Calcitonin in Prostate Growth and Neoplasia

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We and others have shown that all primary prostate cancers (PCs) and PC cell lines express calcitonin (CT) and/or its receptor (CTR), and their co-expression positively correlates with the tumor grade of primary PCs and growth/invasiveness of PC cell lines. Moreover, activation of the CT-CTR axis in non-invasive, nontumorigenic LNCaP cells induces an invasive and tumorigenic phenotype. In contrast, silencing of the CT/CTR expression in highly metastatic PC-3M cells remarkably reduces their tumorigenicity and abolishes their ability to form distant metastases in nude mice. We have discovered that the cytoplasmic (C) tail of the CTR contains the canonical class I type PSD-95, Discs-large, Zona Occludens-1 (PDZ) ligand and a mutation in the PDZ ligand abrogates the CT-elicited increase in growth and invasiveness of PC cell lines. In a second major discovery, we have shown that the PDZ ligand of the CTR binds to a PDZ domain of the membrane protein, zonula occludens (ZO-1), to form ¿metastasis receptosome¿. Our studies have also identified that CTR activates cyclic AMP-dependent protein kinase (PKA), and activated PKA facilitates disassembly of TJs by phosphorylating key TJ proteins. In a third important discovery, we have identified that A kinase anchoring protein 2 (AKAP2) plays a key role in CTR-mediated oncogenic actions by targeting the PKA to the CTR in a localized area of TJ complex. Our working hypothesis is that the CTR-ZO-1 interaction and localized action of PKA within the TJ complex is required for CT-induced disassembly of junctional complexes and increase in invasion of PC cell lines. We will test the hypothesis that CTR interacts with ZO-1 to activate AKAP2-tethered PKA within the TJ complex to facilitate TJ disassembly in three Specific Aims: 1) Using site-directed mutagenesis, we will identify the key amino acid(s) of the CTR-C-PDZ ligand for ZO-1 binding, and investigate the effect PDZ mutation(s) on the actions of CT on TJ assembly, invasion, and in vivo tumor growth/metastasis; 2) Using ?PDZ deletion constructs of ZO-1, we will identify which of the three PDZ domains of ZO-1 binds to the CTR and investigate the role of ZO-1 in mediating the actions of CTR on TJ assembly, invasion, and in vivo tumor growth/metastasis ; 3) We will investigate the role of AKAP2 in targeting the PKA to the TJ complex, and phosphorylation of ZO-1 and claudin 3 by PKA, which may play a key role in TJ disassembly, invasion, in vivo tumor growth/ metastasis. We have identified a novel mechanism for CTR-activated oncogenic signaling in PC cell lines, generated a variety of research tools, cell lines and experimental models, and assembled a team of investigators to examine the role of TJs in CTR-mediated PC growth/metastasis. We believe this study will uncover important intracellular mechanism(s) associated with PC progression, and provide new targets for the development of diagnostic tools and therapeutic models for the treatment of advanced PCs.