V600E Cross-Regulation between Oncogenic BRAF Kinase and the MST1 Pathway in Papillary Thyroid Carcinoma Seong Jin Lee1,3., Min Hee Lee1., Dong Wook Kim1., SeongEun Lee1, Songmei Huang 2 , Min Jeong Ryu 1 , Yong Kyung Kim1, Sung Jin Kim1, Soung Jung Kim1, Jung Hwan Hwang1,4, Sangphil Oh 5 , Heeyeong Cho3, Jin Man Kim2, Dae-Sik Lim5, Young Suk Jo6*, Minho Shong 1 * 1 Department of Internal Medicine, Chungnam National University School of Medicine, Daejeon, Republic of Korea, 2 Department of Pathology, Chungnam National University School of Medicine, Daejeon, Republic of Korea, 3 Pharmacology Research Center, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea, 4 Animal Model Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea, 5 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea, 6 Research Center for Endocrine and Metabolic Diseases, Chungnam National University Hospital, Daejeon, Republic of Korea Abstract Background: The BRAFV600E mutation leading to constitutive signaling of MEK-ERK pathways causes papillary thyroid cancer (PTC). Ras association domain family 1A (RASSF1A), which is an important regulator of MST1 tumor suppressor pathways, is inactivated by hypermethylation of its promoter region in 20 to 32% of PTC. However, in PTC without RASSF1A methylation, the regulatory mechanisms of RASSF1A-MST1 pathways remain to be elucidated, and the functional cooperation or cross regulation between BRAFV600E and MST1,which activates Foxo3,has not been investigated. Methodology/Principal Findings: The negative regulators of the cell cycle, p21 and p27, are strongly induced by transcriptional activation of FoxO3 in BRAFV600E positive thyroid cancer cells. The FoxO3 transactivation is augmented by RASSF1A and the MST1 signaling pathway. Interestingly, introduction of BRAFV600Emarkedly abolished FoxO3 transactivation and resulted in the suppression of p21 and p27 expression. The suppression of FoxO3 transactivation by BRAFV600Eis strongly increased by coexpression of MST1 but it is not observed in the cells in which MST1, but not MST2,is silenced. Mechanistically, BRAFV600Ewas able to bind to the C-terminal region of MST1 and resulted in the suppression of MST1 kinase activities. The induction of the G1-checkpoint CDK inhibitors, p21 and p27,by the RASSF1A-MST1-FoxO3 pathway facilitates cellular apoptosis, whereasaddition of BRAFV600E inhibits the apoptotic processes through the inactivation of MST1. Transgenic induction of BRAFV600Ein the thyroid gland results in cancers resembling human papillary thyroid cancers. The development of BRAFV600Etransgenic mice with the MST1 knockout background showed that these mice had abundant foci of poorly differentiated carcinomas and large areas without follicular architecture or colloid formation. Conclusions/Significance: The results of this study revealed that the oncogenic effect of BRAFV600E is associated with the inhibition of MST1 tumor suppressor pathways, and that the activity of RASSF1A-MST1-FoxO3 pathways determines the phenotypes of BRAFV600E tumors. Citation: Lee SJ, Lee MH, Kim DW, Lee S, Huang S, et al. (2011) Cross-Regulation between Oncogenic BRAFV600E Kinase and the MST1 Pathway in Papillary Thyroid Carcinoma. PLoS ONE 6(1): e16180. doi:10.1371/journal.pone.0016180 Editor: Sean Bong Lee, The National Institute of Diabetes and Digestive and Kidney Diseases, United States of America Received September 8, 2010; Accepted December 7, 2010; Published January 13, 2011 Copyright: 2011 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by the second phase of the BK 21 Program of the Ministry of Education, by the Research Program for New Drug Target Discovery (M10748000319-07N4800-31910) grant from the Ministry of Science & Technology and by a grant of the Korea Healthcare Technology R&D Project (A100588), Ministry for Health, Welfare & Family Affairs, Republic of Korea. MHL, SEL, SH, MJR, SJK, JMK and MS were supported in part by the second phase of the Brain Korea 21 program of the Ministry of Education. JMK, YSJ and MS were supported by the Research Program for New Drug Target Discovery (M10748000319- 07N4800-31910) grant from the Ministry of Science & Technology, Republic of Korea. MHL, SEL,MJR, YKK, SJK, SJK, YSJ and MS were supported in part by a grant of the Korea Healthcare Technology R&D Project (A100588), Ministry for Health, Welfare & Family Affairs, Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: minhos@cnu.ac.kr (MS); ysmrj@cnuh.ac.kr (YSJ) . These authors contributed equally to this work. Introduction Activating mutations in the BRAF gene are found at high frequency in various human cancers, and BRAFV600E is the most common of these activating mutations, especially in papillary thyroid cancer, where it is found at a frequency of 40–70% [1,2,3]. In BRAFV600E-positive thyroid cancer cell lines and BRAFV600E transgenic mice, this mutation is responsible for tumor initiation, transformation, growth, proliferation and dedifferentiation [4,5,6]. Research into the molecular mechanisms of BRAFV600E-positive tumors has revealed that the missense valine to glutamic acid mutation increases kinase activity, promoting the constitutive activation of MEK-ERK signaling [7,8,9,10,11] and enhancing ERK-dependent transcriptional output [12,13]. However, other signaling pathways except MEK-ERK [14,15]regulated in BRAFV600E tumors are not fully characterized [16]. Moreover, PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16180