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TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase
Biochemical and Biophysical Research Communications xxx (2017) 1e6
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TEAD4-YAP interaction regulates tumoral growth by controlling cellcycle arrest at the G1 phase Shin Takeuchi a, Atsushi Kasamatsu b, *, Masanobu Yamatoji b, Dai Nakashima a, Yosuke Endo-Sakamoto b, Nao Koide a, Toshikazu Takahara a, Toshihiro Shimizu c, Manabu Iyoda d, Katsunori Ogawara e, Masashi Shiiba f, Hideki Tanzawa a, b, Katsuhiro Uzawa a, b, ** a
Department of Oral Science, Graduate School of Medicine, Chiba University, Chiba, Japan Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, Chiba, Japan c Division of Oral Surgery, Kashima Rosai Hospital, Ibaraki, Japan d Division of Oral Surgery, Chiba Rosai Hospital, Chiba, Japan e Division of Oral Surgery and Oral Implant Center, Funabashi Central Hospital, Chiba, Japan f Department of Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 March 2017 Accepted 12 March 2017 Available online xxx
TEA domain transcription factor 4 (TEAD4), which has critical functions in the process of embryonic development, is expressed in various cancers. However, the important role of TEAD4 in human oral squamous cell carcinomas (OSCCs) remain unclear. Here we investigated the TEAD4 expression level and the functional mechanism in OSCC using quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. Furthermore, TEAD4 knockdown model was used to evaluate cellular proliferation, cell-cycle analysis, and the interaction between TEAD4 and Yes-associated protein (YAP) which was reported to be a transcription coactivator of cellular proliferation. In the current study, we found that TEAD4 expression increased significantly in vitro and in vivo and correlated with tumoral size in OSCC patients. TEAD4 knockdown OSCC cells showed decreased cellular proliferation resulting from cell-cycle arrest in the G1 phase by down-regulation of cyclins, cyclin-dependent kinases (CDKs), and up-regulation of CDK inhibitors. We also found that the TEAD4-YAP complex in the nuclei may be related closely to transcriptions of G1 arrest-related genes. Taken together, we concluded that TEAD4 might play an important role in tumoral growth and have potential to be a therapeutic target in OSCCs. © 2017 Elsevier Inc. All rights reserved.
Keywords: Human oral squamous cell carcinoma TEA domain transcription factor 4 Yes-associated protein (YAP)
1. Introduction The TEA domain transcription factor (TEAD) family member, TEAD1-4, has the same domain structures, i.e., a TEA domain for DNA binding and a transactivation domain [1,2]. TEADs are required for the coactivators to participate in transmittance of the signaling pathways to the downstream processes [3]. To date, several
* Corresponding author. Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. ** Corresponding author. Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail addresses: [email protected] (A. Kasamatsu), uzawak@ faculty.chiba-u.jp (K. Uzawa).
candidate coactivators for TEADs have been identified including Yes-associated protein (YAP) [3,4]. Since YAP does not bind directly to DNA, YAP couples with TEADs to activate the transcriptional events. The TEAD-YAP complex enhances multiple processes important in cellular proliferation, transformation, migration, and invasion [5e7]. Among the TEAD family members, TEAD4 has critical roles in tumoral progression in several cancers, such as breast and colorectal cancers, and promotes cellular growth, metastasis, and poor survival rates, suggesting that it has the potential to be a key molecule in cancer therapy [8,9]. However, the critical roles of TEAD4 in human oral squamous cell carcinomas (OSCCs) remain unknown. The current study found that TEAD4 expression increased
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Please cite this article in press as: S. Takeuchi, et al., TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.050
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S. Takeuchi et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
significantly in OSCCs and that TEAD4 was correlated closely with cell-cycle arrest in the G1 phase. Our data indicated that TEAD4 might potentially be a biomarker for tumoral growth. 2. Materials and methods 2.1. Ethics statement The Chiba University Ethical Review Board approved the study protocol (approval number 236), which was performed according to the tenets of the Helsinki Declaration. All patients provided written informed consent before participating in the study. 2.2. OSCC-derived cell lines and tissue specimens The Human Science Research Resources Bank (Osaka, Japan) and the RIKEN Bio Resource Center (Ibaraki, Japan) provided immortalized human OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, Sa3, Ca9-22, Ho-1-N-1, Ho-1-u-1, KOSC-2, and SAS). We donated primary culture human normal oral keratinocytes (HNOKs) from the donor who was 3 healthy human and it served as a normal control. As previously described, all OSCC-derived cells and HNOKs were cultured [10e13]. 2.3. mRNA expression analysis Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed as described previously [14e19]. The primer sets were TEAD4 (50 -CTCCACGAAGGTCTGCTCTT-30 and 50 GTCCATTCTCATAGCGAGCATA-30 ) and universal probe #22.
according to the manufacturer's protocol (Thermo Fisher Scientific, Peabody, MA, USA). Briefly, we added 200 ml of CER I buffer to the cell pellet and incubated it on ice. We then added 11 ml of CER II buffer, centrifuged a tube for 5 min at 16,000g, and transferred the supernatant (cytoplasmic extract) to a new sampling tube. We added 100 ml of NER buffer to the pellet and incubated it on ice for 10 min. Finally, we centrifuged the tube for 10 min and transferred the supernatant (nuclear extract). 2.9. Cell-cycle analysis We analyzed the preparation for a cell-cycle as described before [14e17,19]. We used nocodazole (Sigma) to synchronize the cellcycle at the M transition. 2.10. Statistical analysis The c2 test, Mann-Whitney U test, Student's t-test, and Fisher's exact test were performed to identify significant correlations. We used receiver operating characteristic (ROC) curves to detect a cutoff point to judge whether TEAD4 was positive or negative for the classified clinical parameters. As previously described, we also determined the area under the ROC curve values to confirm the usefulness of this experimentation [20,21]. The data are expressed as the mean ± the standard error of the mean. 3. Results 3.1. Up-regulation of TEAD4 in OSCC-Derived cell lines
2.4. Western blot analysis As previously described, Western blot analysis was performed [14e19]. The antibodies used were rabbit anti-TEAD4 antibody (Abcam, Cambridge, UK), murine anti-GAPDH antibody, murine YAP antibody, murine anti-cyclin D1 antibody, murine anti-cyclin E antibody, and murine anti-cyclin-dependent kinase 6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit antiphosphorylated-YAP (p-YAP) antibody, rabbit anti-CDK2 antibody, rabbit anti-CDK4 antibody, rabbit anti-p21cip1 antibody, and murine anti-p27kip1 antibody (Cell Signaling Technology, Danvers, MA, USA). 2.5. Immunohistochemistry (IHC) As previously described, we performed IHC and used the IHC scoring system [14e19]. 2.6. Transfection with shRNA plasmid As previously described, to establish knockdown transfectants, the cell lines HSC3 and Sa3 were transfected with TEAD4 shRNA (shTEAD4) or control shRNA (shMock) vectors (Santa Cruz Biotechnology) [14e19].
To evaluate TEAD4 expression, qRT-PCR and Western blot analysis were performed using nine OSCC-derived cell lines and HNOKs. TEAD4 mRNA and protein expressions were up-regulated significantly in all OSCC cell lines compared with the HNOKs (Fig. 1A and B, p < 0.05). Both transcription and translational products of TEAD4 were greater in all OSCC cell lines than in the HNOKs. 3.2. Evaluation of TEAD4 expression in primary OSCCs Positive and negative stainings for TEAD4 were seen in primary OSCC samples and normal tissue samples, respectively (Fig. 1C and D). We used the IHC scoring system with the tissue samples from 100 patients with OSCC to study the clinical correlations between the TEAD4 expression in primary OSCC and the pathological characteristics. The TEAD4 expression levels in the OSCC tissues were significantly higher than in normal tissues (p < 0.05). The TEAD4 IHC scores in the adjacent normal tissues and OSCC tissues ranged from 43.4 to 228.6 (median, 85.0) and 13.6 to 95.8 (median, 39.2), respectively (Fig. 1E). In the clinical classifications, TEAD4-positive OSCCs were associated significantly with the primary tumoral size (Table 1, p < 0.05).
2.7. Cellular growth analysis
3.3. Establishment of TEAD4 knockdown cells
As previously described, Cellular proliferation analysis was performed [14e19]. We used nocodazole (Sigma, St. Louis, MO, USA) to synchronize the cell-cycle at the M transition.
Since significant up-regulation of TEAD4 occurred in OSCCderived cells (Fig. 1A and B), the HSC-3 and Sa3 cells were transfected with TEAD4 shRNA and shMock vectors (control). To verify the efficiency of the transfections, we performed qRT-PCR and Western blot analysis (Fig. 2A and B). The mRNA expression and protein levels of shTEAD4 cells were down-regulated significantly compared with shMock cells (Fig. 2A, B, p < 0.05).
2.8. Preparation of cytoplasmic and nuclear extracts To separate cytoplasmic and nuclear extracts, we used NE-PER
Please cite this article in press as: S. Takeuchi, et al., TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.050
S. Takeuchi et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
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Fig. 1. Evaluation of TEAD4 expression in OSCC-derived cell lines and primary OSCCs. (A) Quantification of TEAD4 mRNA expression in OSCC-derived cell lines by qRT-PCR analysis. Significant (*p < 0.05, Student's t-test) up-regulation of TEAD4 mRNA is seen in nine OSCC-derived cell lines compared with the HNOKs. (B) Western blot analysis of TEAD4 protein in OSCC-derived cell lines and HNOCKs. TEAD4 protein expression is up-regulated in OSCC-derived cell lines compared with that in HNOKs. Densitometric data are normalized to GAPDH protein levels. The values are expressed as a percentage of HNOKs. (C, D) Representative IHC results for TEAD4 protein in normal oral tissue (C) and primary OSCC tissue (D). Original magnification, 100. (E) The status of TEAD4 protein expression in primary OSCCs (n ¼ 100) and the normal counterparts. The TEAD4 IHC scores of the normal oral tissues range from 13.6 to 95.8 (median, 39.2); and those of the OSCCs range from 43.4 to 228.6 (median, 85.0). TEAD4 protein expression levels in OSCCs are significantly (*p < 0.05, Student's t-test) greater than in normal oral tissues.
3.4. Cellular proliferation of TEAD4 knockdown cells Table 1 Correlation between TEAD4 expression and clinical classification in OSCCs. Clinical classification
Total
Immunostaining results No. patients TEAD4-negative
Age at surgery (years) <70 56 29 S70 44 12 Gender Male 62 26 Female 38 15 Primary tumoral size T1þT2 53 31 T3þT4 47 10 Regional lymph node metastasis Negative 67 26 Positive 33 15 Vascular invasion Negative 81 35 Positive 19 6 Stage I þ II 43 24 III þ IV 57 17 * 2
c test. Fisher's exact test. zP < 0.05. y
p-value
We confirmed the effect of TEAD4 knockdown on cellular proliferation and found a significant decrease of cellular growth in shTEAD4 cells compared with shMock cells (Fig. 2C, p < 0.05). 3.5. Localizations of YAP in TEAD4 knockdown cells
TEAD4-positive 27 32
0.305*
36 23
0.932y
22 37
0.028yz
41 18
0.489y
46 13
0.378y
19 40
0.102y
Since TEAD4 forms a complex with YAP in nuclei, and the complex regulates tumoral progression in several cancers [3,4,7,8], we analyzed the YAP and p-YAP expression levels using nuclear and cytoplasmic fractions. YAP was down-regulated in the nuclear fraction, and p-YAP was up-regulated in the cytoplasmic fraction of shTEAD4 cells compared with that in shMock cell (Fig. 3). 3.6. Effect of TEAD4 knockdown on cell-cycle distribution CDK6, a cell-cycle-related gene in the G1 phase, is a downstream molecule of the TEAD4-YAP complex [22]. Therefore, we evaluated the expression levels of the G1 phase-related proteins and cellcycle distribution. The expression levels of the cyclin-dependent kinase inhibitors (p21cip1 and p27kip1) were up-regulated; in contrast, cyclin D1 and cyclin E and CDK2, CDK4, and CDK6 were down-regulated significantly in shTEAD4 cells (Fig. 4, p < 0.05). We also assessed the cell-cycle distribution in the TEAD4 knockdown
Please cite this article in press as: S. Takeuchi, et al., TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.050
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Fig. 2. Establishment of TEAD4 knockdown cells and evaluation of cell proliferation ability of TEAD4 knockdown cells. (A) qRT-PCR shows that mRNA levels of TEAD4 are significantly (*p < 0.05, Student's t-test) down-regulated in shTEAD4 cells (Sa3 and HSC-3-derived transfectants) compared with shMock cells. (B) Western blot analysis and the densitometric data indicate that TEAD4 protein levels are decreased markedly (*p < 0.05, Student's t-test) compared with shMock cells. The data was normalized to GAPDH protein levels. (C) In shTEAD4 cells, the cellular growth is inhibited significantly (*p < 0.05, Student's t-test) after 168 h. The results are expressed as the means ± the standard error of the mean values from three assays.
cells by flow cytometry. The ratio of the cells in the G1 phase in shTEAD4 cells was significantly higher than in shMock cells (Fig. 4, p < 0.05), suggesting that TEAD4 expression is related closely to cell-cycle regulation of the G1 phase. 4. Discussion In the current study, we found that TEAD4 expression increased significantly in OSCC-derived cell lines and clinical OSCC samples (Fig. 1). TEAD4 knockdown OSCC cells showed decreased cellular
proliferation (Fig. 2C) resulting from cell-cycle arrest in the G1 phase. We also found that the TEAD4-YAP complex in the nuclei was related closely to transcriptions of G1 arrest-related genes (Figs. 3 and 4). Similar to the current data, TEAD4 protein along with YAP was found frequently in the nuclei of gastric cancer cells. In addition, TEAD4 knockdown resulted in decreased cellular growth of gastric cancer cells in vitro and in vivo [23]. Although there was no relationship between the TEAD4 expression status and regional lymph node metastasis in OSCCs, breast cancer and malignant melanoma
Please cite this article in press as: S. Takeuchi, et al., TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.050
S. Takeuchi et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
Fig. 3. Localizations of YAP in TEAD4 knockdown cells. Western blot analysis is performed using nuclear and cytoplasmic fractions. YAP is down-regulated in the nuclear fraction of shTEAD4 cells (Sa3 and HSC-3-derived transfectants) compared with that of shMock cells. p-YAP is up-regulated in the cytoplasmic fraction compared with the counterparts. N, nuclear fraction; C, cytoplasmic fraction.
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present the metastatic potentials of those cells by increasing TEAD transcriptional activity [24]. Therefore, TEAD4 plays pivotal roles in tumoral progression and metastasis of several cancers. The TEAD4-YAP complex regulates multiple functions of cancers, i.e., cellular growth, migration, and invasiveness. Phosphorylation of YAP by LATS1/2 tumor suppressor kinase leads to cytoplasmic translocation and inactivation of the YAP [25e30]. Interestingly, our observations (Fig. 3) showed that TEAD4 knockdown resulted in highly phosphorylated levels of YAP and decreased YAP in the nuclei. Therefore, TEAD4 expression levels might control not only DNA binding and transcriptional activities with YAP but also the phosphorylation levels of YAP. CDK6 is the downstream target of the TEAD4-YAP complex for cellular senescence [22]. Since CDK6 is a key molecule of the cellcycle in the G1 phase, we speculated that the inactivated TEAD4YAP complex (TEAD4 knockdown) showed cell-cycle arrest in the G1 phase. Consistent with our hypothesis, less tumoral progression occurred in shTEAD4 cells and patients with OSCC negative for TEAD4. We concluded that TEAD4 has a biologic role in human oral cancer. Overexpression of TEAD4 especially increased tumoral size in primary OSCCs via regulation of the cell-cycle via the activity of the TEAD4-YAP complex. While further studies are required, TEAD4 might be a critical biomarker and a therapeutic target for OSCCs. Acknowledgements We thank Lynda C. Charters for editing this manuscript. The authors received no financial support. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.03.050. Disclosure statement The authors declare no conflicts of interest. References
Fig. 4. Effect of TEAD4 knockdown on cell-cycle distribution. (A) Western blot analysis shows up-regulation of p21cip1 and p27kip1 and down-regulation of cyclin D1, cyclin E, CDK2, CDK4, and CDK6 in shTEAD4 cells (Sa3 and HSC-3-derived transfectants) compared with shMock cells. (B) Flow cytometric analysis is performed to investigate cell-cycle progression in shTEAD4 and shMock cells after synchronization at the M phase using nocodazole. The percentage of cells in the G1 phase in shTEAD4 cells is increased compared with shMock cells.
[1] T.R. Bürglin, The TEA domain: a novel, highly conserved DNA-binding motif, Cell 66 (1991) 11e12, http://dx.doi.org/10.1016/0092-8674(91)90132-I. [2] B. Zhao, X. Ye, J. Yu, L. Li, W. Li, S. Li, J. Yu, J.D. Lin, TEAD mediates YAPdependent gene induction and growth control TEAD mediates YAPdependent gene induction and growth control, Genes Dev. (2008) 1962e1971, http://dx.doi.org/10.1101/gad.1664408. [3] Y. Zhou, T. Huang, A.S.L. Cheng, J. Yu, W. Kang, K.F. To, The TEAD family and its oncogenic role in promoting tumorigenesis, Int. J. Mol. Sci. 17 (2016) 1e15, http://dx.doi.org/10.3390/ijms17010138. [4] K. Zhang, H.X. Qi, Z.M. Hu, Y.N. Chang, Z.M. Shi, X.H. Han, Y.W. Han, R.X. Zhang, Z. Zhang, T. Chen, W. Hong, YAP and TAZ take center stage in cancer, Biochemistry 54 (2015) 6555e6566, http://dx.doi.org/10.1021/ acs.biochem.5b01014. [5] Z. Li, B. Zhao, P. Wang, F. Chen, Z. Dong, H. Yang, Structural insights into the YAP and TEAD complex service Structural insights into the YAP and TEAD complex, Genes Dev. (2010) 235e240, http://dx.doi.org/10.1101/ gad.1865810. [6] T. Yoshida, MCAT elements and the TEF-1 family of transcription factors in muscle development and disease, Arterioscler. Thromb. Vasc. Biol. 28 (2008) 8e17, http://dx.doi.org/10.1161/ATVBAHA.107.155788. [7] B. Zhao, B. Zhao, X. Wei, X. Wei, W. Li, W. Li, R.S. Udan, R.S. Udan, Q. Yang, Q. Yang, J. Kim, J. Kim, J. Xie, J. Xie, T. Ikenoue, T. Ikenoue, J. Yu, J. Yu, L. Li, L. Li, P. Zheng, P. Zheng, K. Ye, K. Ye, A. Chinnaiyan, A. Chinnaiyan, G. Halder, G. Halder, Z. Lai, Z. Lai, K.-L. Guan, K.-L. Guan, Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control, Genes Dev. 21 (2007) 2747e2761, http://dx.doi.org/10.1101/ gad.1602907.Hpo/Sav. [8] C. Wang, Z. Nie, Z. Zhou, H. Zhang, R. Liu, J. Wu, J. Qin, Y. Ma, L. Chen, S. Li, W. Chen, F. Li, P. Shi, Y. Wu, J. Shen, C. Chen, The interplay between TEAD4 and KLF5 promotes breast cancer partially through inhibiting the transcription of p27Kip1, Oncotarget 6 (2015), http://dx.doi.org/10.18632/oncotarget.3779.
Please cite this article in press as: S. Takeuchi, et al., TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.050
6
S. Takeuchi et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
[9] Y. Liu, G. Wang, Y. Yang, Z. Mei, Z. Liang, A. Cui, T. Wu, C.-Y. Liu, L. Cui, Increased TEAD4 expression and nuclear localization in colorectal cancer promote epithelial-mesenchymal transition and metastasis in a YAPindependent manner, Oncogene (2015) 1e12, http://dx.doi.org/10.1038/ onc.2015.342. [10] A. Kasamatsu, K. Uzawa, D. Nakashima, H. Koike, M. Shiiba, H. Bukawa, H. Yokoe, H. Tanzawa, Galectin-9 as a regulator of cellular adhesion in human oral squamous cell carcinoma cell lines, Int. J. Mol. Med. 16 (2005) 269e273. [11] Y. Endo, K. Uzawa, Y. Mochida, M. Shiiba, H. Bukawa, H. Yokoe, H. Tanzawa, Sarcoendoplasmic reticulum Ca(2þ) ATPase type 2 downregulated in human oral squamous cell carcinoma, Int. J. Cancer 231 (2004) 225e231, http:// dx.doi.org/10.1002/ijc.20118. [12] M. Yamatoji, A. Kasamatsu, Y. Yamano, K. Sakuma, K. Ogoshi, M. Iyoda, K. Shinozuka, K. Ogawara, Y. Takiguchi, M. Shiiba, H. Tanzawa, K. Uzawa, State of homeobox A10 expression as a putative prognostic marker for oral squamous cell carcinoma, Oncol. Rep. 23 (2010) 61e67, http://dx.doi.org/10.3892/ or-00000606. [13] K. Sakuma, A. Kasamatsu, M. Yamatoji, Y. Yamano, K. Fushimi, M. Iyoda, K. Ogoshi, K. Shinozuka, K. Ogawara, M. Shiiba, H. Tanzawa, K. Uzawa, Expression status of Zic family member 2 as a prognostic marker for oral squamous cell carcinoma, J. Cancer Res. Clin. Oncol. 136 (2010) 553e559, http://dx.doi.org/10.1007/s00432-009-0689-y. [14] Y. Minakawa, A. Kasamatsu, H. Koike, M. Higo, D. Nakashima, Y. Kouzu, Y. Sakamoto, K. Ogawara, M. Shiiba, H. Tanzawa, K. Uzawa, Kinesin family member 4A: a potential predictor for progression of human oral cancer, PLoS One 8 (2013) 1e8, http://dx.doi.org/10.1371/journal.pone.0085951. [15] T. Koyama, K. Ogawara, A. Kasamatsu, A. Okamoto, H. Kasama, Y. Minakawa, K. Shimada, H. Yokoe, M. Shiiba, H. Tanzawa, K. Uzawa, ANGPTL3 is a novel biomarker as it activates ERK/MAPK pathway in oral cancer, Cancer Med. 4 (2015) 759e769, http://dx.doi.org/10.1002/cam4.418. [16] C. Fukumoto, D. Nakashima, A. Kasamatsu, M. Unozawa, T. Shida-Sakazume, M. Higo, K. Ogawara, H. Yokoe, M. Shiiba, H. Tanzawa, K. Uzawa, WWP2 is overexpressed in human oral cancer, determining tumor size and poor prognosis in patients: downregulation of WWP2 inhibits the AKT signaling and tumor growth in mice, Oncoscience 1 (2014) 807e820, http://dx.doi.org/ 10.18632/oncoscience.101. [17] I. Miyamoto, A. Kasamatsu, M. Yamatoji, D. Nakashima, K. Saito, M. Higo, Y. Endo-Sakamoto, M. Shiiba, H. Tanzawa, K. Uzawa, Kinesin family member 14 in human oral cancer: a potential biomarker for tumoral growth, Biochem. Biophys. Rep. 3 (2015) 26e31, http://dx.doi.org/10.1016/j.bbrep.2015.07.008. [18] Y. Kimura, A. Kasamatsu, D. Nakashima, M. Yamatoji, Y. Minakawa, K. Koike, K. Fushimi, M. Higo, Y. Endo-Sakamoto, M. Shiiba, H. Tanzawa, K. Uzawa, ARNT2 regulates tumoral growth in oral squamous cell carcinoma, J. Cancer 7 (2016) 702e710, http://dx.doi.org/10.7150/jca.14208. [19] T. Saito, A. Kasamatsu, K. Ogawara, I. Miyamoto, K. Saito, M. Iyoda, T. Suzuki, Y. Endo-Sakamoto, M. Shiiba, H. Tanzawa, K. Uzawa, Semaphorin7A
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27] [28] [29]
[30]
promotion of tumoral growth and metastasis in human oral cancer by regulation of g1 cell cycle and matrix metalloproteases: possible contribution to tumoral angiogenesis, PLoS One 10 (2015) 1e20, http://dx.doi.org/10.1371/ journal.pone.0137923. K. Uzawa, T. Baba, F. Uchida, M. Yamatoji, A. Kasamatsu, Y. Sakamoto, K. Ogawara, M. Shiiba, H. Bukawa, H. Tanzawa, Circulating tumor-derived mutant mitochondrial DNA: a predictive biomarker of clinical prognosis in human squamous cell carcinoma, Oncotarget 3 (2012) 670e677 doi:523 [pii]. K. Uzawa, A. Kasamatsu, T. Baba, Y. Kimura, D. Nakashima, M. Higo, Y. Sakamoto, K. Ogawara, M. Shiiba, H. Tanzawa, Quantitative detection of circulating tumor-derived mitochondrial NADH subunit variants as a potential prognostic biomarker for oral cancer, Int. J. Oncol. (2015) 1077e1083, http:// dx.doi.org/10.3892/ijo.2015.3083. Q. Xie, J. Chen, H. Feng, S. Peng, U. Adams, Y. Bai, L. Huang, J. Li, J. Huang, S. Meng, Z. Yuan, YAP/TEAD-mediated transcription controls cellular senescence, Cancer Res. 73 (2013) 3615e3624, http://dx.doi.org/10.1158/00085472.CAN-12-3793. B. Lim, J.L. Park, H.J. Kim, Y.K. Park, J.H. Kim, H.A. Sohn, S.M. Noh, K.S. Song, W.H. Kim, Y.S. Kim, S.Y. Kim, Integrative genomics analysis reveals the multilevel dysregulation and oncogenic characteristics of TEAD4 in gastric cancer, Carcinogenesis 35 (2014) 1020e1027, http://dx.doi.org/10.1093/carcin/bgt409. J.M. Lamar, P. Stern, H. Liu, J.W. Schindler, Z.-G. Jiang, R.O. Hynes, The Hippo pathway target, YAP, promotes metastasis through its TEAD-interaction domain, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) E2441eE2450, http:// dx.doi.org/10.1073/pnas.1212021109. J.-W. Jang, M.-K. Kim, Y.-S. Lee, J.-W. Lee, D.-M. Kim, S.-H. Song, J.-Y. Lee, B.Y. Choi, B. Min, X.-Z. Chi, S.-C. Bae, RAC-LATS1/2 signaling regulates YAP activity by switching between the YAP-binding partners TEAD4 and RUNX3, Oncogene (2016) 1e13, http://dx.doi.org/10.1038/onc.2016.266. N. Nishioka, K. ichi Inoue, K. Adachi, H. Kiyonari, M. Ota, A. Ralston, N. Yabuta, S. Hirahara, R.O. Stephenson, N. Ogonuki, R. Makita, H. Kurihara, E.M. MorinKensicki, H. Nojima, J. Rossant, K. Nakao, H. Niwa, H. Sasaki, The hippo signaling pathway components lats and yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass, Dev. Cell. 16 (2009) 398e410, http://dx.doi.org/10.1016/j.devcel.2009.02.003. F.X. Yu, K.L. Guan, The Hippo pathway: regulators and regulations, Genes Dev. 27 (2013) 355e371, http://dx.doi.org/10.1101/gad.210773.112. E. Lever, D. Sheer, The role of nuclear organization in cancer, J. Pathol. 220 (2010) 114e125, http://dx.doi.org/10.1002/path. B. Zhao, L. Li, Q. Lu, L.H. Wang, C.Y. Liu, Q. Lei, K.L. Guan, Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein, Genes Dev. 25 (2011) 51e63, http://dx.doi.org/10.1101/gad.2000111. B.Z. Stanger, Quit your YAPing: a new target for cancer therapy, Genes Dev. 26 (2012) 1263e1267, http://dx.doi.org/10.1101/gad.196501.112.specificity.
Please cite this article in press as: S. Takeuchi, et al., TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.050
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TEAD4-YAP interaction regulates tumoral growth by controlling cellcycle arrest at the G1 phase Shin Takeuchi a, Atsushi Kasamatsu b, *, Masanobu Yamatoji b, Dai Nakashima a, Yosuke Endo-Sakamoto b, Nao Koide a, Toshikazu Takahara a, Toshihiro Shimizu c, Manabu Iyoda d, Katsunori Ogawara e, Masashi Shiiba f, Hideki Tanzawa a, b, Katsuhiro Uzawa a, b, ** a
Department of Oral Science, Graduate School of Medicine, Chiba University, Chiba, Japan Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, Chiba, Japan c Division of Oral Surgery, Kashima Rosai Hospital, Ibaraki, Japan d Division of Oral Surgery, Chiba Rosai Hospital, Chiba, Japan e Division of Oral Surgery and Oral Implant Center, Funabashi Central Hospital, Chiba, Japan f Department of Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 March 2017 Accepted 12 March 2017 Available online xxx
TEA domain transcription factor 4 (TEAD4), which has critical functions in the process of embryonic development, is expressed in various cancers. However, the important role of TEAD4 in human oral squamous cell carcinomas (OSCCs) remain unclear. Here we investigated the TEAD4 expression level and the functional mechanism in OSCC using quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. Furthermore, TEAD4 knockdown model was used to evaluate cellular proliferation, cell-cycle analysis, and the interaction between TEAD4 and Yes-associated protein (YAP) which was reported to be a transcription coactivator of cellular proliferation. In the current study, we found that TEAD4 expression increased significantly in vitro and in vivo and correlated with tumoral size in OSCC patients. TEAD4 knockdown OSCC cells showed decreased cellular proliferation resulting from cell-cycle arrest in the G1 phase by down-regulation of cyclins, cyclin-dependent kinases (CDKs), and up-regulation of CDK inhibitors. We also found that the TEAD4-YAP complex in the nuclei may be related closely to transcriptions of G1 arrest-related genes. Taken together, we concluded that TEAD4 might play an important role in tumoral growth and have potential to be a therapeutic target in OSCCs. © 2017 Elsevier Inc. All rights reserved.
Keywords: Human oral squamous cell carcinoma TEA domain transcription factor 4 Yes-associated protein (YAP)
1. Introduction The TEA domain transcription factor (TEAD) family member, TEAD1-4, has the same domain structures, i.e., a TEA domain for DNA binding and a transactivation domain [1,2]. TEADs are required for the coactivators to participate in transmittance of the signaling pathways to the downstream processes [3]. To date, several
* Corresponding author. Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. ** Corresponding author. Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail addresses: [email protected] (A. Kasamatsu), uzawak@ faculty.chiba-u.jp (K. Uzawa).
candidate coactivators for TEADs have been identified including Yes-associated protein (YAP) [3,4]. Since YAP does not bind directly to DNA, YAP couples with TEADs to activate the transcriptional events. The TEAD-YAP complex enhances multiple processes important in cellular proliferation, transformation, migration, and invasion [5e7]. Among the TEAD family members, TEAD4 has critical roles in tumoral progression in several cancers, such as breast and colorectal cancers, and promotes cellular growth, metastasis, and poor survival rates, suggesting that it has the potential to be a key molecule in cancer therapy [8,9]. However, the critical roles of TEAD4 in human oral squamous cell carcinomas (OSCCs) remain unknown. The current study found that TEAD4 expression increased
http://dx.doi.org/10.1016/j.bbrc.2017.03.050 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: S. Takeuchi, et al., TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.050
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S. Takeuchi et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
significantly in OSCCs and that TEAD4 was correlated closely with cell-cycle arrest in the G1 phase. Our data indicated that TEAD4 might potentially be a biomarker for tumoral growth. 2. Materials and methods 2.1. Ethics statement The Chiba University Ethical Review Board approved the study protocol (approval number 236), which was performed according to the tenets of the Helsinki Declaration. All patients provided written informed consent before participating in the study. 2.2. OSCC-derived cell lines and tissue specimens The Human Science Research Resources Bank (Osaka, Japan) and the RIKEN Bio Resource Center (Ibaraki, Japan) provided immortalized human OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, Sa3, Ca9-22, Ho-1-N-1, Ho-1-u-1, KOSC-2, and SAS). We donated primary culture human normal oral keratinocytes (HNOKs) from the donor who was 3 healthy human and it served as a normal control. As previously described, all OSCC-derived cells and HNOKs were cultured [10e13]. 2.3. mRNA expression analysis Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed as described previously [14e19]. The primer sets were TEAD4 (50 -CTCCACGAAGGTCTGCTCTT-30 and 50 GTCCATTCTCATAGCGAGCATA-30 ) and universal probe #22.
according to the manufacturer's protocol (Thermo Fisher Scientific, Peabody, MA, USA). Briefly, we added 200 ml of CER I buffer to the cell pellet and incubated it on ice. We then added 11 ml of CER II buffer, centrifuged a tube for 5 min at 16,000g, and transferred the supernatant (cytoplasmic extract) to a new sampling tube. We added 100 ml of NER buffer to the pellet and incubated it on ice for 10 min. Finally, we centrifuged the tube for 10 min and transferred the supernatant (nuclear extract). 2.9. Cell-cycle analysis We analyzed the preparation for a cell-cycle as described before [14e17,19]. We used nocodazole (Sigma) to synchronize the cellcycle at the M transition. 2.10. Statistical analysis The c2 test, Mann-Whitney U test, Student's t-test, and Fisher's exact test were performed to identify significant correlations. We used receiver operating characteristic (ROC) curves to detect a cutoff point to judge whether TEAD4 was positive or negative for the classified clinical parameters. As previously described, we also determined the area under the ROC curve values to confirm the usefulness of this experimentation [20,21]. The data are expressed as the mean ± the standard error of the mean. 3. Results 3.1. Up-regulation of TEAD4 in OSCC-Derived cell lines
2.4. Western blot analysis As previously described, Western blot analysis was performed [14e19]. The antibodies used were rabbit anti-TEAD4 antibody (Abcam, Cambridge, UK), murine anti-GAPDH antibody, murine YAP antibody, murine anti-cyclin D1 antibody, murine anti-cyclin E antibody, and murine anti-cyclin-dependent kinase 6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit antiphosphorylated-YAP (p-YAP) antibody, rabbit anti-CDK2 antibody, rabbit anti-CDK4 antibody, rabbit anti-p21cip1 antibody, and murine anti-p27kip1 antibody (Cell Signaling Technology, Danvers, MA, USA). 2.5. Immunohistochemistry (IHC) As previously described, we performed IHC and used the IHC scoring system [14e19]. 2.6. Transfection with shRNA plasmid As previously described, to establish knockdown transfectants, the cell lines HSC3 and Sa3 were transfected with TEAD4 shRNA (shTEAD4) or control shRNA (shMock) vectors (Santa Cruz Biotechnology) [14e19].
To evaluate TEAD4 expression, qRT-PCR and Western blot analysis were performed using nine OSCC-derived cell lines and HNOKs. TEAD4 mRNA and protein expressions were up-regulated significantly in all OSCC cell lines compared with the HNOKs (Fig. 1A and B, p < 0.05). Both transcription and translational products of TEAD4 were greater in all OSCC cell lines than in the HNOKs. 3.2. Evaluation of TEAD4 expression in primary OSCCs Positive and negative stainings for TEAD4 were seen in primary OSCC samples and normal tissue samples, respectively (Fig. 1C and D). We used the IHC scoring system with the tissue samples from 100 patients with OSCC to study the clinical correlations between the TEAD4 expression in primary OSCC and the pathological characteristics. The TEAD4 expression levels in the OSCC tissues were significantly higher than in normal tissues (p < 0.05). The TEAD4 IHC scores in the adjacent normal tissues and OSCC tissues ranged from 43.4 to 228.6 (median, 85.0) and 13.6 to 95.8 (median, 39.2), respectively (Fig. 1E). In the clinical classifications, TEAD4-positive OSCCs were associated significantly with the primary tumoral size (Table 1, p < 0.05).
2.7. Cellular growth analysis
3.3. Establishment of TEAD4 knockdown cells
As previously described, Cellular proliferation analysis was performed [14e19]. We used nocodazole (Sigma, St. Louis, MO, USA) to synchronize the cell-cycle at the M transition.
Since significant up-regulation of TEAD4 occurred in OSCCderived cells (Fig. 1A and B), the HSC-3 and Sa3 cells were transfected with TEAD4 shRNA and shMock vectors (control). To verify the efficiency of the transfections, we performed qRT-PCR and Western blot analysis (Fig. 2A and B). The mRNA expression and protein levels of shTEAD4 cells were down-regulated significantly compared with shMock cells (Fig. 2A, B, p < 0.05).
2.8. Preparation of cytoplasmic and nuclear extracts To separate cytoplasmic and nuclear extracts, we used NE-PER
Please cite this article in press as: S. Takeuchi, et al., TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.050
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Fig. 1. Evaluation of TEAD4 expression in OSCC-derived cell lines and primary OSCCs. (A) Quantification of TEAD4 mRNA expression in OSCC-derived cell lines by qRT-PCR analysis. Significant (*p < 0.05, Student's t-test) up-regulation of TEAD4 mRNA is seen in nine OSCC-derived cell lines compared with the HNOKs. (B) Western blot analysis of TEAD4 protein in OSCC-derived cell lines and HNOCKs. TEAD4 protein expression is up-regulated in OSCC-derived cell lines compared with that in HNOKs. Densitometric data are normalized to GAPDH protein levels. The values are expressed as a percentage of HNOKs. (C, D) Representative IHC results for TEAD4 protein in normal oral tissue (C) and primary OSCC tissue (D). Original magnification, 100. (E) The status of TEAD4 protein expression in primary OSCCs (n ¼ 100) and the normal counterparts. The TEAD4 IHC scores of the normal oral tissues range from 13.6 to 95.8 (median, 39.2); and those of the OSCCs range from 43.4 to 228.6 (median, 85.0). TEAD4 protein expression levels in OSCCs are significantly (*p < 0.05, Student's t-test) greater than in normal oral tissues.
3.4. Cellular proliferation of TEAD4 knockdown cells Table 1 Correlation between TEAD4 expression and clinical classification in OSCCs. Clinical classification
Total
Immunostaining results No. patients TEAD4-negative
Age at surgery (years) <70 56 29 S70 44 12 Gender Male 62 26 Female 38 15 Primary tumoral size T1þT2 53 31 T3þT4 47 10 Regional lymph node metastasis Negative 67 26 Positive 33 15 Vascular invasion Negative 81 35 Positive 19 6 Stage I þ II 43 24 III þ IV 57 17 * 2
c test. Fisher's exact test. zP < 0.05. y
p-value
We confirmed the effect of TEAD4 knockdown on cellular proliferation and found a significant decrease of cellular growth in shTEAD4 cells compared with shMock cells (Fig. 2C, p < 0.05). 3.5. Localizations of YAP in TEAD4 knockdown cells
TEAD4-positive 27 32
0.305*
36 23
0.932y
22 37
0.028yz
41 18
0.489y
46 13
0.378y
19 40
0.102y
Since TEAD4 forms a complex with YAP in nuclei, and the complex regulates tumoral progression in several cancers [3,4,7,8], we analyzed the YAP and p-YAP expression levels using nuclear and cytoplasmic fractions. YAP was down-regulated in the nuclear fraction, and p-YAP was up-regulated in the cytoplasmic fraction of shTEAD4 cells compared with that in shMock cell (Fig. 3). 3.6. Effect of TEAD4 knockdown on cell-cycle distribution CDK6, a cell-cycle-related gene in the G1 phase, is a downstream molecule of the TEAD4-YAP complex [22]. Therefore, we evaluated the expression levels of the G1 phase-related proteins and cellcycle distribution. The expression levels of the cyclin-dependent kinase inhibitors (p21cip1 and p27kip1) were up-regulated; in contrast, cyclin D1 and cyclin E and CDK2, CDK4, and CDK6 were down-regulated significantly in shTEAD4 cells (Fig. 4, p < 0.05). We also assessed the cell-cycle distribution in the TEAD4 knockdown
Please cite this article in press as: S. Takeuchi, et al., TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.050
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Fig. 2. Establishment of TEAD4 knockdown cells and evaluation of cell proliferation ability of TEAD4 knockdown cells. (A) qRT-PCR shows that mRNA levels of TEAD4 are significantly (*p < 0.05, Student's t-test) down-regulated in shTEAD4 cells (Sa3 and HSC-3-derived transfectants) compared with shMock cells. (B) Western blot analysis and the densitometric data indicate that TEAD4 protein levels are decreased markedly (*p < 0.05, Student's t-test) compared with shMock cells. The data was normalized to GAPDH protein levels. (C) In shTEAD4 cells, the cellular growth is inhibited significantly (*p < 0.05, Student's t-test) after 168 h. The results are expressed as the means ± the standard error of the mean values from three assays.
cells by flow cytometry. The ratio of the cells in the G1 phase in shTEAD4 cells was significantly higher than in shMock cells (Fig. 4, p < 0.05), suggesting that TEAD4 expression is related closely to cell-cycle regulation of the G1 phase. 4. Discussion In the current study, we found that TEAD4 expression increased significantly in OSCC-derived cell lines and clinical OSCC samples (Fig. 1). TEAD4 knockdown OSCC cells showed decreased cellular
proliferation (Fig. 2C) resulting from cell-cycle arrest in the G1 phase. We also found that the TEAD4-YAP complex in the nuclei was related closely to transcriptions of G1 arrest-related genes (Figs. 3 and 4). Similar to the current data, TEAD4 protein along with YAP was found frequently in the nuclei of gastric cancer cells. In addition, TEAD4 knockdown resulted in decreased cellular growth of gastric cancer cells in vitro and in vivo [23]. Although there was no relationship between the TEAD4 expression status and regional lymph node metastasis in OSCCs, breast cancer and malignant melanoma
Please cite this article in press as: S. Takeuchi, et al., TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.050
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Fig. 3. Localizations of YAP in TEAD4 knockdown cells. Western blot analysis is performed using nuclear and cytoplasmic fractions. YAP is down-regulated in the nuclear fraction of shTEAD4 cells (Sa3 and HSC-3-derived transfectants) compared with that of shMock cells. p-YAP is up-regulated in the cytoplasmic fraction compared with the counterparts. N, nuclear fraction; C, cytoplasmic fraction.
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present the metastatic potentials of those cells by increasing TEAD transcriptional activity [24]. Therefore, TEAD4 plays pivotal roles in tumoral progression and metastasis of several cancers. The TEAD4-YAP complex regulates multiple functions of cancers, i.e., cellular growth, migration, and invasiveness. Phosphorylation of YAP by LATS1/2 tumor suppressor kinase leads to cytoplasmic translocation and inactivation of the YAP [25e30]. Interestingly, our observations (Fig. 3) showed that TEAD4 knockdown resulted in highly phosphorylated levels of YAP and decreased YAP in the nuclei. Therefore, TEAD4 expression levels might control not only DNA binding and transcriptional activities with YAP but also the phosphorylation levels of YAP. CDK6 is the downstream target of the TEAD4-YAP complex for cellular senescence [22]. Since CDK6 is a key molecule of the cellcycle in the G1 phase, we speculated that the inactivated TEAD4YAP complex (TEAD4 knockdown) showed cell-cycle arrest in the G1 phase. Consistent with our hypothesis, less tumoral progression occurred in shTEAD4 cells and patients with OSCC negative for TEAD4. We concluded that TEAD4 has a biologic role in human oral cancer. Overexpression of TEAD4 especially increased tumoral size in primary OSCCs via regulation of the cell-cycle via the activity of the TEAD4-YAP complex. While further studies are required, TEAD4 might be a critical biomarker and a therapeutic target for OSCCs. Acknowledgements We thank Lynda C. Charters for editing this manuscript. The authors received no financial support. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.03.050. Disclosure statement The authors declare no conflicts of interest. References
Fig. 4. Effect of TEAD4 knockdown on cell-cycle distribution. (A) Western blot analysis shows up-regulation of p21cip1 and p27kip1 and down-regulation of cyclin D1, cyclin E, CDK2, CDK4, and CDK6 in shTEAD4 cells (Sa3 and HSC-3-derived transfectants) compared with shMock cells. (B) Flow cytometric analysis is performed to investigate cell-cycle progression in shTEAD4 and shMock cells after synchronization at the M phase using nocodazole. The percentage of cells in the G1 phase in shTEAD4 cells is increased compared with shMock cells.
[1] T.R. Bürglin, The TEA domain: a novel, highly conserved DNA-binding motif, Cell 66 (1991) 11e12, http://dx.doi.org/10.1016/0092-8674(91)90132-I. [2] B. Zhao, X. Ye, J. Yu, L. Li, W. Li, S. Li, J. Yu, J.D. Lin, TEAD mediates YAPdependent gene induction and growth control TEAD mediates YAPdependent gene induction and growth control, Genes Dev. (2008) 1962e1971, http://dx.doi.org/10.1101/gad.1664408. [3] Y. Zhou, T. Huang, A.S.L. Cheng, J. Yu, W. Kang, K.F. To, The TEAD family and its oncogenic role in promoting tumorigenesis, Int. J. Mol. Sci. 17 (2016) 1e15, http://dx.doi.org/10.3390/ijms17010138. [4] K. Zhang, H.X. Qi, Z.M. Hu, Y.N. Chang, Z.M. Shi, X.H. Han, Y.W. Han, R.X. Zhang, Z. Zhang, T. Chen, W. Hong, YAP and TAZ take center stage in cancer, Biochemistry 54 (2015) 6555e6566, http://dx.doi.org/10.1021/ acs.biochem.5b01014. [5] Z. Li, B. Zhao, P. Wang, F. Chen, Z. Dong, H. Yang, Structural insights into the YAP and TEAD complex service Structural insights into the YAP and TEAD complex, Genes Dev. (2010) 235e240, http://dx.doi.org/10.1101/ gad.1865810. [6] T. Yoshida, MCAT elements and the TEF-1 family of transcription factors in muscle development and disease, Arterioscler. Thromb. Vasc. Biol. 28 (2008) 8e17, http://dx.doi.org/10.1161/ATVBAHA.107.155788. [7] B. Zhao, B. Zhao, X. Wei, X. Wei, W. Li, W. Li, R.S. Udan, R.S. Udan, Q. Yang, Q. Yang, J. Kim, J. Kim, J. Xie, J. Xie, T. Ikenoue, T. Ikenoue, J. Yu, J. Yu, L. Li, L. Li, P. Zheng, P. Zheng, K. Ye, K. Ye, A. Chinnaiyan, A. Chinnaiyan, G. Halder, G. Halder, Z. Lai, Z. Lai, K.-L. Guan, K.-L. Guan, Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control, Genes Dev. 21 (2007) 2747e2761, http://dx.doi.org/10.1101/ gad.1602907.Hpo/Sav. [8] C. Wang, Z. Nie, Z. Zhou, H. Zhang, R. Liu, J. Wu, J. Qin, Y. Ma, L. Chen, S. Li, W. Chen, F. Li, P. Shi, Y. Wu, J. Shen, C. Chen, The interplay between TEAD4 and KLF5 promotes breast cancer partially through inhibiting the transcription of p27Kip1, Oncotarget 6 (2015), http://dx.doi.org/10.18632/oncotarget.3779.
Please cite this article in press as: S. Takeuchi, et al., TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.050
6
S. Takeuchi et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
[9] Y. Liu, G. Wang, Y. Yang, Z. Mei, Z. Liang, A. Cui, T. Wu, C.-Y. Liu, L. Cui, Increased TEAD4 expression and nuclear localization in colorectal cancer promote epithelial-mesenchymal transition and metastasis in a YAPindependent manner, Oncogene (2015) 1e12, http://dx.doi.org/10.1038/ onc.2015.342. [10] A. Kasamatsu, K. Uzawa, D. Nakashima, H. Koike, M. Shiiba, H. Bukawa, H. Yokoe, H. Tanzawa, Galectin-9 as a regulator of cellular adhesion in human oral squamous cell carcinoma cell lines, Int. J. Mol. Med. 16 (2005) 269e273. [11] Y. Endo, K. Uzawa, Y. Mochida, M. Shiiba, H. Bukawa, H. Yokoe, H. Tanzawa, Sarcoendoplasmic reticulum Ca(2þ) ATPase type 2 downregulated in human oral squamous cell carcinoma, Int. J. Cancer 231 (2004) 225e231, http:// dx.doi.org/10.1002/ijc.20118. [12] M. Yamatoji, A. Kasamatsu, Y. Yamano, K. Sakuma, K. Ogoshi, M. Iyoda, K. Shinozuka, K. Ogawara, Y. Takiguchi, M. Shiiba, H. Tanzawa, K. Uzawa, State of homeobox A10 expression as a putative prognostic marker for oral squamous cell carcinoma, Oncol. Rep. 23 (2010) 61e67, http://dx.doi.org/10.3892/ or-00000606. [13] K. Sakuma, A. Kasamatsu, M. Yamatoji, Y. Yamano, K. Fushimi, M. Iyoda, K. Ogoshi, K. Shinozuka, K. Ogawara, M. Shiiba, H. Tanzawa, K. Uzawa, Expression status of Zic family member 2 as a prognostic marker for oral squamous cell carcinoma, J. Cancer Res. Clin. Oncol. 136 (2010) 553e559, http://dx.doi.org/10.1007/s00432-009-0689-y. [14] Y. Minakawa, A. Kasamatsu, H. Koike, M. Higo, D. Nakashima, Y. Kouzu, Y. Sakamoto, K. Ogawara, M. Shiiba, H. Tanzawa, K. Uzawa, Kinesin family member 4A: a potential predictor for progression of human oral cancer, PLoS One 8 (2013) 1e8, http://dx.doi.org/10.1371/journal.pone.0085951. [15] T. Koyama, K. Ogawara, A. Kasamatsu, A. Okamoto, H. Kasama, Y. Minakawa, K. Shimada, H. Yokoe, M. Shiiba, H. Tanzawa, K. Uzawa, ANGPTL3 is a novel biomarker as it activates ERK/MAPK pathway in oral cancer, Cancer Med. 4 (2015) 759e769, http://dx.doi.org/10.1002/cam4.418. [16] C. Fukumoto, D. Nakashima, A. Kasamatsu, M. Unozawa, T. Shida-Sakazume, M. Higo, K. Ogawara, H. Yokoe, M. Shiiba, H. Tanzawa, K. Uzawa, WWP2 is overexpressed in human oral cancer, determining tumor size and poor prognosis in patients: downregulation of WWP2 inhibits the AKT signaling and tumor growth in mice, Oncoscience 1 (2014) 807e820, http://dx.doi.org/ 10.18632/oncoscience.101. [17] I. Miyamoto, A. Kasamatsu, M. Yamatoji, D. Nakashima, K. Saito, M. Higo, Y. Endo-Sakamoto, M. Shiiba, H. Tanzawa, K. Uzawa, Kinesin family member 14 in human oral cancer: a potential biomarker for tumoral growth, Biochem. Biophys. Rep. 3 (2015) 26e31, http://dx.doi.org/10.1016/j.bbrep.2015.07.008. [18] Y. Kimura, A. Kasamatsu, D. Nakashima, M. Yamatoji, Y. Minakawa, K. Koike, K. Fushimi, M. Higo, Y. Endo-Sakamoto, M. Shiiba, H. Tanzawa, K. Uzawa, ARNT2 regulates tumoral growth in oral squamous cell carcinoma, J. Cancer 7 (2016) 702e710, http://dx.doi.org/10.7150/jca.14208. [19] T. Saito, A. Kasamatsu, K. Ogawara, I. Miyamoto, K. Saito, M. Iyoda, T. Suzuki, Y. Endo-Sakamoto, M. Shiiba, H. Tanzawa, K. Uzawa, Semaphorin7A
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27] [28] [29]
[30]
promotion of tumoral growth and metastasis in human oral cancer by regulation of g1 cell cycle and matrix metalloproteases: possible contribution to tumoral angiogenesis, PLoS One 10 (2015) 1e20, http://dx.doi.org/10.1371/ journal.pone.0137923. K. Uzawa, T. Baba, F. Uchida, M. Yamatoji, A. Kasamatsu, Y. Sakamoto, K. Ogawara, M. Shiiba, H. Bukawa, H. Tanzawa, Circulating tumor-derived mutant mitochondrial DNA: a predictive biomarker of clinical prognosis in human squamous cell carcinoma, Oncotarget 3 (2012) 670e677 doi:523 [pii]. K. Uzawa, A. Kasamatsu, T. Baba, Y. Kimura, D. Nakashima, M. Higo, Y. Sakamoto, K. Ogawara, M. Shiiba, H. Tanzawa, Quantitative detection of circulating tumor-derived mitochondrial NADH subunit variants as a potential prognostic biomarker for oral cancer, Int. J. Oncol. (2015) 1077e1083, http:// dx.doi.org/10.3892/ijo.2015.3083. Q. Xie, J. Chen, H. Feng, S. Peng, U. Adams, Y. Bai, L. Huang, J. Li, J. Huang, S. Meng, Z. Yuan, YAP/TEAD-mediated transcription controls cellular senescence, Cancer Res. 73 (2013) 3615e3624, http://dx.doi.org/10.1158/00085472.CAN-12-3793. B. Lim, J.L. Park, H.J. Kim, Y.K. Park, J.H. Kim, H.A. Sohn, S.M. Noh, K.S. Song, W.H. Kim, Y.S. Kim, S.Y. Kim, Integrative genomics analysis reveals the multilevel dysregulation and oncogenic characteristics of TEAD4 in gastric cancer, Carcinogenesis 35 (2014) 1020e1027, http://dx.doi.org/10.1093/carcin/bgt409. J.M. Lamar, P. Stern, H. Liu, J.W. Schindler, Z.-G. Jiang, R.O. Hynes, The Hippo pathway target, YAP, promotes metastasis through its TEAD-interaction domain, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) E2441eE2450, http:// dx.doi.org/10.1073/pnas.1212021109. J.-W. Jang, M.-K. Kim, Y.-S. Lee, J.-W. Lee, D.-M. Kim, S.-H. Song, J.-Y. Lee, B.Y. Choi, B. Min, X.-Z. Chi, S.-C. Bae, RAC-LATS1/2 signaling regulates YAP activity by switching between the YAP-binding partners TEAD4 and RUNX3, Oncogene (2016) 1e13, http://dx.doi.org/10.1038/onc.2016.266. N. Nishioka, K. ichi Inoue, K. Adachi, H. Kiyonari, M. Ota, A. Ralston, N. Yabuta, S. Hirahara, R.O. Stephenson, N. Ogonuki, R. Makita, H. Kurihara, E.M. MorinKensicki, H. Nojima, J. Rossant, K. Nakao, H. Niwa, H. Sasaki, The hippo signaling pathway components lats and yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass, Dev. Cell. 16 (2009) 398e410, http://dx.doi.org/10.1016/j.devcel.2009.02.003. F.X. Yu, K.L. Guan, The Hippo pathway: regulators and regulations, Genes Dev. 27 (2013) 355e371, http://dx.doi.org/10.1101/gad.210773.112. E. Lever, D. Sheer, The role of nuclear organization in cancer, J. Pathol. 220 (2010) 114e125, http://dx.doi.org/10.1002/path. B. Zhao, L. Li, Q. Lu, L.H. Wang, C.Y. Liu, Q. Lei, K.L. Guan, Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein, Genes Dev. 25 (2011) 51e63, http://dx.doi.org/10.1101/gad.2000111. B.Z. Stanger, Quit your YAPing: a new target for cancer therapy, Genes Dev. 26 (2012) 1263e1267, http://dx.doi.org/10.1101/gad.196501.112.specificity.
Please cite this article in press as: S. Takeuchi, et al., TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.050