Nucera, Nehs, Nagarkatti et al. B-RafV600E) showed significantly lower cell prolifera- tion, migration, and invasion. PLX4720 treatment did not block cell invasion in TPC-1 cells with wild-type B- Raf, which showed very low and delayed in vivo tu- mor growth. In vivo, PLX4720 treatment of 8505c orthotopic thyroid tumors inhibited tumor aggres- siveness and significantly upregulated the thyroid dif- ferentiation markers thyroid transcription factor 1 and paired box gene 8. Conclusions. Here, we have shown that PLX4720 INTRODUCTION The incidence of well-differentiated thyroid cancers has been increasing [1]. The majority of patients with these tu- mors have a low risk for recurrence or death with standard therapy (surgery, radioiodine, and thyroid-stimulating hor- mone [TSH] suppression). However, a subset of patients with aggressive thyroid cancer, especially anaplastic thy- roid carcinoma (ATC), present with locally advanced dis- ease that recurs and metastasizes [2]. Mutations in key regulatory genes are likely to play an important role. Muta- tions known to be important in papillary thyroid cancers (PTCs) include: RET/PTC translocation (10%–50% of PTCs) [3, 4], Ras mutations (about 12% of PTCs) [5], and B-RafV600E mutations (29%– 83% of PTCs) [6, 7]. B-RafV600E is the second most common somatic mutation in human cancer, with a frequency of approximately 8% [8]. The B-RafV600E protein (valine-to-glutamate, position 600) results from a transversion in the gene (T1799A) and results in increased mitogen-activated protein kinase (e.g., mito- gen-activated protein kinase/extracellular signal–related kinase kinase [MEK]-1/MEK-2, extracellular signal–re- lated kinase [ERK]-1/ERK-2) activity [8, 12]. When PTC and ATC occur together in the same thyroid malignant le- sion, they generally share the B-RafV600E mutation, imply- ing that many ATCs are derived from malignant degeneration of pre-existing well-differentiated PTCs [13]. Our study focuses on further elucidating the role of this mu- tation in aggressive thyroid cancers. PLX4720 (7-azaindole derivative) is a selective small molecule inhibitor of B-RafV600E [14]. PLX4720 was de- signed to block the ATP-binding site of oncogenic B-RafV600E [14]. PLX4720 binds selectively to the active B-RafV600E protein conformation. Consistent with the high degree of selectivity, PLX4720 inhibits B-RafV600E kinase activity both in vitro and in vivo in melanoma and colorec- tal tumor cell models [14]. We performed in vitro testing of PLX4720 on human thy- roid cancer cell lines harboring the B-RafV600E mutation or the www.TheOncologist.com 297 preferentially inhibits migration and invasion of B-RafV600E thyroid cancer cells and tumor aggressive- ness. Normal thyroid cells were generated to be heterozygous for wild-type B-Raf/B-RafV600E, mimick- ing the condition found in most human thyroid cancers. PLX4720 was effective in reducing cell proliferation, migration, and invasion in this heterozygous model. PLX4720 therapy should be tested and considered for a phase I study for the treatment of patients with B-RafV600E ATC. The Oncologist 2011;16:296 –309 RET/PTC-1 translocation (with wild-type [wt] B-Raf in both alleles) and on primary human normal thyroid (NT) follicular cells engineered to express B-RafV600E. We furthermore used an orthotopic mouse model of ATC harboring B-RafV600E to test in detail the in vivo activity of PLX4720 on tumor aggres- siveness using an early intervention model. MATERIALS AND METHODS Antibodies Antibodies for Western blot (WB) and immunohistochem- istry (IHC) are reported in the supplemental online data. PLX4720 Preparation PLX4720 was dissolved in dimethyl sulfoxide (DMSO) at 1 M or 10 M for in vitro assays. For the in vivo prepara- tion, PLX4720 was dissolved in DMSO (120 mg/ml) and then suspended in a 1% solution of carboxymethycellulose (Sigma, St. Louis). Human Thyroid Cancer Cell Lines and NT Cells We used the TPC-1 cell line (human PTC, RET/PTC-1/wt for B-Raf), provided by Dr. F. Frasca (University of Catania, Ca- tania, Italy), and the 8505c cell line (human ATC harboring B-RafV600E), purchased from DSMZ (German collection of microorganisms and cell culture) [15]. NT cells were obtained according to Fischer et al. [16]. All in vitro assays were per- formed by growing these cell lines with the specific growth medium supplemented with 1% fetal bovine serum. More de- tails are reported in the supplemental online data. Cell Transfections for Retrovirus Production Sixty-millimeter plates of HEK 293T cells (5 105) were transfected using Fugene-6 (Roche, Indianapolis) in Op- tiMEM (Invitrogen, Carlsbad, CA) for 48 hours. B-RafV600E Transduction in NT Cells B-RafV600E-pBABE-puro and pBABE-puro (empty vector) retroviral constructs were provided by Dr. W.C. Hahn (Dana