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Role of PKD1 in production of MMP-2, MMP-9 and E-cadherin shedding in prostate cancer cells
Md. Helal U. Biswas, Ph.D., Chaunyou Zhang, Ph.D., Cheng Du, Ph.D., K. C. Balaji, MD.
University of Massachusetts Medical School, Worcester, MA, USA.

BACKGROUND: We have previously demonstrated that Protein Kinase D1 (PKD1), founding member of PKD family of serine threonine kinases, is down regulated in advanced prostate cancer (PC). PKD1 interacts with E-cadherin, a major cell adhesion protein, and influences cell motility, aggregation and proliferation. E-cadherin function in cells is regulated in cell through several mechanisms including proteolysis. Matrix Metalloproteinases (MMPs) are neutral proteinases that catalyze degradation of various transmembrane and extracellular matrix proteins including E-cadherin after activation at cell surface. Because of complex interplay between these proteins which play a role in PC, we explored the effect of PKD1 dysregulation on MMP secretion and its consequence on E-cadherin function.
METHODS: Expression of GFP and PKD1-GFP, PKD1 knockdown, phosphorylation of MEK, Erk and Akt as well as expression of MMP-2 were confirmed by western blot analysis. Gelatin-zymography or western blot was performed to detect the secretion of MMP-2, MMP-9 and sE-cadherin using conditioned media. Cell proliferation was measured by MTS assay and RT-PCR was done by using manufacturers’ protocol.
RESULTS: Overexpression of wild-type PKD1 in DU145 and PC3 PC cells increased the production of MMP-2 and MMP-9 compared to control. In addition, we also observed PKD1 enhanced the activation of kinases known to regulate MMP secretion including Akt, Erk and Erk kinase, Mek in DU145 and PC3 cells. Increased MMP-2 and MMP-9 secretion in PC3 cells by PKD1 overexpression were inhibited by treatment with U0126, a MEK inhibitor and LY294002, PI3K inhibitor, which confirmed PKD1 increased MMP-2 and MMP-9 secretion through MEK-Erk1/2 and PI3K-Akt pathways. In contrast, we demonstrated that downregulation of PKD1 by shRNA could suppress the phosphorylation of Erk and Akt as well as MMP-2 and MMP-9 secretion in DU145 cells. Interestingly, we also demonstrated that PKD1 enhanced the secretion of soluble fragment of E-cadherin in conditioned media of DU145 cells, which was suppressed when the cells were treated with MMP-2 and MMP-9 inhibitor. Finally, the suppression of cell proliferation by PKD1 was rescued by MMP-2 and MMP-9 inhibitor in DU145 cells, suggesting an antiproliferative role for MMP-2 and 9 in PC cells.
CONCLUSIONS: Our results demonstrate that PKD1 increases production of MMP-2 and MMP-9 in PC cells in Erk-MAPK and PI3K-Akt dependent manner with consequent increased shedding of E-cadherin; MMPs in turn seem to mediate PKD1 dependent decreased cell proliferation. Our study has discovered an interesting paradigm where the tumor suppressive effects of PKD1 are mediated by MMP 2 and 9, which are commonly known to play a role in tumor progression, and may involve E-cadherin shedding.


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