in G1 from 42% (DMSO control) to 71% after only 24 hours (Fig. 5A). Co-treatment with BEZ235 and temsirolimus led to a slight increase of cells in G1 compared to temsirolimus alone at 24 hours (Fig. 5A). In contrast, treatment of Hec50co cells with temsirolimus alone caused a 4% increase in the G1 population, which required 72 hours of prolonged exposure to demonstrate (Fig. 5B). However, when temsirolimus was combined with BEZ235, the G1 population increased to 60% by 72 hours (Fig. 5B). Further, ZSTK474 effects in combination with temsirolimus (Fig. 5C) replicated those of BEZ235 (Fig. 5A), suggesting that G1 arrest is a common method by which combined inhibition of PI3K and mTOR achieves the decrease in cell viability observed in Figure 4. Also of note, the G1 block was achieved rapidly in sensitive cells, but eventually was observed even in resistant cells. Expression of the cyclin dependent kinase inhibitor, p27, was next tested in order to explore the cell cycle regulatory proteins downstream of mTOR that might explain the G1 block. As shown in Figure 5D, single and combined drug treatments promoted increased expression of p27 at the protein level, implicating induction of p27 in the mechanism of G1 arrest. Combined treatment induces autophagy as a mechanism of cell death We next characterized the mechanism of cell death in response to combination therapy. Examination of several apoptotic markers, including caspase 3, revealed that the cells did not undergo caspase-dependent apoptosis (data not shown). However, several studies have demonstrated that both down-regulation of Akt [28,29] and treatment with rapalogs [26] promote autophagy, which can lead to caspase-independent apoptosis characterized by poly (ADP-ribose) polymerase (PARP) cleavage [30,31]. There- fore, we first tested processing of microtubule-associated protein light chain 3-I (LC3-I), which is a common marker for autophagy [32]. As noted in Figure 6A, under our experimental conditions, no loss of LC3-I occurred upon temsirolimus treatment when compared with control. In contrast, BEZ235 alone dramatically reduced the level of LC3-I in all tested cells. Furthermore, combination of temsirolimus with ZSTK474 reduced levels of LC3-I compared with ZSTK474 alone. The reduction in LC3-I levels was accompanied by the expected increase or maintenance of LC3-II in some cell lines (Ishikawa H and Hec50co), further confirming that the cells are undergoing autophagy (Fig. 6A). Since others have reported that BEZ235 induces caspase- independent apoptosis through PARP cleavage [30], we treated cells for longer periods of time (48 to 72 hrs) and examined PARP cleavage as a marker of apoptotic cell death. In both sensitive and resistant cell lines, we observed PARP cleavage following treatment with BEZ235 or ZSTK474, and addition of temsir- olimus did not substantially increase the effect (Fig. 6B). Collectively, these data suggest that the mechanism of cell death involves massive autophagy which proceeds into caspase-indepen- dent apoptosis. Mechanism of synergy between PI3K and mTOR inhibitors Recently, several groups reported that 4E-BP1, not rS6, which are both downstream of mTORC1, is critical for controlling oncogenic Akt signaling [10,11]. Additionally, it was demonstrated the BEZ235 is more effective at down-regulating 4E-BP1 phosphorylation as compared to rapamycin and second-genera- tion mTOR inhibitors [11]. ZSTK474 targets PI3K, which is upstream of PI3K/Akt/mTOR signaling, while temsirolimus mTOR and PI3 Kinase Inhibitor Synergy inhibits mTORC1, which is downstream of this pathway. This strategy, targeting both ends of the same pathway, clearly demonstrated synergy in cell proliferation assays (Fig. 4B). However, BEZ235 itself is a dual PI3K/mTOR inhibitor that functions both upstream and downstream of the PI3K/Akt/ mTOR pathway and should be sufficient to completely block signaling; nevertheless, a synergistic effect was observed when BEZ235 was combined with another mTOR inhibitor, temsir- olimus. This finding was unexpected, and we sought an explanation for why blocking mTORC1 with two different agents produced synergy. We addressed this question by examining the individual effect of BEZ235 and temsirolimus on downstream components of mTORC1 pathway signaling (Fig. 7A, B). The downstream components tested were 4E-BP1 and rS6 (Fig. 7C). Surprisingly, temsirolimus had little or no effect on inhibiting 4E- BP1 phosphorylation compared with control despite its ability to fully block phosphorylation of rS6 (Fig. 7B). On the other hand, BEZ235 completely blocked 4E-BP1 phosphorylation in all tested cell lines but had less effect on rS6 phosphorylation (Fig. 7A, B). When testing the phosphorylation of rS6, we discovered that temsirolimus inhibited it much more effectively (at 1 nM) than BEZ235 at the same concentration (Fig. 7B and Supporting Fig. S3). In summary, to fully block both of the important downstream effectors, both drugs were required. Figure 7C is a schematic model to summarize our findings on the specific mechanistic functions of PI3K/Akt/mTOR inhibitors in the treatment of cancers that are reliant on this pathway. Discussion Temsirolimus is currently in phase II trials for advanced endometrial cancer and has shown some promise. However, lack of initial response to therapy as well as the development of acquired drug resistance continues to be problematic. To more fully understand the therapeutic potential of mTOR inhibition in endometrial cancer, we first examined the effect of temsirolimus alone on the viability of a panel of endometrial cancer cell lines. We sought to distinguish between cellular events which predict for primary resistance as well as those events which are linked to the eventual development of acquired resistance. Consistent with other types of cancer, primary resistance to temsirolimus is found in a subset of these cell lines. Our data suggest that primarily resistant cells lack robust Akt signaling, are unable to phosphor- ylate Akt at baseline, and express PTEN. In contrast, the most sensitive cell lines have lost PTEN expression and have high baseline phosphorylation of Akt. Our data demonstrate that in these cells, temsirolimus treatment promotes a further increase in Akt phosphorylation, indicating that signaling through the pro- survival PI3K/Akt pathway is likely how these endometrial cancer cell lines eventually circumvent mTOR inhibition. These results are consistent with previous reports in other types of cancers documenting compensatory Akt phosphorylation in response to other rapalogs [7,16,24]. This has been observed in xenograft models of lung cancer [24] as well as in advanced colon and breast cancer tissues following rapalog therapy [16]. The elevated Akt phosphorylation is thought to be a predominant driving force in resistance to temsirolimus treatment in these cancers [7]. To overcome resistance, we adopted a combination strategy. Dual treatment with temsirolimus and the PI3K inhibitor ZSTK474 or the PI3K/mTOR inhibitor BEZ235 overcame the temsirolimus-induced Akt hyper-phosphorylation, which is a marker for developing acquired resistance; furthermore, this treatment strategy synergistically decreased viability and promoted G1 cell cycle arrest even in the cell lines that were primarily PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e26343