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Objectives: Akt/Protein Kinase B, an integral mediator of cancer cell survival and tumor progression, is activated by phosphorylation. This results in activation of mammalian target of rapamycin (mTOR), a pro-survival mediator and inactivation of BAD, an inducer of mitochondrial apoptosis signaling. BAD inactivation prevents the cleavage and activation of caspases, which are ultimately responsible for apoptosis. Since several pathways can result in Akt activation, the identification of signals downstream of Akt, specific to endometrial cancers, may provide a rationale for targeting these signals for treatment. Our objectives are to identify pro-survival intermediaries in endometrial tumors through phosphoprotein arrays. Methods: Twenty-three frozen endometrial tumors were obtained following IRB approval, sectioned and the epithelium microdissected from the underlying stroma. Protein lysates were made and arrayed onto RPPA’s. One array was stained for total protein and 6 different arrays were separately probed with antibodies for total and phosphospecific forms of BAD, and mTOR as well as total and cleaved Caspase-3. Arrays were analyzed using Image Quant software and after normalization to total protein, relative fold change in the phosphorylated and cleaved forms were determined. Results: Ten of the 23 (43%) tumors analyzed demonstrated significant phosphorylation of BAD compared to total BAD. Caspase-3 cleavage was absent in all (100%) tumors examined and mTOR was phosphorylated in only 3 of the 23 (13%) tumors analyzed. mTOR and BAD phosphorylation was observed in 2 of the 3 tumors both of which were FIGO Grade 1. Conclusions: Although Akt expression has been shown to be important in cancer survival, signaling downstream of Akt has not been examined in endometrial tumors to date. Our preliminary results suggest that loss of Caspase-3 cleavage may play a role in endometrial cancer cell survival and appears to proceed through BAD and non-BAD dependent mechanisms. Furthermore, a subset of low grade endometrial tumors has an additional mechanism of survival through mTOR. After validation of our results, Caspase-3 and phospho-mTOR targeted therapy will be investigated in endometrial cancer cell lines to determine their effectiveness in apoptosis induction and potential use in advanced or recurrent disease.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]