Thyroid Cancer and NVP-BEZ235 Figure 2. mTORC1downstream signaling is inhibited and p-ERK1/2 is activated by BEZ235 at 100 nmol/L. p-AKT (Thr308) and p-AKT (Ser473) was increased in 8505C for more than 24 hours. p-AKT (Thr308) and p-AKT (Ser473) was decreased transiently in TT and BHP7-13. p-4E-BP1 (Thr70), p-4E-BP1 (Thr37/46) and p-S6 ribosomal protein (Ser235/236) were consistently reduced from 2 hours through over 24 hours in three cell lines. BEZ235 caused a rapid increase of p- ERK1/2 (Thr202/Tyr204) by 2 to 4 hours in three cell lines. doi:10.1371/journal.pone.0046726.g002 BEZ235 did not significantly reduce body weight between the two groups during the study period (Figure 5C). Oral BEZ235 reduced phosphorylation of AKT and S6 ribosomal protein by 2 hours and achieved its greatest inhibitory effects at 4 hours with just 2% and 10% of pAKT and pS6 remaining, respectively. This inhibitory effect persisted for 24 hours in xenograft tumors (Figure 5D, Figure S5). PCNA was slightly and transiently repressed from 2 to 4 hours. However, significant degradation of caspase-3 occurred by 4 hours, with only 4% of caspase-3 remaining by 24 hours. Immunohistochemistry of p-S6 ribosomal protein (Ser235/236) in tumors treated with BEZ235 similarly demonstrated a reduction in staining, most markedly from 2 to 6 hours (Figure 5E). Interaction of BEZ235 and chemotherapy in ATC cells We studied the combination of chemotherapeutic agents and BEZ235 against ATC. Five chemotherapeutic agents (paclitaxel, irinotecan, etoposide, 5-FU and doxorubicin) demonstrated cytotoxic effects in a dose- and time-dependent manner in four ATC cell lines (Figure S6). The data were used to calculate the median effect dose (Dm) on day 4 using CompuSyn software (Figure 6A). These Dm of paclitaxel, irinotecan and etoposide are achievable in patients’ serum, and are clinically relevant [32–34]. Although doxorubicin is used clinically for anaplastic thyroid cancer, the relatively high Dm in these cell lines implied that clinically relevant dosing is not achievable in patients serum and therefore was excluded from these studies. Interactions between BEZ235 and paclitaxel, irinotecan, and etoposide were evaluated (Figure 6B). The combination of BEZ235 and paclitaxel significantly improved cytotoxicity over single agent therapy in four cell lines. BEZ235 combined with irinotecan also enhanced therapeutic efficacy, particularly when more cells were affected. BEZ235 plus etoposide only slightly increased cytotoxicity. Interactions between BEZ235 and each chemotherapeutic agent were determined by calculating the CI by Chou-Talalay equation (Figure 6C). Synergistic effects were identified for the combination of BEZ235 and paclitaxel in all ATC lines (CI, 8305C = 0.77–0.86, 8505C = 0.43–0.59, KAT4C = 0.66–0.81 and KAT18 = 0.64–0.71). The combination of BEZ235 with irinotecan ranged from synergistic to antagonist in 8305C, 8505C and KAT18 (CI, 0.86– 1.34, 0.97–1.23 and 0.74–1.14) and was antagonist in KAT4C (CI, 1.09–1.36). The combination of BEZ235 with etoposide was slightly synergistic to antagonist in 8305C (CI, 0.95–1.15) and antagonist in 8505C, KAT4C and KAT18 (CI, 1.16–1.17, 1.13– 2.05 and 1.05–1.19, respectively). These results demonstrate that BEZ235 and paclitaxel had the best combined effects in treating ATC. The calculated DRI of the chemotherapy agent means the fold of the drug dose that could be reduced in the presence of BEZ235 (Figure 6D). Among three chemotherapeutic agents, paclitaxel had the greatest DRIs in 8505C (2.55–4.2), KAT4C (2.25–3.19) and KAT18 (2.59–2.63), and also demonstrated favorable DRIs in 8305C (1.55–2.16). Discussion 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 [22]. 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 (,44 nmol/L) suggest that BEZ235 may have utility for treating a spectrum of thyroid malignancy. Refractory cancers that develop activation of PI3K/ PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e46726