Translation initiation is a critical step in the regulation of cell growth and, therefore, represents an attractive target for cancer therapy. Experiments with clotrimazole and its analogs have uncovered a novel approach to inhibit translation initiation that has proven very efficient in inhibiting growth of cancer cell lines. Remarkably, clotrimazole is also a potent inhibitor of tumor growth in conventional animal models of cancer. Clotrimazole inhibits translation initiation by sustained depletion of intracellular Ca++ stores resulting in activation of double stranded RNA dependent protein kinase and in inhibition of eukaryotic initiation factor 2-alpha. The activity of eIF2-alpha is rate limiting for translation initiation and its inhibition by clotrimazole reduces the rate of protein synthesis. As a consequence, the expression of cyclins and other growth promoting proteins are preferentially affected by clotrimazole which causes cell cycle arrest in G1 and inhibits cell growth. The goal of this application is to evaluate the use of inhibitors of translation initiation for cancer therapy. To this end, the following Specific Aims are proposed: 1) to evaluate the efficacy of the translation initiation inhibitor clotrimazole in genetically engineered animals that spontaneously develop cancers due to the absence of functional p53, the most commonly mutated gene in human cancers; 2) to study the molecular mechanism responsible for the anti-cancer activity of clotrimazole; in particular, the in vivo effect of clotrimazole on the phosphorylation state of eIF2-alpha and on the expression of cyclins and other cell cycle regulatory proteins. The relative contribution of inhibition of cell growth, induction of apoptosis and inhibition of angiogenesis to the anti-cancer effect of clotrimazole will be determined; 3) the molecular target(s) of clotrimazole that mediate Ca++ depletion responsible for inhibition of translation initiation will be identified by affinity purification and cloning. It is expected that as a result of this research, inhibitors of translation initiation such as clotrimazole and its analogs will emerge as a novel class of anti-cancer agents. This work, therefore, sets the stage for the future testing of inhibitors of translation initiation for human cancer therapy.
