Nucera, Nehs, Nagarkatti et al. novel therapies. We chose to study this both in vitro and in vivo using PLX4720, a potent and selective B-RafV600E in- hibitor. In one previous report, in vitro treatment of B-RafV600E-positive thyroid cancer cell lines with PLX4720 (50% inhibitory concentration in the range of 78 –113 nM) showed cell cycle arrest, whereas RET/PTC-1 and Ras mutated thyroid cancer cell lines were resistant to inhibition even at 500 nM [27]. In contrast, we found that only higher doses (both 1 M and 10 M) of PLX4720 caused cell cycle arrest and inhib- ited tumor cell migration and invasion in 8505c cells (B- RafV600E). Interestingly, by overexpressing B-RafV600E in NT cells, we were able to create a heterozygous model that genetically reproduces human thyroid cancers that have both a B-RafV600E allele and a wt B-Raf allele. With this overexpression of B-RafV600E, the ability of NT cells to in- vade and migrate was much greater than in controls. The gain of these functions was effectively inhibited by 10 M PLX4720, suggesting a strong role for B-RafV600E in thy- roid cancer progression. Therefore, this is the first in vitro translational demonstration that PLX4720 might be a useful compound in clinical trials for patients with thyroid cancers harboring the B-RafV600E mutation. Recently, it was shown that PLX4720 could have unex- pected effects in some cell contexts (e.g., presence of wt B- Raf and mutated Ras) as a result of ERK-1/ERK-2 activation [25, 33, 34]. However, in our study, PLX4720 treatment of NT cells (wt B-Raf/wt B-Raf) transduced with empty vector did not result in greater proliferation, migra- tion, or invasion despite a higher level of phospho-ERK-1/ ERK-2. Somewhat unexpectedly, cell proliferation and migration were also inhibited in TPC-1 cells (RET/PTC-1 translocation, PIK3CA H1047R mutation, and wt B-Raf). These results indicate that certain wt B-Raf thyroid tumors (perhaps those that harbor RET/PTC-1 and/or PIK3CA H1047R) might be somewhat responsive to PLX4720 treat- ment as well. Further preclinical validation is needed to un- derstand this phenomenon prior to human testing. PLX4720 treatment downregulated genes involved in cell proliferation, such as CCND1, and certain genes known to be important in migration, invasion, and metastasis, such as TSP-1 and ITGA6 [35]. We have previously shown that knockdown of TSP-1 decreases phospho-ERK-1/ERK-2 levels and reduces thyroid cancer progression [19], perhaps through binding of the N-terminal domain of TSP-1 to in- tegrins such as 3 1 or 6 1 [36, 37]. TSP-1 is known to be intimately involved in the regulation of cellular prolifer- ation by affecting early response genes [38], and our data are consistent with those results [19]. Our in vitro studies show higher phospho-ERK-1/ ERK-2 levels when TPC-1 cells are treated with PLX4720. www.TheOncologist.com 307 Recent findings by Hatzivassiliou et al. [34] showed that ATP-competitive Raf inhibitors, including PLX4720, have opposing roles as inhibitors and activators of ERK-1/ ERK-2 phosphorylation depending on the cellular and ge- notypic context. Furthermore, other authors recently reported that inhibition of wt B-Raf by PLX4720 in Ras mutant (e.g., N-Ras) human cancer cells (i.e., melanoma cells) leads to MEK–ERK-1/ERK-2 hyperactivation through C-Raf [25]. These aforementioned studies [25, 33, 34, 39] and others [40] highlight the importance of individualized genomic profiling to guide patient selection for inclusion in targeted therapy tri- als. We also found that PLX4720 treatment in vitro and in vivo in 8505c cells upregulated important thyroid-related transcrip- tion factors, such as TTF-1 and PAX-8, that have been widely described as markers of thyroid follicular cell differentiation, thus suggesting that B-RafV600E might play a biological role in thyroid follicular cell dedifferentiation. In vivo, we demonstrated a dramatic reduction in tumor aggressiveness in our orthotopic mouse model of ATC. PLX4720 treatment reduced tumor volume by 97% and vir- tually eliminated metastatic spread to the lungs. Though the ATC cell line 8505c also harbors a TP53 mutation [41], the selective inhibition of B- RafV600E was sufficient to dramat- ically reduce tumor volume, extrathyroidal extension, and metastasis without any obvious toxicity. Both in vitro and in vivo results point to the potential therapeutic utility of this drug in patients with advanced B-RafV600E thyroid cancers. In conclusion, our study shows that inhibition of B-RafV600E with the novel agent PLX4720 results in dra- matic changes in cell proliferation, migration, and invasion in vitro, and early intervention with this drug results in marked inhibition of tumor aggressiveness in an in vivo model of ATC harboring B-RafV600E. Our in vitro model us- ing primary human NT follicular cells (heterozygous wt B-Raf/B-RafV600E) recapitulates the genetic status of this mutation in human thyroid cancer, and is the first preclini- cal model to show that PLX4720 inhibits cell proliferation, migration, and invasion in a B-RafV600E heterozygous model. PLX4720 might be an effective therapy for appro- priately selected patients with metastatic or refractory thy- roid cancers that harbor the B-RafV600E mutation. SUMMARY AND CLINICAL RELEVANCE Despite considerable enthusiasm about targeted therapies and understanding the underlying reasons why the B-RafV600E mutation may lead to more aggressive clinical behavior in some patients with thyroid cancer, there have not been many studies on the inhibition of B-RafV600E. Our study shows that inhibition of B-RafV600E with the novel agent PLX4720 can decrease cell proliferation, migration, and invasion in thyroid cancer cell lines and markedly in-