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Small molecule targeted at specific signal transduction pathways hold great promise for creating a new approach to prostate cancer treatment. However, resistance to these small molecule agents occurs through multiple mechanisms and restricts their therapeutic activity. Developing potential targets that can overcome this resistance by novel mechanisms is essential. In this proposal, the applicant research team demonstrates that resistance to small molecule AKT protein kinase inhibitors is potentially mediated by the Pim-1 protein kinase, and that unique Pim protein kinase inhibitors that can in turn synergize with AKT inhibitors to block prostate cancer growth overcome this resistance. The knowledge gained through the studies proposed in this application is essential for the development of this combined chemotherapeutic strategy., The original document contains color images.

This proposal has evaluated the activity of the Pim protein kinase. This enzyme has been shown to function with the c-Myc protein and enhance tumor growth. It is possible that Pim enhanced the growth of tumor cells by stimulating growth and then also decreasing proteins that would normally inhibit the growth of cells. The goals of this research were to study the ability of Pim to regulate hepatocyte growth factor/ cMet signaling, examine the regulation of the p27 cell cycle inhibitory protein by Pim, and investigate the interaction of the Pim and AKT pathways in driving Pim mediated growth. The results of these studies have led to an understanding of Pim-1 mechanism of action and three publications in peer-reviewed journals. In this report, these findings are detailed with a focus on the ability of Pim to upregulate c-Met, downregulate p27, and enhance protein synthesis in the face of AKT inhibition., The original document contains color images.

Small molecule targeted at specific signal transduction pathways hold great promise for creating a new approach to prostate cancer treatment. However, resistance to these small molecule agents occurs through multiple mechanisms and restricts their therapeutic activity. Developing potential targets that can overcome this resistance by novel mechanisms is essential. In this proposal, the applicant research team demonstrates that resistance to small molecule AKT protein kinase inhibitors is potentially mediated by the Pim-1 protein kinase, and that unique Pim protein kinase inhibitors that can in turn synergize with AKT inhibitors to block prostate cancer growth overcome this resistance. The knowledge gained through the studies proposed in this application is essential for the development of this combined chemotherapeutic strategy., The original document contains color images.

The Pim protein kinase is over expressed in prostate cancer. To clarify the role of this protein in regulating normal prostate and cancer growth we have investigated its mechanism of action. We demonstrate that inhibition of AKT in prostate cancer cell lines induces the expression of multiple receptor tyrosine kinases (RTKs), but increases the protein levels of serine threonine protein kinase Pim-1. Pim-1 activity is identified as essential in the feedback regulation of RTK levels by AKT inhibition. Pim-1 appears to control RTKs by regulating the translation of these proteins in a cap-independent mechanism. Both tissue culture and animal experiments demonstrate that the combination of AKT and Pim inhibitors provide synergistic inhibition of tumor growth. Thus, this combination therapy has the potential activity to be developed as a prostate cancer treatment., The original document contains color images.

The Pim protein kinase is over expressed in prostate cancer. To clarify the role of this protein in regulating prostate cancer growth we have investigated its mechanism of action. We have examined the ability of small molecule inhibitors of this kinase to combine with other agents and have developed two rationally developed dual therapies. First, because overexpression of Bcl-2 family members is implicated in chemotherapeutic resistance in prostate cancer, we investigated the cooperative effects of Pim kinase inhibition with ABT-737, a small molecule antagonist of Bcl-2 family members. Strikingly, the addition of ABT-737 to Pim inhibitors triggered a robust apoptosis of prostate cancer cells in vitro and in vivo. Second, we demonstrate that inhibition of AKT in prostate cancer cell lines not only induces the expression of multiple RTKs, but increases the protein levels of serine threonine protein kinase Pim-1. Pim-1 activity is identified as essential in the feedback regulation of RTK levels by AKT inhibition. Both tissue culture and animal experiments demonstrate that the combination of AKT and Pim inhibitors provides synergistic inhibition of tumor growth. Thus Pim inhibitor has potential activity to treat this cancer.

The Pim protein kinase is over expressed in prostate cancers. To clarify the role of this protein in regulating prostate cancer growth we have investigated its mechanism of action. Additionally, we have studied the ability of small molecule inhibitors of Pim developed in our laboratory to block the growth stimulatory affects of this protein kinase. We find that Pim-1 is able to decrease the levels of the cell cycle inhibitory protein p27 by phosphorylating Skp-2, the protein that regulates the level of p27,. Phosphorylation of Skp-2 prolongs the half-life of Skp-2, increases its levels, and enhances its ability to degrade p27. Pim also functions to stimulate the phosphorylation of the Cdc27 protein also inhibiting its function. Since active Cdc27 is needed for the destruction of Skp-2, this also increases Skp-2 and decreases the levels of p27. Low levels of p27 allow prostate cancer cells to rapidly transit the cell cycle. We find that our chemical inhibitor of Pim, SMI-4a, is able to reverse the activity of Pim-1 and increase the levels of p27. This data suggests that inhibition of Pim-1 by these agents would function to block prostate cancer growth by inhibiting transit through the cell cycle. Thus, our agent has potential activity to treat this cancer.

Pim is a protein kinase that has been implicated to play a role in prostate cancer. Kaplan-Meier analysis demonstrated that those patients with high Pim-1 expression are at a significantly greater risk for developing metastatic cancer. In transgenic animal models, the levels of Pim-1 protein kinase are elevated in prostate tumors caused by over expression of the c-myc ongoene, a gene overexpressed in human prostate cancer. In this proposal, we will examine whether Pim mimics Akt and TOR or modulates additional biochemical pathways and use knockout mice to dissect how myc and Pim collaborate to induce transformation and growth or prostate cancers. Pim inhibitors alone or in combination with TOR inhibitors will make a powerful chemotherapeutic strategy.

TRAIL-TNF-related apoptosis inducing ligand) is a protein that induces apoptotic cell death in cancerous but not normal tissues. TRAIL has the potential to be a novel prostate cancer treatment. However, we have found that the majority of prostate cancer cell lines are insensitive to this agent. The goal of this proposal is decipher the nature of this resistance and develop biochemical tools to enhance the sensitivity of prostate cancer to TRAIL killing. Scope This proposal examines normal prostate epithelia, prostate cancer cell lines, and animal tumors for sensitivity to TRAIL.