Angiomotin Decreases Lung Cancer Progression by Sequestering Oncogenic YAP/TAZ and Decreasing Cyr61 Expression

ORIGINAL ARTICLE Angiomotin decreases lung cancer progression by sequestering oncogenic YAP/TAZ and decreasing Cyr61 expression

Y-L Hsu1, J-Y Hung2,3, S-H Chou4, M-S Huang2,3, M-J Tsai1,2, Y-S Lin2, S-Y Chiang5, Y-W Ho5, C-Y Wu2 and P-L Kuo5,6,7

Lung cancer is the leading cause of cancer death worldwide, with metastasis underlying majority of related deaths. Angiomotin (AMOT), a scaffold protein, has been shown to interact with oncogenic Yes-associated protein/transcriptional co-activator with a PDZ-binding motif (YAP/TAZ) proteins, suggesting a potential role in tumor progression. However, the functional role of AMOT in lung cancer remains unknown. This study aimed to identify the patho-physiological characteristics of AMOT in lung cancer progression. Results revealed that AMOT expression was signiﬁcantly decreased in clinical lung cancer specimens. Knockdown of AMOT in a low metastatic CL1-0 lung cancer cell line initiated cancer proliferation, migration, invasion and epithelial–mesenchymal transition. The trigger of cancer progression caused by AMOT loss was transduced by decreased cytoplasmic sequestration and increased nuclear translocation of oncogenic co-activators YAP/TAZ, leading to increased expression of the growth factor, Cyr61. Tumor promotion by AMOT knockdown was reversed when YAP/TAZ or Cyr61 was absent. Further, AMOT knockdown increased the growth and spread of Lewis lung carcinoma in vivo. These ﬁndings suggest that AMOT is a crucial suppressor of lung cancer metastasis and highlight its critical role as a tumor suppressor and its potential as a prognostic biomarker and therapeutic target for lung cancer.

Oncogene (2015) 34, 4056–4068; doi:10.1038/onc.2014.333; published online 10 November 2014

INTRODUCTION Yes-associated protein (YAP) and its paralogue, transcriptional Lung cancer, particularly non-small cell lung cancer, is the leading co-activator with a PDZ-binding motif (TAZ), are transcription cause of cancer-related deaths worldwide.1,2 The vast majority of co-activators and major downstream targets regulated by the 14–17 deaths occur because patients are diagnosed in the advanced Hippo pathway. Sustained or elevated YAP/TAZ expression levels has been noted in human breast cancer and lung cancer cell stage but even those with the early stages have high rates of 14,18,19 relapse after effective therapy.3,4 Moreover, up to 40% of patients lines. The major regulatory control of YAP/TAZ activity is via develop local or distal metastasis, resulting in dramatically alterations in their subcellular distribution and stability. The increased mortality rates.5 Thus understanding the processes that relationship between AMOT and YAP/TAZ is controversial: AMOT contribute to cancer progression, including cancer growth and p130 can prevent the inactivating phosphorylation of YAP, resulting in the promotion of cell growth and tumorigenesis in metastasis, is crucial for developing effective therapeutic 20 strategies and improving the cure rate. liver cells, whereas AMOT can regulate the Hippo pathway by ﬁ ﬁ sequestrating YAP/TAZ in the cytoplasm and increasing the Angiomotin (AMOT) was rst identi ed as an angiostatin- 12,13,21–23 binding protein that enhanced endothelial cell motility by actitvity of tumor-suppressor LATS1/2. However, the role reducing tight junctions and altering actin dynamics.6–8 The of AMOT in lung cancer is still poorly understood. This study was AMOT family of proteins has three members: AMOT (p80 and p130 designed to identify the patho-physiological characteristics of isoforms), AMOT-like protein 1 (AMOTL1) and AMOTL2.9 The AMOT in lung cancer progression. AMOTL1 and AMOTL2 genes also generate two splicing isoforms in humans.9 AMOT has been shown to block the activity of Rich1, a small GTPase activating protein, leading to increased Rac1-GTP RESULTS levels and altering growth signaling, tight junction and cell Reduced AMOT (p130) expression in highly proliferative and migration.10,11 AMOT p80 has been shown to promote cell growth invasive lung cancer cells in mouse and human lung cancer by increasing mitogen-activated protein kinase activation in cell lines breast cancer.12 In contrast to the stimulatory role of AMOT on To investigate the candidate genes involved in lung cancer cell growth, AMOT p130 decreased cell proliferation in breast metastasis in vivo, Lewis lung carcinoma (LLC) cells were cancer MDA-MB-468 cells in a recent study.13 However, the repeatedly transplanted into mice by tail vein injection, thereby functional role of AMOT in the development of lung cancer is creating the LLC-1, LLC-2, LLC-3, LLC-4 and LLC-5 cell line series. poorly understood. Because our previous study demonstrated that migratory ability

1Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 2Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; 3School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 4Division of Chest Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; 5Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 6Research Center for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan and 7Institute of Medical Science and Technology, National Sun Yat-Sen University, Kaohsiung, Taiwan. Correspondence: Professor P-L Kuo, Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, No. 100, Shih-Chuan First Road, Kaohsiung 807, Taiwan. E-mail: [email protected] Received 22 January 2014; revised 18 August 2014; accepted 22 August 2014; published online 10 November 2014 Role of angiomotin in lung cancer Y-L Hsu et al 4057 was enhanced by increasing the time of transplantations mRNA transcript (Supplementary Figure S3A), while AMOT (p130) (LLC-54LLC-44LLC-34LLC-24LLC-1),24 the proliferative capacity protein expression was completely inhibited in all stable of these cell strains were assessed. The cell proliferative ability of clones (Supplementary Figure S3B). Knockdown of AMOT sig- the LLC cells was significantly enhanced after a series of five nificantly increased CL1-0 cell proliferation, as determined by transplantations (relative change to LLC-1 was 128.67 ± 9.90% and bromodeoxyuridine (BrdU) incorporation (relative change to 186.69 ± 8.99% for LLC-3 and LLC-5, respectively) (Supplementary control shRNA plasmid-transfected CL1-0 cells: 211.78 ± 18.27% Figure S1A). The invasiveness of LLC cells was also significantly and 190.72 ± 23.40% for clones 17 and 18, respectively, Po0.001) enhanced by repeated transplantation (relative change to LLC-1 and WST-1 assays (relative change to control shRNA plasmid- was 198.40 ± 13.27% and 238.75 ± 27.80% for LLC-3 and LLC-5, transfected CL1-0 cells: 192.56 ± 32.60% and 180.86 ± 21.20% for respectively) (Supplementary Figure S1B), suggesting that the clones 17 and 18, respectively, Po0.001) (Figure 2a; Supplemen- LLC-1/LLC-5 isogenic cell lines were useful for the study of cancer tary Figure S3C). progression. Scratch wound-healing assay analysis showed that knockdown To investigate which candidate genes contributed to cancer of AMOT remarkably increased the migratory ability of CL1-0 cells fi progression, the RNA transcription pro les of LLC-1 and LLC-5 was (Supplementary Figure S3D). Trans-well migration assay indicated assessed by microarray. The transcript levels of AMOT was 11.78- that the inhibition of AMOT significantly increased cell motility fold higher in LLC-1 cells than in LLC-5 cells (Supplementary Figure (194.88 ± 16.40% and 189.78 ± 8.04% for stable clones 17 and 18, S2A). The AMOT transcription level, as measured by quantitative o – fi respectively, P 0.001), as compared with CL1-0 cells transfected real-time PCR (qRT PCR), was signi cantly reduced in LLC-3 and with the control shRNA plasmid (Figure 2b). Similarly, invasion LLC-5 cells (by 0.15 ± 0.04 and 0.04 ± 0.02-fold, respectively, assay revealed that AMOT-knockdown cells exhibited significantly Po0.05), compared with that in LLC-1 (Supplementary Figure higher invasiveness than control cells (218.89 ± 27.47% and S2B). The expression of AMOT was also negatively associated with 208.54 ± 24.12% for stable clones 17 and 18, respectively, the time of transplantations. Immunoblot analysis revealed that Po0.001) (Figure 2c). AMOT p130 protein levels were lower in LLC-5 cells than in LLC-1 Because epithelial–mesenchymal transition (EMT) was impor- and LLC-3 cells (Figure 1a). 27,28 To further assess the association between reduced AMOT levels tant for cancer cells to metastasize, the role of AMOT in EMT and malignant cellular properties, AMOT levels were compared was assessed. The inhibition of AMOT by shRNA changed CL1-0 from a cobblestone-like cell morphology to a spindle-like between the paired isogenic lung cancer cell lines CL1-0 (low fi invasiveness) and CL1-5 (high invasiveness).25,26 The level of broblastic morphology (Supplementary Figure S3E). In addition, AMOT mRNA was 3.44-fold higher in CL1-0 cells than in CL1-5 cells knockdown of AMOT expression decreased epithelial marker (Figure 1b). Immunoblot analysis also revealed higher levels of E-cadherin and increased mesenchymal markers such as N-cad- AMOT p130 (118 kDa) in CL1-0 cells, consistent with the results of herin and vimentin in the CL1-0 cells (Figure 2d), suggesting that qRT–PCR. Notably, the AMOT p80 isoform (72 kDa) was undetect- AMOT was involved in the regulation of EMT. Furthermore, AMOT able in both CL1-0 and CL1-5 cells (Figure 1a). inhibition increased the level of snail, a critical regulator of EMT, in the nuclear fraction of CL1-0 cells (Figure 2d). To further validate the role of AMOT p130 in cancer proliferation Reduced AMOT expression and elevated YAP in the nuclei of lung and migration, AMOT p130 expression was inhibited by shRNA cancer cells transfection at different concentrations (3, 6 and 9 μg AMOT Analysis of AMOT mRNA transcript from a lung cancer cDNA array shRNA plasmid/6 cm dish). The AMOT shRNA plasmid significantly was confirmatory for the pathogenic role of AMOT downregula- fi inhibited the level of AMOT protein in a dose-dependent manner tion. The levels of AMOT mRNA transcript was signi cantly lower in CL1-0 (Supplementary Figure S4A). The silencing of AMOT also in the human tumor sections than in the non-tumor sections in 11 significantly enhanced cell proliferation, migration and EMT in a of the 24 (45.8%) of human lung cancer patients. Of the 11 low dose-dependent manner (Supplementary Figures S4B and D), mRNA transcript samples, four had undetectable AMOT mRNA which were consistent with the results of AMOT-knockdown transcripts by q–PCR in tumor sections but was detected in the stable clones. corresponding normal region. Nine of the 24 samples (37.5%) demonstrated no marked difference between tumor sections and matched non-tumor sections (Supplementary Table S1). Ectopic AMOT (p130) expression decreased cell proliferation and The negative association of AMOT with nuclear YAP/TAZ was migration and restored epithelial protein expression noted in clinical lung patient specimens (n = 15). Compared with To further investigate the role of AMOT, CL1-5 cells were the normal regions, AMOT levels were decreased in the cancer transfected with AMOT (p130) cDNA. Immunoblot analysis reveals regions, with high YAP/TAZ levels, in the nuclei of lung cancer cells that AMOT (p130) was expressed in CL1-5 cells (Figure 3a). (Figure 1c). The AMOT and YAP/TAZ levels were also assessed by Overexpression of AMOT significantly decreased CL1-5 cell quantitative immunofluorescence and the results revealed that the proliferation (relative change to control pCMV plasmid- amount of AMOT in the tumor regions were significantly reduced transfected CL1-5 cells: 70.86 ± 3.87% and 68.78 ± 14.19% for 4 4 4 4 (389.73 × 10 ± 209.51 × 10 and 269.26 × 10 ± 125.94 × 10 for clones 4 and 8, respectively, Po0.01) (Figure 3b). In addition, the normal and tumor regions, respectively, P = 0.047). In addition, migration of AMOT-overexpressing CL1-5 cells was significantly the level of nuclear YAP/TAZ proteins was increased 4 4 4 4 suppressed compared with the control cells (Figure 3c). (5.86 × 10 ± 10.92 × 10 and 56.53 × 10 ± 28.84 × 10 for normal Cells with enhanced AMOT expression exhibited significantly and tumor regions, respectively, P = 0.0002) (Figure 1d). Cytosolic lower invasiveness compared with the control cells (74.97 ± 3.70% fi AMOT protein signi cantly but negatively correlated with the and 61.34 ± 8.26% for stable clones 4 and 8, respectively, nuclear YAP/TAZ level (r = 0.69) in tumor region (Figure 1e). Po0.001) (Figure 3d). Ectopic expression of AMOT changed CL1-5 from spindle-like fibroblastic morphology to a cobblestone- Knockdown of AMOT increased cell proliferation, migration, like cell phenotype (Supplementary Figure S5A). The expression invasion and mesenchymal protein expression levels of epithelial protein E-cadherin and mesenchymal markers To investigate whether decreased AMOT expression was (N-cadherin, vimentin and snail) were significantly increased associated with lung adenocarcinoma cell progression, AMOT and decreased, respectively, in AMOT-overexpressing cells expression was reduced by shRNA transfection in CL1-0 cells. The (Figure 3e). Furthermore, the ectopic expression of AMOT AMOT shRNA transfection showed 498% inhibition of AMOT decreased cell proliferation, migration and N-cadherin but

Figure 1. Decreased AMOT expression in highly invasive lung cancer cell lines. (a) Reduced AMOT levels in the LLC-5 and CL1-5 cells. (b) Decreased AMOT mRNA expression in the CL1-5 cells. With the CL1-0 level as reference, the relative AMOT expression was calculated using the 2–ΔΔCT method. (c) Decreased AMOT and increased nuclear YAP/TAZ in the tumor regions of lung cancer patients. (d) Quantitative data of AMOT and nuclear YAP/TAZ levels in tissue sections of lung cancer patients. (e) The correlation of cytosolic AMOT and nuclear YAP/TAZ protein. The mRNA and protein expression was assessed by qRT–PCR and immunoblotting, respectively. The AMOT and YAP/TAZ protein levels in the tumor region and non-tumor regions (n = 15) were analyzed by immunoﬂuorescence staining and confocal microscopy (×40). Red, YAP/TAZ; green, AMOT; blue, DAPI nuclear stain; AMOT and YAP/TAZ levels were indicated as signal intensities (pixel count) in normal and tumor regions of lung cancer. The relationship of cytosolic AMOT and nuclear YAP/TAZ was analyzed using linear regression. *Signiﬁcant difference with the controls, by Student’s t-test (Po0.05). All experiments were performed independently at least three times.

increased E-cadherin expression in a dose-dependent manner oncogenic potential,23 the interaction of AMOT with YAP and TAZ (Supplementary Figures S5B–E). was investigated. With more times of transplantation of LLC cells (form LLC-1, LLC-3 to LLC-5), the cytosolic YAP/TAZ level AMOT decreased the nuclear location of YAP/TAZ by sequestering decreased and the nuclear YAP/TAZ level increased progressively them in the cytoplasm (Figure 4a). The cytoplasmic association of AMOT with YAP/TAZ Because previous studies reported that the AMOT protein could was also remarkably higher in LLC-1 cells than in LLC-5 cells bind to the YAP and TAZ proteins, thereby inhibiting their (Figure 4a). Similarly, the YAP/TAZ level in the nuclei was higher in

Figure 2. Inhibition of AMOT promoted cancer progression in lung cancer cells. Knockdown of AMOT increased CL1-0 cell (a) proliferation, (b) migration and (c) invasion. (d) Reduced AMOT expression caused the cells to undergo EMT and increased the EMT regulator snail expression. AMOT-knockdown CL1-0 cell lines were established by transfecting AMOT shRNA plasmid or control plasmid vector into CL1-0 cells and stably transfected colonies were selected by puromycin. The cell proliferations of the control and AMOT-knockdown CL1-0 cells were determined by BrdU incorporation analysis after 72 h of incubation. Control plasmid or AMOT-shRNA plasmid-transfected CL1-0 cells were seeded in the upper insert in serum-free conditions and culture medium containing 10% fetal bovine serum was added into the lower well to act as a chemo-attractant for 24 h. The migratory and invasive cells were quantified by fluorescence dye staining. The expression of EMT markers was assessed by immunoblot. *Significant difference with the controls, by Student’s t-test (Po0.05). All experiments were performed independently at least three times.

CL1-5 cells than in CL1-0 cells, and the cytoplasmic interaction of CL1-5 cells (Figure 4c). The role of YAP and TAZ was also confirmed AMOT with YAP/TAZ was higher in CL1-0 cells than in CL1-5 cells by specific siRNA transfection. We transfected AMOT-knockdown (Figure 4b). CL1-0 with control siRNA, YAP, TAZ or both siRNA and checked that Knockdown of AMOT by shRNA transfection increased the the transfections inhibited the targets specifically (Supplementary nuclear localization of YAP/TAZ in CL1-0 cells (Figure 4c). On the Figures S6A and B). Silencing YAP or TAZ resulted in a partial other hand, overexpression of AMOT decreased the amount of YAP/ reversion of cell proliferation and migration, while inhibition of both TAZ in the nuclei of CL1-5 cells (Figure 4c). The inhibition of AMOT YAP and TAZ exhibited a complete restoration in AMOT-knockdown also decreased AMOT/YAP/TAZ association in CL1-0 cells, whereas CL1-0 cells (Figure 4d). Mesenchymal markers in AMOT-knockdown enhanced AMOT level increased AMOT/YAP/TAZ interaction in CL1-0 cells had similar results (Figure 4e).

Figure 3. Overexpression of AMOT (p130) decreased cell proliferation, migration and invasion and restored epithelial markers in CL1-5 cells. (a) The AMOT protein level in AMOT (p130) overexpressing CL1-5 cells. Enhanced AMOT expression decreased (b) proliferation, (c) migration and (d) invasion of CL1-5 cells. (e) Increased AMOT expression caused the cells to undergo mesenchymal–epithelial transition (MET) and decreased the EMT regulator snail expression. CL1-5 cells were transfected with control or AMOT (p130 cDNA) plasmid, and the stable clones were created by G418 selection. The cell proliferation of controls and AMOT-overexpressing CL1-5 cells were determined by BrdU incorporation analysis after 72 h incubation. Control pCMV plasmid or pCMV-AMOT p130-cDNA plasmid-transfected CL1-5 cells were seeded in the upper insert in serum-free conditions, and culture medium containing 10% fetal bovine serum was added into the lower well to act as a chemo-attractant for 24 h. Migratory and invasive cells were quantified by fluorescence dye staining and EMT marker expressions were assessed by immunoblotting. *Significant difference with the controls, by Student’s t-test (Po0.05). All experiments were performed independently at least three times.

Figure 4. AMOT decreased the nuclear location of YAP/TAZ by sequestering them in the cytoplasm. (a) Progressively increased YAP/TAZ nuclear location and decreased the association of AMOT with YAP/TAZ in the cytoplasmic fraction with more times of transplantations in the LLC cells. (b) Reduced association of AMOT with YAP/TAZ in the cytosol of CL1-5 cells. (c) The influence of AMOT knockdown or overexpression on nuclear translocation of YAP/TAZ. Knockdown of YAP or TAZ prevented the enhancement of (d) cell proliferation, migration and (e)EMT mediated by AMOT knockdown in CL1-0 cells. Cell lysates were immunoprecipitated with anti-YAP or TAZ antibody, and the co-immunoprecipitated AMOT was detected by immunoblotting with anti-AMOT antibody. AMOT-knockdown CL1-0 cells were transfected with either non-target, YAP or TAZ siRNA (10 nM). After 48 h transfection, the cell biological behaviors were evaluated. The knockdown efficiency of specific siRNA was determined by qRT–PCR, while cell proliferation was determined by BrdU assay. Cell migration was analyzed as previously described. #Denotes statistically significant difference from control siRNA-transfected cells, by Student’s t-test (Po0.05). All experiments were performed independently at least three times.

Oncogene (2015) 4056 – 4068 © 2015 Macmillan Publishers Limited Role of angiomotin in lung cancer Y-L Hsu et al 4061 Involvement of YAP and TAZ on cell proliferation, migration and growth factor (CTGF), Cyr61, BICR2 and ANKRD1 were well- invasion characterized YAP target genes,29,30 this study assessed whether Because the major function of YAP/TAZ was transcription co- YAP and TAZ increased the expression of these genes in the LLC activation to stimulate gene expression and connective tissue cell line series. The levels of Cyr61, ANKRD1 and BICR2 mRNA

© 2015 Macmillan Publishers Limited Oncogene (2015) 4056 – 4068 Role of angiomotin in lung cancer Y-L Hsu et al 4062 transcripts were 9.8-, 4.1- and 2.3-fold higher, respectively, in LLC-5 (Supplementary Figures S8B and C). However, the level of YAP/ than in LLC-1 cells (Figure 5a). Transfection of YAP or TAZ siRNA TAZ-inducible gene CTGF was not dependent on YAP/TAZ in lung decreased YAP or TAZ mRNA transcript by 466% in LLC-5 cancer (data not shown). (Supplementary Figure S7). Knockdown of YAP expression in the To validate the regulatory role of YAP and TAZ on Cyr61 LLC-5 cells significantly decreased the levels of Cyr61, ANKRD1 expression, YAP, TAZ or both were inhibited in AMOT-knockdown and BICR2 mRNA transcripts by 72.7, 54.3 and 65.4%, respectively, LLC-1 cells. Inhibition of YAP or TAZ alone partially decreased while the inhibition of TAZ by siRNA significantly reduced the Cyr61 mRNA trascripts while silencing both YAP and TAZ levels of Cyr61, ANKRD1 and BICR2 mRNA transcripts by 60.9, 61.8 completely eliminated Cyr61 enhancement, suggesting that both and 76.1%, respectively, in LLC-5 (Figure 5b). YAP and TAZ had critical modulating roles on Cyr61 expression To further investigate whether Cyr61 act as downstream of (Figure 5d). AMOT, the role of AMOT, LLC-1 cells were transfected with AMOT The role of YAP/TAZ was also assessed by siRNA knockdown in shRNA plasmid. Transfection of AMOT shRNA decreased AMOT LLC-5 and CL1-5 cells. Transfection of YAP or TAZ siRNA decreased mRNA transcript by 490% in LLC-1 (Supplementary Figure S8A). YAP or TAZ mRNA transcript by 465% in both LLC-5 and CL1-5 Inhibiting AMOT in LLC-1 cells increased the levels of Cyr61, (Supplementary Figure S7 and S9A). Knockdown of YAP or TAZ ANKRD1 and BICR2 mRNA transcripts (10.77-, 4.02- and 2.38-fold, significantly decreased the proliferation and migration of LLC-5 respectively, in clone 1 and 9.17-, 4.01- and 2.71-fold, respectively, and CL1-5 cells (Supplementary Figures S9B and C) while the in clone 2; Po0.001) (Figure 5c). Knockdown of AMOT increased inhibition of YAP and TAZ increased E-cadherin expression and the level of Cyr61 mRNA transcript in human lung cancer CL1-0 decreased N-cadherin expression in CL1-5 and LLC-5 cells cells, whereas AMOT overexpression decreased it in CL1-5 cells (Supplementary Figure S9D).

Figure 5. YAP/TAZ had an oncogenic role in AMOT-knockdown lung cancer. (a) The levels of Cyr61, ANKRD1 and BICR2 RNA transcripts in LLC cell lines series. (b) Knockdown of YAP or TAZ by siRNA decreased Cyr61, ANKRD1 and BICR2 mRNA transcripts in LLC5. (c) Silencing of AMOT increased Cyr61 mRNA transcript in LLC1. (d) Inhibition of YAP or TAZ decreased Cyr61 upregulation that was induced by AMOT knockdown. AMOT-knockdown LLC1 cells were transfected with either non-target, YAP, TAZ or YAP plus TAZ siRNA (10 nM) for 48 h. The Cyr61 mRNA transcript levels were then determined by qRT–PCR. LLC1 cells were transfected with control shRNA or AMOT shRNA plasmid, and the stable clones were established by puromycin selection. The relative mRNA levels of Cyr61, ANKRD1 and BICR2 in LLC1 and LLC5 were assessed by microarray. *Signiﬁcant difference with the controls, by Student’s t-test (Po0.05). #Denotes statistically signiﬁcant difference between control siRNA-transfected cells (Po0.05). All experiments were performed independently at least three times.

Figure 6. Cyr61 was involved in cancer progression in AMOT-knockdown lung cancer. Knockdown of Cyr61 decreased (a) cell proliferation and (b) migration in AMOT-knockdown LLC-1 cells. (c) Silencing of Cyr61 caused MET. (d) Inhibition of Cyr61 reversed FAK and AKT phosphorylation induced by AMOT knockdown in lung cancer cells. (e) AKT and FAK inhibitors decreased AMOT-knockdown-enhanced cell migration. AMOT-knockdown LLC-1 cells were transfected with non-target or Cyr61 siRNA (10 nM) for 48 h. Their cell biological behaviors were then evaluated. The knockdown efficiency of specific siRNA was determined by qRT–PCR, while cell proliferation was determined by BrdU assay. Cell migration was analyzed as previously described. The levels of various proteins were assessed by immunoblotting. *Significant difference with the controls, by the Student’s t-test (Po0.05). #Denotes statistically significant difference between control siRNA-transfected cells (Po0.05). All experiments were performed independently at least three times.

© 2015 Macmillan Publishers Limited Oncogene (2015) 4056 – 4068 Role of angiomotin in lung cancer Y-L Hsu et al 4064 Cyr61 contributed to cell proliferation, migration and EMT in specific set of genes that facilitate tumorigenesis in the liver by AMOT-knockdown lung cancer preventing its phosphorylation. The DNA vaccination targeting We assessed whether Cyr61 was involved in lung cancer AMOT are reported to decrease tumor angiogenesis, resulting in 31,32 progression. Transfection of Cyr61 siRNA decreased Cyr61 mRNA cancer growth suppression. However, recent studies indicate transcript by 487% in AMOT-knockdown LLC-1 cells. This genetic that phosphorylated AMOT caused by tumor-suppressor LATS1/2 13 inhibition did not affect the CTGF mRNA transcript kinases restricts YAP-mediated gene response and cell growth. (Supplementary Figure S10). Inhibition of Cyr61 significantly Moreover, different isoforms of AMOT have been shown to decreased the proliferation and migration of AMOT-knockdown exhibit different functions on cell migration: AMOT p80 increases LLC-1 (Figures 6a and b). Moreover, blocking Cyr61 also increased cell migration, whereas AMOT p130 binds to and stabilizes tight junction ZO-1, which is an important marker of the epithelial E-cadherin expression and decreased N-cadherin expression in 33,34 AMOT-knockdown LLC-1 (Figure 6c). phenotype. In this study, the knockdown of AMOT p130 Assessing the mechanism of Cy61 on cell proliferation and increases the properties of lung cancer progression, including migration, the inhibition of AMOT increased the phosphorylation proliferation, migration, invasion and EMT. The inhibition of AMOT of focal adhesion kinase (FAK; Tyr397) and AKT, which were p130 expression also increases lung cancer metastasis in vivo, and fi reduced by Cyr61 siRNA transfection (Figure 6d). Both FAK and a signi cant loss of AMOT p130 is observed in tumor regions in AKT inhibitors decreased cell migration in AMOT-knockdown clinical lung cancer samples, further supporting that AMOT p130 LLC-1, suggesting that FAK/AKT might be involved in cell may act as a tumor suppressor in lung cancer. Future studies fi migration induced by AMOT knockdown (Figure 6e). investigating whether the expression pattern and modi cation of AMOT and isoforms influence their function in different types of tissue are warranted to elucidate the mechanisms of tumor Knockdown of AMOT increased lung cancer growth and repression by AMOT. metastasis in vivo Increasing evidences demonstrate that YAP/TAZ are oncogenes To assess the tumor growth of human lung cancer cells in a nude in mammalian cells.35,36 YAP/TAZ genes have been reported to be mice model, AMOT-knockdown CL1-0 cells was delivered by overexpressed at a high frequency in various human cancers and subcutaneous injection into the flanks of mice. The rate of tumor are associated with poor prognosis.36 Furthermore, YAP over- growth of AMOT-knockdown CL1-0 was enhanced compared with expression has been associated with non-small cell lung cancer, that of the control plasmid-transfected CL1-0 cells (Figure 7a). This especially lung adenocarcinoma.37,38 Elevated YAP/TAZ levels are was consistent with the results of the in vitro data. reported to induce target gene expression, resulting in cell To further validate the regulation of AMOT on cell proliferation, proliferation, evasion of apoptosis and amplification of progenitor AMOT-knockdown LLC was delivered by subcutaneous injection or stem cells, thereby promoting organ size.39,40 YAP/TAZ have into the flanks of mice. Before AMOT-knockdown LLC1 transplan- also been reported to facilitate the processes of cancer metastasis, tation, this study assessed whether AMOT silencing increased the including inducing the loss of cell contact inhibition, migration, ability of LLC in cell proliferation, migration and EMT. Inhibition of invasion and EMT.41,42 AMOT increased the cell proliferation, migration and EMT (with The activity of YAP/TAZ is regulated by physical interactions E-cadherin downregulation and N-cadherin upregulation) (Supple- with other proteins such as AMOT,23,43,44 which sequesters mentary Figures S11A–C). These data were consistent with human YAP/TAZ in the cytoplasm and inactivates it. In the present study, lung cancer data. The rate of tumor growth of AMOT-knockdown a loss of AMOT in cells leads to the accumulation of nuclear LLC increased significantly, as compared with that of the control YAP/TAZ, because AMOT sequesters them in the cytoplasm. The LLC in vivo (Figure 7b). knockdown of YAP/TAZ also prevents cancer cell proliferation, To further test whether AMOT acted as a suppressor of lung migration and EMT caused by the loss of AMOT, suggesting that cancer metastasis in vivo, LLC and AMOT-knockdown LLC cells YAP/TAZ has a critical role as an oncogenic promoter in lung were injected into the tail veins of mice. The mice were killed cancer. 24 days after, and the metastatic tumor nodules that formed in Cyr61 (also known as CCN1) is a secretory extracellular matrix- the lungs were examined. Knockdown of AMOT increased lung associated molecule of the matricellular CCN family. It is a multi- metastasis (tumor nodules, AMOT-knockdown clone 1: functional protein that has an important role in regulating cellular 138.33 ± 39.34; clone 2: 136.50 ± 14.97; control: 97.00 ± 21.07; activities, such as cell attachment, migration, survival, growth, Po0.05; Figure 7c). angiogenesis and gene expression.45 Dysregulation of Cyr61 Analysis of proliferation by Ki-67 immunohistochemical staining expression is associated with various types of tumors, including showed that the level of nuclear Ki-67 increased by 2.83- and 1.87- lung cancer.45,46 Blocking Cyr61 by neutralizing antibodies can fold in the tumors of AMOT-knockdown clone 1 and clone 2 LLC-1 decrease the cancer growth and metastasis in mice.47 Cyr61 has cells in mice, respectively, as compared with the tumors of control also been shown to be a downstream target of YAP/TAZ and is plasmid-transfected LLC cells. Furthermore, YAP/TAZ were located involved in cancer growth, migration and metastasis owing to the in the cytosol of the tumors of control plasmid-transfected LLC dysregulation of Hippo signaling in lung cancer, ovarian cancer cells, whereas they were prominently located in the nuclear cells and breast cancer.48 fraction of the tumors of AMOT-knockdown clones 1 and 2 LLC-1 In this study, knockdown of AMOT increases Cyr61 expression. cells (Figures 7d and e). In contrast, YAP/TAZ inhibition decreases the transcription of Cyr61. Inhibition of Cyr61 by siRNA transfection prevents cancer progression mediated by AMOT knockdown, suggesting that DISCUSSION AMOT can inhibit YAP/TAZ transcriptional activation and decrease Lung cancer has been one of the most common causes of death Cyr61-mediated cancer progression in lung cancer. worldwide for several decades.1,2 A series of in vitro and in vivo Cyr61 has been reported to regulate cell proliferation and investigations have assessed the role of AMOT in the regulation of migration by directly binding with integrin ανβ3 and ανβ5.49 lung cancer cell proliferation, migration and invasion, and the Interaction of Cyr61 with integrin increases FAK phosphorylation, results clearly show that AMOT functions as a scaffold protein in which enhances AKT activation.50 AKT has been a critical mediator modulating the oncogenic potential of YAP/TAZ and its target of cell proliferation, survival and metastasis in a variety of cell Cyr61 signaling by restricting its activity in lung cancer. types.51 Results of the present study show that AMOT down- The role of AMOT in cancer is controversial. Yi et al.20 report that regulation increases FAK and AKT phosphorylation, which are AMOT acts as a YAP co-factor, augmenting its activity towards a inhibited by Cyr61 silencing. Inhibition of AKT or FAK reduces the

Figure 7. Knockdown of AMOT increased lung cancer growth and metastasis in vivo. Inhibition of AMOT increased tumor growth of (a) CL1-0 and (b) LLC-1 cells in mice. (c) The number of tumor nodules in the lungs of AMOT-knockdown LLC transplanted mice. (d) Levels of Ki-67 and YAP/TAZ in the tumor sections. (e) The number of Ki-67 and YAP/TAZ in cell nuclei in lung tumor nodules. AMOT-knockdown LLC-1 and CL1-0 cells were transplanted into C57BL/6 (for LLC-1 cells) or BALB/c athymic nude mice (for AMOT-knockdown CL1-0 cells) by subcutaneous injection. Tumor volume was measured using a caliper and recorded (for CL1-0 cells) every day. For metastasis experiment, AMOT-knockdown LLC-1 cells were delivered into mice by tail vein injection. Mice were killed at 22 days after injection, and tumor nodules on the surface of lungs were counted. Tumor sections were subjected to Ki-67 or YAP/TAZ and histological staining. Tumor cells with positive Ki-67 nuclear staining were counted and normalized to total tumor in a field. *Significant difference with the controls, by the Student’s t-test (Po0.05). influence of AMOT knockdown on cell migration, which supports prognostic biomarker, and further investigations on AMOT the hypothesis that Cyr61-medated FAK/AKT activation is involved functions may provide a potential treatment strategy for lung in the regulation of cancer migration in AMOT-knockdown lung cancer. cancer. In summary, this study provides compelling evidence that AMOT has a major role in tumor suppression in lung cancer. AMOT MATERIALS AND METHODS exerts its repressive function through its action as an inhibitory Cell cultures partner of oncogenic YAP/TAZ signaling. Restricting YAP/TAZ in The less invasive (CL1-0) and highly invasive (CL1-5) human lung the cytoplasm decreases Cyr61 expression, which reduces the adenocarcinoma cell lines were provided by Dr Pan-Chyr Yang (Depart- metastatic phenotypes of tumor cells. Taken together, this study ment of Internal Medicine, National Taiwan University Hospital, Taipei, delineates the functional role of AMOT in lung cancer progression. Taiwan) and cultured in RPMI 1640 supplemented with 10% fetal bovine AMOT expression in lung cancer specimens may be a valuable serum and 1% penicillin–streptomycin (Gibco BRL (Grand Island, NY, USA),

© 2015 Macmillan Publishers Limited Oncogene (2015) 4056 – 4068 Role of angiomotin in lung cancer Y-L Hsu et al 4066 Life Technologies (Grand Island, NY, USA)).25,26 The mouse LLC (CLR1642) For immunoprecipitation, cell lysates (200 μg of total protein) were cell line was obtained from the American Type Culture Collection incubated with 2 μg of anti-YAP/TAZ overnight and then with 20 μlof (Manassas, VA, USA) and cultured in Dulbecco’s Modiﬁed Eagle’s Medium protein A-agarose beads (Millipore) for 2 h at 4 °C. YAP and AMOT were supplemented with 10% fetal bovine serum plus 1% antibiotics. detected by incubating the blots with speciﬁc antibodies.

Microarray Quantitative real-time PCR Microarray experimental procedures were performed following the RNA isolation was performed using TRIzol reagent (Invitrogen, Carlsbad, manufacturer’s protocols. Total RNA (1 μg) was amplified by an Agilent CA, USA). cDNA was prepared using an oligo (dT) primer and reverse Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA, USA). LLC-5 transcriptase (Takara, Shiga, Japan) following the standard protocols. cells were labeled with Cy5, and LLC-1 cells were labeled with Cy3 in an RT–PCR was performed using SYBR Green on an ABI 7500 Real-Time PCR in vitro transcription process. Cy-labeled cRNA (0.825 μg) was cleaved to an System (Applied Biosystems, Foster City, CA, USA). Each PCR reaction average size of about 50–100 nucleotides by incubation with fragmenta- mixture contained 200 nM of each primer, 10 μl of 2 × SYBR Green PCR tion buffer (Agilent Technologies) at 60 °C for 30 min. An equal amount of Master Mix (Applied Biosystems), 5 μl cDNA and RNase-free water for a Cy-labeled cRNA was pooled and hybridized to an Agilent SurePrint G3 total volume of 20 μl. The PCR reaction was performed with a denaturation Mouse GE 8 × 60 K microarray (Agilent Technologies) at 65 °C for 17 h. step at 95 °C for 10 min and then 40 cycles at 95 °C for 15 s and 60 °C for After washing and drying by nitrogen gun blowing, the microarrays 1 min. All PCRs were performed in triplicate and normalized to the internal control GAPDH mRNA. Relative expression levels were presented using the were scanned by an Agilent microarray scanner (Agilent Technologies) at –ΔΔCT 535 nm for Cy3 and 625 nm for Cy5. The scanned images were analyzed by 2 method. The AMOT level in Lung Cancer cDNA Array I (Origene) was the Feature Extraction Software 10.5 (Agilent Technologies), an image also measured by PCR. analysis and normalization software used to quantify signal and back- ground intensity for each feature. The data were then substantially Immunofluorescence fi normalized by the rank-consistency- ltering LOWESS method. Non-cancer and cancer lung tissue specimens obtained from human lung cancer patients were embedded in optimal cutting temperature and Gene knockdown and overexpression frozen in liquid nitrogen. Sections (3–5 μm) were fixed with acetone at − Knockdown of AMOT in CL1-0 and YAP/TAZ in CL1-5 was performed using 20 °C and stained with AMOT antibody (Abcam) or co-stained by anti- a lentiviral expression system provided by the National RNAi Core Facility YAP/TAZ antibody (Cell Signaling Technology). After washing with phosphate-buffered saline containing 0.1% Tween-20, the slides were (Taipei, Taiwan). The lentiviruses were produced by co-transfecting incubated with Dylight 488- or Dylight 549-conjugated secondary HEK293T with pLKO-AS2, pLKO-AS3-AMOT-1 and two packaging plasmids antibodies (Rockland, Gilbertsville, PA, USA), with or without DAPI (4,6- (pCMVVDR8.91 and pMD.G). The stable clones were established by diamidino-2-phenylindole), for 1 h at room temperature. puromycin selection. The efficacy of the AMOT shRNA plasmid was Spectral image acquisition was performed using a Zeiss LSM 700 assessed by qRT–PCR. Knockdown of YAP, TAZ and Cyr61 was performed confocal microscope and quantitated by the Zen 2011 software (Carl Zeiss by ON-TARGET plus siRNA reagents transfection (Dharmacon, Chicago, MicroImaging LLC, Thornwood, NY, USA). Representative pixels from each IL, USA). of these images were selected, creating the emission spectra of each For AMOT (p130) overexpression, cells were transfected with pCMV or component. These spectra were then used to linearly unmix the images pCMV-AMOT (p130) plasmid (Origene, Rockville, MD, USA).The stable using the same setting in the Zen software. Two representative regions of clones were established by treatment with G418. interest (defined as areas with robust immunostaining, ~ 100 μm2 each) For mouse AMOT inhibition, LLC cells were transfected with either per slide were chosen, and the cumulative florescence intensity specific for control or AMOT shRNA plasmid provided by the National RNAi Core Dyligh 488 (green) was determined.52 Facility. The efficacy of siRNA was assessed by qRT–PCR. Animal model/immunohistochemical reactions Cell proliferation, migration and invasion The LLC cells were transplanted into C57BL/6 mice by tail vein injection. Cell proliferation was determined by BrdU incorporation, which was The animals were then killed on day 20 after LLC transplantation, and the performed using a BrdU Cell Proliferation Assay kit (Millipore Corp., LLC cells in the lungs were isolated by mincing, collagenase type I Bedford, MA, USA). Cell migration was assessed by scratch wound-healing digestion and filtering. LLC cells were cultured in a growth medium assay. The cells were allowed to grow into full confluence in 24-well plates. containing 10% fetal bovine serum and 1% penicillin–streptomycin and The following day, a uniform scratch was made down the center of the expanded for second-round transplantation. The sub-line of the first-round well using a micropipette tip, followed by washing once with phosphate- transplantation was designated as LLC-1 and sub-lines from 2, 3, 4 and 5 buffered saline. Photographic imaging was performed using an Olympus rounds of transplantation were designated as LLC-2, -3, -4 and -5, 1 × 50 inverted microscope (Olympus, Tokyo, Japan). Cell migration and respectively. invasion were also assessed by QCM 24-well Cell Migration Assay and The LLC-1, AMOT-knockdown LLC-1, CL1-0, and AMOT-knockdown Invasion Systems (Millipore), according to the manufacturer’s instructions. CL1-0 cells were transplanted into C57BL/6 or nude mice by subcutaneous injection or tail vein injection. After transplantation, tumor size was Immunoblot/immunoprecipitation measured using calipers, while tumor volume was estimated using the formula: The cells were lysed on ice for 15 min in M-PER solution. The cell lysate was ÀÁÀÁ centrifuged at 14 000 g for 15 min, and the supernatant fraction was Tumor volume mm3 ¼ long diameter ´ short diameter2 =2 collected for immunoblotting. Equivalent amounts of protein were The animals were killed on day 24, and the number of tumor nodules resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was recorded for analysis of the incidence of lung cancer. The tumor – fl (10 12%) and transferred to polyvinylidene di uoride membranes. After samples were fixed in 10% buffered formalin, dehydrated and embedded blocking for 1 h in 5% non-fat dry milk in Tris-buffered saline, the in paraffin blocks from which hematoxylin and eosin-stained slides were membranes were incubated with the desired primary antibody for 1–16 h. made and used for verification of the histopathological diagnosis. All The membranes were then treated with the appropriate peroxidase- immunohistochemical reactions were performed on 5-μm-thick paraffin conjugated secondary antibody and the immunoreactive proteins were sections. Briefly, the sections were de-paraffinized in xylene and detected using an enhanced chemiluminescence kit (Millipore) according rehydrated and then incubated in target retrieval solution (DAKO, Glostrup, to the manufacturer’s instructions. Denmark) in an autoclave for 8 min to retrieve the antigens. The activity The primary antibodies used to target YAP/TAZ, N-cadherin, E-cadherin, of endogenous peroxidase was blocked by incubation with 3% H2O2 snail, vimentin, FAK (total and phosphorylated forms), AKT (total and for 10 min. phosphorylated forms) and tubulin were from Cell Signaling Technology The YAP/TAZ and Ki-67 antigen expression levels were demonstrated (Danvers, MA, USA), whereas AMOT, YAP and TAZ antibodies were from using the mouse monoclonal anti-YAP/TAZ (dilution 1:100) and anti-Ki-67 Abcam Ltd, (Cambridge, UK). Actin antibodies and anti-glyceraldehyde-3- (dilution 1:100) antibodies, respectively. The sections were incubated with phosphate dehydrogenase (GAPDH) were from Millipore. Anti-LaminA/C primary antibodies overnight at 4 °C. The studied antigens were then antibody was from BD Biosciences (Allschwil, Switzerland). visualized using biotinylated antibodies and streptavidin-conjugated with

Oncogene (2015) 4056 – 4068 © 2015 Macmillan Publishers Limited Role of angiomotin in lung cancer Y-L Hsu et al 4067 horseradish peroxidase. Diaminobenzidine (DakoCytomation, Glostrup, 16 Kanai F, Marignani PA, Sarbassova D, Yagi R, Hall RA, Donowitz M et al. TAZ: a Denmark) served as the substrate, and all of the sections were counter- novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ stained with hematoxylin. domain proteins. EMBO J 2000; 19: 6778–6791. 17 Sudol M, Bork P, Einbond A, Kastury K, Druck T, Negrini M et al. Characterization of the mammalian YAP (Yes-associated protein) gene and its role in Statistical analysis defining a novel protein module, the WW domain. J Biol Chem 1995; 270: Data were expressed as means ± s.d., and statistical comparisons were 14733–14741. made using analysis of variance. Significant differences (Po0.05) between 18 Wang Y, Dong Q, Zhang Q, Li Z, Wang E, Qiu X. Over-expression of yes-associated the means of the two test groups were analyzed by the Student’s t-test. protein contributes to progression and poor prognosis of non-small-cell lung cancer. Cancer Sci 2010; 101: 1279–1278. 19 Zhi X, Zhao D, Zhou Z, Liu R, Chen C. YAP promotes breast cell proliferation and CONFLICT OF INTEREST survival partially through stabilizing the KLF5 transcription factor. Am J Pathol 2012; 180: 2452–2461. The authors declare no conflict of interest. 20 Yi C, Shen Z, Stemmer-Rachamimov A, Dawany N, Troutman S, Showe LC et al. The p130 isoform of angiomotin is required for Yap-mediated hepatic epithelial 6 ACKNOWLEDGEMENTS cell proliferation and tumorigenesis. Sci Signal 2013; : ra77. 21 Oka T, Schmitt AP, Sudol M. Opposing roles of angiomotin-like-1 and zona This study was supported by grants from the National Science Council of Taiwan (NSC occludens-2 on pro-apoptotic function of YAP. Oncogene 2012; 31:128–134. 101-2628-B-037-001-MY3; NSC 101-2320-B-037-043-MY3), the Ministry of Science 22 Paramasivam M, Sarkeshik A, Yates JR 3rd, Fernandes MJ, McCollum D. and Technology (MOST 103-2320-B-037-006-MY3; MOST 103-2314-B-037-052), the Angiomotin family proteins are novel activators of the LATS2 kinase tumor Kaohsiung Medical University “Aim for the top 500 universities grant, Grant No. KMU- suppressor. Mol Biol Cell 2011; 22: 3725–3733. DT103008”, the Research Center for Environmental Medicine, Kaohsiung Medical 23 Chan SW, Lim CJ, Chong YF, Pobbati AV, Huang C, Hong W. Hippo pathway- 286 University (KMU-TP103A19; KMU-TP103A20) and the Kaohsiung Medical University independent restriction of TAZ and YAP by angiomotin. J Biol Chem 2011; : – Hospital Research Foundation (KMUH102-2T06). We also thank the Center for 7018 7026. 24 Hsu YL, Wu CY, Hung JY, Lin YS, Huang MS, Kuo PL. 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