Tumor Biol. DOI 10.1007/s13277-016-4934-0
ORIGINAL ARTICLE
Fascin 1 promoted the growth and migration of non-small cell lung cancer cells by activating YAP/TEAD signaling Zhigang Liang 1,2 & Ying Wang 1 & Zhenya Shen 1 & Xiaomei Teng 1 & Xinjian Li 2 & Chenwei Li 2 & Weijie Wu 2 & Zenghui Zhou 2 & Zishan Wang 2
Received: 29 November 2015 / Accepted: 28 January 2016 # International Society of Oncology and BioMarkers (ISOBM) 2016
Abstract Fascin 1 (Fascin actin-bundling protein 1) is an actin-binding protein. Although several studies have reported the dysregulation of Fascin 1 in non-small cell lung cancer (NSCLC), its functions in the progression of NSCLC and the related molecular mechanism were not fully understood. In this study, the expression of Fascin 1 in NSCLC tissues was determined using quantitative PCR (qPCR), and the roles of Fascin 1 in the progression of NSCLC were investigated. It was found that both the messenger RNA (mRNA) level and the protein level of Fascin 1 were upregulated in NSCLC tissues. Forced expression of Fascin 1 promoted the growth and migration of NSCLC cells, while knocking down the expression of Fascin 1 inhibited the growth, migration, and tumorigenesis of NSCLC cells. Mechanism studies showed that Fascin 1 increased the transcriptional activity of the YAP/TEAD (Yes-associated protein/TEA domain transcriptional factor) complex, and knocking down the expression of Fascin 1 attenuated the expression of target genes downstream the YAP/TEAD complex. In addition, MST1 interacted with Fascin 1. Taken together, Fascin 1 plays an oncogenic role in NSCLC by activating the transcriptional activity of the YAP/ TEAD complex.
Zhigang Liang and Ying Wang contributed equally to this work. * Zhenya Shen [email protected]
1
Department of Cardiovascular Surgery of the First Affiliated Hospital & Institute for Cardiovascular Science, Soochow University, Suzhou, Jiangsu, China
2
Department of Thoracic Surgery, Ningbo First Hospital, Ningbo, Zhejiang, China
Keywords NSCLC . Fascin 1 . YAP/TEAD signaling . Cell growth . Migration
Introduction Fascins are actin-binding proteins, which cross-link the filamentous actin to the tightly packed bundles [1]. These actin bundles are involved in the formation of stress fibers, protrusions, and other sub-cellular structures [2, 3]. The Fascins family includes three conserved members, Fascin1, Fascin2, and Fascin3 [4]. Fascin1 and Fascin2 share about 60 % homolog [5], while Fascin1 and Fascin3 only share about 25 % homolog [5]. The F-actin cross-linking activity of Fascin1 in mammalian cells contributes to the formation of actin-based structure [6]. Dynamic alternations of these actin-based structures assist the migration and metastasis of cells [1, 7], suggesting the important roles of Fascin1 in regulating cell motility. Upregulation of Fascin1 has been observed in most of the aggressive human carcinomas [8–10]. The clinical data revealed that the expression of Fascin1 has been reported to correlate with the poor prognosis [8–10]. The functional studies of Fascin1 in cancer cell lines showed that knocking down the expression of Fascin1 inhibited cell migration and metastasis, while overexpression of Fascin1 promoted the motility of the cancer cells [5]. These finding indicated the oncogenic roles of Fascin1 in the tumorigenesis. The Hippo signaling controls the sizes of the organs in the development by regulating a panel of proliferationrelated and apoptosis-related genes expression, such as connective tissue growth factor (CTGF), Cyclin D1, Survivin, and so on [11, 12]. The core of this signaling
Tumor Biol.
pathway is the YAP/TAZ (YAP, Yes-Activated Protein; TAZ, transcriptional co-activator with PDZ binding motif) and its transcriptional factor partner TEAD (TEAD domain transcriptional factor). The activity of YAP/TAZ complex is regulated by the upstream kinase cascade composed of Mst1/2, Lats1/2 (large tumor suppressor kinase 1/2), and the adaptor proteins WW45 (Salvador family WW domain containing protein 1) and Mob1 (MOB kinase activator 1A). Phosphorylation of YAP by Lats1/2 leads to the degradation of YAP through ubiquitin pathway [13]. Recent studies have implicated the regulation of YAP/TEAD signaling by the F-actin. Although the F-actin cross-linking activity of Fascin1 has been recognized, the roles of Fascin1 in the regulation of YAP/TEAD signaling remain unknown. In this study, we examined the expression of Fascin1 in non-small cell lung cancer and its roles in the regulation of YAP/TEAD signaling.
qPCR analysis NSCLC tissues and matched non-cancerous tissues were treated with TRIzol reagent (Invitrogen) for the total RNA extraction. The isolated RNA was used to complementary DNA (cDNA) synthesis using the reverse transcription kit (Promega, Madison, WI), following the manufacturer’s instructions. Real-time PCR was performed using a Stratagene MAXP3000 PCR system and Brilliant Q-PCR Master Mixture. The primer pair used for amplification of the human Fascin 1 gene was as follows: forward primer, 5′AAGAAGAAGCAGATCTGGAC-3′, and reverse primer, 5′-CACGATGAGGAAACGGCAGT-3′. As an internal standard, a fragment of human 18S was amplified by PCR using the following primers: forward primer, 5′-TCAACT TTCG ATGGTAGTCG-3′, and reverse primer, 5′-TCCTCGTT AAAGGATTTAAA-3′. We calculated a Ct (reference-target). Ct(18S-Fascin) = Ct(18S)-Ct(Fascin). Western blot assay
Materials and methods Cell lines and cell culture Two lung cancer cell lines (A549 and SPC-A-1) were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI1640 medium (Invitrogen) supplemented with 10 % fetal bovine serum (FBS) in a normal conditions according to the protocols of ATCC.
Tissue samples A total of 31 non-small cell lung cancer (NSCLC) tissues and paired non-cancerous tissues were collected from patients who received surgery for lung cancer. This study was performed with the informed consent from all of the patients. None of the patients have received chemotherapy before surgery. NSCLC tissues and paired non-cancerous tissues were stored at −80 °C until processed.
Cells were lysed, and the protein concentration was measured by a BCA kit (Thermo). The proteins were separated by 10 % SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA), and probed with specific antibodies. The immunoreactive protein bands were visualized by ECL kit (Pierce). Antibody to Fascin 1 was purchased from Abcam (EP5902); antibodies to c-Myc (9402), c-Jun (9165), CyclinD1 (2978), GSK3beta (12456), and phosphorylated GSK3beta (9323) were purchased from Cell Signaling Technology; and antibody to GAPDH (sc365062) was purchased from Santa Cruz Biotechnology. RNA interference of Fascin 1 in NSCLC cells Quantitative PCR (RNAi) lenti-virus particles (shcon and shFascin 1) were purchased from GeneChem (China). A549 and SPC-A-1 cells were incubated with the lentivirus particles for 24 h and then sorted with GFP using cytometry.
Plasmid construction and transfection
Immunohistochemistry
For the construction of Fascin 1 expression vector, the coding sequence of Fascin 1 was amplified by PCR and inserted into the empty vector pcDNA 3.1-myc. The Fascin 1 expression vector and empty pcDNA3.1 plasmid were transfected into A549 and SPC-A-1 cells using Lipofectamine 2000 reagent (Invitrogen). After the selection with G418 for 2 weeks, the resistant cells were pooled and further confirmed the expression of exogenous Fascin 1 using anti-myc antibody.
Clinical NSCLC tissues and paired non-cancerous tissues were fixed in formalin, embedded in paraffin, and cut as 5-μm-thick consecutive sections. After deparaffin and antigen recovery (in sodium citrate solution, pH 6.0, 20 min, 98 °C), the sections were washed thrice in 0.01 mol/l PBS (8 mmol/l Na2HPO4, 2 mmol/l NaH2PO4, and 150 mmol/l NaCl) for 5 min each, blocked for 1 h in 0.01 mol/l PBS containing 0.3 % Triton X-100 and 5 % BSA, followed by addition of anti-Fascin 1 (1:200) antibody at 4 °C
Tumor Biol.
Fig. 1 The mRNA level and protein level of Fascin 1 was elevated in NSCLC tissues. a The mRNA levels of Fascin 1 in 31 NSCLC samples and 31 adjacent non-cancerous tissues were examined using real-time PCR. The Fascin 1 mRNA level was normalized to that of 18S. Data was calculated from triplicates. b The protein level of Fascin 1 in NSCLC
samples and paired adjacent normal tissues was examined by immunohistochemistry staining. c Western blotting analysis to examine the protein level of Fascin 1 in five pairs of NSCLC samples (C) and adjacent normal tissues (N). **P < 0.01
overnight. After brief washes with 0.01 mol/l PBS, sections were incubated with 0.01 mol/l PBS containing horseradish peroxidase-conjugated rabbit anti-rabbit IgG (1:500) for 2 h, followed by development with 0.003 % H2O2 and 0.03 % 3,30-diaminobenzidine in 0.05 mol/l Tris-HCl (pH 7.6). Immunohistochemistry for each sample was repeated three times.
Cell growth assay
Fig. 2 Forced expression of Fascin 1 promoted the growth and migration of A549 and SPC-A-1 cells. a Forced expression of Fascin 1 in A549 and SPC-A-1 cells. pcDNA3.1 vector or the Fascin 1 expression vector was transfected into A549 and SPC-A-1 cells. The transfected cells were selected with G418. G418-resistant cells were pooled and confirmed the
overexpression of Fascin 1 by Western blot analysis. b Overexpression of Fascin promoted the colony formation of A549 and SPC-A-1 cells, which was confirmed in the crystal violet assay. c Forced expression of Fascin 1 promoted the migration of A549 and SPC-A-1 cells in the migration assay using a Boyden chamber. *P < 0.05; **P < 0.01
Colony formation assay was performed to examine the effects of Fascin 1 on the growth of NSCLC cells. Equal number of control cells and experimental cells were seeded in 12-well plates and cultured in medium supplemented with 10 % FBS at a density of 1000 cells/well.
Tumor Biol.
Fig. 3 Downregulating the expression of Fascin 1 inhibited the growth and migration of A549 and SPC-A-1 cells. a Knocking down the expression of Fascin 1 in A549 and SPC-A-1 cells by RNA interference (RNAi). b Knocking down the expression of Fascin 1
inhibited the colony formation of A549 and SPC-A-1 cells. c Knocking down the expression of Fascin 1 inhibited the migration of A549 and SPC-A-1 cells. *P < 0.05; **P < 0.01
Medium was changed every other day. After 10 days of culture under the standard condition, the medium was removed and the cells were stained with 0.5 % crystal violet solution in 20 % methanol. After staining for 10 min, the fixed cells were washed with phosphate-buffered saline (PBS) and photographed. Cells were dissolved with 1 % SDS, and OD 600 nm was measured.
Immunoprecipitation assay
Cell migration assay Cell migration assay was performed using a modified Boyden chamber. Cells (2 × 105) suspended in 0.05 ml medium containing 1 % FBS were placed in the upper chamber, and the lower chamber was loaded with 0.152 ml medium containing 10 % FBS. Six hours later, cells that migrated to the lower surface of filters was detected with traditional H&E staining. The experiments were repeated three times.
Cells were washed with ice-cold PBS and lysated in Trisbuffered saline (pH 7.4), containing 50 mM Tris, 150 mM NaCl, 1 % NP-40, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 2.5 mg/ml aprotinin and leupeptin, 1 mM betaglycerophosphate and AEBSF (4-(2-aminoethyl ) benzenesulfonyl fluoride hydrochloride), and 10 mM iodoacetate. Lysates were incubated on ice for 15 min before cellular debris and nuclei were removed by centrifugation at 10000g for 20 min. Cell lysates were incubated with the corresponding primary antibody overnight at 4 °C. Protein ASepharose (Amersham Biosciences, Piscataway, NJ, USA) beads in a 50:50 mixture in 50 mM Tris buffer, pH 7.0, were added, and further incubated for another 4 h at 4 °C. The immunoprecipitates were washed four times in Tris-buffered saline and boiled for 5 min in 40 μl Laemmli buffer containing 0.02 % blue bromophenol and 2 % bmercaptoethanol.
GST pull-down assay A549 cells were lysed, and the cell lysates were incubated with 10 μg GST-Fascin 1 protein of GST protein overnight. Then, Sepharose 4B was added to the cell lysates and incubated for another 4 h. After centrifugation, the beads were collected and washed with the lysis buffer for three times. Beads were boiled for 5 min in 40 μl Laemmli buffer containing 0.02 % blue bromophenol and 2 % bmercaptoethanol. The proteins pulled down were examined by Western blot.
Reporter assay Cells were plated at a subconfluent density and co-transfected with 0.05 μg of the reporter plasmid, 0.5 μg of expression vectors, and 0.05 μg of Renilla luciferase pRL-TK as an internal control for transfection efficiency. Twenty-four hours later, cell lysates were prepared and the reporter activity was measured using the dual-luciferase reporter assay system
Tumor Biol.
Fig. 4 Fascin 1 activated YAP/TEAD signaling. a Fascin 1 activated the YAP/TEAD reporter gene in a dose dependent manner in A549 cells. b Knocking down the expression of Fascin 1 inhibited the expression of
YAP, CyclinD1, Cyclin E, and CTGF. c Knocking down the expression of YAP abolished the growth-promoting effects of A549 cells induced by Fascin 1
(Promega). Transfections were performed in triplicate and repeated three times to ensure reproducibility.
of Fascin in 31 NSCLC tissues and paired noncancerous tissues was first examined using quantitative PCR (qPCR). Fascin1 mRNA level was significantly increased in NSCLC tissues compared to that in noncancerous tissues (Fig. 1a). Moreover, immunohistochemistry staining and Western blot analysis showed increased protein level of Fascin in NSCLC tissues (Fig. 1b–c). Taken together, these findings demonstrated that the expression of Fascin 1 was upregulated in the progression of NSCLC.
Result Fascin 1 expression is upregulated in NSCLC tissues Fascin 1 has been reported to be upregulated in various tumor tissues. Here, the messenger RNA (mRNA) level
Tumor Biol.
Fascin 1 promoted the growth and migration of NSCLC cells To explore the biological function of Fascin1 in the progression of NSCLC, A549 and SPC-A-1 cells were transfected with myc-taged Fascine1 plasmid, and the overexpression of exogenous Fascin1 (myc-Fascin 1) was confirmed by the Western blot analysis (Fig. 2a). The effects of Fascin1 on the growth and migration of the A549 and SPC-A-1 cells were examined by crystal violet staining and modified Boyden chamber. Overexpression of Fascin 1 remarkably promoted the growth and migration of A549 and SPC-A-1 cells (Fig. 2b–c). To further study the functions of endogenously expressed Fascin 1, two independent small interfering RNA (siRNA) were used to knock down the expression of Fascin 1 in A549 and SPC-A-1 cells (Fig. 3a). Consistent with the observations shown in Fig. 2b–c, knocking down the expression of Fascin 1 inhibited the growth and migration of A549 and SPC-A-1 cells (Fig. 3b–c). These results demonstrated that Fascin 1 played an oncogenic role in the progression of NSCLC by promoting cell growth and migration.
expression of Fascin 1 downregulated the expression of YAP and several downstream target genes, such as Cyclin D1, Cyclin E, and CTGF at both mRNA level and protein level (Fig. 4b), indicating Fascin 1 activated YAP/TEAD signaling in NSCLC cells. Moreover, knocking down the expression of YAP abolished the promoting effects of Fascin on the growth of A549 cells (Fig. 4c). Collectively, these data suggested that Fascin might promote the growth and migration of NSCLC cells by activating YAP/TEAD signaling. Fascin 1 interacted with MST1 Next, the detail mechanism through which Fascin 1 regulated YAP/TEAD signaling was studied. The interactions between Fascin 1 and several components upstream of YAP were examined. It was found that GST-Fascin fusion protein interacted with MST1 (Fig. 5a). In addition, exogenously expressed Fascin and MST1 were found in the same immunoprecipitation complex (Fig. 5b). Moreover, endogenously expressed Fascin and MST1 was found to interact with each other in A549 cells (Fig. 5c). In summary, these results demonstrated the interaction between MST1 and Fascin 1.
Fascin 1 increased the transcriptional activity of YAP To explore the underlying molecular mechanisms through which Fascin1 promoted the growth and migration of the NSCLC cells, a preliminary screening was performed using reporter gene assay. Interestingly, overexpression of Fascin 1 activated the transcriptional activity of YAP/TEAD in a dosedependent manner, while knocking down the expression of Fascin1 inhibited the transcriptional activity of YAP/TEAD in A549 cells (Fig. 4a). Moreover, knocking down the
Fig. 5 Fascin 1 interacted with MST1. a GST-Fascin 1 fusion protein interacted with MST1 in A549 cells. GST-Fascin 1 fusion protein was incubated with the A549 cell lysates. The interaction was examined by Western blot analysis. b Exogenously expressed Fascin 1 interacted with MST1 in A549 cells. A549 cells were stably transfected with myc-Fascin 1 plasmid, and then perfomed immunoprecipitation using anti-myc antibody. The interaction was examined by Western blot analysis. c Endogenously expressed Fascin 1 interacted with MST1 in A549 cells
Discussion The present study has revealed a novel role of Fascin 1 in the regulation of YAP/TEAD signaling. Although the expression of Fascin 1 has been observed in various cancer types [14, 15], the regulation of YAP/TEAD signaling by Fascin has not been reported. Here, it was found that Fascin 1 was overexpressed in non-small cell lung cancer (NSCLC). Forced expression of Fascin 1 promoted the growth and migration of NSCLC cells, while knocking down the expression of Fascin 1inhibited the growth and migration of cancer cells. The mechanism study showed that Fascin 1 activated the transcriptional activity of YAP/TEAD complex possibly by binding with the kinase MST1. This study provided another explanation for the oncogenic activity of Fascin1. The regulation of YAP/TEAD signaling by Fascin 1 might attribute to its F-actin cross-linking activity. Several regulators of F-actin have been reported to regulate YAP/TEAD signaling [16, 17]. Deletion of actin capping protein led to the actin polymerization from globular (G) actin to filamentous (F) form and the disruption of YAP/TEAD signaling [17]. In mammalian cells, mechanical cues led to the changes in actin cytoskeleton, and thus regulated YAP/TEAD signaling [18–20]. These studies emphasized the pivotal role of Factin in YAP/TEAD signaling. Therefore, Fascin 1 might regulate YAP/TEAD signaling through changing the F-actin level. Recently, much attention has been focused on the potential application of Fascin 1 as the cancer therapeutic target [21].
Tumor Biol.
Migrastain, a small molecule inhibitor of actin bundling by Fascin1, has shown anti-tumor effects on several types of cancer cells [22]. Based on the present study, migrastain might be a potential antagonist against YAP/TEAD signaling. Therefore, our data provided a possible explanation for the anti-tumor activity of migrastain. Although our results are promising, further study using Fascin1 knocking out mice will provide novel insights into its function in tumorigenesis and regulation of YAP/TEAD signaling.
9.
10.
11.
12.
Compliance with ethical standards 13. Conflicts of interest None 14.
15.
References 1.
Adams JC. Roles of fascin in cell adhesion and motility. Curr Opin Cell Biol. 2004;16:590–6. doi:10.1016/j.ceb.2004.07.009. 2. Elkhatib N, Neu MB, Zensen C, et al. Fascin plays a role in stress fiber organization and focal adhesion disassembly. Curr Biol. 2014;24:1492–9. doi:10.1016/j.cub.2014.05.023. 3. Yamashiro S, Yamakita Y, Ono S, Matsumura F. Fascin, an actinbundling protein, induces membrane protrusions and increases cell motility of epithelial cells. Mol Biol Cell. 1998;9:993–1006. 4. Okenve-Ramos P, Llimargas M. Fascin, may the Forked be with you. Fly (Austin). 2014;8:157–64. doi:10.4161/fly.34368. 5. Hashimoto Y, Kim DJ, Adams JC. The roles of fascins in health and disease. J Pathol. 2011;224:289–300. doi:10.1002/path.2894. 6. Kureishy N, Sapountzi V, Prag S, Anilkumar N, Adams JC. Fascins, and their roles in cell structure and function. Bioessays. 2002;24:350–61. doi:10.1002/bies.10070. 7. Bompard G, Sharp SJ, Freiss G, Machesky LM. Involvement of Rac in actin cytoskeleton rearrangements induced by MIM-B. J Cell Sci. 2005;118:5393–403. doi:10.1242/jcs.02640. 8. Omran OM, Al Sheeha M. Cytoskeletal focal adhesion proteins fascin-1 and paxillin are predictors of malignant progression and poor prognosis in human breast cancer. J Environ Pathol Toxicol Oncol. 2015;34:201–12.
16.
17.
18.
19.
20.
21.
22.
Pelosi G, Pastorino U, Pasini F, et al. Independent prognostic value of fascin immunoreactivity in stage I nonsmall cell lung cancer. Br J Cancer. 2003;88:537–47. doi:10.1038/sj.bjc.6600731. Roh YH, Kim YH, Choi HJ, Lee KE, Roh MS. Fascin overexpression correlates with positive thrombospondin-1 and syndecan-1 expressions and a more aggressive clinical course in patients with gallbladder cancer. J Hepatobiliary Pancreat Surg. 2009;16:315– 21. doi:10.1007/s00534-009-0046-1. Fujii M, Toyoda T, Nakanishi H, et al. TGF-beta synergizes with defects in the Hippo pathway to stimulate human malignant mesothelioma growth. J Exp Med. 2012;209:479–94. doi:10.1084/jem. 20111653. Zhao B, Ye X, Yu J, et al. TEAD mediates YAP-dependent gene induction and growth control. Genes Dev. 2008;22:1962–71. doi: 10.1101/gad.1664408. Badouel C, McNeill H. SnapShot: the hippo signaling pathway. Cell. 2011;145:484-484 e1. doi:10.1016/j.cell.2011.04.009. Kim SJ, Kim DC, Kim MC, et al. Fascin expression is related to poor survival in gastric cancer. Pathol Int. 2012;62:777–84. doi:10. 1111/pin.12012. Oh SY, Kim YB, Suh KW, Paek OJ, Moon HY. Prognostic impact of Fascin-1 expression is more significant in advanced colorectal cancer. J Surg Res. 2012;172:102–8. doi:10.1016/j.jss.2010.07. 015. Mana-Capelli S, Paramasivam M, Dutta S, McCollum D. Angiomotins link F-actin architecture to Hippo pathway signaling. Mol Biol Cell. 2014;25:1676–85. doi:10.1091/mbc.E13-11-0701. Fernandez BG, Gaspar P, Bras-Pereira C, Jezowska B, Rebelo SR, Janody F. Actin-capping protein and the Hippo pathway regulate Factin and tissue growth in Drosophila. Development. 2011;138: 2337–46. doi:10.1242/dev.063545. Wang J, Zhang Y, Zhang N, Wang C, Herrler T, Li Q. An updated review of mechanotransduction in skin disorders: transcriptional regulators, ion channels, and microRNAs. Cell Mol Life Sci. 2015;72:2091–106. doi:10.1007/s00018-015-1853-y. Halder G, Dupont S, Piccolo S. Transduction of mechanical and cytoskeletal cues by YAP and TAZ. Nat Rev Mol Cell Biol. 2012;13:591–600. doi:10.1038/nrm3416. Dupont S, Morsut L, Aragona M, et al. Role of YAP/TAZ in mechanotransduction. Nature. 2011;474:179–83. doi:10.1038/ nature10137. Adams JC. Fascin-1 as a biomarker and prospective therapeutic target in colorectal cancer. Expert Rev Mol Diagn. 2015;15:41–8. doi:10.1586/14737159.2015.976557. Nakae K, Nishimura Y, Ohba S, Akamatsu Y. Migrastatin acts as a muscarinic acetylcholine receptor antagonist. J Antibiot (Tokyo). 2006;59:685–92. doi:10.1038/ja.2006.91.
ORIGINAL ARTICLE
Fascin 1 promoted the growth and migration of non-small cell lung cancer cells by activating YAP/TEAD signaling Zhigang Liang 1,2 & Ying Wang 1 & Zhenya Shen 1 & Xiaomei Teng 1 & Xinjian Li 2 & Chenwei Li 2 & Weijie Wu 2 & Zenghui Zhou 2 & Zishan Wang 2
Received: 29 November 2015 / Accepted: 28 January 2016 # International Society of Oncology and BioMarkers (ISOBM) 2016
Abstract Fascin 1 (Fascin actin-bundling protein 1) is an actin-binding protein. Although several studies have reported the dysregulation of Fascin 1 in non-small cell lung cancer (NSCLC), its functions in the progression of NSCLC and the related molecular mechanism were not fully understood. In this study, the expression of Fascin 1 in NSCLC tissues was determined using quantitative PCR (qPCR), and the roles of Fascin 1 in the progression of NSCLC were investigated. It was found that both the messenger RNA (mRNA) level and the protein level of Fascin 1 were upregulated in NSCLC tissues. Forced expression of Fascin 1 promoted the growth and migration of NSCLC cells, while knocking down the expression of Fascin 1 inhibited the growth, migration, and tumorigenesis of NSCLC cells. Mechanism studies showed that Fascin 1 increased the transcriptional activity of the YAP/TEAD (Yes-associated protein/TEA domain transcriptional factor) complex, and knocking down the expression of Fascin 1 attenuated the expression of target genes downstream the YAP/TEAD complex. In addition, MST1 interacted with Fascin 1. Taken together, Fascin 1 plays an oncogenic role in NSCLC by activating the transcriptional activity of the YAP/ TEAD complex.
Zhigang Liang and Ying Wang contributed equally to this work. * Zhenya Shen [email protected]
1
Department of Cardiovascular Surgery of the First Affiliated Hospital & Institute for Cardiovascular Science, Soochow University, Suzhou, Jiangsu, China
2
Department of Thoracic Surgery, Ningbo First Hospital, Ningbo, Zhejiang, China
Keywords NSCLC . Fascin 1 . YAP/TEAD signaling . Cell growth . Migration
Introduction Fascins are actin-binding proteins, which cross-link the filamentous actin to the tightly packed bundles [1]. These actin bundles are involved in the formation of stress fibers, protrusions, and other sub-cellular structures [2, 3]. The Fascins family includes three conserved members, Fascin1, Fascin2, and Fascin3 [4]. Fascin1 and Fascin2 share about 60 % homolog [5], while Fascin1 and Fascin3 only share about 25 % homolog [5]. The F-actin cross-linking activity of Fascin1 in mammalian cells contributes to the formation of actin-based structure [6]. Dynamic alternations of these actin-based structures assist the migration and metastasis of cells [1, 7], suggesting the important roles of Fascin1 in regulating cell motility. Upregulation of Fascin1 has been observed in most of the aggressive human carcinomas [8–10]. The clinical data revealed that the expression of Fascin1 has been reported to correlate with the poor prognosis [8–10]. The functional studies of Fascin1 in cancer cell lines showed that knocking down the expression of Fascin1 inhibited cell migration and metastasis, while overexpression of Fascin1 promoted the motility of the cancer cells [5]. These finding indicated the oncogenic roles of Fascin1 in the tumorigenesis. The Hippo signaling controls the sizes of the organs in the development by regulating a panel of proliferationrelated and apoptosis-related genes expression, such as connective tissue growth factor (CTGF), Cyclin D1, Survivin, and so on [11, 12]. The core of this signaling
Tumor Biol.
pathway is the YAP/TAZ (YAP, Yes-Activated Protein; TAZ, transcriptional co-activator with PDZ binding motif) and its transcriptional factor partner TEAD (TEAD domain transcriptional factor). The activity of YAP/TAZ complex is regulated by the upstream kinase cascade composed of Mst1/2, Lats1/2 (large tumor suppressor kinase 1/2), and the adaptor proteins WW45 (Salvador family WW domain containing protein 1) and Mob1 (MOB kinase activator 1A). Phosphorylation of YAP by Lats1/2 leads to the degradation of YAP through ubiquitin pathway [13]. Recent studies have implicated the regulation of YAP/TEAD signaling by the F-actin. Although the F-actin cross-linking activity of Fascin1 has been recognized, the roles of Fascin1 in the regulation of YAP/TEAD signaling remain unknown. In this study, we examined the expression of Fascin1 in non-small cell lung cancer and its roles in the regulation of YAP/TEAD signaling.
qPCR analysis NSCLC tissues and matched non-cancerous tissues were treated with TRIzol reagent (Invitrogen) for the total RNA extraction. The isolated RNA was used to complementary DNA (cDNA) synthesis using the reverse transcription kit (Promega, Madison, WI), following the manufacturer’s instructions. Real-time PCR was performed using a Stratagene MAXP3000 PCR system and Brilliant Q-PCR Master Mixture. The primer pair used for amplification of the human Fascin 1 gene was as follows: forward primer, 5′AAGAAGAAGCAGATCTGGAC-3′, and reverse primer, 5′-CACGATGAGGAAACGGCAGT-3′. As an internal standard, a fragment of human 18S was amplified by PCR using the following primers: forward primer, 5′-TCAACT TTCG ATGGTAGTCG-3′, and reverse primer, 5′-TCCTCGTT AAAGGATTTAAA-3′. We calculated a Ct (reference-target). Ct(18S-Fascin) = Ct(18S)-Ct(Fascin). Western blot assay
Materials and methods Cell lines and cell culture Two lung cancer cell lines (A549 and SPC-A-1) were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI1640 medium (Invitrogen) supplemented with 10 % fetal bovine serum (FBS) in a normal conditions according to the protocols of ATCC.
Tissue samples A total of 31 non-small cell lung cancer (NSCLC) tissues and paired non-cancerous tissues were collected from patients who received surgery for lung cancer. This study was performed with the informed consent from all of the patients. None of the patients have received chemotherapy before surgery. NSCLC tissues and paired non-cancerous tissues were stored at −80 °C until processed.
Cells were lysed, and the protein concentration was measured by a BCA kit (Thermo). The proteins were separated by 10 % SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA), and probed with specific antibodies. The immunoreactive protein bands were visualized by ECL kit (Pierce). Antibody to Fascin 1 was purchased from Abcam (EP5902); antibodies to c-Myc (9402), c-Jun (9165), CyclinD1 (2978), GSK3beta (12456), and phosphorylated GSK3beta (9323) were purchased from Cell Signaling Technology; and antibody to GAPDH (sc365062) was purchased from Santa Cruz Biotechnology. RNA interference of Fascin 1 in NSCLC cells Quantitative PCR (RNAi) lenti-virus particles (shcon and shFascin 1) were purchased from GeneChem (China). A549 and SPC-A-1 cells were incubated with the lentivirus particles for 24 h and then sorted with GFP using cytometry.
Plasmid construction and transfection
Immunohistochemistry
For the construction of Fascin 1 expression vector, the coding sequence of Fascin 1 was amplified by PCR and inserted into the empty vector pcDNA 3.1-myc. The Fascin 1 expression vector and empty pcDNA3.1 plasmid were transfected into A549 and SPC-A-1 cells using Lipofectamine 2000 reagent (Invitrogen). After the selection with G418 for 2 weeks, the resistant cells were pooled and further confirmed the expression of exogenous Fascin 1 using anti-myc antibody.
Clinical NSCLC tissues and paired non-cancerous tissues were fixed in formalin, embedded in paraffin, and cut as 5-μm-thick consecutive sections. After deparaffin and antigen recovery (in sodium citrate solution, pH 6.0, 20 min, 98 °C), the sections were washed thrice in 0.01 mol/l PBS (8 mmol/l Na2HPO4, 2 mmol/l NaH2PO4, and 150 mmol/l NaCl) for 5 min each, blocked for 1 h in 0.01 mol/l PBS containing 0.3 % Triton X-100 and 5 % BSA, followed by addition of anti-Fascin 1 (1:200) antibody at 4 °C
Tumor Biol.
Fig. 1 The mRNA level and protein level of Fascin 1 was elevated in NSCLC tissues. a The mRNA levels of Fascin 1 in 31 NSCLC samples and 31 adjacent non-cancerous tissues were examined using real-time PCR. The Fascin 1 mRNA level was normalized to that of 18S. Data was calculated from triplicates. b The protein level of Fascin 1 in NSCLC
samples and paired adjacent normal tissues was examined by immunohistochemistry staining. c Western blotting analysis to examine the protein level of Fascin 1 in five pairs of NSCLC samples (C) and adjacent normal tissues (N). **P < 0.01
overnight. After brief washes with 0.01 mol/l PBS, sections were incubated with 0.01 mol/l PBS containing horseradish peroxidase-conjugated rabbit anti-rabbit IgG (1:500) for 2 h, followed by development with 0.003 % H2O2 and 0.03 % 3,30-diaminobenzidine in 0.05 mol/l Tris-HCl (pH 7.6). Immunohistochemistry for each sample was repeated three times.
Cell growth assay
Fig. 2 Forced expression of Fascin 1 promoted the growth and migration of A549 and SPC-A-1 cells. a Forced expression of Fascin 1 in A549 and SPC-A-1 cells. pcDNA3.1 vector or the Fascin 1 expression vector was transfected into A549 and SPC-A-1 cells. The transfected cells were selected with G418. G418-resistant cells were pooled and confirmed the
overexpression of Fascin 1 by Western blot analysis. b Overexpression of Fascin promoted the colony formation of A549 and SPC-A-1 cells, which was confirmed in the crystal violet assay. c Forced expression of Fascin 1 promoted the migration of A549 and SPC-A-1 cells in the migration assay using a Boyden chamber. *P < 0.05; **P < 0.01
Colony formation assay was performed to examine the effects of Fascin 1 on the growth of NSCLC cells. Equal number of control cells and experimental cells were seeded in 12-well plates and cultured in medium supplemented with 10 % FBS at a density of 1000 cells/well.
Tumor Biol.
Fig. 3 Downregulating the expression of Fascin 1 inhibited the growth and migration of A549 and SPC-A-1 cells. a Knocking down the expression of Fascin 1 in A549 and SPC-A-1 cells by RNA interference (RNAi). b Knocking down the expression of Fascin 1
inhibited the colony formation of A549 and SPC-A-1 cells. c Knocking down the expression of Fascin 1 inhibited the migration of A549 and SPC-A-1 cells. *P < 0.05; **P < 0.01
Medium was changed every other day. After 10 days of culture under the standard condition, the medium was removed and the cells were stained with 0.5 % crystal violet solution in 20 % methanol. After staining for 10 min, the fixed cells were washed with phosphate-buffered saline (PBS) and photographed. Cells were dissolved with 1 % SDS, and OD 600 nm was measured.
Immunoprecipitation assay
Cell migration assay Cell migration assay was performed using a modified Boyden chamber. Cells (2 × 105) suspended in 0.05 ml medium containing 1 % FBS were placed in the upper chamber, and the lower chamber was loaded with 0.152 ml medium containing 10 % FBS. Six hours later, cells that migrated to the lower surface of filters was detected with traditional H&E staining. The experiments were repeated three times.
Cells were washed with ice-cold PBS and lysated in Trisbuffered saline (pH 7.4), containing 50 mM Tris, 150 mM NaCl, 1 % NP-40, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 2.5 mg/ml aprotinin and leupeptin, 1 mM betaglycerophosphate and AEBSF (4-(2-aminoethyl ) benzenesulfonyl fluoride hydrochloride), and 10 mM iodoacetate. Lysates were incubated on ice for 15 min before cellular debris and nuclei were removed by centrifugation at 10000g for 20 min. Cell lysates were incubated with the corresponding primary antibody overnight at 4 °C. Protein ASepharose (Amersham Biosciences, Piscataway, NJ, USA) beads in a 50:50 mixture in 50 mM Tris buffer, pH 7.0, were added, and further incubated for another 4 h at 4 °C. The immunoprecipitates were washed four times in Tris-buffered saline and boiled for 5 min in 40 μl Laemmli buffer containing 0.02 % blue bromophenol and 2 % bmercaptoethanol.
GST pull-down assay A549 cells were lysed, and the cell lysates were incubated with 10 μg GST-Fascin 1 protein of GST protein overnight. Then, Sepharose 4B was added to the cell lysates and incubated for another 4 h. After centrifugation, the beads were collected and washed with the lysis buffer for three times. Beads were boiled for 5 min in 40 μl Laemmli buffer containing 0.02 % blue bromophenol and 2 % bmercaptoethanol. The proteins pulled down were examined by Western blot.
Reporter assay Cells were plated at a subconfluent density and co-transfected with 0.05 μg of the reporter plasmid, 0.5 μg of expression vectors, and 0.05 μg of Renilla luciferase pRL-TK as an internal control for transfection efficiency. Twenty-four hours later, cell lysates were prepared and the reporter activity was measured using the dual-luciferase reporter assay system
Tumor Biol.
Fig. 4 Fascin 1 activated YAP/TEAD signaling. a Fascin 1 activated the YAP/TEAD reporter gene in a dose dependent manner in A549 cells. b Knocking down the expression of Fascin 1 inhibited the expression of
YAP, CyclinD1, Cyclin E, and CTGF. c Knocking down the expression of YAP abolished the growth-promoting effects of A549 cells induced by Fascin 1
(Promega). Transfections were performed in triplicate and repeated three times to ensure reproducibility.
of Fascin in 31 NSCLC tissues and paired noncancerous tissues was first examined using quantitative PCR (qPCR). Fascin1 mRNA level was significantly increased in NSCLC tissues compared to that in noncancerous tissues (Fig. 1a). Moreover, immunohistochemistry staining and Western blot analysis showed increased protein level of Fascin in NSCLC tissues (Fig. 1b–c). Taken together, these findings demonstrated that the expression of Fascin 1 was upregulated in the progression of NSCLC.
Result Fascin 1 expression is upregulated in NSCLC tissues Fascin 1 has been reported to be upregulated in various tumor tissues. Here, the messenger RNA (mRNA) level
Tumor Biol.
Fascin 1 promoted the growth and migration of NSCLC cells To explore the biological function of Fascin1 in the progression of NSCLC, A549 and SPC-A-1 cells were transfected with myc-taged Fascine1 plasmid, and the overexpression of exogenous Fascin1 (myc-Fascin 1) was confirmed by the Western blot analysis (Fig. 2a). The effects of Fascin1 on the growth and migration of the A549 and SPC-A-1 cells were examined by crystal violet staining and modified Boyden chamber. Overexpression of Fascin 1 remarkably promoted the growth and migration of A549 and SPC-A-1 cells (Fig. 2b–c). To further study the functions of endogenously expressed Fascin 1, two independent small interfering RNA (siRNA) were used to knock down the expression of Fascin 1 in A549 and SPC-A-1 cells (Fig. 3a). Consistent with the observations shown in Fig. 2b–c, knocking down the expression of Fascin 1 inhibited the growth and migration of A549 and SPC-A-1 cells (Fig. 3b–c). These results demonstrated that Fascin 1 played an oncogenic role in the progression of NSCLC by promoting cell growth and migration.
expression of Fascin 1 downregulated the expression of YAP and several downstream target genes, such as Cyclin D1, Cyclin E, and CTGF at both mRNA level and protein level (Fig. 4b), indicating Fascin 1 activated YAP/TEAD signaling in NSCLC cells. Moreover, knocking down the expression of YAP abolished the promoting effects of Fascin on the growth of A549 cells (Fig. 4c). Collectively, these data suggested that Fascin might promote the growth and migration of NSCLC cells by activating YAP/TEAD signaling. Fascin 1 interacted with MST1 Next, the detail mechanism through which Fascin 1 regulated YAP/TEAD signaling was studied. The interactions between Fascin 1 and several components upstream of YAP were examined. It was found that GST-Fascin fusion protein interacted with MST1 (Fig. 5a). In addition, exogenously expressed Fascin and MST1 were found in the same immunoprecipitation complex (Fig. 5b). Moreover, endogenously expressed Fascin and MST1 was found to interact with each other in A549 cells (Fig. 5c). In summary, these results demonstrated the interaction between MST1 and Fascin 1.
Fascin 1 increased the transcriptional activity of YAP To explore the underlying molecular mechanisms through which Fascin1 promoted the growth and migration of the NSCLC cells, a preliminary screening was performed using reporter gene assay. Interestingly, overexpression of Fascin 1 activated the transcriptional activity of YAP/TEAD in a dosedependent manner, while knocking down the expression of Fascin1 inhibited the transcriptional activity of YAP/TEAD in A549 cells (Fig. 4a). Moreover, knocking down the
Fig. 5 Fascin 1 interacted with MST1. a GST-Fascin 1 fusion protein interacted with MST1 in A549 cells. GST-Fascin 1 fusion protein was incubated with the A549 cell lysates. The interaction was examined by Western blot analysis. b Exogenously expressed Fascin 1 interacted with MST1 in A549 cells. A549 cells were stably transfected with myc-Fascin 1 plasmid, and then perfomed immunoprecipitation using anti-myc antibody. The interaction was examined by Western blot analysis. c Endogenously expressed Fascin 1 interacted with MST1 in A549 cells
Discussion The present study has revealed a novel role of Fascin 1 in the regulation of YAP/TEAD signaling. Although the expression of Fascin 1 has been observed in various cancer types [14, 15], the regulation of YAP/TEAD signaling by Fascin has not been reported. Here, it was found that Fascin 1 was overexpressed in non-small cell lung cancer (NSCLC). Forced expression of Fascin 1 promoted the growth and migration of NSCLC cells, while knocking down the expression of Fascin 1inhibited the growth and migration of cancer cells. The mechanism study showed that Fascin 1 activated the transcriptional activity of YAP/TEAD complex possibly by binding with the kinase MST1. This study provided another explanation for the oncogenic activity of Fascin1. The regulation of YAP/TEAD signaling by Fascin 1 might attribute to its F-actin cross-linking activity. Several regulators of F-actin have been reported to regulate YAP/TEAD signaling [16, 17]. Deletion of actin capping protein led to the actin polymerization from globular (G) actin to filamentous (F) form and the disruption of YAP/TEAD signaling [17]. In mammalian cells, mechanical cues led to the changes in actin cytoskeleton, and thus regulated YAP/TEAD signaling [18–20]. These studies emphasized the pivotal role of Factin in YAP/TEAD signaling. Therefore, Fascin 1 might regulate YAP/TEAD signaling through changing the F-actin level. Recently, much attention has been focused on the potential application of Fascin 1 as the cancer therapeutic target [21].
Tumor Biol.
Migrastain, a small molecule inhibitor of actin bundling by Fascin1, has shown anti-tumor effects on several types of cancer cells [22]. Based on the present study, migrastain might be a potential antagonist against YAP/TEAD signaling. Therefore, our data provided a possible explanation for the anti-tumor activity of migrastain. Although our results are promising, further study using Fascin1 knocking out mice will provide novel insights into its function in tumorigenesis and regulation of YAP/TEAD signaling.
9.
10.
11.
12.
Compliance with ethical standards 13. Conflicts of interest None 14.
15.
References 1.
Adams JC. Roles of fascin in cell adhesion and motility. Curr Opin Cell Biol. 2004;16:590–6. doi:10.1016/j.ceb.2004.07.009. 2. Elkhatib N, Neu MB, Zensen C, et al. Fascin plays a role in stress fiber organization and focal adhesion disassembly. Curr Biol. 2014;24:1492–9. doi:10.1016/j.cub.2014.05.023. 3. Yamashiro S, Yamakita Y, Ono S, Matsumura F. Fascin, an actinbundling protein, induces membrane protrusions and increases cell motility of epithelial cells. Mol Biol Cell. 1998;9:993–1006. 4. Okenve-Ramos P, Llimargas M. Fascin, may the Forked be with you. Fly (Austin). 2014;8:157–64. doi:10.4161/fly.34368. 5. Hashimoto Y, Kim DJ, Adams JC. The roles of fascins in health and disease. J Pathol. 2011;224:289–300. doi:10.1002/path.2894. 6. Kureishy N, Sapountzi V, Prag S, Anilkumar N, Adams JC. Fascins, and their roles in cell structure and function. Bioessays. 2002;24:350–61. doi:10.1002/bies.10070. 7. Bompard G, Sharp SJ, Freiss G, Machesky LM. Involvement of Rac in actin cytoskeleton rearrangements induced by MIM-B. J Cell Sci. 2005;118:5393–403. doi:10.1242/jcs.02640. 8. Omran OM, Al Sheeha M. Cytoskeletal focal adhesion proteins fascin-1 and paxillin are predictors of malignant progression and poor prognosis in human breast cancer. J Environ Pathol Toxicol Oncol. 2015;34:201–12.
16.
17.
18.
19.
20.
21.
22.
Pelosi G, Pastorino U, Pasini F, et al. Independent prognostic value of fascin immunoreactivity in stage I nonsmall cell lung cancer. Br J Cancer. 2003;88:537–47. doi:10.1038/sj.bjc.6600731. Roh YH, Kim YH, Choi HJ, Lee KE, Roh MS. Fascin overexpression correlates with positive thrombospondin-1 and syndecan-1 expressions and a more aggressive clinical course in patients with gallbladder cancer. J Hepatobiliary Pancreat Surg. 2009;16:315– 21. doi:10.1007/s00534-009-0046-1. Fujii M, Toyoda T, Nakanishi H, et al. TGF-beta synergizes with defects in the Hippo pathway to stimulate human malignant mesothelioma growth. J Exp Med. 2012;209:479–94. doi:10.1084/jem. 20111653. Zhao B, Ye X, Yu J, et al. TEAD mediates YAP-dependent gene induction and growth control. Genes Dev. 2008;22:1962–71. doi: 10.1101/gad.1664408. Badouel C, McNeill H. SnapShot: the hippo signaling pathway. Cell. 2011;145:484-484 e1. doi:10.1016/j.cell.2011.04.009. Kim SJ, Kim DC, Kim MC, et al. Fascin expression is related to poor survival in gastric cancer. Pathol Int. 2012;62:777–84. doi:10. 1111/pin.12012. Oh SY, Kim YB, Suh KW, Paek OJ, Moon HY. Prognostic impact of Fascin-1 expression is more significant in advanced colorectal cancer. J Surg Res. 2012;172:102–8. doi:10.1016/j.jss.2010.07. 015. Mana-Capelli S, Paramasivam M, Dutta S, McCollum D. Angiomotins link F-actin architecture to Hippo pathway signaling. Mol Biol Cell. 2014;25:1676–85. doi:10.1091/mbc.E13-11-0701. Fernandez BG, Gaspar P, Bras-Pereira C, Jezowska B, Rebelo SR, Janody F. Actin-capping protein and the Hippo pathway regulate Factin and tissue growth in Drosophila. Development. 2011;138: 2337–46. doi:10.1242/dev.063545. Wang J, Zhang Y, Zhang N, Wang C, Herrler T, Li Q. An updated review of mechanotransduction in skin disorders: transcriptional regulators, ion channels, and microRNAs. Cell Mol Life Sci. 2015;72:2091–106. doi:10.1007/s00018-015-1853-y. Halder G, Dupont S, Piccolo S. Transduction of mechanical and cytoskeletal cues by YAP and TAZ. Nat Rev Mol Cell Biol. 2012;13:591–600. doi:10.1038/nrm3416. Dupont S, Morsut L, Aragona M, et al. Role of YAP/TAZ in mechanotransduction. Nature. 2011;474:179–83. doi:10.1038/ nature10137. Adams JC. Fascin-1 as a biomarker and prospective therapeutic target in colorectal cancer. Expert Rev Mol Diagn. 2015;15:41–8. doi:10.1586/14737159.2015.976557. Nakae K, Nishimura Y, Ohba S, Akamatsu Y. Migrastatin acts as a muscarinic acetylcholine receptor antagonist. J Antibiot (Tokyo). 2006;59:685–92. doi:10.1038/ja.2006.91.
