Thyroid Cancer and NVP-BEZ235 Figure 5. BEZ235 decreases p-AKT(Thr308), p-S6 ribosomal protein (Ser235/236), caspase-3, and retards the growth of ATC flank xenograft tumors without significant toxicity in nude mice. A, daily oral gavage of BEZ235 (50 mg/kg) represses 8505C tumor growth. The differences of tumor volume between BEZ235 and control group achieved statistical significance on days 21 and 24. Significance was lost on day 28, 4 days after treatment had been discontinued. B, representative mice with 8505C xenograft tumors (arrows) were photographed on last day of treatment (day 24). C, BEZ235 did not result in any weight loss, in treated and control mice (P.0.05 for both comparison). D, BEZ235 represses p-AKT (Thr308) and p-S6 ribosomal protein (Ser235/236) by 2 hours and caspase-3 by 4 hours in vivo. PCNA was slightly reduced at early period. E, expression of p-S6 ribosomal protein in tumor cells is detected by immunostaining at the indicated periods (magnification, 6200). These results were in consistent with Western blot results. doi:10.1371/journal.pone.0046726.g005 suggesting that prolonged treatment may be necessary to maintain therapeutic efficacy. BEZ235 significantly degraded caspase-3 in 8505C xenograft tumors, indicating this compound may induce apoptosis in vivo. After discontinuation of BEZ235, the volume of 8505C xenograft tumors subsequently increased. This enlarge- ment of tumor following cessation of therapy might be due to normalization of cell size and resumption of cell proliferation, as mTOR and its downstream proteins S6 Kinase 1 and 4E-BP1 play pivotal roles in this respect [43]. No significant weight loss or illness was observed during study period, suggesting that this therapy may have a promising safety profile. A prior report demonstrated that the presence of genetic alterations of PTEN, PIK3CA and AKT1 correlated well with sensitivity to an AKT inhibitor, but had weaker correlations with an mTOR inhibitor [44]. This discrepency suggests that mTOR activity does not depend solely on PI3K/AKT activity. The reported data of genetic alterations in 8 thyroid cancer cell lines was summarized (Appendix S2). Limited genetic changes on RAS/RAF/ERK and PI3K/mTOR pathways were identified. The available data do not show any correlation between genetic aberrations of PI3K/mTOR pathways and sensitivity of BEZ235, as no mutation of this pathway in these cell lines reported. Mutations of p53 and BRAF also seem to not be correlated with PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e46726