RASSF1A and hWW45 [20,51]. Because RASSF1A promotes MST1 activation and apoptosis via this hetero-dimerization, we postulated that the direct interaction of MST1 with BRAFV600E might affect MST1 kinase activity. Actually, our in vitro kinase assay clearly showed that MST1 kinase activity on histone H2B was markedly decreased by BRAFV600E. Another possible mechanism of MST1 regulation may be the inhibitory phosphor- ylation of MST1 by BRAFV600E. Prior research has shown that Akt/PKB can phosphorylate the Thr387 residue of MST1 and prevent its proteolytic activity, providing a mechanism for the Akt/PKB-mediated inactivation of MST1-FoxO3 pathways [50]. Although the BRAFV600E-induced MST1 phosphorylation was not assessed, it remains possible that BRAFV600E may phosphorylate and inactivate MST1 through direct interaction with the C- terminal domain of MST1. Few mouse models exist for the study of tumor suppressor systems in PTC. In the present study, a new in vivo model was generated to investigate the cross-regulation between BRAFV600E and MST1, and the results obtained with transgenic mice were consistent with the in vitro generated data. Although MST1 knockout mice did not show any thyroid gland pathological changes compared to wild-type mice, Tg-BRAFV600E/MST1 mice showed an aggressive phenotype, supporting the hypothesis that MST1 might have a modifying effect on BRAFV600E-induced carcinogenesis. Although the lack of an appropriate antibody for the specific immunohisto- chemical detection of serine 207 residue in FOXO3 phosphorylated by MST1 kinaseprevented the detection of FoxO3 activity modified by MST1, these results, coupled with the results of in vitro assays, suggest that BRAFV600E may alleviate the tumor suppressor function of RASSF1A-MST1 and vice versa. In conclusion, the present study suggests that a direct interaction between oncogenic BRAFV600E and MST1 kinase plays a crucial role in determining tumor behavior in PTC. This novel interaction might provide advanced insight into the pathogenesis of the disease, and form the basis for novel drug development for the treatment of BRAFV600E-induced PTC. In light of this, it will be important to delineate the precise regulatory mechanism of the RASSF1A-MST1 pathway and verify the action of BRAFV600E on this signal pathway in human PTC. Supporting Information Figure S1 MST1 significantly increases 3XIRS reporter activ- ities. (A) 293T cells were cultured in 12 well dishes until they reached 80% confluence, and co-transfected with 3XIRS Luc (100 ng/well), FoxO3 (0.5 mg/well), and MST1 (0.5 mg/well) as indicated for 24 hours. To verify transfection efficacy, total lysates were immunoblotted with anti-HA, anti-Flag, and anti-Actin antibodies. (B) Cells were co-transfected with 3XIRS Luc (100 ng/ well), FoxO3 (0.5 mg/well), and MST1 (indicated amount/well). Total lysates were immunoblotted with anti-HA, anti-Flag, and anti-Actin antibodies. For each sample, firefly luciferase activity was normalized to Renilla luciferase activity and expressed as References 1. Cohen Y, Xing M, Mambo E, Guo Z, Wu G, et al. (2003) BRAF mutation in papillary thyroid carcinoma. J Natl Cancer Inst 95: 625–627. 2. Jo YS, Li S, Song JH, Kwon KH, Lee JC, et al. (2006) Influence of the BRAF V600E mutation on expression of vascular endothelial growth factor in papillary thyroid cancer. J ClinEndocrinolMetab 91: 3667–3670. 3. Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, et al. (2003) High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res 63: 1454–1457. 4. Knauf JA, Ma X, Smith EP, Zhang L, Mitsutake N, et al. (2005) Targeted expression of BRAFV600E in thyroid cells of transgenic mice results in Cross-Regulation between MST1 and BRAFV600E relative-fold change compared to basal luciferase activity. All data are presented as mean6SD: (*) P,0.01 between two groups. (TIF) Figure S2 BRAFV600E inhibits MST1-induced nuclear translo- cation of FoxO1. 293T cells were cultured in a six-well dish until they reached 80% confluence and co-transfected with FoxO3- GFP (0.5 mg/well), Flag-MST1 (0.5 mg/well), and Myc- BRAFV600E (0.5 mg/well) as indicated. Twenty-four hours after transfection, the cells were prepared for subcellular fractionation using the Nuclear/Cytosol Fractionation kit (BioVision, Inc. CA). The markers, origin recognition complex subunit 1 (ORC1) and b-tubulin, were used to verify the identity and purity of the nuclear and cytosolic fractions, respectively. Based on these markers, a good overall yield was obtained without mixing of the fractions. (TIF) Figure S3 BRAFV600E mediated FoxO3 inhibition was not altered by RAF-1. 293T cells were cultured in 12 well dishes until they reached 80% confluence and co-transfected with 3XIRS Luc (100 ng/well), FoxO3 (0.5 mg/well), BRAFV600E (0.5 mg/well), and SiRAF-1 (20 mM/well StealthTM RNA) for 24 h as indicated. Total lysates were immunoblotted with anti-HA, anti-BRAF, anti-RAF-1, and anti-Actin antibodies. For each sample, firefly luciferase activity was normalized to Renilla luciferase activity and expressed as relative-fold change compared to basal luciferase activity. All data are presented as mean6SD: (*) P,0.01 between two groups. (TIF) Figure S4 BRAFV600E suppresses FoxO3 transactivation via a MEK/ERK-, PI3 kinase-independent pathway. 293T cells were cultured in 12 well dishes until they reached 80% confluence, and co-transfected with 3XIRS Luc (100 ng/well), FoxO3 (0.5 mg/ well), and BRAFV600E (0.5 mg/well) as indicated for 24 h. MEK inhibitor (lane 3, U0126 20 mM/well) and PI3 kinase inhibitors (lane 4, Wortmannin 200 nM/well, and lane 5, LY294002 20 mM/well) were added. Total lysates were immunoblotted with anti-HA, anti-BRAF, anti-pERK, anti-ERK, anti-pAkt/PKB, anti-Akt/PKB, and anti-Actin antibodies. For each sample, firefly luciferase activity was normalized to Renilla luciferase activity and expressed as relative fold change compared to basal luciferase activity. All data are presented as mean6SD. Abbreviations: U0, U0126; WT, Wortmannin; LY, LY294002; and Con, control. (TIF) Author Contributions Conceived and designed the experiments: DSL YSJ MS. Performed the experiments: SJL MHL DWK SEL SH. Analyzed the data: SJL MJR YKK JHH JMK. Contributed reagents/materials/analysis tools: SJK SO HC JMK. papillary thyroid cancers that undergo dedifferentiation. Cancer Res 65: 4238–4245. 5. Mitsutake N, Knauf JA, Mitsutake S, Mesa C, Jr., Zhang L, et al. (2005) Conditional BRAFV600E expression induces DNA synthesis, apoptosis, dedifferentiation, and chromosomal instability in thyroid PCCL3 cells. Cancer Res 65: 2465–2473. 6. Xu X, Quiros RM, Gattuso P, Ain KB, Prinz RA (2003) High prevalence of BRAF gene mutation in papillary thyroid carcinomas and thyroid tumor cell lines. Cancer Res 63: 4561–4567. 7. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, et al. (2002) Mutations of the BRAF gene in human cancer. Nature 417: 949–954. PLoS ONE | www.plosone.org 10 January 2011 | Volume 6 | Issue 1 | e16180