MOLECULAR EVENTS IN PROSTATE CANCER: LENTIVIRUS INDUCED KNOCKDOWN OF HUMAN MKP-2 ENHANCES ERK SIGNALLING IN LNCaP (AI) CELLS

The MAP kinase phosphatases regulate the magnitude and kinetics of MAP kinase signalling within the cell and as a consequence cell cycle progression and division. Therefore, these enzymes have been implicated in the regulation of number of disease conditions. One member of this family, MKP-2, has been linked to cancers of the breast and prostate. However, in human cells correlating changes in MKP-2 expression and effects upon ERK signalling, the main substrate for MKP-2 has been difficult, due to lack of convincing cellular analysis. Therefore, we utilized a novel lentivirus shRNA MKP-2 construct (L.001) to examine the consequences of knockdown on the regulation of ERK MAP kinase signalling. Infection of LNCaP AI cells with lentivirus shRNA MKP-2 reduced endogenous MKP-2 mRNA by approximately 80% in LNCaP cells. Immunofluorescent GFP labelling revealed strong cellular expression of MKP-2, approximately 70% of the cells were infected. MKP-2 knockdown reduced adenoviral induced hMKP-2 expression by approximately 50%. Both EGF and FCS induced strong but transient activation of ERK phosphorylation in LNCaP (AI), EGF giving a more rapid activation of the two agents. However, following MKP-2 knockdown ERK phosphorylation was enhanced and more sustained relative to control cells. These findings demonstrate a key role for MKP-2 in the regulation of MAP kinase (ERK) in human cancer cells and its potential role in controlling cell proliferation in cancer and metastasis.