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 phosphorylate 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 
. This has been observed in xenograft models of lung cancer 
as well as in advanced colon and breast cancer tissues following rapalog therapy 
. The elevated Akt phosphorylation is thought to be a predominant driving force in resistance to temsirolimus treatment in these cancers 
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 resistant to temsirolimus alone. These findings are consistent with a recent study in melanoma cells in which dual treatment with the PI3K inhibitor PI-103 and rapamycin reversed compensatory Akt phosphorylation and induced cell cycle arrest, and xenograft studies demonstrated reduced tumor growth with this combination strategy 
. We extend these findings herein to define a potential mechanism by which the combination therapy promotes cell death.
We found that BEZ235 alone blocked PI3K, mTORC1, and mTORC2 activity, in particular 4E-BP1 phosphorylation at a dose of 100 nM. However, BEZ235 was less effective in blocking rS6 phosphorylation. In comparison, temsirolimus completely abrogated phosphorylation of rS6 at 1 nM. Thus, combining both agents (BEZ235 and temsirolimus) completely inhibited signaling throughout the pathway and synergistically induced cell death.
Currently, combinatorial therapies are being applied to prevent resistance to single-agent treatments such as rapalogs. Examples of targeted small-molecule inhibitors under investigation include BEZ235 (dual PI3K/mTOR inhibitor) 
, AZD2171 (dual VEGF2/PDGFR inhibitor); LBH589 (histone deacetylase inhibitor) 
, LY294002 (PI3K inhibitor) 
, AZD6244 (MEK inhibitor) 
, and ZSTK474 (PI3K inhibitor) 
. BEZ235 is a novel orally bioavailable inhibitor originally designed as a pan-PI3K family inhibitor based on the p110γ (catalytic subunit of PI3K) kinase domain structure 
. Interestingly, when this compound was evaluated in preclinical studies, in vitro
kinase assays revealed it also targets mTOR at a concentration of 20.7 nM 
. Therefore, BEZ235 is classified as a dual inhibitor that is capable 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 
, including breast cancer cells with mutant or amplified PIK3CA 
. BEZ235 showed antitumor activity in nude mice with few side effects 
. 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 
. 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 
. 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 
; 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 
. 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 
. 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 
. 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 
, and inhibition of mTOR with the alkylphospholipid perifosine induces autophagic cell death 
. BEZ235 has also been shown to induce caspase-independent apoptosis in a mechanism that includes PARP cleavage 
, 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.
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 
. 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 
. 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 
. 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 
. 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 
. Studies involving PTEN+/−
mice or cell lines devoid of PTEN show that PTEN-deficient tumors are sensitive to mTOR inhibition 
. 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 
. 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
mutations by other investigators 
. These studies reported herein promote a further understanding 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.