Thyroid Cancer and NVP-BEZ235 Figure 3. BEZ235 inhibits cell cycle progression at G0/G1 phase, and induces apoptosis in KAT4C and KAT18. A, analysis of cell cycle by measurements of DNA content revealed increasing doses of BEZ235 accumulates cells at G0/G1 by 24 hours (KAT4C, 8505C, BHP7-13 and WRO82-1) and 72 hours (TT). B, a representative cell line, BHP7-13 demonstrated cell cycle arrest at G0/G1 phase. C, cell cycle regulating proteins were evaluated using immunoblot in cells treated with BEZ235 at 100 nmol/L. p53 was decreased by 2 to 4 hours in 8505C and TT. BEZ235 gradually reduced p21 in TT and BHP7-13. p27 was increased in all cell lines. Cyclin D1 gradually decreased in 8505C and BHP7-13. D, apoptosis measured by flow cytometry to measure sub-G1 cells revealed that BEZ235 increases proportions of sub-G1 cells in KAT4C and KAT18 at 96 hours. E, immunoblot showed BEZ235 induced degradation of executioner caspase-3 in KAT4C and KAT18. doi:10.1371/journal.pone.0046726.g003 mTOR signaling in the process of tumor de-differentiation may be particularly attractive targets for therapy. The failure of BEZ235 to repress p-AKT (Ser473) in 8505C and KAT4C may be related to a negative feedback inhibition. It has been previously shown that inhibition of mTORC1 leads to inactivation of S6 kinase 1, which may subsequently overwhelms the inhibitory effect of BEZ235 on mTORC2, activates mTORC2, and increases p-AKT (Ser473) in 8505C [35]. Prior reports also showed that lower doses of BEZ235 fail to inhibit p- AKT (Ser473) in some cell lines, and higher doses of BEZ235 may overcome the negative feedback of mTORC1/S6 kinase 1 feedback loop [18,20]. Although p-AKT (Ser473) was activated in 8505C and KAT4C, BEZ235 had better inhibitory effects in these cell lines as compared to TT and BHP7-13, suggesting that other molecules affected by BEZ235 play a more important role in determining therapeutic outcome. We found that the expression of p-S6 ribosomal protein (Ser235/236) and p27 correlate with the sensitivity of BEZ235 in thyroid cancer. S6 ribosomal protein is a downstream of S6 kinase 1, which is activated by mTORC1 [6–8]. Phosphorylation of S6 ribosomal protein increases translational control of protein synthesis and enhances cell growth. Cells with higher levels of p-S6 ribosomal protein (Ser235/236) are more susceptible to BEZ235, suggesting that the inhibition of mTORC1 and S6 ribosomal protein is the major therapeutic effect of BEZ235. p-S6 ribosomal protein (Ser235/236) has also recently been recognized as a marker to predict therapeutic effect of an mTOR inhibitor in sarcoma [36]. In this study, thyroid cancer lines with higher expressions of p-S6 ribosomal protein (Ser235/236) also showed lower levels of p27. This finding suggests S6 ribosomal protein is a suppressor of p27 in thyroid cancer, and may explain why both p-S6 ribosomal protein (Ser235/236) and p27 were predictors of sensitivity to BEZ235. Interestingly, p27 was previously noted to have an inverse association with the activity of the PI3K/mTOR pathway in thyroid cancer cells, and repression of this pathway increases p27 [37]. PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e46726