Prostate cancer (PC) is the most commonly diagnosed cancer type and the second leading cause of male cancer deaths in the United States. We and others have shown that all primary 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, non-tumorigenic LNCaP cells induces an invasive and tumorigenic phenotype. In contrast, silencing of CT/CTR expression in highly metastatic PC-3M cells markedly reduces their tumorigenicity and abolishes their ability to form distant metastases in nude mice. Furthermore, we made the key discovery that the cytoplasmic (C) tail of CTR contains a canonical class I type PSD-95, Discs-large, Zona Occludens-1 (PDZ) ligand motif, mutation of which abrogates the CT-elicited increase in growth and invasiveness of PC cell lines. In a second major discovery, we showed that the PDZ ligand of the CTR binds to a PDZ domain of the membrane protein zonula occludens (ZO-1) to form a "metastasis receptosome". Our studies also showed that CTR activates cyclic AMP-dependent protein kinase (PKA), and that activated PKA facilitates disassembly of tight junctions (TJs) by phosphorylating key TJ proteins. In a third important discovery, we showed that A kinase anchoring protein 2 (AKAP2) plays a key role in CTR-mediated oncogenic actions by targeting PKA to CTR within a localized sub-region of the TJ complex. Our central 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, permitting a loosening of cell-cell contacts and facilitating increased invasiveness of PC cell lines. We will test this hypothesis in three Specific Aims: 1) Using site-directed mutagenesis, we will identify the key amino acid(s) of the CTR-C-PDZ ligand required for ZO-1 binding, and investigate the effect of PDZ mutation(s) on the actions of CTR 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 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 both targeting PKA to the TJ complex and its phosphorylation of ZO-1 and claudin 3, which may play a key role in TJ disassembly, invasion, and 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 successfully accomplish these aims. We believe this study will uncover important intracellular mechanisms associated with PC progression, and provide new targets for the development of diagnostic tools and therapeutic agent for the treatment of advanced PCs.

Public Health Relevance

Prostate Cancer (PC) is the most commonly diagnosed cancer and the second leading cause of cancer deaths in men in America. The mechanisms associated with the progression from treatable, localized PC to relatively untreatable, metastatic form have not been elucidated. Our results suggest that the activation of the calcitonin (CT)-CT receptor (CTR) axis plays a major role in tumor growth and metastasis of prostate cancer. Recently, we have discovered that the interaction between CTR and tight junctions of PC cell lines is prerequisite for oncogenic actions of CTR, and it leads to the disassembly of tight junctions. The goal of this application is to elucidate early molecular events associated with CTR-stimulated TJ disassembly that lead to increased tumor growth/metastasis.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Tumor Microenvironment Study Section (TME)
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Sathyamoorthy, Neeraja
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University of Louisiana at Monroe
Schools of Pharmacy
United States
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Shah, Girish V; Muralidharan, Anbalagan; Thomas, Shibu et al. (2009) Identification of a small molecule class to enhance cell-cell adhesion and attenuate prostate tumor growth and metastasis. Mol Cancer Ther 8:509-20
Mudit, Mudit; Khanfar, Mohammad; Muralidharan, Anbalagan et al. (2009) Discovery, design, and synthesis of anti-metastatic lead phenylmethylene hydantoins inspired by marine natural products. Bioorg Med Chem 17:1731-8
Sayed, Khalid A El; Khanfar, Mohammad A; Shallal, Hassan M et al. (2008) Latrunculin A and its C-17-O-carbamates inhibit prostate tumor cell invasion and HIF-1 activation in breast tumor cells. J Nat Prod 71:396-402
Shah, Girish V; Thomas, Shibu; Muralidharan, Anbalagan et al. (2008) Calcitonin promotes in vivo metastasis of prostate cancer cells by altering cell signaling, adhesion, and inflammatory pathways. Endocr Relat Cancer 15:953-64
El Sayed, Khalid A; Laphookhieo, Surat; Baraka, Hany N et al. (2008) Biocatalytic and semisynthetic optimization of the anti-invasive tobacco (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol. Bioorg Med Chem 16:2886-93
Thomas, Shibu; Muralidharan, A; Shah, Girish V (2007) Knock-down of calcitonin receptor expression induces apoptosis and growth arrest of prostate cancer cells. Int J Oncol 31:1425-37
Thomas, Shibu; Chiriva-Internati, Maurizio; Shah, Girish V (2007) Calcitonin receptor-stimulated migration of prostate cancer cells is mediated by urokinase receptor-integrin signaling. Clin Exp Metastasis 24:363-77
Thomas, Shibu; Chigurupati, Srinivasulu; Anbalagan, Muralidharan et al. (2006) Calcitonin increases tumorigenicity of prostate cancer cells: evidence for the role of protein kinase A and urokinase-type plasminogen receptor. Mol Endocrinol 20:1894-911
Iczkowski, Kenneth A; Omara-Opyene, A Levi; Shah, Girish V (2006) The predominant CD44 splice variant in prostate cancer binds fibronectin, and calcitonin stimulates its expression. Anticancer Res 26:2863-72
Sabbisetti, Venkata; Chigurupati, Srinivasulu; Thomas, Shibu et al. (2006) Calcitonin stimulates the secretion of urokinase-type plasminogen activator from prostate cancer cells: its possible implications on tumor cell invasion. Int J Cancer 118:2694-702

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