Nucera, Nehs, Nagarkatti et al. 26 6.7 cells/field (p .001) (Fig. 3A, 3B). Invasion was also approximately twofold lower using 1 M PLX4720 (27.2 6.7 cells/field versus 52.7 2.1 cells/field; p .001) and fourfold lower using 10 M (13.6 4.0 cells/ field versus 59.5 8.8 cells/field; p .001) (Fig. 3A, 3B). PLX4720 CAUSES SIGNIFICANT REDUCTION IN CELL PROLIFERATION AND MIGRATION IN TPC-1 CELLS TPC-1 is another authentic human thyroid carcinoma cell line [24] with a known RET/PTC-1 translocation [27] that we have confirmed to have wt B-Raf and found to harbor a PIK3CA H1047R mutation (Fig. 1A) (by mass spectromet- ric genotyping); TPC-1 cells were found to be negative for other mutations (e.g., H-, N-, and K-RAS mutations, etc.). Though TPC-1 cells grew rapidly in vitro, they showed very low in vivo tumorigenicity, with a mean tumor size 1 mm3 after 60 days post-orthotopic injection (supplemental online Fig. 2). Paradoxically, in TPC-1 cells, treatment with 1 M PLX4720 for 1 hour resulted in higher phospho-ERK-1/ ERK-2 protein levels (by approximately 80%); however, 1 hour of treatment with 10 M PLX4720 reduced phospho- ERK-1/ERK-2 by about 45% (Fig. 1B). Seventy-two hours of treatment with 1 M or 10 M PLX4720 resulted in per- sistently elevated levels of phospho-ERK-1/ERK-2 protein (95% and 400%, respectively) (Fig. 1B, 1C). Both 1 M and 10 M PLX4720 caused lower cell proliferation as measured by BrdU uptake (27.9% 6.0% in controls ver- sus 12.9% 2.9% with 1 M PLX4720; p .01 and 36.7% 2.2% with control treatment versus 14.9% 0.2% with 10 M PLX4720; p .001) (Fig. 2A–2C). Nei- ther 1 M nor 10 M PLX4720 lead to apoptosis (absence of sub-G1 cell population) in TPC-1 cells evaluated by flow cytometric analysis (Fig. 2D). In addition, we found that only 10 M PLX4720 re- duced the migration of TPC-1 cells after 72 hours of treat- ment. Migration was about 1.6-fold lower (42.6 16.7 cells/field versus 78.6 6.8 cells/field) with 10 M PLX4720 (Fig. 3A). No significant differences were found for invasion with either 1 M or 10 M PLX4720 (Fig. 3B). There was no significant difference in the levels of phospho-Akt ser473 (supplemental online Fig. 1A) or C- Raf with PLX4720, compared with controls (supplemental online Fig. 1B). 301 overexpressed B-RafV600E in NT cells (passage 0). These NT cells have two wt B-Raf alleles, are positive for cyto- keratin, and are negative for desmin, indicating epithelial origin. They are also positive for the following thyroid- specific markers: TSH receptor, thyroperoxidase (TPO), thyroid transcription factor (TTF)-1, and paired box gene 8 (PAX-8) (supplemental online Fig. 3). Stably overexpress- ing B-RafV600E in NT cells (B-RafV600E–NT cells) resulted in greater B-Raf mRNA and protein expression: 4.24 0.1 copies in empty vector NT cells versus 52.4 6.1 copies in B-RafV600E–NT cells (p .001) (Fig. 3C). Stable B-RafV600E overexpression (detected by anti-tag-c-myc-B- RafV600E antibody) in these otherwise normal early-passage thyroid follicular cells, even in the presence of a wt B-Raf allele (heterozygous wt B-Raf/B-RafV600E), resulted in pro- found functional changes including: (a) significantly higher phospho-ERK-1/ERK-2 protein levels than in NT cells transduced with the empty vector (Fig. 3C), (b) greater cell migration (35.2 5.2 cells/field in overexpressed B-RafV600E NT cells versus 6.7 2.7 cells/field in the con- trol; p .001) (Fig. 3D), and (c) greater invasion (60.2 8.0 cells/field in overexpressed B-RafV600E cells versus 12.2 2.0 cells/field in the control; p .001) (Fig. 3D). PLX4720 treatment (1 M) of B-RafV600E–NT cells re- sulted in lower migration—21.2 7.4 cells/field versus 35.2 5.2 cells/field in cells treated with the vehicle (p .05)—and 10 M was even more effective at reducing mi- gration—10.7 3.4 cells/field in cells treated with PLX4720 versus 37 12.6 cells/field in cells treated with the vehicle; p .01 (Fig. 3D). One micromolar PLX4720 also resulted in lower B-RafV600E–NT cell invasion—21 0.8 cells/field versus 60.2 8.0 cells/field in cells treated with the vehicle (p .001). Ten micromolar PLX4720 was even more effective at reducing invasion—9.5 1.9 cells/ field in cells treated with PLX4720 versus 59 11.1 cells/ field in cells treated with the vehicle; p .001 (Fig. 3D). B-RafV600E–NT cells treated with 10 M PLX4720 (but not with 1 M) showed significantly lower cell proliferation— 17.3% 1.8% in cells treated with 10 M PLX4720 versus 27.3% 0.7% in cells treated with vehicle; p .01 (sup- plemental online Fig. 4). No significant differences in cell proliferation (supplemental online Fig. 4), migration, or in- vasion were found in NT cells transduced with the empty vector treated with PLX4720 (either 1 M or 10 M), com- pared with the vehicle (Fig. 3D). PLX4720 INHIBITS CELL PROLIFERATION, MIGRATION, AND INVASION IN NT CELLS WITH B-RAFV600E OVEREXPRESSION To understand the role of B-RafV600E in thyroid cell migra- tion and invasion in the heterozygous setting, we stably PLX4720 Inhibits Tumor Aggressiveness In Vivo Sixteen animals had 8505c cells tagged with GFP orthoto- pically implanted into the right thyroid lobe. One week after tumor implantation, mice were randomized and treatment was initiated; eight animals were treated with PLX4720 (30 www.TheOncologist.com