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 ICI 182780 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 Niltubacin supply 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 Hec1Bcells 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 +/2 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 and/or PTEN 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.