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Cancer upregulated gene (CUG)2 elevates YAP1 expression, leading to enhancement of epithelial-mesenchymal transition in human lung cancer cells
Biochemical and Biophysical Research Communications 511 (2019) 122e128
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Cancer upregulated gene (CUG)2 elevates YAP1 expression, leading to enhancement of epithelial-mesenchymal transition in human lung cancer cells Sirichat Kaowinn a, Natpaphan Yawut a, Sang Seok Koh b, Young-Hwa Chung a, * a b
BK21 Plus, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan, 46241, Republic of Korea Department of Biosciences, Dong-A University, Busan, 49315, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 January 2019 Accepted 7 February 2019 Available online 14 February 2019
Although our previous studies have showed that a novel oncogene, cancer upregulated gene (CUG)2 induced epithelial-mesenchymal transition (EMT), the detailed molecular mechanism remains unknown. Because several lines of evidence documented that Yes-Associated Protein (YAP)1 is closely associated with cancer stem cell (CSC)-like phenotypes including EMT, stemness, and drug resistance, we wondered if YAP1 is involved in CUG2-induced EMT. We herein found that the overexpression of CUG2 increased YAP1 expression at the transcriptional as well as protein levels. Chromatin immunoprecipitation assay revealed that the elevated YAP1 transcripts are attributed to c-Jun and AP2 bindings to the YAP1 promoter. Akt and MAPK kinases including ERK, JNK, and p38 MAPK enhanced the level of YAP1 protein. In spite of a close relationship between b-catenin and YAP1, not b-catenin but NEK2 played the role in increasing YAP1 expression. Silencing YAP1 inhibited CUG2-induced cell migration and invasion. Ncadherin and vimentin expressions were decreased during YAP1 knockdown. The suppression of YAP1 diminished TGF-b transcriptional activity and expression as well as phosphorylation level of Smad2 and Twist protein. Conversely, LY2109761 or Smad2 siRNA treatment reduced YAP1 protein levels, indicating a close interplay between YAP1 and TGF-b signaling. Taken together, we suggest that CUG2 induces upregulation of YAP1 expression, leading to enhancing CUG2-induced EMT via a close crosstalk between YAP1 and TGF-b signaling. © 2019 Elsevier Inc. All rights reserved.
Keywords: CUG2 YAP1 Epithelial-mesenchymal transition NEK2 TGF-b
1. Introduction Because cancer upregulated gene 2 (CUG2) has commonly upregulated in various tumor tissues, such as ovary, liver, lung, and colon [1], it has been suggested to play a role in oncogenesis. Murine cells overexpressing CUG2 increase cell proliferation, migration, and induce tumor formation in nude mice, similar to H-RAS oncogene activities [1]. CUG2 overexpression induces Ras activation and enhances phosphorylation of ERK, JNK, and p38MAPK, which eventually enables oncolytic reoviral replication [2]. CUG2 confers EMT through TGF-b signaling [3]. Our study has reported that CUG2 also enhances the epidermal growth factor receptor (EGFR) expression, resulting in the resistance to doxorubicin by
* Corresponding author. BK21 Plus, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan, 46241, Republic of Korea. E-mail address: [email protected] (Y.-H. Chung). https://doi.org/10.1016/j.bbrc.2019.02.036 0006-291X/© 2019 Elsevier Inc. All rights reserved.
activating the Stat1-HDAC4 signaling axis [4]. Further study has revealed that Stat1-HDAC4 signaling furthermore induces malignant tumor features such as EMT and sphere formation in CUG2overexpressing cancer cells [5]. The Hippo signaling pathway controls organ size by regulation of cell proliferation, apoptosis and stem cell self-renewal [6,7]. Many lines of evidence document that the dysregulation of the Hippo pathway contributes to cancer development [8e11]. Once the activation of large tumor suppressor (LATS1/2) occurs, it inhibits the translocation of YAP and transcriptional co-activator with PDZ-binding motif (TAZ) by phosphorylation. YAP/TAZ function as transcriptional co-activators, when dephosphorylation occurs. They thus translocate into the nucleus, induce the expression of genes that promote cell proliferation and inhibit apoptosis via interaction with the TEAD family of transcription factors [12]. The elevated YAP expression and nuclear translocation lead to induction of CSC properties in different cancer types, such as breast cancer [13], lung cancer [14], and pancreatic cancer [15]. In cancer
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cells, the Hippo pathway regulates YAP/TAZ activation through a wide range of upstream stimuli, such as extracellular ligands, environmental stress, cell-cell contact, and mechanotransduction [16]. Growing data suggest that two pathways between Hippo and Wnt/b-catenin influence each other in many ways to regulate cell growth [17,18]. In this study, we initiated to explore the molecular mechanism of CUG2-induced EMT. We found that CUG2 upregulates YAP1 expression through NEK2 and furthermore YAP1 plays a role in CUG2-induced EMT by activation of TGF-b signaling. 2. Materials and methods 2.1. Cell culture Human lung cancer A549 and immortalized human bronchial BEAS-2B cells were obtained from American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI-1640 and 50% DMEM/50% F12, respectively. The cells were stably transfected with either vector alone (A549-Vec; BEAS-Vec) or wild-type CUG2 (A549-CUG2; BEAS-CUG2). The media were supplemented with 10% FBS, 1% penicillin, 1% streptomycin, and G418 (Sigma-Aldrich, St. Louis, MO, USA; 0.5 mg/mL) at 37 C and 5% CO2. 2.2. Reagents and antibodies Antibodies against YAP1, phospho-LATS, E-cadherin, N-cadherin, vimentin, CUG2 and Twist were acquired from Abcam (Cambridge, MA, USA). Anti-b-catenin, -Smad2/3, -AKT, -ERK, -JNK, -p38 MAPK and their specific phospho-antibodies were purchased from Cell Signaling Biotechnology (Danvers, MA, USA). Anti-b-actin, -LATS, and -TGF-b antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against NEK2 for performing western blotting and immunofluorescence microscopy were purchased from BD Biosciences (San Jose, CA, USA) and those for performing immunoprecipitation acquired from Santa Cruz Biotechnology. The protein kinase inhibitors, wortmannin, PD98059, SP600125, and SB203580 were obtained from Calbiochem (San Diego, CA, USA). LY2109761, as the TGF-b inhibitor, was purchased from Cayman Chemical (Ann Arbor, MI, USA) and Wnt3a was purchased from R&D Systems Inc. (Minneapolis, MN, USA). 2.3. Western blot analysis For performing Western blotting, proteins from whole cell lysates were separated by SDS-polyacrylamide gel electrophoresis (8%, 10% or 12% gel), followed by transferring to nitrocellulose membranes. The expression of each protein was detected using a dilution of 1:1000 or 1:2000 primary antibody. After incubation with a dilution of 1:2000 horseradish peroxidase-conjugated secondary antibody in 5% nonfat dry milk, the membrane was developed with an enhanced chemiluminescence assay using Image Quant LAS 4000 Mini (GE-Healthcare, Tokyo, Japan).
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incubated with 25 mL of Protein A-Sepharose resin (Santa Cruz Biotechnology) for 2 h. DNA fragments were eluted in TE buffer (10 mM Tris-HCl [pH8.0], 1 mM EDTA) by heating at 90 C for 10 min and analyzed by semi-quantitative PCR using YAP1 promoter-specific primers. YAP1 promoter primers were as follows: sense 50 -AGA CAG AGT CTC GCT GTG TTG-30 , antisense 50 -CCA AAA TGG TGA TAC CCT GTC-30 . 2.5. siRNA transfection A549-CUG2 and BEAS-CUG2 cells were transfected with siRNAs targeting YAP1, b-catenin, NEK2, Smad2 gene or nonsilencing control (Bioneer, Daejeon, Korea) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Six hours after transfection, cells were rinsed with a medium containing 10% FBS and incubated at 37 C for 42 h before harvesting. 2.6. Reverse transcription-polymerase chain reaction The total RNA was prepared from cultured cells by using RNeasy protect cell mini kit (Qiagen, Valencia, CA, USA), following the manufacturer's instructions. The cDNA was synthesized by using Superscript II reverse transcriptase (Invitrogen). PCR was performed using an optimized cycle with YAP1 primers. Housekeeping b-actin was used as the endogenous control. The following primers were obtained from Bioneer: YAP1 primers: sense 50 -GCA GTT GGG AGC TGT TTC TC-30 , antisense 50 -GCC ATG TTG TTG TCT GAT CG-30 . b-actin primers: sense 50 -ACC AAC TGG GAC GAC ATG GAG AAA-30 , and antisense 50 -TTA ATG TCA CGC ACG ATT TCC CGC-30 . 2.7. Immunofluorescence microscopy The cells grown on coverslips were fixed with 4% at room temperature for 10 min. The cells were added with 0.2% TritonX100 for 5 min after washing. After blocking with 10% goat serum, the cells were treated with anti-YAP1, -NEK2, -vimentin, and -phosphorylated Smad2 (dilution, 1:100) for 1 h. Subsequently, the cells were incubated with the appropriate secondary antibody coupled to Alexa Fluor 488 or Alexa Fluor 594 (dilution, 1:500; Thermo Fisher Scientific Inc., Waltham, MA, USA) at room temperature for 45 min. 4ʹ,6-Diamidino-2-phenylindole (DAPI) was used for DNA stain in the dark place for 5 min. After mounting with 10% glycerol, the images were obtained using a fluorescence microscope. 2.8. Luciferase reporter assays A549-CUG2 and BEAS-CUG2 cells were transiently transfected with TGF-b promoter (pTG5) or mutant (pTG7) vector [19] by using Lipofectamine 2000. The cells were incubated for 48 h after transfection and luciferase activity was measured using the Luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer's instructions. All luciferase activities were normalized to b-galactosidase activity.
2.4. Chromatin immunoprecipitation (ChIP) assay 2.9. Invasion assay Cells were crosslinked with 1% formaldehyde at room temperature for 15 min, and the reaction was stopped by adding glycine (125 mM) at room temperature for 10 min. Fixed cells were washed twice with PBS and resuspended in SDS lysis buffer, and sheared by sonication to generate 300- to 800-bp fragments of DNA. After nuclear extract preparation, the nuclear extract was immunoprecipitated overnight at 4 C with the antibody against c-Jun or AP2. Mouse and rabbit IgGs were used as negative controls. For capturing the protein-DNA-antibody complexes, samples were
The cells in a serum-free medium were seeded into 48-well Boyden chambers (Neuroprobe, Gaithersburg, MD, USA). The chamber was assembled using polycarbonate filters (Neuroprobe) coated with matrigel. Lower wells of the chamber were filled with 10% FBS medium as a chemoattractant. After 24 h of incubation, cells that invaded through the membrane were fixed, stained with hematoxylin-eosin, and quantified by counting the number under a phase-contrast microscope (CKX31-11 PHP; Olympus, Tokyo,
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Japan) at 100 magnification. 2.10. Wound healing assay Cells were cultured in six-well plates (5 105 cells/well) to achieve 90% confluence and scratched in the center of the cell monolayer using a P-200 pipette tip. The image was analyzed at 0 and 24 h after incubation using a phase-contrast microscope (Olympus) at 100 magnification. 2.11. Statistical analysis All data are presented as means ± standard deviation (SD). An unpaired t-test or one-way ANOVA was performed by GraphPad Prism program for statistical analysis. A p-value of <0.05 was considered to denote statistical significance. 3. Results 3.1. CUG2 enhances expression of YAP1 protein Becuase many lines of evidence have reported that the elevated expression of YAP1 protein is closely associated with oncogenesis [8e11], we wonder if the overexpression of CUG2 increases YAP1 expression. When expression levels of YAP1 in A549-CUG2 and BEAS-CUG2 cells were compared with those in A549-Vec and BEASVec cells, as control cells, the overexpression of CUG2 enhanced YAP1 protein levels in both A549-CUG2 and BEAS-CUG2 cells compared with the control cells (Fig. 1A). Supporting this observation, YAP1 proteins stained with stronger green fluorescence were detected in the nucleus of A549-CUG2 and BEAS-CUG2 cells than those in the nucleus of A549-Vec and BEAS-Vec cells (Fig. 1B). Because it is well known that LATS, as an upstream molecule of
Hippo signaling [6], regulates YAP1 expression and activity [6,12], this result raised a question that the increased expression of YAP1 is attributed to the decrease of LATS expression and activity. We herein found that the overexpression of CUG2 did not increase phosphorylation and expression levels of LATS, indicating that LATS does not affect YAP1 the expression and activity in A549-CUG2 and BEAS-CUG2 cells at least. Next, as we observed the elevated levels of YAP1 protein by overexpression of CUG2, we tested one of possibilities that the overexpression of CUG2 increases YAP1 transcript levels. The overexpression of CUG2 elevated mRNA levels of YAP1 in both A549-CUG2 and BEAS-CUG2 cells (Fig. 1C). Because other studies reported that c-Jun and AP2 transcription factors were involved in the synthesis of YAP1 transcripts in the YAP1 promoter [12,20], we examined whether c-Jun and AP2 transcription factors bind to the YAP1 promoter region under CUG2 overexpression with ChIp assay. In A549-CUG2 and BEAS-CUG2 cells, both c-Jun and AP2 protein much more bound to YAP1 promoter compared with those in A549-Vec and BEAS-Vec cells. The result indicates that the overexpression of CUG2 induces more interaction of c-Jun and AP2 proteins to YAP1 promoter, leading to the increase of YAP1 transcript synthesis. 3.2. Akt and MAPK kinases are involved in the increase of YAP1 protein expression under overexpression of CUG2 As our previous studies showed that the overexpression of CUG2 activates Akt and MAPK kinases including ERK, JNK and p38 MAPK [3], we wonder if these kinases are involved in the increase of YAP1 protein expression. To test this question, A549-CUG2 and BEASCUG2 cells were treated with wortmannin, PD98059, SP600125 and SB203580 to inhibit activities of Akt, ERK, JNK, and p38MAPK, respectively. Treatment with wortmannin decreased protein levels
Fig. 1. CUG2 enhances expression of YAP1 protein. (A) The expression of CUG2, YAP1, phospho-LATS, and LATS was detected by immunoblotting using corresponding antibodies. (B) The expressions of YAP1 in A549-Vec, A549-CUG2, BEAS-Vec, and BEAS-CUG2 cells were detected by immunofluorescence using Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). DAPI was used for DNA stain before mounting in glycerol. Scale bars indicate 10 mm. (C) Total RNAs were isolated from A549-Vec, A549-CUG2, BEAS-Vec, and BEAS-CUG2 cells. cDNAs were synthesized using specific YAP1 primers. b-Actin was used as an internal control. (D) ChIP assays determined the interaction of c-Jun and AP2 with the YAP1 promoter. Chromatin fragments were pulled down with a specific antibody and analyzed by semi-quantitative PCR using YAP1 promoter primers. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 2. Akt and MAPK kinases are involved in the increase of YAP1 protein in CUG2 overexpressing cells. A549-CUG2 and BEAS-CUG2 cells were treated with (A) wortmannin (Wort; 10 mM), (B) PD98059 (PD; 30 mM), (C) SP600125 (SP; 20 mM), or SB203580 (SB; 30 mM) for 24 h. Western blotting demonstrates the inhibition of YAP1 expression by these inhibitors.
of YAP1 in both cells (Fig. 2A). In addition, treatment with PD98059, SP600125 and SB203580 reduced protein levels of YAP1 in A549CUG2 cells (Fig. 2B, C, and D). Although PD98059 treatment slightly decreased YAP1 protein levels in BEAS-CUG2, other SP600125 and SB203580 kinase inhibitors significantly diminished the protein levels (Fig. 2B, C, and D). These results indicate that the elevated Akt and MAPK kinases induced by overexpression of CUG2 are involved in the increase of YAP1 protein expression. 3.3. Not b-catenin but NEK2 is involved in increase of YAP1 protein expression under overexpression of CUG2 Other studies reported that Wnt/b-catenin signaling enhances
YAP1 protein levels [21] and moreover, that b-catenin and TBX5 form a complex with YAP1 [22]. Our recent study showed that overexpression of CUG2 elevates b-catenin expression and activity through NEK2 (in press). On the basis of these documents, we set up a hypothesis that the elevated b-catenin by overexpression of CUG2 contributes to the upregulation of YAP1 expression. When A549-CUG2, BEAS-CUG2, and their control cells were treated with Wnt3a, b-catenin as well as YAP1 protein levels were increased in A549-Vec and BEAS-Vec cells during Wnt3a treatment as expected (Fig. 3A). However, A549-CUG2 and BEAS-CUG2 cells did not show the increase of b-catenin and YAP1 protein levels during Wnt3a treatment (Fig. 3A). Thus, our hypothesis appeared not to work in this model. Instead, the result indicates that the overexpression of
Fig. 3. NEK2 is involved in the increase of YAP1 protein expression in CUG2 overexpressing cells. (A) Cell lysates treated with Wnt3a (100 ng/mL) or PBS were determined by performing immunoblotting with antibodies against YAP1, b-catenin and phosphorylated Ser33/Ser37/Thr41 of b-catenin. A549-CUG2 and BEAS-CUG2 cells were treated with (B) bcatenin siRNA, (C) YAP1 siRNA, (D) NEK2 siRNA, or control siRNA (500 nM) for 48 h and the cell lysates were determined by performing immunoblotting using corresponding antibodies.
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CUG2 confers YAP1 independence from Wnt/b-catenin signaling. Supporting the new proposal, silencing b-catenin did not decrease YAP1 protein levels (Fig. 3B). Moreover, silencing YAP1 diminished b-catenin protein levels, which can be explained by the increased phosphorylation levels of b-catenin at Ser33/Ser37, leading to its ubiquitination and proteasomal degradation (Fig. 3C). Because we observed that CUG2 induced the upregulation of NEK2 involved in cell mitosis (In press), NEK2 protein levels were examined. YAP1 suppression reduced protein levels of NEK2, a binding partner of bcatenin, and silencing NEK2 conversely decreased YAP1 protein levels (Fig. 3C and D). Taken together, these results suggest that YAP1 expression is influenced by NEK2 rather than b-catenin under the overexpression of CUG2. 3.4. YAP1 is involved in CUG2-induced EMT Next, to explore roles of the elevated YAP1 protein under CUG2 overexpression, YAP1 siRNA was introduced in A549-CUG2 and BEAS-CUG2 cells. We found that silencing YAP1 protein decreased cell migration in A549-CUG2 and BEAS-CUG2 cells (Fig. 4A). In addition, the suppression of YAP1 reduced cell invasion compared with control siRNA treatment (Fig. 4B). Silencing YAP1 also inhibited the ability of CUG2-induced sphere formation
(Supplementary Fig. 1). Furthermore, when we examined EMT protein markers such as E-cadherin, N-cadherin and vimentin during YAP1 knockdown in A549-CUG2 and BEAS-CUG2 cells, we found that at least N-cadherin and vimentin expressions were diminished (Fig. 4C). Supporting the immunoblotting result, immunofluorescent intensity of vimentin was reduced after YAP1 siRNA treatment (Fig. 4D). However, the E-cadherin expression was not increased (Fig. 4C). Because TGF-b signaling plays a critical role in EMT [23,24], TGF-b transcriptional activity was examined during YAP1 siRNA treatment. We found that the YAP1 suppression reduced TGF-b transcriptional activity in A549-CUG2 and BEASCUG2 cells with phTG5 luciferase reporter vector bearing wild type of TGF-b promoter but not with phTG7 luciferase reporter vector bearing mutant type of TGF-b promoter (Fig. 4E). Silencing YAP1 diminished TGF-b and Twist protein levels, and reduced phosphorylation level of Smad2 (Fig. 4F). We also observed the decreased immunofluorescent intensity of phosphorylated Smad2 during YAP1 suppression in A549-CUG2 and BEAS-CUG2 cells (Supplementary Fig. 2A). Conversely, when we treated A549-CUG2 and BEAS-CUG2 with LY2109761, as a TGF-b inhibitor, YAP1 protein levels were decreased. Supporting this result, silencing Smad2 diminished the immunofluorescent intensity of YAP1 localized in the nucleus but control siRNA treatment did not (Supplementary
Fig. 4. YAP1 is involved in CUG2-induced EMT. (A) After post-transfection with YAP1 (500 nM) or control siRNA for 48 h, cell migration was measured by a wound healing assay. The wound closure areas were monitored by phase-contrast microscopy at a magnification of 100x. (B) An invasion assay was performed after 48 h post-transfection with YAP1 siRNA. Scale bar indicates 100 mm. The assays were repeated twice. Each assay was performed in triplicate and error bars indicate SD (***; p < 0.001). (C) Expressions of EMT proteins were detected by immunoblotting using the corresponding antibodies at 48 h post-transfection with YAP1 or control siRNA. (D) Expressions of vimentin in A549-CUG2 and BEASCUG2 cells were detected at 48 h post-transfection with YAP1 or control siRNA by immunofluorescence using Alexa Fluor 488-conjugated donkey anti-goat IgG (green). DAPI was used for DNA stain before mounting in glycerol. Scale bars indicate 10 mm. (E) A549-CUG2 and BEAS-CUG2 cells were transfected with YAP1 siRNA (500 nM) and subsequently transfected with TGF-b promoter vectors (phTG5 and 7; 1 mg). After transfection for 48 h, luciferase activity was measured. Transfection efficiency was normalized with bgalactosidase activity. The results showed the average of triplicate wells and error bars indicate SD. (**; p < 0.01, ***; p < 0.001, compared with control siRNA treatment). (G) Expressions of P-Smad2, Smad2/3, and YAP1 were detected by immunoblotting after treated with LY2109761 for 24 h. (F) At 48 h post-transfection with YAP1 siRNA, expressions of TGF-b, P-Smad2, Smad2/3, and Twist were detected by immunoblotting. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 2B). These results suggest that the interplay occurs between YAP1 and TGF-b signaling. These results suggest that there is a cross-talk between TGF-b signaling and YAP1 under the overexpression of CUG2. 4. Discussion Many lines of evidence documented a close relationship between YAP1 and b-catenin, affecting each other, for the development of tumor [21,25,26]. Herein, we found that the overexpression of CUG2 bypassed a unidirectional road from b-catenin to YAP1. Supporting the result, silencing b-catenin did not enhance the YAP1 expression under the overexpression of CUG2. However, YAP1 still affected b-catenin expression in spite of the overexpression of CUG2. Recent study showed that YAP1 can be activated by acute loss of APC but not by activation of b-catenin in a colon cancer mouse model [26], supporting our results. As our recent report (In press) and other studies showed that NEK2, involved in mitosis, interacts with b-catenin [27], NEK2 protein attracted our attention. We first reported that NEK2 affects YAP1 expression and vice versa. Thus, the next assignment will be given to illustrate how NEK2 protein regulates the YAP1 expression without involvement of bcatenin. Recent other studies showed that YAP1 is involved in cell migration and invasion through Slug [28]. Similarly, we also found that the upregulation of YAP1 induced by CUG2 overexpression played a critical role in cell migration and invasion through TGF-b signaling. Conversely, YAP1 expression was influenced by TGF-b signaling because it was diminished by LY2109761 treatment and silencing Smad2. Clinical data show that patients with lower expression of phospho-Smad2 and YAP1 had significantly longer time for metastasis and a longer overall survival during osteosarcoma progression [29], which support our observation on the interplay between YAP1 and TGF-b signaling. However, some studies reported that in hepatocellular carcinoma, TGF-b treatment inhibited YAP1 activity in spite of the activation of Smad2, leading to suppression of cell proliferation [30]. This might be due to different cellular context regarding a response to TGF-b. Conflicts of interest All authors declare that there is no financial conflict of interest regarding this study.
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[3]
[4]
[5]
[6] [7] [8] [9]
[10]
[11]
[12]
[13]
[14]
[15]
[16] [17] [18]
Acknowledgements [19]
This study was supported by the Basic Research Program of the National Research Foundation funded by the Korean government (NRF-2016R 1D1A1B03930168). This study was financially supported by the "2018 Post-Doc. Development Program" of Pusan National University. Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.036. Transparency document
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Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.036.
[24]
References [1] S. Lee, J. Gang, S.B. Jeon, S.H. Choo, B. Lee, Y.G. Kim, Y.S. Lee, J. Jung, S.Y. Song,
[25]
[26]
127
S.S. Koh, Molecular cloning and functional analysis of a novel oncogene, cancer-upregulated gene 2 (CUG2), Biochem. Biophys. Res. Commun. 360 (2007) 633e639. E.H. Park, E.H. Park, I.R. Cho, R. Srisuttee, H.J. Min, M.J. Oh, Y.J. Jeong, B.H. Jhun, R.N. Johnston, S. Lee, S.S. Koh, Y.H. Chung, CUG2, a novel oncogene confers reoviral replication through Ras and p38 signaling pathway, Cancer Gene Ther. 17 (2010) 307e314. S. Kaowinn, J. Kim, J. Lee, D.H. Shin, C.D. Kang, D.K. Kim, S. Lee, M.K. Kang, S.S. Koh, S.J. Kim, Y.H. Chung, Cancer upregulated gene 2 induces epithelialmesenchymal transition of human lung cancer cells via TGF-beta signaling, Oncotarget 8 (2017) 5092e5110. S. Kaowinn, S.W. Jun, C.S. Kim, D.M. Shin, Y.H. Hwang, K. Kim, B. Shin, C. Kaewpiboon, H.H. Jeong, S.S. Koh, O.H. Kramer, R.N. Johnston, Y.H. Chung, Increased EGFR expression induced by a novel oncogene, CUG2, confers resistance to doxorubicin through Stat1-HDAC4 signaling, Cell. Oncol. 40 (2017) 549e561. S. Kaowinn, C. Kaewpiboon, S.S. Koh, O.H. Kramer, Y.H. Chung, STAT1HDAC4 signaling induces epithelialmesenchymal transition and sphere formation of cancer cells overexpressing the oncogene, CUG2, Oncol. rep. 40 (2018) 2619e2627. F.X. Yu, K.L. Guan, The Hippo pathway: regulators and regulations, Genes Dev. 27 (2013) 355e371. H. Liu, D. Jiang, F. Chi, B. Zhao, The Hippo pathway regulates stem cell proliferation, self-renewal, and differentiation, Protein & cell 3 (2012) 291e304. J. Avruch, D. Zhou, N. Bardeesy, YAP oncogene overexpression supercharges colon cancer proliferation, Cell Cycle 11 (2012) 1090e1096. C.A. Hall, R. Wang, J. Miao, E. Oliva, X. Shen, T. Wheeler, S.G. Hilsenbeck, S. Orsulic, S. Goode, Hippo pathway effector Yap is an ovarian cancer oncogene, Cancer Res. 70 (2010) 8517e8525. T. Pei, Y. Li, J. Wang, H. Wang, Y. Liang, H. Shi, B. Sun, D. Yin, J. Sun, R. Song, S. Pan, Y. Sun, H. Jiang, T. Zheng, L. Liu, YAP is a critical oncogene in human cholangiocarcinoma, Oncotarget 6 (2015) 17206e17220. T. Muramatsu, I. Imoto, T. Matsui, K. Kozaki, S. Haruki, M. Sudol, Y. Shimada, H. Tsuda, T. Kawano, J. Inazawa, YAP is a candidate oncogene for esophageal squamous cell carcinoma, Carcinogenesis 32 (2011) 389e398. W. Hong, K.L. Guan, The YAP and TAZ transcription co-activators: key downstream effectors of the mammalian Hippo pathway, Semin. Cell Dev. Biol. 23 (2012) 785e793. T. Kim, S.J. Yang, D. Hwang, J. Song, M. Kim, S. Kyum Kim, K. Kang, J. Ahn, D. Lee, M.Y. Kim, S. Kim, J. Seung Koo, S. Seok Koh, S.Y. Kim, D.S. Lim, A basallike breast cancer-specific role for SRF-IL6 in YAP-induced cancer stemness, Nat. Commun. 6 (2015) 10186. B. Zhou, P. Flodby, J. Luo, D.R. Castillo, Y. Liu, F.X. Yu, A. McConnell, B. Varghese, G. Li, N.O. Chimge, M. Sunohara, M.N. Koss, W. Elatre, P. Conti, J.M. Liebler, C. Yang, C.N. Marconett, I.A. Laird-Offringa, P. Minoo, K. Guan, B.R. Stripp, E.D. Crandall, Z. Borok, Claudin-18-mediated YAP activity regulates lung stem and progenitor cell homeostasis and tumorigenesis, J. Clin. Invest. 128 (2018) 970e984. B. Yan, Z. Jiang, L. Cheng, K. Chen, C. Zhou, L. Sun, W. Qian, J. Li, J. Cao, Q. Xu, Q. Ma, J. Lei, Paracrine HGF/c-MET enhances the stem cell-like potential and glycolysis of pancreatic cancer cells via activation of YAP/HIF-1alpha, Exp. Cell Res. 371 (2018) 63e71. J.H. Park, J.E. Shin, H.W. Park, The role of hippo pathway in cancer stem cell biology, Mol. Cell. 41 (2018) 83e92. W.M. Konsavage Jr., G.S. Yochum, Intersection of Hippo/YAP and Wnt/betacatenin signaling pathways, Acta Biochim. Biophys. Sin. 45 (2013) 71e79. M. Imajo, K. Miyatake, A. Iimura, A. Miyamoto, E. Nishida, A molecular mechanism that links Hippo signalling to the inhibition of Wnt/beta-catenin signalling, EMBO J. 31 (2012) 1109e1122. S.J. Kim, A. Glick, M.B. Sporn, A.B. Roberts, Characterization of the promoter region of the human transforming growth factor-beta 1 gene, J. Biol. Chem. 264 (1989) 402e408. Y. Qiao, Y. Qian, J. Wang, X. Tang, Melanoma cell adhesion molecule stimulates yes-associated protein transcription by enhancing CREB activity via c-Jun/cFos in hepatocellular carcinoma cells, Oncology letters 11 (2016) 3702e3708. W.I. Seo, S. Park, J. Gwak, B.G. Ju, J.I. Chung, P.M. Kang, S. Oh, Wnt signaling promotes androgen-independent prostate cancer cell proliferation through up-regulation of the hippo pathway effector YAP, Biochem. Biophys. Res. Commun. 486 (2017) 1034e1039. J. Rosenbluh, D. Nijhawan, A.G. Cox, X. Li, J.T. Neal, E.J. Schafer, T.I. Zack, X. Wang, A. Tsherniak, A.C. Schinzel, D.D. Shao, S.E. Schumacher, B.A. Weir, F. Vazquez, G.S. Cowley, D.E. Root, J.P. Mesirov, R. Beroukhim, C.J. Kuo, W. Goessling, W.C. Hahn, beta-Catenin-driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis, Cell 151 (2012) 1457e1473. M.K. Wendt, M. Tian, W.P. Schiemann, Deconstructing the mechanisms and consequences of TGF-beta-induced EMT during cancer progression, Cell Tissue Res. 347 (2012) 85e101. J. Fuxe, T. Vincent, A. Garcia de Herreros, Transcriptional crosstalk between TGF-beta and stem cell pathways in tumor cell invasion: role of EMT promoting Smad complexes, Cell Cycle 9 (2010) 2363e2374. W.M. Konsavage Jr., S.L. Kyler, S.A. Rennoll, G. Jin, G.S. Yochum, Wnt/betacatenin signaling regulates Yes-associated protein (YAP) gene expression in colorectal carcinoma cells, J. Biol. Chem. 287 (2012) 11730e11739. J. Cai, A. Maitra, R.A. Anders, M.M. Taketo, D. Pan, beta-Catenin destruction
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complex-independent regulation of Hippo-YAP signaling by APC in intestinal tumorigenesis, Genes Dev. 29 (2015) 1493e1506. [27] S. Bahmanyar, D.D. Kaplan, J.G. Deluca, T.H. Giddings Jr., E.T. O'Toole, M. Winey, E.D. Salmon, P.J. Casey, W.J. Nelson, A.I. Barth, beta-Catenin is a Nek2 substrate involved in centrosome separation, Genes Dev. 22 (2008) 91e105. [28] M. Yu, Y. Chen, X. Li, R. Yang, L. Zhang, L. Huangfu, N. Zheng, X. Zhao, L. Lv, Y. Hong, H. Liang, H. Shan, YAP1 contributes to NSCLC invasion and migration by promoting Slug transcription via the transcription co-factor TEAD, Cell
Death Dis. 9 (2018) 464. [29] A.K. Luu, C.R. Schott, R. Jones, A.C. Poon, B. Golding, R. Hamed, B. Deheshi, A. Mutsaers, G.A. Wood, A.M. Viloria-Petit, An evaluation of TAZ and YAP crosstalk with TGFbeta signalling in canine osteosarcoma suggests involvement of hippo signalling in disease progression, BMC Vet. Res. 14 (2018) 365. [30] X. Zhang, Q. Fan, Y. Li, Z. Yang, L. Yang, Z. Zong, B. Wang, X. Meng, Q. Li, J. Liu, H. Li, Transforming growth factor-beta1 suppresses hepatocellular carcinoma proliferation via activation of Hippo signaling, Oncotarget 8 (2017) 29785e29794.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Cancer upregulated gene (CUG)2 elevates YAP1 expression, leading to enhancement of epithelial-mesenchymal transition in human lung cancer cells Sirichat Kaowinn a, Natpaphan Yawut a, Sang Seok Koh b, Young-Hwa Chung a, * a b
BK21 Plus, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan, 46241, Republic of Korea Department of Biosciences, Dong-A University, Busan, 49315, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 January 2019 Accepted 7 February 2019 Available online 14 February 2019
Although our previous studies have showed that a novel oncogene, cancer upregulated gene (CUG)2 induced epithelial-mesenchymal transition (EMT), the detailed molecular mechanism remains unknown. Because several lines of evidence documented that Yes-Associated Protein (YAP)1 is closely associated with cancer stem cell (CSC)-like phenotypes including EMT, stemness, and drug resistance, we wondered if YAP1 is involved in CUG2-induced EMT. We herein found that the overexpression of CUG2 increased YAP1 expression at the transcriptional as well as protein levels. Chromatin immunoprecipitation assay revealed that the elevated YAP1 transcripts are attributed to c-Jun and AP2 bindings to the YAP1 promoter. Akt and MAPK kinases including ERK, JNK, and p38 MAPK enhanced the level of YAP1 protein. In spite of a close relationship between b-catenin and YAP1, not b-catenin but NEK2 played the role in increasing YAP1 expression. Silencing YAP1 inhibited CUG2-induced cell migration and invasion. Ncadherin and vimentin expressions were decreased during YAP1 knockdown. The suppression of YAP1 diminished TGF-b transcriptional activity and expression as well as phosphorylation level of Smad2 and Twist protein. Conversely, LY2109761 or Smad2 siRNA treatment reduced YAP1 protein levels, indicating a close interplay between YAP1 and TGF-b signaling. Taken together, we suggest that CUG2 induces upregulation of YAP1 expression, leading to enhancing CUG2-induced EMT via a close crosstalk between YAP1 and TGF-b signaling. © 2019 Elsevier Inc. All rights reserved.
Keywords: CUG2 YAP1 Epithelial-mesenchymal transition NEK2 TGF-b
1. Introduction Because cancer upregulated gene 2 (CUG2) has commonly upregulated in various tumor tissues, such as ovary, liver, lung, and colon [1], it has been suggested to play a role in oncogenesis. Murine cells overexpressing CUG2 increase cell proliferation, migration, and induce tumor formation in nude mice, similar to H-RAS oncogene activities [1]. CUG2 overexpression induces Ras activation and enhances phosphorylation of ERK, JNK, and p38MAPK, which eventually enables oncolytic reoviral replication [2]. CUG2 confers EMT through TGF-b signaling [3]. Our study has reported that CUG2 also enhances the epidermal growth factor receptor (EGFR) expression, resulting in the resistance to doxorubicin by
* Corresponding author. BK21 Plus, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan, 46241, Republic of Korea. E-mail address: [email protected] (Y.-H. Chung). https://doi.org/10.1016/j.bbrc.2019.02.036 0006-291X/© 2019 Elsevier Inc. All rights reserved.
activating the Stat1-HDAC4 signaling axis [4]. Further study has revealed that Stat1-HDAC4 signaling furthermore induces malignant tumor features such as EMT and sphere formation in CUG2overexpressing cancer cells [5]. The Hippo signaling pathway controls organ size by regulation of cell proliferation, apoptosis and stem cell self-renewal [6,7]. Many lines of evidence document that the dysregulation of the Hippo pathway contributes to cancer development [8e11]. Once the activation of large tumor suppressor (LATS1/2) occurs, it inhibits the translocation of YAP and transcriptional co-activator with PDZ-binding motif (TAZ) by phosphorylation. YAP/TAZ function as transcriptional co-activators, when dephosphorylation occurs. They thus translocate into the nucleus, induce the expression of genes that promote cell proliferation and inhibit apoptosis via interaction with the TEAD family of transcription factors [12]. The elevated YAP expression and nuclear translocation lead to induction of CSC properties in different cancer types, such as breast cancer [13], lung cancer [14], and pancreatic cancer [15]. In cancer
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cells, the Hippo pathway regulates YAP/TAZ activation through a wide range of upstream stimuli, such as extracellular ligands, environmental stress, cell-cell contact, and mechanotransduction [16]. Growing data suggest that two pathways between Hippo and Wnt/b-catenin influence each other in many ways to regulate cell growth [17,18]. In this study, we initiated to explore the molecular mechanism of CUG2-induced EMT. We found that CUG2 upregulates YAP1 expression through NEK2 and furthermore YAP1 plays a role in CUG2-induced EMT by activation of TGF-b signaling. 2. Materials and methods 2.1. Cell culture Human lung cancer A549 and immortalized human bronchial BEAS-2B cells were obtained from American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI-1640 and 50% DMEM/50% F12, respectively. The cells were stably transfected with either vector alone (A549-Vec; BEAS-Vec) or wild-type CUG2 (A549-CUG2; BEAS-CUG2). The media were supplemented with 10% FBS, 1% penicillin, 1% streptomycin, and G418 (Sigma-Aldrich, St. Louis, MO, USA; 0.5 mg/mL) at 37 C and 5% CO2. 2.2. Reagents and antibodies Antibodies against YAP1, phospho-LATS, E-cadherin, N-cadherin, vimentin, CUG2 and Twist were acquired from Abcam (Cambridge, MA, USA). Anti-b-catenin, -Smad2/3, -AKT, -ERK, -JNK, -p38 MAPK and their specific phospho-antibodies were purchased from Cell Signaling Biotechnology (Danvers, MA, USA). Anti-b-actin, -LATS, and -TGF-b antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against NEK2 for performing western blotting and immunofluorescence microscopy were purchased from BD Biosciences (San Jose, CA, USA) and those for performing immunoprecipitation acquired from Santa Cruz Biotechnology. The protein kinase inhibitors, wortmannin, PD98059, SP600125, and SB203580 were obtained from Calbiochem (San Diego, CA, USA). LY2109761, as the TGF-b inhibitor, was purchased from Cayman Chemical (Ann Arbor, MI, USA) and Wnt3a was purchased from R&D Systems Inc. (Minneapolis, MN, USA). 2.3. Western blot analysis For performing Western blotting, proteins from whole cell lysates were separated by SDS-polyacrylamide gel electrophoresis (8%, 10% or 12% gel), followed by transferring to nitrocellulose membranes. The expression of each protein was detected using a dilution of 1:1000 or 1:2000 primary antibody. After incubation with a dilution of 1:2000 horseradish peroxidase-conjugated secondary antibody in 5% nonfat dry milk, the membrane was developed with an enhanced chemiluminescence assay using Image Quant LAS 4000 Mini (GE-Healthcare, Tokyo, Japan).
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incubated with 25 mL of Protein A-Sepharose resin (Santa Cruz Biotechnology) for 2 h. DNA fragments were eluted in TE buffer (10 mM Tris-HCl [pH8.0], 1 mM EDTA) by heating at 90 C for 10 min and analyzed by semi-quantitative PCR using YAP1 promoter-specific primers. YAP1 promoter primers were as follows: sense 50 -AGA CAG AGT CTC GCT GTG TTG-30 , antisense 50 -CCA AAA TGG TGA TAC CCT GTC-30 . 2.5. siRNA transfection A549-CUG2 and BEAS-CUG2 cells were transfected with siRNAs targeting YAP1, b-catenin, NEK2, Smad2 gene or nonsilencing control (Bioneer, Daejeon, Korea) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Six hours after transfection, cells were rinsed with a medium containing 10% FBS and incubated at 37 C for 42 h before harvesting. 2.6. Reverse transcription-polymerase chain reaction The total RNA was prepared from cultured cells by using RNeasy protect cell mini kit (Qiagen, Valencia, CA, USA), following the manufacturer's instructions. The cDNA was synthesized by using Superscript II reverse transcriptase (Invitrogen). PCR was performed using an optimized cycle with YAP1 primers. Housekeeping b-actin was used as the endogenous control. The following primers were obtained from Bioneer: YAP1 primers: sense 50 -GCA GTT GGG AGC TGT TTC TC-30 , antisense 50 -GCC ATG TTG TTG TCT GAT CG-30 . b-actin primers: sense 50 -ACC AAC TGG GAC GAC ATG GAG AAA-30 , and antisense 50 -TTA ATG TCA CGC ACG ATT TCC CGC-30 . 2.7. Immunofluorescence microscopy The cells grown on coverslips were fixed with 4% at room temperature for 10 min. The cells were added with 0.2% TritonX100 for 5 min after washing. After blocking with 10% goat serum, the cells were treated with anti-YAP1, -NEK2, -vimentin, and -phosphorylated Smad2 (dilution, 1:100) for 1 h. Subsequently, the cells were incubated with the appropriate secondary antibody coupled to Alexa Fluor 488 or Alexa Fluor 594 (dilution, 1:500; Thermo Fisher Scientific Inc., Waltham, MA, USA) at room temperature for 45 min. 4ʹ,6-Diamidino-2-phenylindole (DAPI) was used for DNA stain in the dark place for 5 min. After mounting with 10% glycerol, the images were obtained using a fluorescence microscope. 2.8. Luciferase reporter assays A549-CUG2 and BEAS-CUG2 cells were transiently transfected with TGF-b promoter (pTG5) or mutant (pTG7) vector [19] by using Lipofectamine 2000. The cells were incubated for 48 h after transfection and luciferase activity was measured using the Luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer's instructions. All luciferase activities were normalized to b-galactosidase activity.
2.4. Chromatin immunoprecipitation (ChIP) assay 2.9. Invasion assay Cells were crosslinked with 1% formaldehyde at room temperature for 15 min, and the reaction was stopped by adding glycine (125 mM) at room temperature for 10 min. Fixed cells were washed twice with PBS and resuspended in SDS lysis buffer, and sheared by sonication to generate 300- to 800-bp fragments of DNA. After nuclear extract preparation, the nuclear extract was immunoprecipitated overnight at 4 C with the antibody against c-Jun or AP2. Mouse and rabbit IgGs were used as negative controls. For capturing the protein-DNA-antibody complexes, samples were
The cells in a serum-free medium were seeded into 48-well Boyden chambers (Neuroprobe, Gaithersburg, MD, USA). The chamber was assembled using polycarbonate filters (Neuroprobe) coated with matrigel. Lower wells of the chamber were filled with 10% FBS medium as a chemoattractant. After 24 h of incubation, cells that invaded through the membrane were fixed, stained with hematoxylin-eosin, and quantified by counting the number under a phase-contrast microscope (CKX31-11 PHP; Olympus, Tokyo,
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Japan) at 100 magnification. 2.10. Wound healing assay Cells were cultured in six-well plates (5 105 cells/well) to achieve 90% confluence and scratched in the center of the cell monolayer using a P-200 pipette tip. The image was analyzed at 0 and 24 h after incubation using a phase-contrast microscope (Olympus) at 100 magnification. 2.11. Statistical analysis All data are presented as means ± standard deviation (SD). An unpaired t-test or one-way ANOVA was performed by GraphPad Prism program for statistical analysis. A p-value of <0.05 was considered to denote statistical significance. 3. Results 3.1. CUG2 enhances expression of YAP1 protein Becuase many lines of evidence have reported that the elevated expression of YAP1 protein is closely associated with oncogenesis [8e11], we wonder if the overexpression of CUG2 increases YAP1 expression. When expression levels of YAP1 in A549-CUG2 and BEAS-CUG2 cells were compared with those in A549-Vec and BEASVec cells, as control cells, the overexpression of CUG2 enhanced YAP1 protein levels in both A549-CUG2 and BEAS-CUG2 cells compared with the control cells (Fig. 1A). Supporting this observation, YAP1 proteins stained with stronger green fluorescence were detected in the nucleus of A549-CUG2 and BEAS-CUG2 cells than those in the nucleus of A549-Vec and BEAS-Vec cells (Fig. 1B). Because it is well known that LATS, as an upstream molecule of
Hippo signaling [6], regulates YAP1 expression and activity [6,12], this result raised a question that the increased expression of YAP1 is attributed to the decrease of LATS expression and activity. We herein found that the overexpression of CUG2 did not increase phosphorylation and expression levels of LATS, indicating that LATS does not affect YAP1 the expression and activity in A549-CUG2 and BEAS-CUG2 cells at least. Next, as we observed the elevated levels of YAP1 protein by overexpression of CUG2, we tested one of possibilities that the overexpression of CUG2 increases YAP1 transcript levels. The overexpression of CUG2 elevated mRNA levels of YAP1 in both A549-CUG2 and BEAS-CUG2 cells (Fig. 1C). Because other studies reported that c-Jun and AP2 transcription factors were involved in the synthesis of YAP1 transcripts in the YAP1 promoter [12,20], we examined whether c-Jun and AP2 transcription factors bind to the YAP1 promoter region under CUG2 overexpression with ChIp assay. In A549-CUG2 and BEAS-CUG2 cells, both c-Jun and AP2 protein much more bound to YAP1 promoter compared with those in A549-Vec and BEAS-Vec cells. The result indicates that the overexpression of CUG2 induces more interaction of c-Jun and AP2 proteins to YAP1 promoter, leading to the increase of YAP1 transcript synthesis. 3.2. Akt and MAPK kinases are involved in the increase of YAP1 protein expression under overexpression of CUG2 As our previous studies showed that the overexpression of CUG2 activates Akt and MAPK kinases including ERK, JNK and p38 MAPK [3], we wonder if these kinases are involved in the increase of YAP1 protein expression. To test this question, A549-CUG2 and BEASCUG2 cells were treated with wortmannin, PD98059, SP600125 and SB203580 to inhibit activities of Akt, ERK, JNK, and p38MAPK, respectively. Treatment with wortmannin decreased protein levels
Fig. 1. CUG2 enhances expression of YAP1 protein. (A) The expression of CUG2, YAP1, phospho-LATS, and LATS was detected by immunoblotting using corresponding antibodies. (B) The expressions of YAP1 in A549-Vec, A549-CUG2, BEAS-Vec, and BEAS-CUG2 cells were detected by immunofluorescence using Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). DAPI was used for DNA stain before mounting in glycerol. Scale bars indicate 10 mm. (C) Total RNAs were isolated from A549-Vec, A549-CUG2, BEAS-Vec, and BEAS-CUG2 cells. cDNAs were synthesized using specific YAP1 primers. b-Actin was used as an internal control. (D) ChIP assays determined the interaction of c-Jun and AP2 with the YAP1 promoter. Chromatin fragments were pulled down with a specific antibody and analyzed by semi-quantitative PCR using YAP1 promoter primers. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 2. Akt and MAPK kinases are involved in the increase of YAP1 protein in CUG2 overexpressing cells. A549-CUG2 and BEAS-CUG2 cells were treated with (A) wortmannin (Wort; 10 mM), (B) PD98059 (PD; 30 mM), (C) SP600125 (SP; 20 mM), or SB203580 (SB; 30 mM) for 24 h. Western blotting demonstrates the inhibition of YAP1 expression by these inhibitors.
of YAP1 in both cells (Fig. 2A). In addition, treatment with PD98059, SP600125 and SB203580 reduced protein levels of YAP1 in A549CUG2 cells (Fig. 2B, C, and D). Although PD98059 treatment slightly decreased YAP1 protein levels in BEAS-CUG2, other SP600125 and SB203580 kinase inhibitors significantly diminished the protein levels (Fig. 2B, C, and D). These results indicate that the elevated Akt and MAPK kinases induced by overexpression of CUG2 are involved in the increase of YAP1 protein expression. 3.3. Not b-catenin but NEK2 is involved in increase of YAP1 protein expression under overexpression of CUG2 Other studies reported that Wnt/b-catenin signaling enhances
YAP1 protein levels [21] and moreover, that b-catenin and TBX5 form a complex with YAP1 [22]. Our recent study showed that overexpression of CUG2 elevates b-catenin expression and activity through NEK2 (in press). On the basis of these documents, we set up a hypothesis that the elevated b-catenin by overexpression of CUG2 contributes to the upregulation of YAP1 expression. When A549-CUG2, BEAS-CUG2, and their control cells were treated with Wnt3a, b-catenin as well as YAP1 protein levels were increased in A549-Vec and BEAS-Vec cells during Wnt3a treatment as expected (Fig. 3A). However, A549-CUG2 and BEAS-CUG2 cells did not show the increase of b-catenin and YAP1 protein levels during Wnt3a treatment (Fig. 3A). Thus, our hypothesis appeared not to work in this model. Instead, the result indicates that the overexpression of
Fig. 3. NEK2 is involved in the increase of YAP1 protein expression in CUG2 overexpressing cells. (A) Cell lysates treated with Wnt3a (100 ng/mL) or PBS were determined by performing immunoblotting with antibodies against YAP1, b-catenin and phosphorylated Ser33/Ser37/Thr41 of b-catenin. A549-CUG2 and BEAS-CUG2 cells were treated with (B) bcatenin siRNA, (C) YAP1 siRNA, (D) NEK2 siRNA, or control siRNA (500 nM) for 48 h and the cell lysates were determined by performing immunoblotting using corresponding antibodies.
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CUG2 confers YAP1 independence from Wnt/b-catenin signaling. Supporting the new proposal, silencing b-catenin did not decrease YAP1 protein levels (Fig. 3B). Moreover, silencing YAP1 diminished b-catenin protein levels, which can be explained by the increased phosphorylation levels of b-catenin at Ser33/Ser37, leading to its ubiquitination and proteasomal degradation (Fig. 3C). Because we observed that CUG2 induced the upregulation of NEK2 involved in cell mitosis (In press), NEK2 protein levels were examined. YAP1 suppression reduced protein levels of NEK2, a binding partner of bcatenin, and silencing NEK2 conversely decreased YAP1 protein levels (Fig. 3C and D). Taken together, these results suggest that YAP1 expression is influenced by NEK2 rather than b-catenin under the overexpression of CUG2. 3.4. YAP1 is involved in CUG2-induced EMT Next, to explore roles of the elevated YAP1 protein under CUG2 overexpression, YAP1 siRNA was introduced in A549-CUG2 and BEAS-CUG2 cells. We found that silencing YAP1 protein decreased cell migration in A549-CUG2 and BEAS-CUG2 cells (Fig. 4A). In addition, the suppression of YAP1 reduced cell invasion compared with control siRNA treatment (Fig. 4B). Silencing YAP1 also inhibited the ability of CUG2-induced sphere formation
(Supplementary Fig. 1). Furthermore, when we examined EMT protein markers such as E-cadherin, N-cadherin and vimentin during YAP1 knockdown in A549-CUG2 and BEAS-CUG2 cells, we found that at least N-cadherin and vimentin expressions were diminished (Fig. 4C). Supporting the immunoblotting result, immunofluorescent intensity of vimentin was reduced after YAP1 siRNA treatment (Fig. 4D). However, the E-cadherin expression was not increased (Fig. 4C). Because TGF-b signaling plays a critical role in EMT [23,24], TGF-b transcriptional activity was examined during YAP1 siRNA treatment. We found that the YAP1 suppression reduced TGF-b transcriptional activity in A549-CUG2 and BEASCUG2 cells with phTG5 luciferase reporter vector bearing wild type of TGF-b promoter but not with phTG7 luciferase reporter vector bearing mutant type of TGF-b promoter (Fig. 4E). Silencing YAP1 diminished TGF-b and Twist protein levels, and reduced phosphorylation level of Smad2 (Fig. 4F). We also observed the decreased immunofluorescent intensity of phosphorylated Smad2 during YAP1 suppression in A549-CUG2 and BEAS-CUG2 cells (Supplementary Fig. 2A). Conversely, when we treated A549-CUG2 and BEAS-CUG2 with LY2109761, as a TGF-b inhibitor, YAP1 protein levels were decreased. Supporting this result, silencing Smad2 diminished the immunofluorescent intensity of YAP1 localized in the nucleus but control siRNA treatment did not (Supplementary
Fig. 4. YAP1 is involved in CUG2-induced EMT. (A) After post-transfection with YAP1 (500 nM) or control siRNA for 48 h, cell migration was measured by a wound healing assay. The wound closure areas were monitored by phase-contrast microscopy at a magnification of 100x. (B) An invasion assay was performed after 48 h post-transfection with YAP1 siRNA. Scale bar indicates 100 mm. The assays were repeated twice. Each assay was performed in triplicate and error bars indicate SD (***; p < 0.001). (C) Expressions of EMT proteins were detected by immunoblotting using the corresponding antibodies at 48 h post-transfection with YAP1 or control siRNA. (D) Expressions of vimentin in A549-CUG2 and BEASCUG2 cells were detected at 48 h post-transfection with YAP1 or control siRNA by immunofluorescence using Alexa Fluor 488-conjugated donkey anti-goat IgG (green). DAPI was used for DNA stain before mounting in glycerol. Scale bars indicate 10 mm. (E) A549-CUG2 and BEAS-CUG2 cells were transfected with YAP1 siRNA (500 nM) and subsequently transfected with TGF-b promoter vectors (phTG5 and 7; 1 mg). After transfection for 48 h, luciferase activity was measured. Transfection efficiency was normalized with bgalactosidase activity. The results showed the average of triplicate wells and error bars indicate SD. (**; p < 0.01, ***; p < 0.001, compared with control siRNA treatment). (G) Expressions of P-Smad2, Smad2/3, and YAP1 were detected by immunoblotting after treated with LY2109761 for 24 h. (F) At 48 h post-transfection with YAP1 siRNA, expressions of TGF-b, P-Smad2, Smad2/3, and Twist were detected by immunoblotting. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 2B). These results suggest that the interplay occurs between YAP1 and TGF-b signaling. These results suggest that there is a cross-talk between TGF-b signaling and YAP1 under the overexpression of CUG2. 4. Discussion Many lines of evidence documented a close relationship between YAP1 and b-catenin, affecting each other, for the development of tumor [21,25,26]. Herein, we found that the overexpression of CUG2 bypassed a unidirectional road from b-catenin to YAP1. Supporting the result, silencing b-catenin did not enhance the YAP1 expression under the overexpression of CUG2. However, YAP1 still affected b-catenin expression in spite of the overexpression of CUG2. Recent study showed that YAP1 can be activated by acute loss of APC but not by activation of b-catenin in a colon cancer mouse model [26], supporting our results. As our recent report (In press) and other studies showed that NEK2, involved in mitosis, interacts with b-catenin [27], NEK2 protein attracted our attention. We first reported that NEK2 affects YAP1 expression and vice versa. Thus, the next assignment will be given to illustrate how NEK2 protein regulates the YAP1 expression without involvement of bcatenin. Recent other studies showed that YAP1 is involved in cell migration and invasion through Slug [28]. Similarly, we also found that the upregulation of YAP1 induced by CUG2 overexpression played a critical role in cell migration and invasion through TGF-b signaling. Conversely, YAP1 expression was influenced by TGF-b signaling because it was diminished by LY2109761 treatment and silencing Smad2. Clinical data show that patients with lower expression of phospho-Smad2 and YAP1 had significantly longer time for metastasis and a longer overall survival during osteosarcoma progression [29], which support our observation on the interplay between YAP1 and TGF-b signaling. However, some studies reported that in hepatocellular carcinoma, TGF-b treatment inhibited YAP1 activity in spite of the activation of Smad2, leading to suppression of cell proliferation [30]. This might be due to different cellular context regarding a response to TGF-b. Conflicts of interest All authors declare that there is no financial conflict of interest regarding this study.
[2]
[3]
[4]
[5]
[6] [7] [8] [9]
[10]
[11]
[12]
[13]
[14]
[15]
[16] [17] [18]
Acknowledgements [19]
This study was supported by the Basic Research Program of the National Research Foundation funded by the Korean government (NRF-2016R 1D1A1B03930168). This study was financially supported by the "2018 Post-Doc. Development Program" of Pusan National University. Appendix A. Supplementary data
[20]
[21]
[22]
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.036. Transparency document
[23]
Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.036.
[24]
References [1] S. Lee, J. Gang, S.B. Jeon, S.H. Choo, B. Lee, Y.G. Kim, Y.S. Lee, J. Jung, S.Y. Song,
[25]
[26]
127
S.S. Koh, Molecular cloning and functional analysis of a novel oncogene, cancer-upregulated gene 2 (CUG2), Biochem. Biophys. Res. Commun. 360 (2007) 633e639. E.H. Park, E.H. Park, I.R. Cho, R. Srisuttee, H.J. Min, M.J. Oh, Y.J. Jeong, B.H. Jhun, R.N. Johnston, S. Lee, S.S. Koh, Y.H. Chung, CUG2, a novel oncogene confers reoviral replication through Ras and p38 signaling pathway, Cancer Gene Ther. 17 (2010) 307e314. S. Kaowinn, J. Kim, J. Lee, D.H. Shin, C.D. Kang, D.K. Kim, S. Lee, M.K. Kang, S.S. Koh, S.J. Kim, Y.H. Chung, Cancer upregulated gene 2 induces epithelialmesenchymal transition of human lung cancer cells via TGF-beta signaling, Oncotarget 8 (2017) 5092e5110. S. Kaowinn, S.W. Jun, C.S. Kim, D.M. Shin, Y.H. Hwang, K. Kim, B. Shin, C. Kaewpiboon, H.H. Jeong, S.S. Koh, O.H. Kramer, R.N. Johnston, Y.H. Chung, Increased EGFR expression induced by a novel oncogene, CUG2, confers resistance to doxorubicin through Stat1-HDAC4 signaling, Cell. Oncol. 40 (2017) 549e561. S. Kaowinn, C. Kaewpiboon, S.S. Koh, O.H. Kramer, Y.H. Chung, STAT1HDAC4 signaling induces epithelialmesenchymal transition and sphere formation of cancer cells overexpressing the oncogene, CUG2, Oncol. rep. 40 (2018) 2619e2627. F.X. Yu, K.L. Guan, The Hippo pathway: regulators and regulations, Genes Dev. 27 (2013) 355e371. H. Liu, D. Jiang, F. Chi, B. Zhao, The Hippo pathway regulates stem cell proliferation, self-renewal, and differentiation, Protein & cell 3 (2012) 291e304. J. Avruch, D. Zhou, N. Bardeesy, YAP oncogene overexpression supercharges colon cancer proliferation, Cell Cycle 11 (2012) 1090e1096. C.A. Hall, R. Wang, J. Miao, E. Oliva, X. Shen, T. Wheeler, S.G. Hilsenbeck, S. Orsulic, S. Goode, Hippo pathway effector Yap is an ovarian cancer oncogene, Cancer Res. 70 (2010) 8517e8525. T. Pei, Y. Li, J. Wang, H. Wang, Y. Liang, H. Shi, B. Sun, D. Yin, J. Sun, R. Song, S. Pan, Y. Sun, H. Jiang, T. Zheng, L. Liu, YAP is a critical oncogene in human cholangiocarcinoma, Oncotarget 6 (2015) 17206e17220. T. Muramatsu, I. Imoto, T. Matsui, K. Kozaki, S. Haruki, M. Sudol, Y. Shimada, H. Tsuda, T. Kawano, J. Inazawa, YAP is a candidate oncogene for esophageal squamous cell carcinoma, Carcinogenesis 32 (2011) 389e398. W. Hong, K.L. Guan, The YAP and TAZ transcription co-activators: key downstream effectors of the mammalian Hippo pathway, Semin. Cell Dev. Biol. 23 (2012) 785e793. T. Kim, S.J. Yang, D. Hwang, J. Song, M. Kim, S. Kyum Kim, K. Kang, J. Ahn, D. Lee, M.Y. Kim, S. Kim, J. Seung Koo, S. Seok Koh, S.Y. Kim, D.S. Lim, A basallike breast cancer-specific role for SRF-IL6 in YAP-induced cancer stemness, Nat. Commun. 6 (2015) 10186. B. Zhou, P. Flodby, J. Luo, D.R. Castillo, Y. Liu, F.X. Yu, A. McConnell, B. Varghese, G. Li, N.O. Chimge, M. Sunohara, M.N. Koss, W. Elatre, P. Conti, J.M. Liebler, C. Yang, C.N. Marconett, I.A. Laird-Offringa, P. Minoo, K. Guan, B.R. Stripp, E.D. Crandall, Z. Borok, Claudin-18-mediated YAP activity regulates lung stem and progenitor cell homeostasis and tumorigenesis, J. Clin. Invest. 128 (2018) 970e984. B. Yan, Z. Jiang, L. Cheng, K. Chen, C. Zhou, L. Sun, W. Qian, J. Li, J. Cao, Q. Xu, Q. Ma, J. Lei, Paracrine HGF/c-MET enhances the stem cell-like potential and glycolysis of pancreatic cancer cells via activation of YAP/HIF-1alpha, Exp. Cell Res. 371 (2018) 63e71. J.H. Park, J.E. Shin, H.W. Park, The role of hippo pathway in cancer stem cell biology, Mol. Cell. 41 (2018) 83e92. W.M. Konsavage Jr., G.S. Yochum, Intersection of Hippo/YAP and Wnt/betacatenin signaling pathways, Acta Biochim. Biophys. Sin. 45 (2013) 71e79. M. Imajo, K. Miyatake, A. Iimura, A. Miyamoto, E. Nishida, A molecular mechanism that links Hippo signalling to the inhibition of Wnt/beta-catenin signalling, EMBO J. 31 (2012) 1109e1122. S.J. Kim, A. Glick, M.B. Sporn, A.B. Roberts, Characterization of the promoter region of the human transforming growth factor-beta 1 gene, J. Biol. Chem. 264 (1989) 402e408. Y. Qiao, Y. Qian, J. Wang, X. Tang, Melanoma cell adhesion molecule stimulates yes-associated protein transcription by enhancing CREB activity via c-Jun/cFos in hepatocellular carcinoma cells, Oncology letters 11 (2016) 3702e3708. W.I. Seo, S. Park, J. Gwak, B.G. Ju, J.I. Chung, P.M. Kang, S. Oh, Wnt signaling promotes androgen-independent prostate cancer cell proliferation through up-regulation of the hippo pathway effector YAP, Biochem. Biophys. Res. Commun. 486 (2017) 1034e1039. J. Rosenbluh, D. Nijhawan, A.G. Cox, X. Li, J.T. Neal, E.J. Schafer, T.I. Zack, X. Wang, A. Tsherniak, A.C. Schinzel, D.D. Shao, S.E. Schumacher, B.A. Weir, F. Vazquez, G.S. Cowley, D.E. Root, J.P. Mesirov, R. Beroukhim, C.J. Kuo, W. Goessling, W.C. Hahn, beta-Catenin-driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis, Cell 151 (2012) 1457e1473. M.K. Wendt, M. Tian, W.P. Schiemann, Deconstructing the mechanisms and consequences of TGF-beta-induced EMT during cancer progression, Cell Tissue Res. 347 (2012) 85e101. J. Fuxe, T. Vincent, A. Garcia de Herreros, Transcriptional crosstalk between TGF-beta and stem cell pathways in tumor cell invasion: role of EMT promoting Smad complexes, Cell Cycle 9 (2010) 2363e2374. W.M. Konsavage Jr., S.L. Kyler, S.A. Rennoll, G. Jin, G.S. Yochum, Wnt/betacatenin signaling regulates Yes-associated protein (YAP) gene expression in colorectal carcinoma cells, J. Biol. Chem. 287 (2012) 11730e11739. J. Cai, A. Maitra, R.A. Anders, M.M. Taketo, D. Pan, beta-Catenin destruction
128
S. Kaowinn et al. / Biochemical and Biophysical Research Communications 511 (2019) 122e128
complex-independent regulation of Hippo-YAP signaling by APC in intestinal tumorigenesis, Genes Dev. 29 (2015) 1493e1506. [27] S. Bahmanyar, D.D. Kaplan, J.G. Deluca, T.H. Giddings Jr., E.T. O'Toole, M. Winey, E.D. Salmon, P.J. Casey, W.J. Nelson, A.I. Barth, beta-Catenin is a Nek2 substrate involved in centrosome separation, Genes Dev. 22 (2008) 91e105. [28] M. Yu, Y. Chen, X. Li, R. Yang, L. Zhang, L. Huangfu, N. Zheng, X. Zhao, L. Lv, Y. Hong, H. Liang, H. Shan, YAP1 contributes to NSCLC invasion and migration by promoting Slug transcription via the transcription co-factor TEAD, Cell
Death Dis. 9 (2018) 464. [29] A.K. Luu, C.R. Schott, R. Jones, A.C. Poon, B. Golding, R. Hamed, B. Deheshi, A. Mutsaers, G.A. Wood, A.M. Viloria-Petit, An evaluation of TAZ and YAP crosstalk with TGFbeta signalling in canine osteosarcoma suggests involvement of hippo signalling in disease progression, BMC Vet. Res. 14 (2018) 365. [30] X. Zhang, Q. Fan, Y. Li, Z. Yang, L. Yang, Z. Zong, B. Wang, X. Meng, Q. Li, J. Liu, H. Li, Transforming growth factor-beta1 suppresses hepatocellular carcinoma proliferation via activation of Hippo signaling, Oncotarget 8 (2017) 29785e29794.