The overall direction of the Molecular Mechanisms of Tumor Promotion Section is to understand the regulation of the signalling pathways downstream from the lipophilic second messenger diacylglycerol, to elucidate the basis for heterogeneity of response to different ligands which function through this pathway, and to exploit this understanding for developing novel ligands with unique behaviour that function through this pathway. A complementary direction is to understand the regulation and structure activity relations for the vanilloid receptor. The vanilloid receptor is a downstream target of the diacylglycerol signalling pathway, shares partial homology in its ligands to this pathway, and shares with the diacylglycerol signalling pathway an important role in inflammation. Both directions impact both our understanding of biological regulation and the potential development of therapeutic agents. Protein kinase C, the best studied downstream target for diacylglycerol, represents the classic system for tumor promotion and is a therapeutic target for cancer chemotherapy. The vanilloid receptor represents a promising therapeutic target for cancer pain, among other indications, and thus represents an important direction in palliative care for cancer patients. The C1 domain, the interaction domain of diacylglycerol in protein kinase C or RasGRP, exists in the context of the protein structure. For PKC delta, we show that the C1b domain is the primary contributor to ligand response, independent of its position within the regulatory domain. For beta2-chimaerin, in contrast, we show in collaborative studies with Marcelo Kazanietz that interactions between the C1 domain and other structural elements lead to inaccessibility, reducing responsiveness. Exploiting strong collaborations with groups in computational chemistry, synthetic chemistry, and membrane biochemistry, we continue to improve our understanding of the structural basis for ligand - protein kinase C interactions. In collaboration with the groups of Victor Marquez and Raz Jelinek, we find marked diversity in the nature of interaction of DAG lactones with membranes. In collaboration with the chemistry group of Gary Keck, we have shown that a close analog of bryostatin 1 fails to show this antagonism on U937 leukemia cells although it retains comparable potency to bryostatin 1 on protein kinase C. Other derivatives retain the unique behavior of bryostatin 1, focusing attention on critical structural features of the molecule responsible for the bryostatin like behavior. A critical structural conclusion is that the A,B ring system in the bryostatin structure is NOT simply a linker region, as had previously been hypothesized. In further studies, we have shown that, for different responses, the same bryostatin analog may give variable proportions on antagonism, ranging from virtually none to partial. We can thus conclude that the antagonistic behavior is not an all-or-none phenomenon. These results imply that bryostatin analogs can be designed to antagonize a desired subset of protein kinase C responses. PEP005 is another protein kinase C activator in clinical trials for actinic keratosis and non-melanotic skin cancer. Our studies reveal that, whereas it differs from the typical phorbol ester in its modulation of the NFkB response pathway, similar or even more dramatic differences are seen for some other non-promoting phorbol esters. Since PEP005 is in clinical trials, our findings suggest that some of these other derivatives might have similar or superior effect. RasGRP3 is an activator of the Ras pathway directly stimulated by diacylglycerol and phorbol esters. We find that it is expressed at somewhat elevated levels in both prostate and melanoma. Suppression of the endogenous expression of RasGRP3 with siRNA in both prostate and melanoma cell lines inhibits proliferation, growth in soft agar, and tumor formation in mouse xenografts. In the development of therapeutics targeted to TRPV1, a major problem is designing sufficient specificity of action. We find that its ligand recognition depends on its state of regulatory control. This finding implies that ligands can be designed that will be specific for the vanilloid receptor in a specific regulatory environment, such as at a site of inflammation. Current studies are assessing the roles of individual regulatory elements in the pattern of ligand recognition.
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