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 Alpha-smooth muscle actin (ACTA2) is required for metastatic potential of human

2 lung adenocarcinoma

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

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

16 *These authors contributed equally to this work.

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

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.

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

1 Abstract

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.

1 Translational Relevance

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.

1 Introduction

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

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.

1 Materials and Methods

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. Postoperative follow-up was scheduled at 1 and 2 months, every 3

9 months during the first 2 years after surgery, and then every 6 months thereafter, or more

10 frequently if needed. The patients consisted of 140 males (53%) and 123 females (47%),

11 36~80 years of age. The mean age at surgical resection was 58.9 years. Of the 263 samples,

12 71, 134, 41, and 7 were stage I, II, III, and IV, respectively.

14 Immunohistochemistry and immunofluorescence staining

15 Tissue microarray sections generated from the above-mentioned surgical samples were

16 immunostained with mouse monoclonal antibodies against human α-SMA (N1584; Dako,

17 clone 1A4 or MA5-15806; Thermo Scientific Pierce, clone 4F4). Briefly, endogenous

18 peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide. The antigen was

19 retrieved by heating sections in 10 mM sodium citrate (pH 6.0) at 95°C for 30 minutes (mins).

20 The primary antibodies, biotinylated secondary antibody (Vector Laboratories), and then

21 avidin-biotin complex (Vector Laboratories) were incubated with the sections at 4°C

22 overnight, for 1 hour (h) at room temperature (RT), and for 1 h at RT, respectively. ACTA2

23 immunoreactivity was categorized as low (<50% ACTA2-positive tumor cells, ACTA2-Low)

1 or high grade (>50% ACTA2-positive tumor cells, ACTA2-High) in areas with maximal

2 staining.

3 To confirm the expression of ACTA2 in lung ADC tumor cells, immunofluorescence was

4 performed with the mouse monoclonal anti-ACTA2 (Dako, 1:250) and rabbit polyclonal anti-

5 thyroid transcription factor 1 (TTF-1) (abcam, 1:200) antibody against antigen-retrieved lung

6 ADC paraffin sections. TTF-1 has been reported to be one of several markers differentiating

7 ADC from squamous cell carcinoma of the lung (13). The antibodies were visualized by

8 Alexa Fluor® 488-labeled goat anti-mouse IgG and Alexa Fluor® 594-labeled goat anti-rabbit

9 IgG antibody (Life Technologies), respectively. Samples were then incubated with DAPI

10 (1:1,000) for 5 mins at RT to reveal cell nuclei.

12 Cell culture, transfection, and generation of stable cell lines

13 PC14PE6 cells were established from a pleural effusion developed in a nude mouse

14 injected intravenously with parental human lung ADC PC14 cells (Dr. I. J. Fidler, M. D.

15 Anderson Cancer Center, Houston, TX). The karyotypic analysis of the PC14PE6 cells ruled

16 out contamination with murine cells. PC14PE6 cells were maintained at 37°C and 5% CO2 in

17 DMEM (Hyclone) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-

18 antimycotic solution (GIBCO). H322, H1299, H460, and A549 human NSCLC cell lines were

19 purchased from American Type Culture Collection (ATCC, Manassas, VA) and all

20 experiments were performed within 6 months of purchase. Authentication of these cell lines

21 was performed by Short Tandem Repeat (STR) profiling to exclude cross-contamination

22 between the cell lines. H322, H1299, H460, and A549 cells were grown in RPMI (GIBCO)

23 supplemented with 10% FBS and penicillin/streptomycin (GIBCO). For transfection, human

24 NSCLC cells were plated at a density of 5×105 cells/dish and transfected with the indicated

1 siRNAs using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

2 SiRNAs directed against ACTA2, Focal adhesion kinase (FAK), and c-MET were purchased

3 from Santa Cruz Biotechnology. Control siRNA (siCTRL) was synthesized by Genolution.

4 For stable cell lines, cells were transfected with an ACTA2-specific shRNA (shACTA2,

5 Sigma) or empty pLKO vector (shCTRL) using Lipofectamine 2000, and then selected by

6 culturing in puromycin (5 μg/mL).

8 Western blot analysis and quantitative real-time PCR (RT-qPCR)

9 For western blot analysis, cells were lysed with RIPA buffer containing protease inhibitors

10 and phosphatase inhibitors (Roche). Equal amounts of protein were subjected to SDS-

11 polyacrylamide gels electrophoresis and transferred to polyvinylidene difluoride (PVDF)

12 membranes (Millipore). After blocking in 5% bovine serum albumin (BSA), membranes were

13 incubated with the indicated primary antibodies overnight, and then with the appropriate

14 secondary antibodies. For RT-qPCR, total RNA was isolated using TRIzol reagent

15 (Invitrogen) according to the manufacturer's instructions, and then used to synthesize cDNA

16 with Superscript III Reverse Transcriptase (Invitrogen). Expression of mRNA was quantified

17 by RT-qPCR (ABI Prism 7600) using power SYBR® Green PCR Master Mix (Applied

18 Biosystems). The primers used in this study were summarized in supplementary table S1.

20 Detection of metastatic activity in vitro

22 Migration and invasion assay

1 Migratory activity was measured by the wound closure assay. Briefly, a confluent cell

2 surface was scratched with a pipette tip and migration distance was measured by wound

3 closure. For the invasion assay, Matrigel coated transwells (BD) were utilized. Equal

4 numbers of cells in serum free media were added to the upper chamber. The addition of

5 complete media containing 10% FBS to the lower compartment stimulated the cells to

6 invade. After 24 h incubation, cells were fixed with 100% methanol and stained with H&E.

7 The number of invading cells on the lower surface of the membrane was counted.

9 Transendothelial migration assay

10 Human umbilical vein endothelial cells (HUVECs) were seeded (at 1×105 cells/well) in

11 fibronectin-coated 24-well transwell inserts with a pore size of 8 μm (Corning Costar Corp.),

12 and grown to confluence. Calcein AM-labeled cancer cells (1×105 cells) were suspended in

13 serum free medium and added to the endothelial monolayer. After 24 h incubation, cells

14 remaining in the upper chamber were completely removed and the tumor cells that migrated

15 through the endothelial monolayer to the lower face of the filter were ﬁxed with 4%

16 paraformaldehyde (PFA). The transmigrated tumor cells were counted in 10 random fields at

17 100× magnification.

19 Focus forming assay

20 Cells were seeded (at 300 cells/well) in 6-well plates and maintained in complete medium

21 for 3 weeks. The number of viable colonies per well was counted after staining with 0.2%

22 crystal violet.

1 In vivo tumorigenesis and metastasis assay

2 All animals received humane care in compliance with the “Guide for the Care and Use of

3 Laboratory Animals” prepared by the Institute of Laboratory Animal Resources published by

4 the National Institutes of Health and according to the Animal Experiment Guidelines of

5 Samsung Biomedical Research Institute.

7 Tumorigenicity assay

8 The effect of ACTA2 silencing on tumorigenic potential of lung ADC cells was analyzed in

9 two different in vivo models. For subcutaneous model, 1x106 cancer cells in 100 μL Hank's

10 balanced salt solution (HBSS) were subcutaneously injected into the posterior ﬂank of 6~8

11 week-old female BALB/c nude mice (Orient, 7 mice per group). The volume of subcutaneous

12 tumor (0.5 × length × width2, mm3) was measured on 14, 17, 21, 24, and 28 days after the

13 implantation. At 28 days post-implantation subcutaneous tumors were excised,

14 photographed, and weighted. For orthotopic model, female 6~8 week-old female BALB/c

15 nude mice were anesthetized in the right lateral decubitus position. A 3 mm incision was

16 made at the lateral dorsal axillary line, 1.5 cm above the lower rib line, just below the inferior

17 border of the scapula. Cancer cells (1×106) in 40 μL HBSS were injected into the left lung

18 with a 1 mL insulin syringe and a 30-gauge needle, and then the incision was repaired. The

19 body weights of the animals were measured every day, and an autopsy was performed

20 immediately after a 25% weight loss. Lungs were fixed in formalin, embedded in paraffin,

21 and then stained with H&E.

23 Metastasis assay

1 For experimental metastasis in vivo model, 2×105 cancer cells in 100 μL of HBSS were

2 directly injected into the left ventricle of the heart of 6~8 week-old female BALB/c nude mice

3 with a 1mL insulin syringe. Four weeks later, the animals were sacrificed. Brains, lungs,

4 lower leg bones, adrenal glands, and abnormal organs were harvested, fixed in formalin, and

5 embedded in paraffin. Paraffin-embedded tissues were stained with H&E.

7 Statistical analysis

8 Overall survival and metastasis-free survival were calculated by the Kaplan-Meier method

9 and compared by the log-rank test. Group comparisons were analyzed with Student’s two-

10 tailed t-test. Tumor metastasis rates were compared by Fisher’s exact test, two-tailed.

11 Differences with P values less than 0.05 were considered statistically significant.

1 Results

3 Clinical implications of ACTA2 in lung ADC

4 To confirm the role of ACTA2 in the metastasis of lung ADC, we assayed ACTA2

5 expression in 263 lung ADCs by immunohistochemistry (Fig. 1A). When more than 50% of

6 tumor cells expressed ACTA2, the patients were considered as ACTA2-High (n=14).

7 Specific detection of ACTA2 protein was verified by same staining patterns of two different

8 antibodies against human ACTA2 (Supplementary Fig. S1). Moreover, ACTA2 protein in

9 lung ADC cells was confirmed by co-localization of ACTA2 with a lung ADC specific marker,

10 TTF-1 (Fig. 1B). The stromal ACTA2 expression in immunohistochemistry (Fig. 1A) and the

11 presence of ACTA2-positive, TTF-1-negative cells in immunofluorescence staining (Fig. 1B)

12 suggested concomitant ACTA2 expression in tumor stromal cells (14). ACTA2 staining was

13 also observed in close proximity to the nucleus and/or nuclear domain in TTF-1 positive

14 cancer cells in agreement with previous report (15), although its functional roles need to be

15 further elucidated. Kaplan–Meier survival analysis was conducted to determine the

16 prognostic significance of ACTA2 expression in patients with lung ADCs. ACTA2-High

17 patients had significantly worse overall survival [OS; median (95% CI): 28.0 (6.0-50.0)

18 months, P=0.011; Fig. 1C] and metastasis-free survival [MFS; 16.0 (7.6-24.4) months,

19 P=0.022, Fig. 1D], compared to those of ACTA2-Low patients [n=249; OS=71.0 (55.8-86.2)

20 months; MFS=53.0 (37.4-68.6) months]. These results indicate that not only ACTA2 gene

21 amplification (8), but also ACTA2 overexpression in lung ADC cells are significantly

22 associated with enhanced metastatic potential and worse clinical prognosis of lung ADC.

24 In vitro effects of shRNA-mediated downregulation of ACTA2

1 We generated 7 PC14PE6 lung ADC cell subclones that stably express 6 different ACTA2

2 shRNAs or a control vector (pLKO) to assess the effect of ACTA2 silencing on the metastatic

3 potential of lung ADC cells. Downregulation of ACTA2 mRNA and protein was confirmed by

4 RT-qPCR (Fig. 2A and Supplementary Fig. S2) and western blot analysis (Fig. 2B),

5 respectively. When ACTA2 expression was downregulated by shRNA, the migration (Fig. 2C

6 and Supplementary Fig. S2) and invasion (Fig. 2D and Supplementary Fig. S2) of

7 PC14PE6 cells were significantly inhibited. Since PC14PE6 cells transfected with

8 shACTA2#6 showed the lowest expression of ACTA2 mRNA and strong suppression of

9 migration and invasion, PC14PE6/shACTA2#6 cells (hereafter, PC14PE6/shACTA2) were

10 used for all subsequent experiments. The inhibitory effects of ACTA2 silencing on the in vitro

11 migration potential of lung ADC cells were confirmed using various lung ADC cell lines and a

12 specific siRNA targeting ACTA2 (Supplementary Fig. S3).

13 ACTA2 downregulation also significantly decreased transendothelial migration (TEM, Fig.

14 3A), and clonogenicity (Fig. 3B) of PC14PE6 cells, properties that are required for distant

15 metastasis of lung cancer. In contrast, proliferation (Supplementary Fig. S4A), anoikis-

16 resistance (Supplementary Fig. S4B), and adhesion to fibronectin [an extracellular matrix

17 (ECM) component] (Supplementary Fig. S4C) of PC14PE6 cells were not affected by the

18 shRNA-mediated ACTA2 downregulation. Therefore, the inhibitory effects of ACTA2

19 silencing on the migration and invasion of PC14PE6 cells did not result from decreased

20 proliferation and/or cellular growth.

22 Specific implications of ACTA2 in the metastasis of lung ADC in vivo

23 Metastasis is a sequential, multi-step process that includes invasion, intravasation,

24 survival in circulation, extravasation, and colonization. In vitro downregulation of ACTA2

1 significantly affected those processes, which indicated that ACTA2 could be required for

2 distant metastasis of lung ADC. To investigate whether ACTA2 has the effect on primary

3 lung tumorigenesis in vivo, 1×106 control (PC14PE6/shCTRL) or ACTA2 knockdown

4 (PC14PE6/shACTA2) cells were implanted into the subcutaneous tissue (PC14PE6/shCTRL,

5 n=7; PC14PE6/shACTA2, n=7) or left lung (PC14PE6/shCTRL, n=7; PC14PE6/shACTA2,

6 n=6) of nude mice. In the subcutaneous model, both tumor growth rate (P=0.43) and final

7 tumor weight (P=0.49) were not significantly affected by ACTA2 downregulation (Fig. 4A).

8 ACTA2 downregulation also made no effects on the tumor formation in the lung cancer

9 orthotopic model, since the survival of mice with intralung injection of PC14PE6/shACTA2

10 cells (median survival length=32 days) was comparable to that of the control group (median

11 survival length=25 days) (Fig. 4B, P=0.318). These results are consistent with that of the in

12 vitro cell proliferation assay, which firmly validates no significant role of ACTA2 in lung

13 tumorigenesis.

14 To further examine the in vivo biological role of ACTA2 in the metastatic potential of lung

15 ADC cells, we injected 1×106 PC14PE6/shCTRL or PC14PE6/shACTA2 cells into the left

16 ventricle [systemic metastasis animal model; PC14PE6/shCTRL (n=9), PC14PE6/shACTA2

17 (n=7)] of nude mice. In contrast to the tumorigenicity assays, significantly less metastatic

18 potential was observed in mice that received an intracardiac injection of PC14PE6/shACTA2

19 cells (Fig. 4C). These mice had a sole metastatic tumor in the adrenal gland (14% incidence,

20 1/7 mice), whereas mice that received an intracardiac injection of PC14PE6/shCTRL cells

21 had a 78% incidence (7/9 mice) of metastatic tumors, and the metastatic sites varied [lung,

22 adrenal gland, bone, brain, and eyeball] (Fig. 4C). Each metastatic tumor was confirmed

23 pathologically (Fig. 4D). These in vivo assays indicated that ACTA2 promotes lung ADC

24 metastasis without affecting primary tumor formation potential, which would be mediated by

25 increased migration, invasion, and clonogenicity of lung ADC cells.

26 15

1 Effects of ACTA2 on FAK and c-MET expression

2 From the above results, we determined that ACTA2 is a critical factor for the acquisition of

3 metastatic potential in lung ADC cells. To address the mechanism whereby ACTA2

4 regulates metastatic potential, we analyzed the expression of EMT-associated proteins, such

5 as FAK and c-MET, since EMT is a critical process for acquisition of metastatic potential.

6 PC14PE6 cells were transfected with an ACTA2-specific siRNA. Forty eight hours after the

7 transfection, mRNA and protein of both FAK and c-MET were significantly decreased (Fig.

8 5A). ACTA2 silencing also significantly increased E-cadherin expression and decreased

9 vimentin expression (Fig. 5B). These results indicated that the expression of FAK and c-

10 MET, which are major signaling molecules for metastasis, is suppressed by ACTA2

11 downregulation, and that ACTA2 expression is required for maintenance of the

12 mesenchymal characteristics of PC14PE6 cells. To ensure that ACTA2 is associated with

13 FAK and c-MET expression, we checked the downregulation effects again with several

14 shRNAs specifically targeting ACTA2. As shown in Supplemental Fig. S5, shRNA-elicited

15 knockdown of ACTA2 also reduced the expression of FAK and c-MET.

16 To determine if FAK and c-MET are required for ACTA2 silencing-mediated suppression

17 of migration and invasion, PC14PE6 cells were transfected with FAK and c-MET-specific

18 siRNAs (Supplementary Fig. S5). Similarly to ACTA2 silencing, silencing of either FAK and

19 c-MET inhibited migration (Fig. 5C) and invasion (Fig. 5D) of PC14PE6 cells. In accordance

20 with data obtained from PC14PE6, ACTA2 silencing also inhibited migratory and invasive

21 activity of lung ADC H322 cells through downregulation of FAK and c-MET (Supplemental

22 Fig. S6 and S7). These results suggest that decreased expression of FAK and c-MET is

23 involved in ACTA2 downregulation-mediated suppression of metastatic potential.

1 Discussion

3 Distant metastasis decreases the survival of patients with NSCLC. While targeted

4 molecular therapies have greatly improved the management of primary NSCLC, these

5 therapies are ineffective for the treatment of distant metastases. Because systemic

6 metastasis is induced by additional metastasis-specific genetic alterations, identifying key

7 molecules that contribute to lung cancer cell dissemination is essential for the development

8 of new predictive biomarkers and/or anti-metastatic therapeutic strategies. In this study, we

9 demonstrated that ACTA2 plays a critical role in lung ADC metastasis. Clinically, high

10 expression of ACTA2 in lung ADC cells was significantly associated with worse prognostic

11 outcome and early distant metastasis of lung ADC patients. Experimentally, ACTA2

12 expression was closely correlated with the metastatic potential of human lung ADC cell line,

13 PC14PE6 both in vitro and in vivo. Moreover, our results suggested that the decreased

14 metastatic potential induced by ACTA2 silencing is mediated by downregulation of FAK and

15 c-MET expression.

16 Although ACTA2 is generally expressed in the smooth muscle cells and activated cancer-

17 associated fibroblasts (CAFs), tumor cells could also use actin bundles to allow them to

18 break away from a primary tumor and invade the surrounding tissue (9, 10). In many

19 epithelial cancers, transforming growth factor (TGF)-β-elicited EMT induces the expression

20 of ACTA2 (16), increases tumor invasion, and worsens patient survival (17). The connection

21 between EMT and epithelial stemness also suggests that EMT can confer clonogenicity and

22 resistance to apoptosis on primary tumor cells (18). Finally, the presence of ACTA2 in the

23 nuclear proximity of some cells indicated that ACTA2 relate the change of mechanical force

24 to an altered gene expression via nuclear transcription regulators (15, 19). Therefore,

25 ACTA2 expression and/or amplification of the ACTA2 gene in lung ADC cells could be used 17

1 to predict clinical aggressiveness of lung ADC, which were demonstrated in this report and

2 our previous study (8), respectively.

3 However, the possibility that ACTA2 expressing CAFs could synergistically contribute the

4 progression and metastasis of lung ADCs with adjacent ACTA2 positive cancer cells could

5 not be ruled out. In recent studies, lung CAFs has a significant role in the metastatic

6 potential of NSCLC via the direct regulation of gene expression at the invasive front of

7 cancer nests and induction of EMT (20, 21). Although the present study and previous reports

8 highlight the importance of ACTA2 expression of both cancer cells and CAFs in NSCLC

9 progression and metastasis, further studies are needed to elucidate detail mechanisms how

10 ACTA2 contributes to tumor promoting effects in CAFs and lung ADC cells.

11 Inhibition of ACTA2 expression in lung ADC PC14PE6 and H322 cells downregulated the

12 transcription of c-MET and FAK, which decreased the in vitro migration and invasion of

13 PC14PE6 and H322 cells. Hepatocyte growth factor (HGF)/c-MET signaling activates a

14 number of downstream pathways, including Mitogen-activated protein kinase (MAPK),

15 Phosphoinositide 3-kinase (PI3K)/Protein Kinase B (PKB/Akt), and nuclear factor kappa-

16 light-chain-enhancer of activated B cells (NF-κB), in epithelial cancer cells (17), and induces

17 proliferation, motility, cell survival, and EMT. Recent studies demonstrated that HGF is an

18 indicator of poor prognosis in lung ADC (22). FAK is activated by integrins that connect the

19 cytoskeleton to the extracellular matrix and act as nucleation sites for the assembly of cell

20 adhesions (23, 24). In addition, FAK plays a critical role in TGF-β1-induced EMT. Its

21 overexpression was signiﬁcantly associated with positive lymph node metastasis and worse

22 overall survival of patients with lung ADC (25). Because ACTA2 not only mediatesalterations

23 in the mechanical properties of cancer cells, but also regulates the expression of signaling

24 proteins that significantly influence the clinical outcome of lung ADC, this protein could be

25 used as a therapeutic target for lung ADCs. 18

1 Maintenance of stemness is critical for lung ADC cells to make distant metastases since

2 establishment of new colonies in unfamiliar microenvironments is the final step of sequential

3 metastatic processes. According to elaborate analysis of genomic features of primary and

4 metastatic tumors, metastasis is a result from clonal selection of heterogeneous cancer cells

5 in the primary tumor (26, 27). Therefore, clonogenicity is one of key phenotypes of stemness,

6 which is required for distant metastasis. Our results indicate that ACTA2 is involved in the

7 clonogenicity of lung ADC PC14PE6 cells. Moreover EMT, a transdifferentiating process,

8 was also affected by ACTA2 expression, in this study. One study postulated that EMT and

9 stem cell properties are combined in invasive cancer cells–which often coexpress EMT and

10 stem cell markers (28). Moreover, EMTs additionally equip more differentiated epithelial cells

11 with stem-cell traits through molecular linking of EMT-inducing transcription factors to self-

12 renewal programs (29, 30). This connection between EMT and epithelial stemness indicates

13 that by imparting mesenchymal traits to carcinoma cells, an EMT can confer motility,

14 invasiveness, are resistance to apoptosis and metastatic dissemination from primary tumors

15 (18). Accordingly, stemness of lung ADC cells might be regulated by ACTA2, which could in

16 turn influence the metastatic potential of lung ADC cells. However, the detailed mechanism

17 of how ACTA2 mediates this regulation remains to be elucidated further.

18 In this study, we demonstrated for the first time that ACTA2 regulates FAK and c-MET

19 expression in lung ADC cells, which positively influences in vitro and in vivo metastatic

20 potential. Since ACTA2 expression in lung ADC cells is significantly associated with poor

21 clinical outcome and early distant metastasis, ACTA2 could be utilized as a possible

22 therapeutic target and a prognostic biomarker for metastasis.

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1 Figure Legends

3 Figure 1. ACTA2 expression is closely associated with early metastasis and poor

4 prognosis of lung adenocarcinoma (ADC). A, Representative results of

5 immunohistochemical staining against ACTA2 in 263 lung ADCs (top, low-expression,

6 ACTA2-Low; bottom, high-expression, ACTA2-High). B, Colocalization of ACTA2 (green)

7 witha lung ADC specific marker, Thyroid transcription factor 1 (TTF-1, red) in lung ADC cells.

8 ACTA2+/TTF-1+ lung ADC cells were indicated by arrows. C and D, Kaplan-Meier analysis

9 of overall survival (OS) and metastasis free survival (MFS) of 263 patients with lung ADC.

10 Immunohistochemical staining of ACTA2 was performed using Tissue Microarray (TMA)

11 sections from 263 patients. Lung ADCs were categorized by the frequency of ACTA2-

12 stained cancer cells (>50% tumor cells = ACTA2-High). P values were determined by the

13 log-rank test.

15 Figure 2. Short hairpin (sh) RNA-mediated ACTA2 knockdown inhibited the migratory

16 and invasive activity of PC14PE6 cells. A and B, Knockdown of ACTA2 mRNA and

17 protein was verified by RT-qPCR (A) and western blot (B), respectively. C and D, The effect

18 of ACTA2 silencing on in vitro migration and invasion was assessed by the wound closure

19 assay (C) and Matrigel coated transwell assay (D), respectively. Migration distance was

20 measured 24 hours (h) after wound formation on a confluent cell surface and invaded cells

21 were counted under a microscope in more than 6 fields for each group. Data shown are the

22 mean plus the standard deviation.*, P<0.05.

1 Figure 3. Effects of shRNA mediated ACTA2 knockdown on transendothelial migration

2 and clonogenicity of PC14PE6 cells. A, For transendothelial migration, cells were added

3 to a endothelial monolayer. The transmigrated cells were counted under a fluorescent

4 microscope in more than 10 fields for each group. B, To assess clonogenicity, cells were

5 seeded in 6-well plates and cultured in complete media for 3 weeks. Colonies were stained

6 with 0.2% crystal violet and counted. Data shown are the mean plus the standard deviation.*,

7 P<0.05.

9 Figure 4. Decreased expression of ACTA2 specifically suppressed systemic

10 metastasis of PC14PE6 cells in vivo. A, Downregulation of ACTA2 had no effect on

11 tumorigenic potential of lung ADC in subcutaneous model. 1×106 PC14PE6 control

12 (PC14PE6/shCTRL) or ACTA2 knockdown (PC14PE6/shACTA2) cells were implanted into

13 the subcutaneous tissue of nude mice (n=7 per group). The volume of xenograft tumors and

14 relative tumor weight at 28 days post-implantation are demonstrated. Data = mean±

15 standard error of means (SEM). Representative photographs of the excised tumors at 28

16 days post-implantation. B, The effect of ACTA2 on primary tumor growth was evaluated in

17 an orthotopic lung cancer animal model by an intra-lung injection of 1×106 of PC14PE6

18 control (PC14PE6/shCTRL) (n=7) or ACTA2-knockdown (PC14PE6/shACTA2) cells (n=6). C,

19 The in vivo effects of ACTA2 on the metastatic potential of lung ADC PC14PE6 cells were

20 evaluated by injection of 2×105 PC14PE6/shCTRL (n=9) or PC14PE6/shACTA2 cells (n=7)

21 into the left ventricle of female BALB/c nude mice. The incidence of systemic metastases to

22 each organ was compared.*, P<0.05. D, Representative illustrations of metastatic foci in

23 each organ.

1 Figure 5. Silencing of ACTA2 suppressed c-MET and FAK expression. A, PC14PE6

2 cells were transfected with control (siCTRL) or ACTA2 siRNA (siACTA2). At 48 hours after

3 transfection, protein and mRNA levels of ACTA2, c-MET, and FAK were determined by

4 western blot (left) and RT-qPCR (right), respectively. GAPDH=internal control. B, The

5 protein and mRNA levels of the EMT makers, E-cadherin and vimentin, were determined by

6 western blot (left) and RT-qPCR (right), respectively. C and D, PC14PE6 cells were

7 transfected with c-MET or FAK siRNA. At 48 hours after transfection, in vitro migration (C)

8 and invasion (D) were determined by wound closure assay and Matrigel transwell assay,

9 respectively. The data shown are the mean plus standard deviation from 3 separate

10 experiments. *, P<0.05.

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 Published OnlineFirst August 30, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-13-1181

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