Thyroid Cancer and NVP-BEZ235 Figure 1. BEZ235 induces dose and time dependent cytotoxicity in 8 thyroid cancer cell lines. A, dose-response curves were obtained daily from cells treated with a range of six 1:1 dilutions of BEZ235. B, the Dm of BEZ235 on day 4 was calculated using CompuSyn software for each cell line. ATC cell lines (KAT4C, KAT18, 8305C and 8505C) had the lowest Dm and thus most sensitive, follow by follicular undifferentiated thyroid cancer (FRO81-2), medullary (TT) and well-differentiated papillary (BHP7-13) and follicular (WRO82-1) cancer. doi:10.1371/journal.pone.0046726.g001 BEZ235 sensitivity correlates with baseline expression of p-S6 ribosomal protein (Ser235/236) and p27 The Dm of BEZ235 spans a broad spectrum across 8 different cell lines, with an 11-fold difference between KAT4C and WRO82-1.To explore potential biomarkers that correlate with sensitivity of BEZ235, the baseline expression of proteins involved in the PI3K/mTOR and RAS/RAF/ERK pathways, and cell cycle-associated proteins were evaluated in 6 cell lines (Figure 4A). The sensitivity of the six cell lines to BEZ235 was ordered according to the Dm value. The expression of p-S6 ribosomal protein (Ser235/236) gradually decreases across increasing Dm, while the level of p-27 gradually increases across increasing Dm. Statistical relationships were analyzed using Pearson’s correlation coefficient (Figure 4B). The expression of p-S6 ribosomal protein (Ser235/236) had a significant correlation (R2 = 0.8475, P = 0.009) with BEZ235 sensitivity, while p27 had a significant inverse correlation (R2 = 0.8117, P = 0.014). A repeated immunoblot of p27 showed consistent results (Appendix S1). The expression of baseline p- AKT (Thr308), p-AKT (Ser473), p-4E-BP1(Thr70), p-4E-BP1 (Thr37/46), p-S6 ribosomal protein (Ser240/244), p-ERK1/2 (Thr202/Tyr204), p53, p21 and cyclin D1 failed to reveal any correlations with BEZ235 sensitivity (data not shown). BEZ235 therapy of murine flank tumors Athymic nude mice with flank xenografts of 8505C were used to study the therapeutic effects and safety of BEZ235 in vivo. Animals with established flank tumors of similar starting volumes were treated with oral BEZ235 (50 mg/kg) or vehicle daily for 25 days and followed until day 31 (Figure 5A). BEZ235 retarded tumor growth and the difference of tumor volumes between BEZ235 and control mice was statistically significant at day 21 (94.1626.4 mm3 and 335.1678.5 mm3, P = 0.009, t-test) and day 24 (109.9629.6 mm3 and 403.16112.8 mm3, P = 0.019, t-test). The difference of tumor volumes lost significance at day 28, 4 days after treatment had been discontinued (402.76123.6 mm3 and 3 750.36198.3 mm , P = 0.15, t-test). Representative mice were photographed on the last day of treatment (Figure 5B). PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e46726