mTOR and PI3 Kinase Inhibitor Synergy Figure 2. Baseline expression of PTEN, phospho-Akt and phospho-PDK1. Eight endometrial cancer cell lines were grown without treatment. Total protein extracts were analyzed by Western blotting for PTEN, phospho-Akt (P-Akt S473 and P-Akt T308), total Akt, phospho-PDK1 (P-PDK1 S241), and total PDK1. b-actin expression served as a loading control. doi:10.1371/journal.pone.0026343.g002 Baseline expression of PTEN as a correlate with Akt activation and primary sensitivity Endometrial cancer is frequently accompanied by the loss of functional PTEN: 30%–83% have PTEN mutations or loss of PTEN expression [17,18,19]. Loss of the PTEN tumor suppressor has been proposed to correlate with mTOR inhibitor sensitivity in some other cancer cell lines and patient tumors [20,21]. As discussed above, Figures 1 and 2 demonstrate the presence of basal phospho-Akt in some of the endometrial cancer cell lines, which we propose is a marker for initial sensitivity to single agent temsirolimus, with compensatory hyper-phosphorylation as a marker for developing acquired resistance. To understand the basis for high baseline Akt phosphorylation, we compared the baseline expression of PTEN and phospho-Akt in eight endometrial cancer cell lines. As shown in Figure 2, five cell lines (RL95-2, AN3CA, SKUT1B, ECC-1, and Ishikawa H) showed PTEN loss, and three cell lines (Hec50co, Hec1A, and KLE) expressed PTEN. For most cell lines, the exception being Hec50co, an inverse relationship was found between Akt phosphorylation at S473 and PTEN loss such that the cells with absent PTEN exhibited higher Akt S473 phosphorylation levels (Fig. 2). Strikingly, the cells with the lowest level of PTEN expression were also the most sensitive and responded with decreased cell proliferation when treated with temsirolimus (Fig. 1A). Conversely, the three cell lines with PTEN expression showed lower Akt S473 phosphorylation and were relatively resistant to mTOR inhibition. It should be noted that Ishikawa H cells have high Akt phosphorylation and loss of PTEN but are relatively resistant to temsirolimus. Hec50co cells, which have high Akt phosphorylation and maintain PTEN expression, are also resistant to temsirolimus. These data confirm the general, but not absolute, correlation between PTEN loss and constitutive Akt baseline activation at S473 as a marker for primary cell sensitivity in the most responsive cells. Dual therapy: PI3K inhibitors reverse temsirolimus- induced Akt phosphorylation To overcome the compensatory Akt phosphorylation induced by temsirolimus treatment as a mechanism of acquired cell resistance, six candidate inhibitors were chosen based on previously reported efficacy (Supporting Table S2). Ishikawa H (type I endometrial cancer cells) and Hec50co cells (type II endometrial cancer cells) [22] were first used to test the efficacy of these agents alone and in combination with temsirolimus. As shown in Figure 3A, Ishikawa H and Hec50co cells displayed a basal level of Akt phosphorylation that was further increased by temsirolimus treatment, with Ishikawa H cells showing higher levels compared with Hec50co cells, as discussed above (also, Fig. 1C, D). BEZ235 (dual mTOR/PI3K inhibitor) and ZSTK474 (PI3K inhibitor) decreased the basal level of Akt phosphorylation in both cell lines tested when used alone (Fig. 3A). Strikingly, when combined with temsirolimus, both BEZ235 and ZSTK474 blocked the temsirolimus-induced hyper-phosphorylation of Akt. We tested four other endometrial cancer cell lines and observed a similar pattern of inhibition of Akt phosphorylation (Fig. 3B, left panel). In order to demonstrate the effect of drug treatment even on resistant KLE and Hec1A cells, which have very low levels of basal phospho-Akt, we exposed the immunoblot for extended periods of time (Fig. 3B). The effect of the treatment regimens was preserved in these cells as well even though the baseline levels of phospho-Akt were substantially reduced. A marker for temsir- olimus activity is the loss of phosphorylation of p70S6K, which is directly downstream of mTORC1. Accordingly, we detected a loss of phospho-S6K whereas total S6K levels were generally unchanged (Fig. 3B, right panel). This result confirms that whether temsirolimus is used alone or in combination, this cellular response is maintained. In contrast to BEZ235 and ZSTK474, other molecular inhibitors including AZD6244 (MEK inhibitor), LBH589 (HDAC inhibitor), LY29004 (PI3K inhibitor), or AZD2171 (VEGFR/ PDGFR inhibitor) did not reverse temsirolimus-mediated com- pensatory Akt phosphorylation in Hec50co and Ishikawa H cells (Fig. 3A and Supporting Fig. S2), though these inhibitors have been previously shown to block rapalog-induced Akt phosphory- lation in other cell lines [23,24,25]. Based upon these data, we thus narrowed our focus to the study of BEZ235 and ZSTK474, which PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26343