BEZ235 effectively inhibited cell proliferation in eight thyroid cancer lines originating from four major histologic types. ATC were the most sensitive, followed by follicular undifferentiated, medullary and well-differentiated thyroid cancer cell lines. Cancer cells harboring a PI3K gain-of-function mutation or a PTEN deletion demonstrate higher PI3K/mTOR pathway activity and greater sensitivity to BEZ235. Our data suggest that ATC relies on PI3K/mTOR activity, and interruption of this pathway with BEZ235 impairs ATC growth more significantly than other thyroid cancer histologies. The relatively low median effect doses of BEZ235 in all of the thyroid cancer lines suggest that BEZ235 may have utility for treating a PTK/ZK structure spectrum of thyroid malignancy. Refractory cancers that develop activation of PI3K/mTOR signaling in the process of tumor de-differentiation may be particularly attractive targets for therapy. The failure of BEZ235 to repress p-AKT 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 107257-28-3 kinase 1, which may subsequently overwhelms the inhibitory effect of BEZ235 on mTORC2, activates mTORC2, and increases p-AKT in 8505C. Prior reports also showed that lower doses of BEZ235 fail to inhibit p-AKT in some cell lines, and higher doses of BEZ235 may overcome the negative feedback of mTORC1/S6 kinase 1 feedback loop. Although p-AKT 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 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. Phosphorylation of S6 ribosomal protein increases translational control of protein synthesis and enhances cell growth. Cells with higher levels of p-S6 ribosomal protein 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 has also recently been recognized as a marker to predict therapeutic effect of an mTOR inhibitor in sarcoma. In this study, thyroid cancer lines with higher expressions of p-S6 ribosomal protein 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 and p27 were predictors of sensitivity to BEZ235.