Epigenetic silencing of PRSS3 provides growth and metastasis advantage for human hepatocellular carcinoma.

Protease, serine, 3 (PRSS3), a member of the trypsin family of serine proteases, has been shown to be aberrantly expressed in several cancer types and to play important roles in tumor progression and metastasis. However, the expression and function of PRSS3 gene in hepatocellular carcinoma (HCC) remain unclear. Here we found that PRSS3 expression was decreased in human HCC cell lines and HCC surgical specimens. This was associated with intragenic methylation of PRSS3 gene. Treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine and/or histone deacetylase inhibitor trichostatin A restored PRSS3 expression in HCC cell lines. Ectopic overexpression of PRSS3 gene in HCC cell lines significantly suppressed cell proliferation and colony formation and arrested cell cycle at G1/S phase, accompanied with downregulation of cyclin D1 (CCND1)/CDK4 and cyclin E1 (CCNE1)/CDK2 complexes. Moreover, PRSS3 overexpression in HCC cells inhibited HCC cell migration and invasion with downregulation of matrix metallopeptidase 2 (MMP2). Further study showed that PRSS3 overexpression diminished the phosphorylation of mitogen-activated protein kinase/extracellular-signal-regulated kinase signaling protein, mitogen-activated protein kinase kinase 1 (MEK1)/mitogen-activated protein kinase kinase 2 (MEK2) and extracellular-signal related kinase 1 (ERK1)/extracellular-signal related kinase 2 (ERK2), in HCC cells. In contrast, knockdown of PRSS3 by small interfering RNA resulted in opposite effects on an HCC cell line SNU-387 which constitutively expresses PRSS3. These results demonstrate that downregulation of PRSS3 by intragenic hypermethylation provides growth and metastasis advantage to HCC cells. The clinical relevance of PRSS3 to human HCC was shown by the intragenic methylation of PRSS3 in HCC specimens and its association with poor tumor differentiation in patients with HCC. Thus, PRSS3 is a potential prognostic biomarker and an epigenetic target for intervention of human HCC.

KEY MESSAGES: • PRSS3 is downregulated by intragenic hypermethylation in HCC. • Epigenetic silencing of PRSS3 facilitates growth, migration, and invasion of HCC. • PRSS3 intragenic methylation has implication in diagnosis of HCC.