Cross-Regulation between MST1 and BRAFV600E Figure 3. BRAFV600E and MST1 kinases competitively regulate FoxO3 transactivation. (a,c) Co-transfection with 3X-IRS Luc (100 ng/well), FoxO3 (0.5 mg/well), BRAFV600E (indicated amount/well), MST1 (indicated amount/well), and/or SiMST1 (20 mM/well StealthTM RNA) was performed as indicated. Twenty-four hours after transfection, a luciferase-based reporter assay was conducted. Total lysates were immunoblotted with anti-Myc, anti-HA, anti-MST1, anti-BRAF, anti-Flag, or anti-Actin antibodies. All data are presented as mean6SD: (*) P,0.01 between two groups, Abbreviations: CTL; control. doi:10.1371/journal.pone.0016180.g003 induction of p21 and p27, which was almost completely reversed by BRAFV600E co-transfection. Notably, these two G1-checkpoint CDK inhibitors have been associated with the initiation and progression of human tumors because p21 and p27 bind and inhibit cyclin-CDK complexes, inducing cell cycle arrest and apoptosis. Therefore, the TUNEL assay was used to determine whether MST1-FoxO3 promotes apoptosis and if BRAFV600E reverses the MST1-FoxO3 effect (Fig. 5C). Transient co- expression of FoxO3 and MST1 increased the number of TUNEL positive 293T cells, but this number was decreased by the introduction of BRAFV600E (see also right panel of Fig. 5C for statistical analysis). These results imply that the MST1-FoxO3 pathway induction of G1-checkpoint CDK inhibitors promotes apoptosis, and oncogenic BRAFV600E inhibits the activity of these tumor suppressor systems through the regulation of MST1. Loss of MST1 aggravates PTC phenotypes in thyroid- specific transgenic BRAFV600E mice To validate the pathophysiological consequences of the interaction between BRAFV600E and MST1 in vivo, animal experiments using thyroid-specific transgenic BRAFV600E mice (Tg-BRAFV600E mice) and MST1 mice were designed. Tg- BRAFV600E mice were crossed with MST1 mice and the phenotypic differences between Tg-BRAFV600E/MST1 mice and their Tg-BRAFV600E littermates were analyzed at 16 weeks [4]. The thyroid glands of MST1 mice with wild-type BRAF were normal (data not shown). However, Tg-BRAFV600E/MST1 mice showed aggressive PTC tumor phenotypes compared with their Tg-BRAFV600E littermates. Although the presence of lymph node metastasis was not observed in either of the two groups, Tg-BRAFV600E/MST1 mice showed abundant foci of undifferentiated carcinomas and large areas without follicular architecture or colloid formation (Figure 6A–H). For the statistical analysis of these results, immunohistochemical staining of cyclin D1 was performed, which showed an intensive cyclin D1 staining area as undifferentiated foci (Fig. 6B&C, F–G). As shown in Figure 6D, the number of undifferentiated foci in female Tg- BRAFV600E/MST1 mice was 3.360.86, but that of their Tg- BRAFV600E littermates was 1.0560.51, reflecting a statistical difference between the two groups (P,0.001). Similarly, male Tg- BRAFV600E/MST1 mice showed a higher number of undifferentiated foci than their Tg-BRAFV600E littermates (3.560.83and 1.3560.67, respectively; P,0.001). The total area composed of sheets of spindle cells without follicular architecture or colloid formation was estimated and ImageJ (http://rsbweb. nih.gov/ij/) was used to quantify these areas and analyze the differences between Tg-BRAFV600E/MST1 mice and their Tg-BRAFV600E littermates (Figure 6H). In female mice, Tg- BRAFV600E/MST1 mice had a significantly larger area without follicular architecture or colloid formation than their Tg- BRAFV600E littermates (75.4763.83% and 52.6967.93%, respec- tively; P = 0.034). In male mice, Tg-BRAFV600E/MST1 mice also showed a larger area without follicular architecture or colloid formation compared to their Tg-BRAFV600E littermates (70.0263.68% and47.3167.93%, respectively; P = 0.021). This in vivo study suggests that the direct interaction between BRAFV600E and MST1 could affect the behavior of papillary thyroid cancer. Another piece of evidence demonstrating the aggressive tumor phenotype in these Tg-BRAFV600E/MST1 mice was the frequent presence of muscle invasion (Figure 6I–L), which was not observed in their Tg-BRAFV600E littermates. Discussion BRAFV600E constitutively activates the MEK-ERK signaling pathway, promoting ERK-dependent transcriptional activity, which regulates cell growth, cell proliferation, apoptosis evasion, replicative potential, angiogenesis, and tissue invasion [44,45]. PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e16180