Attenuation of growth suppression activity of deleted in liver cancer 1 (DLC1) tumor suppressor by Akt phoshorylation

Abstract

Deleted in liver cancer 1 (DLC1) was identified as a putative tumor suppressor in hepatocellular carcinoma (HCC) in 1998. Since its identification, accumulating evidence has shown DLC1 is a bona fide tumor suppressor not only in HCC, but also in diverse human cancers. DLC1 inhibits cancer cell growth, invasion, migration and metastasis, and induces apoptosis. DLC1 is widely expressed in normal human tissues, but it is frequently underexpressed in HCC and other cancers. Heterozygous deletion of DLC1 is commonly found in about 50% of liver, breast, lung and colon cancers. The other allele of DLC1 has been found to be epigenetically silenced. Apart from down regulation of DLC1 expression either by genomic deletion or epigenetic silencing, our study here suggested that phosphorylation of DLC1 by Akt could be the mechanism that functionally deregulates DLC1 in tumors with normal DLC1 expression. Our findings showed that phosphorylation signal of ectopically expressed and endogenous DLC1 was enhanced upon insulin induction or with Akt expression in liver cancer cell lines, while addition of PI3K/Akt pathway inhibitor attenuated the phosphorylation level of DLC1. Analysis of phosphorylation signal of various truncation mutants of DLC1 revealed that phosphorylation residues were resided in the central region of DLC1 where three consensus Akt substrate motifs were found. Site-directed mutagenesis was employed to replace the serine residue of the consensus Akt substrate motifs by alanine. Our data showed that serine-567 of DLC1 was the only target of Akt phosphorylation. We also found that wild-type and constitutively active Akt strongly interacted with DLC1, whereas kinase-dead and inactive Akt failed to interact with DLC1. This suggested that Akt kinase activity is required for the interaction between Akt and DLC1. In accordance with these findings, DLC1 could only be phosphorylated when it associated with Akt. Functionally, phosphomimetic mutant of DLC1 lost its tumor suppressive activity in inhibiting growth of HCC cells. Oncogenic Akt pathway has been implicated in enhancing cell survival. Aberrant expression and activation of Akt have been evident in various cancers. Our findings have provided compelling evidence that Akt is a novel regulator of DLC1 and DLC1 is hyper-phosphorylated when Akt is activated. This suggested that apart from DLC1 underexpression, enhanced phosphorylation of DLC1 could also be an indicator of tumor progression. More importantly, our work revealed the first signaling pathway functionally regulates the biological activities of DLC1. The phosphorylation target residue is well conserved in all DLC family members. Demonstration of Akt phosphorylation of another family member, DLC2 further strengthens the importance of this particular residue and supports a common regulatory mechanism of DLC family. Financial support: RGC GRF (HKU7798/07M) and RGC CRF (HKU1/06C)