20%–63% of patients achieving stable disease for at least four months [15]. Some patients achieve no benefit from therapy (primary resistance), whereas in others, stable disease or an initial response occurs. Nevertheless, most patients eventually experience progression of disease (acquired resistance). More information will be available following the analysis of the phase II trial of temsirolimus for advanced endometrial cancer, Gynecologic Oncology Group trial 248; however, since this trial only recently closed to accrual, the outcome data are not mature. In this current study, we investigated how inhibition of mTOR can be optimized. We examined the growth inhibitory effect of temsirolimus on a panel of endometrial cancer cells and observed differential sensitivity as well as compensatory Akt phosphorylation in a subset of cell lines, which may represent one mechanism for acquired resistance. We identified cells which were primarily resistant to treatment and compared these to other cells which initially responded but employed escape mechanisms to achieve acquired resistance. To overcome both forms of resistance, we applied dual inhibition of PI3K and mTOR to prevent cell survival signaling. Our data reveal that combination treatment of temsirolimus with either BEZ235, a dual PI3K/mTOR inhibitor, or ZSTK474, a pan PI3K inhibitor, blocked Akt activation and inhibited phosphorylation of both 4E-BP1 and the substrate for S6K, ribosomal S6 (rS6), which ultimately resulted in synergistic cell death. Materials and Methods Cell lines Six endometrial cancer cell lines (AN3CA, RL95-2, Hec1A, SKUT1B, ECC-1, and KLE) were obtained from ATCC and grown according to the recommended guidelines. Two endome- trial cancer cell lines, Ishikawa H and Hec50co (gifts from Dr. Erlio Gurpide, New York University), were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin- streptomycin, all from Gibco. Reagents Temsirolimus, NVP-BEZ235, and ZSTK474 were purchased from LC Laboratory and resuspended in DMSO. Antibodies against Akt (#4691), Akt P-S473 (#4060), Akt P-T308 (#2965), rS6 (#2217), rS6 P-S235/S236 (#4858), p70S6K (#9202), p70S6K P-T389 (#9205), p27 (#3686), LC3 (#2775), 4E-BP1 (#9644), 4E-BP1 P-T37/T46 (#2855), and PARP (#9542) were from Cell Signaling. The PTEN (sc-7974) antibody was from Santa Cruz Biotechnology. The b-actin antibody (#A1978) was from Sigma Aldrich. Cell viability assays Ninety-six-well plates were seeded with 10,000–20,000 cells in each well 24 hrs prior to drug treatment. Cells were treated with inhibitors or DMSO control for 3 days. Each experiment was carried out in triplicate. Effects on cell proliferation and loss of cell viability were evaluated with WST-1 (Clontech) assay per manufacturer’s instructions. Western blotting Equal numbers of cells were plated 24 hrs prior to treatment. Unless otherwise noted, cells were grown in the presence of inhibitors for 24 hrs. Cells were solubilized in cold NP-40 cell lysis buffer (150 mM NaCl, 50 mM Tris/HCl, pH 7.4, 1% NP-40, and a protease and phosphatase inhibitor cocktail from Pierce). Lysates were analyzed by Western blotting with specific primary and HRP-conjugated secondary antibodies. mTOR and PI3 Kinase Inhibitor Synergy Cell cycle analysis Cells were grown for 24 hrs in normal growth media followed by drug treatment for an additional 24 or 72 hrs. Floating cells were collected with the media, and attached cells were collected by trypsinization. After centrifugation, the cell pellets were fixed in 70% cold ethanol, treated with RNAase A, stained with propidium iodide, and prepared for flow cytometry. FACScan (BD Bioscience) was used to detect the samples and ModFit was used to analyze the cell cycle alterations. Results Cells display varying responses to temsirolimus To establish a cell-based model system to understand the efficacy of mTOR inhibitors in endometrial cancer patients, we tested the growth-inhibitory properties of temsirolimus on eight endometrial cancer cell lines using in vitro proliferation assays. Proliferation of four endometrial cancer cell lines (SKUT1B, AN3CA, RL95-2, and ECC-1) was inhibited at low nanomolar concentrations of temsirolimus (Fig. 1A). The temsirolimus IC50 for these sensitive cells was approximately 1 nM (Supporting Table S1), indicating a strong growth inhibitory impact. In contrast, Ishikawa H, Hec50co, Hec1A and KLE cells were more resistant to treatment, and the average IC50, when reached, was at least 10-fold higher (Fig. 1B, Supporting Table S1). To understand the signaling pathways responsible for the contrasting effects of temsirolimus on proliferation, we first confirmed functional inhibition of mTOR activity. Surprisingly, even in the sensitive cell lines, phosphorylation of mTOR at S2448 was only partially inhibited by temsirolimus (Supporting Fig. S1), suggesting that loss of mTOR phosphorylation does not distinguish between sensitive and resistant cells. However, temsirolimus completely prevented phosphorylation of rS6, which is downstream of mTORC1, in all the cells regardless of sensitivity and at all tested concentrations (Fig. 1C, D). This finding validates the functional inhibition of mTORC1 activity despite the persistence of some phosphorylation, although reduced, at the S2448 site (Fig. 1C, D and Supporting Fig. S1). We conclude that mTOR inhibition at S2448 and loss of rS6 activation as readouts do not adequately distinguish between cells that are sensitive initially versus those that are primarily resistant to temsirolimus-induced growth inhibition. Akt phosphorylation status distinguishes between sensitive and resistant cells To understand the basis for cell sensitivity to temsirolimus as a single agent, we turned to an analysis of Akt activation, both at baseline and in response to treatment. We found that the baseline constitutive activation of Akt was predictive of cell sensitivity with the most resistant cells having very low basal S473 and T308 phosphorylation (Fig. 1C, D). After treatment with temsirolimus, a compensatory increase in Akt phosphorylation at both sites was detected in the most sensitive endometrial cancer cell lines tested (Fig. 1C), but the primarily resistant cells (KLE) demonstrated no Akt phosphorylation at either site, and another resistant line, Hec50co, showed reduced phosphorylation (Fig. 1D). Thus, in contrast to sensitive cells, primarily resistant cells have low basal Akt phosphorylation and do not respond with compensatory hyper-phosphorylation after temsirolimus treatment. Of note, phospho-PDK1, the kinase responsible for Akt phosphorylation at T308, is low in several resistant cell lines such as Hec1A and KLE (Fig. 2). This indicates primary cell resistance and a general lack of dependence on the Akt signaling pathway for proliferation. On the other hand, the finding of compensatory activation of Akt in responsive cells is consistent with previous reports in the literature PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26343