The working hypothesis of our laboratory focuses on the convergence of prosurvival, angiogenesis and motility signals at common pathways in the local tumor microenvironment for therapeutic targeting and monitoring and the application of the findings to improve diagnosis, prognosis, and treatment of ovarian cancer. We continue to study two pathways we have identified: the BAG-3 stress co-chaperone protein and the ovarian cancer growth and survival factor, progranulin (prgn, previously GEP). Our recent findings confirm the importance of BAG3 as a selective survival protein for pathways in which HSP70 is involved. BAG3 is tightly regulated in the cell requiring both caspase cleavage and proteosomal degradation. Mutation of the putative caspase cleavage site stabilizes the protein and protects the cell from caspase-mediated apoptosis; collaboration with NCI/FCRDC is addressing idenification of the cleavage site. Microarray analysis of cells overexpressing wild type and domain-mutants of BAG3 identified up-regulation of proteins involved in invasion and metastasis, such as CCN1 in cells with more motile and spread phenotype. This fits with our prior report that those cells have upregulation of the integrin/FAK signaling. Domain deletion mutants had the opposite phenotype. The findings in this model were further confirmed using siRNA to downregulate BAG3 expression; this resulted in reduced focal contacts and adhesion. We continue to follow up results of the NCI/Myriad yeast two-hybrid program in which BAG3 was included. Interaction of BAG3 with Hsp27 was identified; this small heat shock protein has been shown to interact with Hsp70 and also when polymerized, to regulate actin. We propose an interaction of Hsp27 with BAG3 and in regulation of its motile/adhesive function. Mutants are being constructed to map those functions and from which to lead into possible drug discovery assay development. Analysis of expression of the BAG family (1, 3, 4, and 6) in ovarian cancer cases with long term follow up (95-C-0055) revealed an association of cytosolic BAG4 with patient outcome. Review of BAG4 expression in the NCI60 cell line screen showed it likely associated with resistance to trabectadin (ET743) an agent shown by Dr. Pommier (LBP/CCR) to inhibit transcription-coupled DNA repair. This suggests that BAG4 may be involved in regulating/chaperoning the cellular response to DNA injury and fits with the clinical study results. This hypothesis will be the basis for a further collaboration. Studies of the Drosophila homolog of BAG 3/4, evil, near completion. Evil has been transfected into MDA-435 hu breast cancer cells and survival phenotype has been shown. We demonstrated that prgn is a growth and survival factor for ovarian cancer. We have recently demonstrated that secretory leukocyte protease inhibitory (SLPI) is a prgn partner protein and itself is necessary for ovarian cancer cells survival. SLPI is amplified and overexpressed in ovarian cancer. We have now shown that SLPI is a protector of prgn, independently of its protease inhibitory activity. Down regulation of SLPI with neutralizing antibody or siRNA induces loss of prgn and apoptosis. Mutations of putative prgn binding sites are under study. Xenograft experiments have shown SLPI and protease inhibition site mutants are more aggressive in extent of disease and invasiveness of disease. The role of SLPI in invasion is now under investigation. Production of GEP and its subsequent activity may be part of a signal amplification cascade in ovarian cancer and is a logical target for molecular therapeutics. Thus, the prgn/SLPI axis and BAG-3 each have potential as molecular therapeutic targets in ovarian cancer and other solid tumors.
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