5 7 8 Authors’ Affiliations: 9 1Cancer Stem Cell Research Center, 2Department of Neurosurgery, 3Department of Health 10 Sciences and Technology, Samsung Advanced Institute for Health Sciences and 11 Technology (SAIHST), 4Samsung Biomedical Research Institute, 5Department of Anatomy 12 and Cell Biology, 6Department of Molecular Cell Biology, 7Center for Genome Research, 13 8Department of Thoracic Surgery, Samsung Medical Center, Sungkyunkwan University 14 School of Medicine, Seoul, Korea 15 16 *These authors contributed equally to this work. 17 18 Running title: ACTA2 confers metastatic potential on lung adenocarcinoma 19 Keywords: Non-small cell lung adenocarcinoma, alpha-smooth muscle actin, migration, 20 invasion, metastasis 1 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 30, 2013; DOI: 10.1158/1078-0432.CCR-13-1181 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 2 1 Financial support: This study was supported by a grant from the Korea Healthcare 2 Technology R&D Project, Ministry for Health & Welfare Affairs, Republic of Korea (A092255), 3 and the Basic Science Research Program, National Research Foundation of Korea by the 4 Ministry of Education, Science, and Technology (2011-009329 to H. H. Kim). 5 Corresponding Author: Hyeon Ho Kim, Department of Health Sciences and Technology, 6 Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan 7 University, 50 Irwon-Dong, Gangnam-Gu, Seoul 135-710, South Korea. Phone: 82-2-3410- 8 1039; Fax: 82-2-3410-0534; E-mail: [email protected]; and Kyeung Min Joo, 9 Department of Anatomy and Cell Biology, Sungkyunkwan University School of Medicine, 10 Samsung Medical Center, 50 Irwon-Dong, Gangnam-Gu, Seoul 135-710, South Korea. 11 Phone: 82-2-2148-9779; Fax: 82-2-2148-9829; E-mail: [email protected] 12 Conflict of interest: The authors have no conflicts of interest to disclose. 13 Word count: 3,657 words 14 Total number of figures and tables: 5 figures (7 supplementary figures and 1 15 supplementary table) 16 Type of manuscript: Research Article 17 Invited manuscript: Not invited 18 2 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 30, 2013; DOI: 10.1158/1078-0432.CCR-13-1181 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 3 1 Abstract 2 3 Purpose: Metastatic relapse of primary lung cancer leads to therapeutic resistance and 4 unfavorable clinical prognosis; therefore, identification of key molecules associated with 5 metastatic conversion has significant clinical implications. We previously identified a link 6 between early brain metastasis of lung adenocarcinoma (ADC) and amplification of the 7 alpha-smooth muscle actin (ACTA2) gene. The aim of present study was to investigate the 8 prognostic and functional significance of ACTA2 expression in cancer cells for the metastatic 9 potential of lung ADCs. Experimental Design: ACTA2 expression was analyzed in tumor 10 cells from 263 patients with primary lung ADCs by immunohistochemistry, and was 11 correlated with clinicopathological parameters. The expression of ACTA2 in human lung 12 ADC cells was modulated with shRNAs and siRNAs specifically targeting ACTA2. Results: 13 The patients with lung ADCs with high ACTA2 expression in tumor cells showed significantly 14 enhanced distant metastasis and unfavorable prognosis. ACTA2 downregulation remarkably 15 impaired in vitro migration, invasion, clonogenicity, and transendothelial penetration of lung 16 ADC cells without affecting proliferation. Consistent with the in vitro results, depletion of 17 ACTA2 in human lung ADC PC14PE6 cells significantly reduced their metastatic potential 18 without altering their tumorigenic potential. Expression of c-MET and FAK in lung ADC cells 19 was also reduced by ACTA2-targeting siRNAs and shRNAs, and was accompanied by a 20 loss of mesenchymal characteristics. Conclusions: These findings indicate that ACTA2 21 regulates c-MET and FAK expression in lung ADC cells, which positively and selectively 22 influence metastatic potential. Therefore, ACTA2 could be a promising prognostic biomarker 23 and/or therapeutic target for metastatic lung ADC. 3 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 30, 2013; DOI: 10.1158/1078-0432.CCR-13-1181 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 4 1 Translational Relevance 2 3 The short latency from the initial diagnosis of lung adenocarcinoma (ADC), the most 4 common subtype of non-small cell lung cancer, to metastatic relapse leads to a high 5 mortality rate. This short latency implies that the cancer cells in a primary lung ADCs have 6 already acquired a number of multi-organ metastatic competencies. In this study, using 263 7 pathologic samples, we determined that ACTA2 expression in lung ADC cells at the primary 8 site is significantly associated with enhanced distant metastasis. This association was 9 translationally interpreted and validated. ACTA2 regulates the expression of the epithelial- 10 mesenchymal transition-associated signaling molecules, c-MET and FAK, and consequently, 11 the in vitro and in vivo metastatic potential of lung ADC cells. These results have high 12 translational and clinical impact because ACTA2 could be utilized for the development of 13 predictive diagnostics and/or anti-metastatic agents for advanced lung ADC. 14 4 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 30, 2013; DOI: 10.1158/1078-0432.CCR-13-1181 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 5 1 Introduction 2 3 Lung cancer is the most common cancer worldwide with the highest mortality rate (1). 4 The identification of therapeutic targets in non-small cell lung cancer (NSCLC), such as 5 epithelial growth factor receptor (EGFR)-activating mutations, has enabled the development 6 of effective targeted therapies for advanced NSCLC (2). However, the 5-year survival rate 7 for NSCLC remains 15% across all stages of the disease. These unfavorable clinical 8 outcomes originate from its high invasive and metastatic potential (3, 4). In particular, lung 9 adenocarcinoma (ADC) is known to establish distant macrometastases in various organs, 10 within months of diagnosis (5). Current targeted therapeutics have limited efficacy for the 11 treatment of distant metastases in lung ADC. 12 The short latency of metastatic relapse in lung ADC implies that the cancer cells in the 13 primary tumor have already acquired numerous multi-organ metastatic competencies, 14 including cell motility, invasiveness, resistance to hypoxia, enhanced angiogenesis, survival 15 after detachment, and evasion of immune surveillance (6, 7). Therefore, it might be possible 16 to identify predictive biomarkers of metastasis and novel therapeutic targets in primary ADCs 17 with high metastatic potential. These novel therapeutic targets could then be used to 18 overcome the resistance of NSCLC to currently available targeted treatments. 19 In our previous study, we compared the gene amplifications and deletions in lung ADCs 20 with synchronous brain metastasis to those in lung ADCs with metachronous brain 21 metastasis to elucidate the genomic alterations associated with early distant metastasis in 22 lung ADC (8). Among several genomic alterations, amplification of alpha-smooth muscle 23 actin (α-SMA; hereafter ACTA2) was significantly associated with synchronous brain 24 metastasis. ACTA2 is known to contribute to cell-generated mechanical tension and 25 maintenance of cell shape and movement. Since cell motility is critically dependent on the 5 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 30, 2013; DOI: 10.1158/1078-0432.CCR-13-1181 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 6 1 actin cytoskeleton, the dynamics of cytoskeletal structures affected by ACTA2 could be 2 essential to invasion and metastasis in lung ADC (9, 10). 3 Herein, we report that high ACTA2 expression in tumor cells in primary lung ADC is 4 closely associated with progression of lung ADC, and provide the first evidence of its novel 5 roles in the metastatic potential of lung ADC. To our knowledge, this is the first report 6 suggesting that ACTA2 is a promising prognostic biomarker and/or therapeutic target for 7 advanced lung ADC. 8 6 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 30, 2013; DOI: 10.1158/1078-0432.CCR-13-1181 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 7 1 Materials and Methods 2 3 Study population 4 Surgical samples were obtained from 263 patients with lung ADC who underwent surgical 5 resection at the Samsung Medical Center (SMC, Seoul, Korea) between November 1994 6 and August 2004 (11, 12). Written informed consent for the use of paraffin-embedded 7 tissues, as approved by the Institutional Review Board at SMC, was obtained from each 8 patient before surgery.
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