M. Beveridge1, D. Delitto1, J. Trevino1, G. A. Sarosi1,2, S. J. Hughes1, R. M. Thomas1,2 1University Of Florida,College Of Medicine, Department Of Surgery,Gainesville, FL, USA 2NF/SG Veterans Health System,Department Of Surgery,Gainesville, FL, USA
Introduction: A large majority of patients with pancreatic ductal adenocarcinoma (PDAC) have metastatic disease at the time of diagnosis. The mechanisms whereby PDAC is able to metastasize are still poorly understood. A-kinase anchor proteins (AKAP) are a family of scaffolding proteins that mediate signal transduction and cellular localization of protein kinase A, an inducer of cell motility. AKAP12 is one member that has shown a role in a variety of highly-metastatic cancers including colon, melanoma, and prostate but its role in PDAC is undefined.
Methods: AKAP12 expression was determined by immunoblot in both commercially available PDAC cell lines as well as from patient-derived PDAC tumors. The effect of AKAP12 knockdown with siRNA on cell proliferation and migration was determined by measurement of Alamar Blue reduction and transwell migration assay to culture media supplemented with 5% fetal bovine serum, respectively. The AKT, ERK, and MEK pathways were interrogated by immunoblot to ascertain changes in signaling associated with abrogation of AKAP12 expression.
Results: A differential expression was noted between PDAC cell lines with a 19, 27, and 328-fold increase in expression of AKAP12 in BxPC3, L3.6pl, and Panc-1 cell lines compared to MiaPaCa. Additionally, a 17.6-fold increased mean expression of AKAP12 was noted in patient-derived PDAC samples compared to normal (p=0.05). While no statistical difference was seen in proliferation after AKAP12 knockdown, migration at 48hrs was significantly reduced by 95.5% (p=0.05) and 86.5% (p=0.02) in BxPC3 and Panc-1, respectively after AKAP12 siRNA knockdown compared to control (Figure 1). Finally, AKAP12 knockdown resulted in the 23-fold, 6-fold, and 4.5-fold increase in phosphorylation of AKT, ERK, and MEK proteins in Panc-1 cells, respectively and 34-fold, 56-fold, and 1.6-fold increase in BxPC-3, respectively.
Conclusions: AKAP12 is differentially expressed in human PDAC tissue samples and cell lines. Inhibition of AKAP12 results in decreased migration in vitro but no effect on proliferation. This may result in compensatory increases in the activation of the AKT and MEK-ERK pathways. AKAP12 may play a role in pancreatic carcinogenesis but further studies are needed to clarify its role.
