There are greater than 1,000,000 new cancer cases per year in the United States and cancer deaths exceed 500,000 annually. The acquisition of drug resistance in tumors is an ongoing problem in cancer therapy. Therefore, our broad aim is to understand the biological signals that confer resistance to apoptosis on tumor cells, with a focus on lymphomas. About one third of all tumors harbor dysfunctional Ras. Therefore, we recently began studies to understand the mechanism of action of a natural tumoricide, Manumycin-A. This compound, is a farnesyltransferase inhibitor (FTI), a family of agents that blocks Ras farnesylation. Farnesylation is a posttranslational process that is required for Ras maturation and insertion into the plasma membrane and is essential for ultimate activation of both normal and dysfunctional Ras. We found that Man-A killed a panel of lymphoid and myeloid tumors irrespective of the activation state of Ras effectors. In all the tumors, Man-A induced reactive oxygen species (ROS) which were upstream of caspase activation. This was followed by selective, ROS and caspase-dependent cleavage of MEK and Akt. We also found strong evidence suggesting a role of the serine/threonine protein phosphatase 1 (PP1) as an initial effector of the Man-A mediated death response. Thus, abrogation of PP1 blocked all downstream effects of Man-A and resulted in stabilization of MEK and Akt. Stabilization of MEK and Akt phosphorylation prevented their cleavage and thus, conferred resistance to the induction of apoptotic death on the otherwise susceptible tumors. Hence, PP1 appears to be a target or proximal player in the death cascade. Therefore, our goals are to determine the interrelationships of ROS, and protein phosphatase activities in the priming and targeting of MEK and Akt for proteolytic cleavage using a combination of biochemical, molecular and proteomic approaches.
Our specific aims are to: 1) establish fine kinetics and interdependence of biochemical responses to Man-A and each other; 2) establish the role of PP1 in FTI-mediated apoptosis using molecular approaches; and 3) to determine the cleavage sites on MEK and Akt and determine the role of oxidation in subsequent processing, using proteomics. These studies will ultimately help in the targeting of these pathways to re-sensitize tumors for therapeutic elimination. ? ? ?
| Porter, Holly A; Carey, Gregory B; Keegan, Achsah D (2012) Insulin receptor substrate 1 expression enhances the sensitivity of 32D cells to chemotherapy-induced cell death. Exp Cell Res 318:1745-58 |
