tumor suppressor systems which may be controlled by BRAFV600E in thyroid cancer remain to be identified. The tumor suppressor gene RASSF1A (Ras association domain family 1A) is epigenetically inactivated through de novo promoter methylation in the early stages of thyroid tumorigenesis [17,18]. Interestingly, RASSF1A has recently been described as an important activator of MST1, which in turn phosphorylates and promotes the nuclear translocation of the forkhead transcription factor FKHRL1 (FoxO3), inducing cell death [19,20,21]. This suggests that FoxO3 transactivation could be induced by the RASSF1A-MST1 pathway and function as a tumor suppressor system in response to specific oncogenic signals, such as BRAFV600E. However, promoter hyper- methylation of RASSF1A could only be detected in a relatively small percentage of PTC (20 to 32%) [18,22]. These observations predict that novel RASSF1A-MST1-FoxO3 pathways regulated by BRAFV600E might function during the development of PTC whichdoes nothave RASSF1Apromoter methylation. FoxO3 transactivation is effectively inhibited by RET/PTC (rearranged in transformation/papillary thyroid carcinomas) kinase [23], the gene rearrangement of which is the most common rearrangement in papillary thyroid cancer. The inactivation of FoxO3 could therefore be a signature molecular event that should also occur in BRAFV600E thyroid tumors. Several molecular mechanisms which are possibly regulated by BRAFV600E may control FoxO3 activity in thyroid cancer. First, as RET/PTC kinase inhibits FoxO3 transactivation through an Akt/PKB dependent pathway, BRAFV600E might also activate Akt/PKB signaling pathway [24]. Second, the constitutive activation of ERK by BRAFV600E could inhibit FoxO3 activation via the ubiquitin- proteasome pathway [25]. Finally, BRAFV600E could act through crosstalk with the RASSF1A-MST1-FoxO3 pathway. Based on these hypotheses, we decided to investigate the regulation of the MST1-FoxO3 pathway by BRAFV600E. This resulted in the identification of novel cross-talksignaling between BRAFV600E and MST1, thereby demonstrating the functional activity of the RASSF1A-MST1-FoxO3 tumor suppressor system. Furthermore, in vivo experiments showed that MST1 knockout mice exhibited more aggressive BRAFV600E tumor phenotypes. Materials and Methods Plasmids The pCMV5-Myc-FoxO3 plasmid was purchased from Addgene (Addgene Inc., Cambridge, MA) and pcDNA3.1/CT-GFP-FoxO3 was constructed using the CT-GFP Fusion TOPO Expression Kit according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). Expression vectors (pME18) for Flag epitope-tagged forms of human MST1, MST1-N (residues 1–326), MST1-C (residues 327–487), HA- RASSF1A and 3X-IRS reporters have been described previously [26,27,28,29]. The cDNA for human BRAF was cloned into pLenti6/V5-DEST using the pDONR221 vector (Invitrogen). For the cloning of BRAF, PCR primers were designed as follows: sense, 59-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAAGAT- GGCGGCGCTGAGCGGT-39, and antisense, 59- GGGGACC- ACTTTGTACAAGAAAGCTGGGTCTCAGTGGACAGGAA- ACGCCC-39. To generate vectors for human BRAFV600E and BRAFG469A, the Site-Directed PCR Mutagenesis Kit (Stratagene, La Jolla, CA) was used to introduce the missense mutation into the wild- type BRAF sequence. SiRNAs for MST1 and RAF-1 were purchased from Invitrogen (Stealth RNAi, Invitrogen). Cross-Regulation between MST1 and BRAFV600E 35 mm dish) were transfected with siRNA at a concentration of 50 nM using the HiPerfect Transfection Reagent (Qiagen, Valencia, CA) and/or with the indicated vectors using Fugen6 Transfection Reagent (Roche Applied Science, Indianapolis, IN). The MEK inhibitor (U0126) and PI3K inhibitors (Ly294002, Wortmannin) were purchased from Cell Signaling Technology (Beverley, MA). Antibodies Primary antibodies against p21, p27, phospho-ERK, total ERK, phospho-Akt, total Akt, MST1, Actin, FoxO3, Myc (Cell Signaling), BRAF (C-19, Santa Cruz Biotechnology, Santa Cruz, CA), RASSF1A (eBioscience, San Diego, CA) and Flag (Sigma, St Louis, MO) were purchased from the indicated companies. Luciferase-based reporter assay To assay the activity of FoxO3, 293T cells were transfected with 3X-IRS-Luc, or p27-Luc and pRL-SV40, which encodes Renilla luciferase (Promega, Madison, WI) using Lipofectamine in OptiMEM (Invitrogen). Luciferase levels were determined using the Dual Luciferase Assay Kit (Promega). Firefly and Renilla luciferase activities were measured sequentially from the same sample using a Fusion Alpha-Microplate Analyzer (Perkin Elmer, Waltham, MA). For each sample, firefly luciferase activity was normalized to Renilla luciferase activity. Immunoprecipitation and Immunoblot analysis All immunoprecipitation and immunoblotting procedures were performed at 4uC. Cells were washed twice with PBS and lysed in lysis buffer containing protease inhibitors. Lysates were centrifuged at 14,000 g for 15 min at 4uC. For the IP analysis, lysates were precleared with protein A/G beads (Santa Cruz Biotechnology) for 30 min, and supernatants were incubated with the indicated primary antibody for 3 h with agitation. Protein A/G beads were then added to the mixture and incubated for at least 2 h at 4uC. Beads were washed with lysis buffer and the immunoprecipitates were subjected to immunoblotting. Cell lysates were separated by SDS-PAGE, and transferred to nitrocellulose membranes. Mem- branes were blocked with TBS containing 5% milk and 0.1% Tween20 for 1 h and then incubated overnight with primary antibody at 4uC. Membranes were washed in TBS/T and incubated with secondary antibodies conjugated to horseradish peroxidase (Cell Signaling) for 1 h at RT. Blots were developed using the LumiGLOChemiluminescent substrate (Cell Signaling). Immunofluorescence staining 293T cells were grown on 6-well plates and transfected with the indicated plasmids using LipofectAmine (Invitrogen). Twenty-four hours after transfection, the cells were washed three times with PBS and fixed in 3.7% formaldehyde for 20 min. Cells were permeabilized with PBS containing 0.1% Triton X-100 and 0.1 M glycine at RT, incubated for 15 min, washed three times with PBS, and blocked with 3% BSA in PBS for 10 min at RT. Cells were incubated with primary anti-Myc and anti-Flag for 1 h at 37uC, washed three times with PBS, and incubated for 1 h at 37uC with Cy5-conjugated anti-Rabbit and TRITC-conjugated anti- Mouse for Myc-BRAFV600E and Flag-MST1, respectively (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were observed with a CellomicsArrayScanV HCS reader (Thermo Scientific, Pittsburgh, PA). Cell culture and transfection FRO cells and 293T cells were cultured in RPMI and DMEM supplemented with 10% fetal bovine serum [20,30]. Cells (26105/ In vitro kinase assay of MST1 MST1 was immunoprecipitated from cell lysates and the washed precipitates were incubated for 30 minutes at 30uC with PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16180