of targeting both upstream (PI3K) and downstream (mTORC1/ mTORC2) of the PI3K/Akt/mTOR axis. BEZ235 has been reported to inhibit growth and proliferation and induce apoptosis in a variety of tumor cell lines [27,35], including breast cancer cells with mutant or amplified PIK3CA [30]. BEZ235 showed antitumor activity in nude mice with few side effects [27]. A recent report from a phase I study of BEZ235 in 59 patients with advanced solid tumors demonstrated antitumor effects and a favorable safety profile [36]. ZSTK474, a pan-class I PI3K inhibitor, also demonstrated high potency against a panel of cancer cell lines and human tumor xenografts without toxicity to major organs [34,37]. As discussed above, among all drugs tested, the agents which produced synergy with temsirolimus in our models were BEZ235 and ZSTK474. A main conclusion of our study is that combination treatment of ZSTK474 or BEZ235 with temsirolimus synergizes to decrease viability in endometrial cancer cell lines. A potential mechanism of synergy from co-treatment with ZSTK474 and temsirolimus is the vertical blockade of hyper-activated PI3K/Akt/mTOR signaling, specifically the simultaneous targeting of the upstream component PI3K by ZSTK474 and the downstream component mTOR (p70S6K) by temsirolimus. Temsirolimus alone only blocks rS6K activity downstream of mTORC1, whereas signaling through the other mTORC1 target 4E-BP1 is left intact. It has been documented in the literature that signaling through 4E-BP1 is required for Akt-mediated oncogenesis [10]; therefore, inhibition of all components of this pathway is necessary to prevent tumor growth. Our data indicate that, in addition to inhibition of Akt activation, BEZ235 effectively blocks this residual signaling through 4E-BP1, which, when combined with temsirolimus inhibition of rS6K, synergistically blocks all arms of the PI3K/ Akt/mTOR pathway. Besides the observed inhibition of 4E-BP1 and rS6 with combined BEZ235 (dual PI3K/mTOR inhibitor) and temsirolimus (mTORC1 inhibitor), another possibility might explain the observed synergy. Temsirolimus and BEZ235 target different structural domains of mTOR: temsirolimus is an allosteric inhibitor that targets the FKBP12-rapamycin-binding (FRB) domain while BEZ235 is a catalytic inhibitor that targets the kinase domain. The inhibitory potential of targeting two structurally distinct regions of the same protein may, therefore, contribute to the synergistic effect we observed when cells were treated with temsirolimus and BEZ235 compared to single agent treatment alone. It has previously been shown that treatment with BEZ235 or ZSTK474 results in cell cycle arrest at G1 [27,37]. Our study demonstrates that cells were more likely to arrest in G1 if they had been treated with either BEZ235 or ZSTK474 with temsirolimus compared to controls or single agent treatment. This may be attributed to the ability of BEZ235 to promote increased expression of the CDKI p27 [27]. Accordingly, we also detected elevated p27 expression when endometrial cancer cells were treated with BEZ235 alone or in combination with temsirolimus. While inhibition of prosurvival Akt signaling is cytotoxic, the mechanism of cell death involves autophagy and apoptosis [26]. We observed a decrease in the autophagy marker, LC-3I, in response to dual mTOR/PI3K inhibition, implicating autophagy. Others have shown that depletion of all three Akt isoforms promoted tumor regression through initiation of autophagy [29], and inhibition of mTOR with the alkylphospholipid perifosine induces autophagic cell death [28]. BEZ235 has also been shown to induce caspase-independent apoptosis in a mechanism that includes PARP cleavage [30], which we also observed in our study. Taken together, these data suggest that the mechanism of cell death is through autophagy and caspase-independent apoptosis. mTOR and PI3 Kinase Inhibitor Synergy Molecular profiling of the endometrial cancer cell lines revealed that sensitivity toward drug treatment correlates with loss of PTEN expression and hyper-activation of Akt. In endometrial tumors, loss of PTEN has previously been shown to correlate with elevated Akt phosphorylation and results in poor outcomes [17,38]. Our findings are consistent with earlier studies showing low expression of PTEN in RL95-2, AN3CA, ECC-1, and Ishikawa H cells and high expression of PTEN in Hec50, Hec1A, and KLE cells [4,39]. Furthermore, RL95-2, AN3CA, ECC-1, and Ishikawa H cells harbor mutant PTEN, whereas KLE, Hec50, Hec1A, and Hec1B (a substrain of Hec1A) cells express wildtype PTEN [40,41]. The fact that Hec50 cells contain both wildtype PTEN and high Akt phosphorylation can be explained by recent data demonstrating that PI3KR1, a regulatory subunit of PI3K, is mutated in Hec50 cells and thus may phenocopy loss of PTEN [4]. Additional investigation is necessary to understand why Ishikawa H cells, which have high Akt phosphorylation and a loss of active PTEN, are relatively resistant to temsirolimus. These data highlight the fact that molecular profiling does not always predict for response due to the complexity of pathways governing tumor initiation and progression. Furthermore, basal Akt phosphorylation correlates with response to another rapalog, RAD001, in a panel of various cancer cell lines [8]. Studies involving PTEN+/2 mice or cell lines devoid of PTEN show that PTEN-deficient tumors are sensitive to mTOR inhibition [20]. A phase I clinical trial demonstrated that 63% of patients with PTEN-negative tumors displayed tumor regression when treated with drugs targeting the PI3K/Akt/ mTOR signaling pathway [42]. These results are consistent with our data that cell lines with little or no PTEN are more sensitive to temsirolimus alone and with the combination of temsirolimus and BEZ235. In accord with our findings, single-agent BEZ235 has been shown to inhibit proliferation of endometrial cancer cells harboring PIK3CA and/or PTEN mutations by other investigators [43]. These studies reported herein promote a further under- standing of the potential underlying mechanisms of both primary cell resistance and the development of acquired resistance after therapy, both of which can be overcome with the combination of mTOR and PI3K inhibitors. Our data underscore the need to inhibit PI3K/Akt/mTOR signaling at multiple levels to achieve sustained cellular responses. These data will enhance the rational use of combinatorial regimens involving temsirolimus and PI3K inhibitors in future clinical trials. Supporting Information Figure S1 Efficacy of temsirolimus in a panel of endometrial cancer cell lines. The indicated cell lines were treated with vehicle or 1 mM temsirolimus for 24 hrs. Lysates were obtained and equal amounts Western blotted for phospho-mTOR, total mTOR, phospho-rS6, or total r6S. (TIF) Figure S2 Effect of various combination therapies on Akt phosphorylation. Ishikawa H (upper panels) or Hec50co (lower panels) cells were treated with temsirolimus in the presence or absence of the indicated molecular inhibitors for 24 hrs at the noted concentrations. Lysates were obtained and equal amounts Western blotted for phospho-Akt or total Akt. (TIF) Figure S3 Effect of BEZ235, ZSTK474, and temsirolimus on rS6 phosphorylation. Phosphorylation of rS6 (P-rS6 S235/236) was assessed after incubating cells with the indicated treatments for 24 hrs. Total rS6 expression serves as a loading control. (TIF) PLoS ONE | www.plosone.org 11 October 2011 | Volume 6 | Issue 10 | e26343