Deleted in Liver Cancer 1 (DLC1) is a negative regulator of metastasis and deregulated by kinase phosphorylation in hepatocellular carcinoma

Abstract

Deleted in liver cancer 1 (DLC1), which encodes a Rho GTPase activity protein (RhoGAP), is a bona fide tumor suppressor not only in hepatocellular carcinoma, but in a wide spectrum of human cancers. Underexpression of DLC1 is commonly found in human cancers and has been attributed to genomic deletion and epigenetic silencing. Recently, mutation of DLC1 identified from cancer patients is shown to inactivate its RhoGAP activity. However, the regulatory mechanism of the tumor suppressive activities of DLC1 is unclear. Recent study about the regulation of activity and compartmentalization of DLC1 by protein kinases has provided the first evidence that DLC1 activity could be regulated by post-translational modification. Moreover, identification of rat homolog of DLC1, p122RhoGAP, as the substrate of Akt has provided insight into the potential regulatory pathway of DLC1. However, the functional significance Akt phosphorylation in p122RhoGAP and its relevance in human DLC1 have not been investigated. In this study, we aimed to elucidate the molecular mechanism by which Akt phosphorylates DLC1 and the functional impact of this phosphorylation on DLC1 activities. In this study, we have shown that Akt interacts with the C-terminus of DLC1 and only Akt with intact kinase activity can interact and phosphorylate DLC1. Although this newly identified residue is distinct from the reported Akt phosphorylated residue in rat p122RhoGAP, it is well conserved in DLC family. Indeed, another family member, DLC2 is also phosphorylated by Akt. This implicates a common regulatory mechanism of DLC family by Akt signaling pathway. To determine the functional significance of phosphorylation of DLC1 by Akt, site-directed mutagenesis is employed to create phosphomimetic and phosphodefective mutant of DLC1. In vitro functional assays have demonstrated that phosphomimetic DLC1 suppresses growth, colony formation and anchorage independent growth of transformed hepatoblasts. Moreover, using subcutaneous injection and orthotopic tumor implantation models, phosphomimetic DLC1 mutant loses its inhibitory activities in tumorigenesis and metastasis of transformed hepatoblasts in nude mice. In conclusion, our findings have identified a novel post-translational modification by which Akt phosphorylation functionally deregulates the tumor suppressive activities of DLC1. Herein we show that apart from underexpression and mutation of DLC1, the enhanced phosphorylation level of DLC1 could be an indicator of deregulated DLC1 in cancers with normal expression level of DLC1.