Abstract 4218: Calcitonin receptor increases invasion of prostate cancer cells by activating cAMP-dependent protein kinase (PKA) and promoting phosphorylation of tight junction proteins
Authors: Shah Girish V. , Thomas Shibu , Thakkar Arvind , Fanning Alan 
Published: August 2012

The role of calcitonin (CT) and its receptor (CTR) in epithelial cancer progression is an emerging concept with great clinical potential. Expression of CT and CTR is frequently elevated in prostate cancers (PCs) and activation of CT-CTR axis in non-invasive PC cells induces an invasive phenotype. Invasive phenotype is acquired by the EMT, a process involving the loss of cell-cell adhesion and cytoskeletal rearrangement. Tight (TJs) and adherens junctions (AJs) are shown to play an important role in this process. Present studies focused on the actions of CTR-mediated intracellular events associated with TJ functionality and the induction of EMT. We have recently identified a type 1 PDZ-binding motif in CTR-C tail, and showed that CTR-C PDZ-binding motif interacts with the PDZ3 domain of ZO-1, a critical scaffolding protein of TJs. CTR-ZO-1 interaction is critical for proinvasive actions CTR on PC cells, and deletion of either CTR-C PDZ-binding motif abolishes the ability of PC cells to form distant metastases in orthotopic xenograft model. Therefore, we first examined the kinetics of CTR-ZO-1 interaction and the effect of CTR on TJ assembly. PC3 cells expressing either CTRwt or CTRαPDZ were acutely treated with CT, and subcellular distribution of TJ proteins was examined by immunocytochemistry and immunoblotting. Since CTR is coupled to stimulatory G protein, we examined effect of PKA inhibitors on CTR actions. Next, we tested whether CTR phosphorylates TJ proteins. Finally, we generated novel peptides and delivered them intracellularly to attenuate CTR-ZO-1 interaction. The results have shown that CTR-ZO-1 interaction occurs rapidly and peaks within 30 seconds of CT stimulus; this is rapidly followed by TJ disassembly and endocytosis of TJ proteins. Endocytosis of ZO-1 occurs first, which is followed with the endocytosis of occludin and claudin 3. We then identified that CTR-mediated TJ disassembly requires PKA-mediated phosphorylation of claudin 3 and ZO-1, and this phosphorylation may cause conformational changes and lead to TJ disassembly. Next, we demonstrated that CTR-mediated PKA phosphorylation must occur within TJ complex, and this spatio-temporal specificity of PKA activation is provided by the CTR-ZO-1 interaction. Finally, we have shown that CTR-ZO-1 interaction as well as CTR-stimulated invasion of PC cells can be attenuated by novel synthetic peptides that we have generated. For the first time, we report here the chain of PKA-mediated events that lead to the disassembly of tight junctions, loss of cell-cell adhesion, induction of EMT, invasion and metastasis of PC cells. We also provide a novel translational approach to generate synthetic peptides to attenuate these events.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4218. doi:1538-7445.AM2012-4218