TP53INP1 negatively regulates ERK1/2 via p73-dependent DUSP10 expression to promote metastasis in hepatocellular carcinoma

Abstract

The recurrence and metastasis of hepatocellular carcinoma (HCC) portends a poor prognosis and represents important clinical challenges. There is a great need to identify critical factors involved in HCC metastasis that will facilitate the development of new therapeutic strategies. We have previously found that the initiation, growth and self-renewal of CD133+ liver tumors to be fine-tuned by a balance of miR-130b overexpression and tumor protein 53 inducible nuclear protein 1 (TP53INP1) down-regulation, suggesting that TP53INP1 is a critical effector driving hepatocarcinogenesis. In this study, we aimed to further investigate the role of TP53INP1 in HCC metastasis. We showed TP53INP1 to be frequently down-regulated in advanced stage IV and metastatic human HCC tumors as compared with early stage (I-III) and primary tumors. Functional studies in immortalized normal liver cell line MIHA and HCC cell line MHCC-97L found TP53INP1 suppression in HCC to promote metastasis in vitro and in vivo. To elucidate the downstream signaling mechanism by which TP53INP1 regulates HCC metastasis, a Proteome Profiler Human Phospho-Kinase Array was utilized. Phospho-ERK was found to be consistently up-regulated in HCC cell lines with TP53INP1 knocked down, with its involvement in TP53INP1-mediated metastasis subsequently validated by rescue experiments using an ERK inhibitor U0126 or shERK1/2 knockdown approach. ERK1/2 is known to be negatively regulated by a family of dual-specificity MAPK phosphatase called DUSP/MKP. Screening of a panel of DUSP/MKP family members by qPCR identified DUSP10 to be commonly down-regulated in both MIHA and MHCC-97L cells with TP53INP1 suppressed as compared to controls. The importance of DUSP10 inactivation on ERK in TP53INP1-mediated HCC was substantiated by rescue experiments whereby DUSP10 ectopic expression in TP53INP1 suppressed cells reversed up-regulated phospho-ERK expression and enhanced HCC aggressiveness. Subsequent analysis of the DUSP10 promoter region by open-access database revealed four putative binding sites for p73, of which transcriptional activity has previously been found to be modulated by TP53INP1. Chromatin immunoprecipitation and luciferase reporter assays collectively demonstrated that TP53INP1 is vital for p73 transcriptional activity of DUSP10. Taken together, TP53INP1 down-regulation promotes HCC metastasis through a p73-dependent DUSP10/ERK signaling pathway.