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Liver cancer is the fifth most common cancer. A highly invasive surgical resection of the liver tumor is the main approach utilized to eliminate the tumor. Mechanisms that terminate liver regeneration when the liver reaches the original size are not known. The aims of this work were to generate the animal model which fails to stop liver regeneration after surgical resections and elucidate mechanisms which are involved in termination of liver regeneration. Because epigenetic control of liver functions has been previously implicated in the regulation of liver proliferation, we have generated C/EBPα-S193A knockin mice, which have alterations in formation of complexes of C/EBP family proteins with chromatin remodeling proteins. The C/EBPα-S193A mice have altered liver morphology and altered liver functions leading to changes of glucose metabolism and blood parameters. Examination of proliferative capacity of C/EBPα-S193A livers showed that livers of S193A mice have a higher rate of proliferation after birth, but stop proliferation at the age of 2 months. These animals have increased liver proliferation in response to liver surgery as well as CCl4-mediated injury. Importantly, livers of C/EBPα-S193A mice fail to stop liver regeneration after surgery when livers reach the original, pre-resection, size. The failure of S193A livers to stop regeneration correlates with the epigenetic repression of key regulators of liver proliferation C/EBPα, p53, FXR, SIRT1, PGC1± and TERT by C/EBPβ-HDAC1 complexes. The C/EBPβ-HDAC1 complexes also repress promoters of enzymes of glucose synthesis PEPCK and G6Pase. Conclusions Our data demonstrate that a proper co-operation of C/EBP and chromatin remodeling proteins is essential for the termination of liver regeneration after surgery and for maintenance of liver functions.

The development of hepatoblastoma (HBL) is associated with failure of hepatic stem cells (HSC) to differentiate into hepatocytes. Despite intensive investigations, mechanisms of the failure of HSC to differentiate are not known. We found that oncogene Gankyrin (Gank) is involved in the inhibition of differentiation of HSC via triggering degradation of tumor suppressor proteins (TSPs) Rb, p53, C/EBPα and HNF4α. Our data show that the activation of a repressor of Gank, farnesoid X receptor, FXR, after initiation of liver cancer by Diethylnitrosamine (DEN) prevents the development of liver cancer by inhibiting Gank and rescuing tumor suppressor proteins. We next analyzed FXR-Gank-Tumor suppressor pathways in a large cohort of HBL patients which include 6 controls and 53 HBL samples. Systemic analysis of these samples and RNA-Seq approach revealed that the FXR-Gank axis is activated; markers of hepatic stem cells are dramatically elevated and hepatocyte markers are reduced in HBL samples. In the course of these studies, we found that RNA binding protein CUGBP1 is a new tumor suppressor protein which is reduced in all HBL samples. Therefore, we generated CUGBP1 KO mice and examined HBL signatures in the liver of these mice. Micro-array studies revealed that the HBL-specific molecular signature is developed in livers of CUGBP1 KO mice at very early ages. Thus, we conclude that FXR-Gank-TSPs-Stem cells pathway is a key determinant of liver cancer in animal models and in pediatric liver cancer. Our data provide a strong basis for development of FXR-Gank-based therapy for treatment of patients with hepatoblastoma.

Summary The development of Non Alcoholic Fatty Liver Disease (NAFLD) is a multiple step process. Here we show that activation of cdk4 triggers the development of NAFLD. We found that cdk4 protein levels are elevated in mouse models of NAFLD and in patients with fatty livers. This increase leads to C/EBPα phosphorylation on Ser193 and formation of C/EBPα-p300 complexes, resulting in hepatic steatosis, fibrosis, and HCC. The disruption of this pathway in cdk4-resistant C/EBPα-S193A mice dramatically reduces development of high fat diet (HFD)-mediated NAFLD. In addition, inhibition of cdk4 by flavopiridol or PD-0332991 significantly reduces development of hepatic steatosis, the first step of NAFLD. Thus, these studies reveal that activation of cdk4 triggers NAFLD, and that inhibitors of cdk4 may be used for the prevention/treatment of NAFLD.

Background: Older patients are more sensitive to drug-mediated development of liver disorders. Results: Age and CCl 4 treatments change expression of C/EBP proteins leading to repression of key regulators of liver biology. Conclusion:The age-associated alterations of C/EBP proteins cause severe liver injury after CCl 4 treatments. Significance: Understanding of mechanisms of age-associated severe liver injury is important for development of therapeutic approaches.

Background & AimsUncontrolled 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.MethodsWe generated Gank liver-specific knock-out (GLKO) mice and examined liver biology and proliferation after surgical resection and liver injury.ResultsGlobal 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.ConclusionsThese 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.

Background: Development of liver cancer involves alterations of multiple pathways of gene expression. Results: Translational activation of C/EBP␤-HDAC1 complexes represses p53, SIRT1, and PGC1␣, leading to liver cancer. Conclusion: Modulation of levels of C/EBP␤-HDAC1 complexes at different stages of cancer is involved in liver cancer. Significance: Understanding the mechanisms of liver cancer is a critical step for the development of therapeutic approaches for cancer.