Cross-Regulation between MST1 and BRAFV600E Figure 6. Microscopic features of Tg-BRAFV600E/MST1 mice. Thyroid tissues were fixed in 10% formalin and embedded in paraffin. Five micrometer-thick sections were prepared and stained with H&E. At least five sections from each mouse were used for ImageJ analysis. Differences between Tg-BRAFV600E/MST1-null mice and Tg BRAFV600E littermates were assessed by Mann-Whitney U-test. All data are presented as mean6SD. (a) Representative figure from Tg-BRAFV600E littermates shows characteristic features of human PTC, including nuclear enlargement, crowding and overlapping (magnification 610). (b&c) Representative immunohistochemical staining results with anti-cyclin D1 antibody in Tg-BRAFV600E littermates (b, magnification 610; c, magnification 6100). Circled area shows intensive staining of cyclin D1 and is magnified in c. (d) Numbers of undifferentiated foci were calculated by counting the number of areas showing increased staining intensity of cyclin D1. (e) Representative image from a Tg-BRAFV600E/MST1 mouse shows a limited area with follicle or colloid formation (e, magnification 610). (f,g) Representative immunohistochemical staining results with anti-cyclin D1 antibody from a Tg-BRAFV600E/MST1 mouse, suggesting abundant undifferentiated foci (f, magnification 610; g, magnification 6100). Circled areas show increased staining intensity of cyclin D1 and are magnified in g. (h) ImageJ analysis of estimated areas without follicular architecture or colloid formation. Tg-BRAFV600E/MST1 mice (n = 10) have larger areas than Tg-BRAFV600E littermates (n = 10). (i,j) Representative images from a Tg-BRAFV600E/MST1 mouse show muscle invasion adjacent to the thyroid gland (f, magnification 640, boxed area is magnified and shown in j (magnification 6100)). (k,l) Representative images from the other Tg-BRAFV600E/MST1 mouse show aggressive invasion to muscle areas surrounding the upper pole of the thyroid gland (k, magnification 640, boxed area is magnified and presented at l (magnification 6100)). Arrows indicate invasive front in each figure. All data are presented as means6SD: (*) P,0.01; (**) P,0.05 between two groups. doi:10.1371/journal.pone.0016180.g006 pathway, whereas RET/PTC inactivates FoxO3 by Akt/PKB activation [23]. In addition, the silencing of RAF-1, which has been reported to bind with MST2, did not alter the inhibition of FoxO3 transactivation by BRAFV600E.Taken together,these data suggest that FoxO3 inactivation might be a common process in the carcinogenesis of PTC and that BRAFV600E inhibits FoxO3 transactivation through a MEK-ERK–, Akt/PKB–, or RAF-1– independent pathway. The results of the present study suggest the existence of a novel FoxO3 inhibitory mechanism that might operate in BRAFV600E-induced PTC. MST1 kinase had a counter-regulatory effect on FoxO3 activation and localization compared with Akt/PKB [49,50]. In contrast to Akt/PKB, MST1 phosphorylation of FoxO3 promotes its nuclear translocation, thereby inducing cell death [19]. A novel inhibitory mechanism of FoxO3 transactivation might be opera- tional in BRAFV600E-induced PTC, which led to the evaluation of BRAFV600E regulation of MST1-FoxO3 signaling pathways. A reporter assay revealed that MST1-induced FoxO3 transactivation was completely inhibited by co-transfection with BRAFV600E. In addition, over-expression of MST1 prevented BRAFV600E-induced FoxO3 inactivation. These data suggested that cross-regulation between MST1 and BRAFV600E might play a critical role in regulating FoxO3 transactivation. Supporting this idea, immuno- fluorescence studies revealed not only the BRAFV600E-mediated inhibition of MST1-induced FoxO3 nuclear translocation, but also a direct interaction between MST1 and BRAFV600E, which was confirmed through IP assays that demonstrated that BRAFV600E binds to the C-terminal dimerization domain of MST1. In fact, the dimerization domain of MST1/2 mediates MST1 kinase activity by interacting with binding partners such as PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e16180