Impact of secretases on metastatic behavior of prostate cancer cells in vitro
Date
2012
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Prostate cancer (PCa) is the most common cancer in North American men. The lifetime risk is 1:6 with about 241,000 new cases and 34,000 deaths from PCa expected in 2011. Deaths from PCa are due largely to metastasis or spreading to other organs, notably bone, where a systemic cytokine imbalance results in global organ failure and death. PCa grows slowly and usually causes no symptoms until urinary obstruction or bone pain occurs. The route for PCa metastasis to bone is proposed to be via blood or lymphatic; however, recent evidence indicates that perineural association and invasion (PNI) by PCa may provide a means for the cancer to spread along nerve or nerve tracts. Voltage sensitive sodium channels (VSSC) are multimeric transmembrane protein complexes comprised of a pore-forming a-subunit and two auxillary ß-subunits. The activity of VSSCs has been implicated in PCa cancer cell migration. The a- subunits of VSSC control intracellular sodium concentration while the ß-subunits modulate surface expression and gating kinetics of the a-subunit. ß-subunits also have homotypic and heterotypic cell adhesion molecule (CAM) function. Likewise, proteases known to cleave transmembrane protein complexes, e.g. the ADAMs and secretases, also have been implicated in enhanced cell motility and invasion. One of the VSSC ß-subunits, ß2 (beta 2), increases in castration resistant and metastatic PCa. Over-expression of beta 2 in LNCaP, a human PCa cell line, resulted in a fibroblastic morphology and enhanced migration. The mechanism by which beta 2 increases PCa metastasis is unknown. I hypothesized that PCa cells cleave ß2 by gamma-secretase and ADAM 10 to increase PCa cell metastasis. To test my hypothesis I generated two aims. First, I over-expressed ß2 in human PCa cancer cell lines, to determine if increased ß2 enhanced PCa cell migration and growth. Then I examined the effect of gamma-secretase and ADAM 10 inhibitors in PCa cell migration and growth in cells over-expressing ß2 as compared to normal PCa cells. For the second aim, I directly tested the impact of mutating the gamma-secretase cleavage site in ß2 on the metastatic phenotype of PCa cells. I successfully mutated these cleavage sites and transfected PCa cells with the constructs. In my migration assays, I observed that over-expression of ß2 increases migration in cells of the LNCaP progression model. Likewise, LNCaP cell lines were sensitive to gamma-secretase inhibitor alone and the combined treatment with inhibitors of gamma-secretase and ADAM 10 inhibitors. Nevertheless, the ADAM 10 inhibitor has no effect on cell migration as compared to Vehicle control suggesting the entire effect was due to gamma-secretase inhibition. In conclusion, functional over-expression of VSSC ß2 in PCa cells may be one mechanism leading to increased metastatic behavior but its involvement with ADAMs and gamma-secretases still needs to be explored.