Translation initiation factors contribute to initiation of cancer and are required for the maintenance of the transformed phenotype, in part because most oncogenes possess highly structured 5'UTRs and their synthesis is, therefore, highly dependent on translation initiation factors such as elF2. The applicants have discovered that a distinct series of compounds that includes clotrimazole and non-imidazolic triphenyl derivatives, the n-3 unsaturated fatty acid EPA and the thiazolidinediones, which have potent anti-cancer activity in vitro and in animal models, downregulate translation initiation by depleting intracellular calcium stores leading to activation of elF2-kinases that phosphorylate and inhibit elF2alpha Suppression of translation initiation preferentially abrogates the synthesis of Gi cyclins and blocks the cell cycle progression in the Gi phase. Thus, translation initiation inhibitors are now considered an emerging class of mechanism-specific anti-cancer drugs. Using an antibody specific for the phosphorylated form of elF2, we have recently documented that Ca++ releasing TI inhibitors induce phosphorylation of elF2 in animal tumors as they do in cancer cells in vitro, and also shown that Ca++ releasing TI inhibitors induce the expression of ER-stress genes such as BiP and CHOP both in cells and xenografts of human cancers. These results validate in vivo the target-specific mechanism of action of Ca++ releasing TI-inhibitors. In this Application, we propose to establish a novel mechanism-specific animal cancer model for accreditation of translation initiation as a bona fide target for cancer therapy. We also propose to validate the mechanism of action of Ca++ releasing TI inhibitors in human cancers conducting a retrospective study in available tumor samples from patients treated with TZDs and a prospective study in human skin cancers treated with an available topical formulation for 3-5 days before biopsy. Since interferons transcriptionally upregulate PKR expression while Ca++ releasing TI inhibitors turn on its enzymatic activity, we will explore the potential synergism between interferons and TI inhibitors in human cancer xenografts implanted in nude mice. We will also evaluate the relative role of the two ER-resident eIF2 kinases, PKR and PERK, and of the ER-stress gene CHOP, as the downstream effectors of Ca-depleting TI inhibitors. The studies proposed here will pave the way for the development of more potent and less toxic TI inhibitors based on our understanding of their molecular effectors, and for the initiation of human clinical trials to evaluate the efficacy of TI inhibitors for cancer therapy either alone or in combination with interferons.
