Background & aims: Uncontrolled liver proliferation is a key characteristic of liver cancer; however, the mechanisms by which this occurs are not well understood. Elucidation of these mechanisms is necessary for the development of better therapy. The oncogene Gankyrin (Gank) is overexpressed in both hepatocellular carcinoma and hepatoblastoma. The aim of this work was to determine the role of Gank in liver proliferation and elucidate the mechanism by which Gank promotes liver proliferation.
Methods: We generated Gank liver-specific knock-out (GLKO) mice and examined liver biology and proliferation after surgical resection and liver injury.
Results: Global profiling of gene expression in GLKO mice showed significant changes in pathways involved in liver cancer and proliferation. Investigations of liver proliferation after partial hepatectomy and CCl4 treatment showed that GLKO mice have dramatically inhibited proliferation of hepatocytes at early stages after surgery and injury. In control LoxP mice, liver proliferation was characterized by Gank-mediated reduction of tumor-suppressor proteins (TSPs). The failure of GLKO hepatocytes to proliferate is associated with a lack of down-regulation of these proteins. Surprisingly, we found that hepatic progenitor cells of GLKO mice start proliferation at later stages and restore the original size of the liver at 14 days after partial hepatectomy. To examine the proliferative activities of Gank in cancer cells, we used a small molecule, cjoc42, to inhibit interactions of Gank with the 26S proteasome. These studies showed that Gank triggers degradation of TSPs and that cjoc42-mediated inhibition of Gank increases levels of TSPs and inhibits proliferation of cancer cells.
Conclusions: These studies show that Gank promotes hepatocyte proliferation by elimination of TSPs. This work provides background for the development of Gank-mediated therapy for the treatment of liver cancer. RNA sequencing data can be accessed in the NCBI Gene Expression Omnibus: GSE104395.
Keywords: 2D, 2-dimensional; BrdU, bromodeoxyuridine; C/EBP, CCAAT/enhancer binding protein; CUGBP1, CUG triplet repeat binding protein 1; Cancer; Co-IP, co-immunoprecipitation; DEN, diethylnitrosamine; FXR, farnesoid X receptor; GLKO, Gankyrin liver-specific knock-out; Gank, Gankyrin; HCC, hepatocellular carcinoma; HNF4α, hepatocyte nuclear factor 4α; LKO, liver-specific knock-out; Liver; Opn, osteopontin; PCNA, proliferating cell nuclear antigen; PH, partial hepatectomy; Progenitor Cells; Proliferation; RT-PCR, reverse-transcriptase polymerase chain reaction; Rb, retinoblastoma; TSP, tumor-suppressor protein; Tumor-Suppressor Proteins; UPS, ubiquitin proteasome system; WT, wild-type; cDNA, complementary DNA; mRNA, messenger RNA.