Thyroid Cancer and NVP-BEZ235 Figure 4. Susceptibility to BEZ235 correlates with baseline expression of p-S6 ribosomal protein (Ser235/236) and p27. A, the baseline levels of proteins associated with signaling transduction and cell cycle were evaluated by immunoblot. The sequence of proteins loaded was according to Dm. The expression of p-S6 ribosomal protein (Ser235/236) gradually decreased from KAT4C to BHP7-13, while p-27 showed the opposite order. The expression of the other proteins showed a random distribution. B, Band densities were imaged and quantified. Relationships between Dm and protein expression were analyzed using Pearson’s correlation coefficients, and graphs were drawn using KAT4C. The expression of p-S6 ribosomal protein (Ser235/236) had a positive correlation, and p27 an inverse correlation, with sensitivity to BEZ235. doi:10.1371/journal.pone.0046726.g004 4E-BP1 is another protein downstream of mTORC1. Inhibition of mTORC1 leads to dephosphorylation (activation) of 4E-BP1, enhancing the binding of 4E-BP1to eIF4E, and blocking protein translation and cell proliferation [7,8]. Although BEZ235 affects both S6 ribosomal protein and 4E-BP1 efficiently, only p-S6 ribosomal protein (Ser235/236) expression predicts for sensitivity to BEZ235 in this study. In ovarian cancer, biomarkers predicting susceptibility of BEZ235 were reported [22], and the expression of p-4E-BP1 (Thr37/46) did correlate with the sensitivity of BEZ235. In addition to inhibiting cell cycle progression, BEZ235 caused apoptosis in two of six cell lines. The inhibition of cell cycle progression is a known effect of BEZ235, even at lower doses (#100 nmol/L). However, apoptosis appears in only some cancer cell lines and is more apparent at higher doses (100–1000 nmol/L) of BEZ235 [17–22]. BEZ235 efficiently inhibited mTORC1, a molecule controlling both cell cycle and apoptosis that might lead to cell cycle arrest and apoptosis in KAT4C and KAT18 [7]. Understanding the mechanisms through which BEZ235 contrib- utes to apoptotic cell death will require further study. ATC is by far the most aggressive of the four major histologic types of thyroid cancer. Chemotherapy has been applied to treat patients with ATC with response rates around 20 to 50%. Novel strategies to improve outcomes are needed. Among three combination therapy regimens, BEZ235 combined with paclitaxel had the best synergistic effect in four ATC cell lines. Cancer cells with activation of PI3K/mTOR signaling are more resistant to paclitaxel, and the co-administration of PI3K/mTOR inhibitors with paclitaxel improves therapeutic effects [38–40]. This finding is of clinical relevance since paclitaxel, a microtubule stabilizer, has shown to achieve a 53% response rate in patients with ATC in a phase II clinical trial [41]. The combination of BEZ235 with a microtubule depolymerizing drug vincristine also revealed prom- ising effect in the treatment of sarcoma [20]. Our data showed that the combinational effects of BEZ235 with inhibitors of DNA topoisomerase type I (irinotecan) or type II (etoposide) in treating ATC were largely antagonistic. Similar antagonistic effects of inhibition both topoisomerase activity and PI3K/AKT pathway have been observed in ovarian cancer cells [42]. Cells in S phase are more susceptible to topoisomearse inhibitors. BEZ235-induced accumulation of cells at G0/G1 phase may therefore reduce the therapeutic advantage of concurrent therapy with topoisomerase inhibitors, explaining the unfavorable combination effects of BEZ235 with irinotecan and etoposide. Daily treatment of BEZ235 significant retarded 8505C xeno- graft tumor growth during the therapeutic period. The inhibitory effect was less prominent after the discontinuation of therapy, PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e46726