Protein phosphatase 2A regulation of markers of extracellular matrix remodelling in hepatocellular carcinoma cells: functional consequences for tumour invasion.

background and purposeA hallmark of tumour invasion is breakdown of the extracellular matrix due to dysregulation of the matrix metalloproteinase (MMP) system. While our understanding of how this is regulated by kinase signalling pathways is well established, its counter-regulation by protein phosphatases (PP) is poorly understood. Therefore, we investigated the effect of PP inhibition on markers of extracellular remodelling and how PP2A activity modulated MMP-9 abundance and function of Hep3B cells. EXPERIMENTAL APPROACH: Cells were exposed to okadaic acid (OA), tautomycetin and cyclosporin A, and the expression profile determined using PCR. Effects of OA and a protein inhibitor of PP2A, CIP2A, on MMP-9 abundance, PP2A activity and cell migration were investigated using ELISA, promoter constructs, siRNA knockdown and transwell migration assays. KEY

resultsOA increased expression and abundance of MMP-9 and the tissue inhibitor of MMP, TIMP-1, without affecting other MMPs, TIMPs and ADAMs. The effect on MMP-9 was mimicked by CIP2A overexpression and knockdown of the PPP2CA catalytic, but not PPP2R1A scaffolding, subunit. Cyclosporin A and PPP1CA silencing did not alter MMP-9 expression, while tautomycetin transiently increased it. Mutation of AP-1, but not NF-κB, binding sites inhibited OA-mediated MMP-9 transcriptional activity. OA and CIP2A decreased PP2A activity and increased cell migration. CONCLUSION AND IMPLICATIONS: OA increased MMP-9 by decreasing PP2A activity and PP2Ac, through AP-1 binding sites on the MMP-9 promoter. The functional consequence of this and CIP2A overexpression was increased cell migration. Hence, PP2A inhibition induced a metastatic phenotype through alterations in MMP-9 in Hep3B cells.