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In the retrorsine (RS)-based model of massive liver repopulation, preexposure to this naturally occurring alkaloid is sufficient to prime normal host parenchymal cells to be slowly replaced by transplanted normal hepatocytes. The basis for this striking effect is yet to be fully elucidated. In the present studies the possible involvement of cell senescence was investigated. Fischer 344 rats were treated according to the RS-based protocol for hepatocyte transplantation, i.e., two doses of RS, 2 weeks apart, and were killed at 4 or 8 weeks after treatment. Control groups were given saline. Expression of senescence-associated beta-galactosidase was greatly induced in hepatocytes exposed to RS. In addition, several other changes that have been related to cell senescence were observed: these included markers of persistent activation of a DNA damage response, an increased expression of mammalian target of rapamycin, and positive regulators of the cell cycle, together with the induction of p21 and p27 cyclin-dependent kinase inhibitors. Furthermore, RS treatment increased levels of interleukin-6 in the liver, consistent with the activation of a senescenceassociated secretory phenotype. Conclusion: These findings indicate that RS induces hepatocyte senescence in vivo. We propose that cell senescence and the associated secretory phenotype can contribute to the selective growth of transplanted hepatocytes in this system. (HEPATOLOGY 2012;56:760-768)

Hepatocyte transplantation to treat liver disease is largely limited by the availability of useful cells. Human amniotic epithelial cells (hAECs) from term placenta express surface markers and gene characteristics of embryonic stem cells and have the ability to differentiate into all three germ layers, including tissues of endodermal origin (i.e., liver). Thus, hAECs could provide a source of stem cell–derived hepatocytes for transplantation. We investigated the differentiation of hAECs in vitro and after transplantation into the livers of severe combined immunodeficient (SCID)/beige mice. Moreover, we tested the ability of rat amniotic epithelial cells (rAECs) to replicate and differentiate upon transplantation into a syngenic model of liver repopulation. In vitro results indicate that the presence of extracellular matrix proteins together with a mixture of growth factors, cytokines, and hormones are required for differentiation of hAECs into hepatocyte-like cells. Differentiated hAECs expressed hepatocyte markers at levels comparable to those of fetal hepatocytes. They were able to metabolize ammonia, testosterone, and 17α-hydroxyprogesterone caproate, and expressed inducible fetal cytochromes. After transplantation into the liver of retrorsine (RS)-treated SCID/beige mice, naive hAECs differentiated into hepatocyte-like cells that expressed mature liver genes such as cytochromes, plasma proteins, transporters, and other hepatic enzymes at levels equal to adult liver tissue. When transplanted in a syngenic animal pretreated with RS, rAECs were able to engraft and generate a progeny of cells with morphology and protein expression typical of mature hepatocytes. Conclusion: Amniotic epithelial cells possess the ability to differentiate into cells with characteristics of functional hepatocytes both in vitro and in vivo, thus representing a useful and noncontroversial source of cells for transplantation. (HEPATOLOGY 2011;)

Concomitant expression of activated forms of v-akt murine thymoma viral oncogene homolog (AKT) and Ras in mouse liver (AKT/Ras) leads to rapid tumor development through strong activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway. mTORC1 functions by regulating p70S6K/ribosomal protein S6 (RPS6) and eukaryotic translation initiation factor 4E-binding protein 1/ eukaryotic translation initiation factor 4E (4EBP1/eIF4E) cascades. How these cascades contribute to hepatocarcinogenesis remains unknown. Here, we show that inhibition of the RPS6 pathway by rapamycin effectively suppressed, whereas blockade of the 4EBP1/eIF4E cascade by 4EBP1A4, an unphosphorylatable form of 4EBP1, significantly delayed, AKT/Ras-induced hepatocarcinogenesis. Combined treatment with rapamycin and 4EBP1A4 completely inhibited AKT/Ras hepatocarcinogenesis. This strong antineoplastic effect was successfully recapitulated by ablating regulatory associated protein of mTORC1, the major subunit of mTORC1, in AKT/Ras-overexpressing livers. Furthermore, we demonstrate that overexpression of eIF4E, the proto-oncogene whose activity is specifically inhibited by 4EBP1, resulted in hepatocellular carcinoma (HCC) development in cooperation with activated Ras. Mechanistically, we identified the ectonucleoside triphosphate diphosphohydrolase 5/ adenylate kinase 1/cytidine monophosphate kinase 1 axis and the mitochondrial biogenesis pathway as targets of the 4EBP1/eIF4E cascade in AKT/Ras and Ras/eIF4E livers as well as in human HCC cell lines and tissues. Conclusions: Complete inhibition of mTORC1 is required to suppress liver cancer development induced by AKT and Ras proto-oncogenes in mice. The mTORC1 effectors, RPS6 and eIF4E, play distinct roles and are both necessary for AKT/Ras hepatocarcinogenesis. These new findings might open the way for innovative therapies against human HCC. (Hepatology 2015;61:200–213)

HuR, a member of the Elav/Hu family, is a gene expression regulator with a central role in mRNA stabilization (1), HuR has been shown to increase cellular division by enhancing the stability of mRNAs encoding important proteins for cell cycle control and proliferation, as well as other factors that influence tumor cell growth (2, 3). HuR expression is elevated in colon, breast, prostate, pancreatic, oral, skin, brain, gastric, lung and ovarian cancers, and correlates with tumor malignancy, supporting a role for HuR in tumorigenesis (4–6). In the liver, HuR plays a crucial role in hepatocyte proliferation, differentiation, and HCC transformation (7). At the transcriptional level, NF-κB can activate HuR transcription downstream of the PI3K-AKT signaling pathway (8). At the post-transcriptional level, the ubiquitin (Ub)-proteasome pathway has been implicated in HuR function, whereas the machinery involved in enhancing HuR protein stability remains obscure (9). The ubiquitin-like molecule NEDD8 is a key regulator of cell growth, viability and development. NEDD8 is ubiquitously expressed and highly conserved among most eukaryotes. At the molecular level, only members of the cullin family of proteins, are well-characterized substrates for NEDDylation (10). However, through direct biological approaches or proteomic techniques, it is evident that NEDD8 proteome is widely diverse. Recent studies have shown that ribonucleoproteins are targets for NEDD8 conjugation which protect them from de-stabilization (11, 12). Conversely, cysteine protease (NEDP1) removes NEDD8 molecules from conjugated substrates (13). Mdm2 acts as an E3 NEDD8 ligase, promoting p53 NEDDylation and stabilization (14), and, of importance, Mdm2 is overexpressed in many human tumors, at least in part due to gene amplification (15). In this study, we show that HuR is overexpressed in proliferative HCC and colon cancer cells and biopsies through Mdm2-mediated NEDDylation. Our data suggests that HuR NEDDylation at K313 and K326 residues stabilize HuR. Mechanistically, Mdm2-mediated NEDDylation of HuR controls the nuclear localization of HuR and protects it from degradation. To our knowledge, this is the first report to implicate NEDDylation in the regulation of HuR stability and localization. Our findings show that NEDDylation is a novel mechanism for HuR regulation that might represent a useful tool for new therapeutic strategies for HCC and colon cancer.

Background & Aims The cell cycle regulators P21 WAF1 , P27 KIP1 , P57 KIP2 , P130, RASSF1A, and FOXO1 are down-regulated during hepatocellular carcinoma (HCC) pathogenesis. We investigated the role of the ubiquitin ligase subunits CKS1 and SKP2, which regulate proteasome degradation of cell cycle regulators, in HCC progression. Methods Human HCC tissues from patients with better (HCCB, >3 years survival) and poorer prognosis (HCCP, Results The promoters of P21 WAF1 , P27 KIP1 , and P57 KIP2 were more frequently hypermethylated in HCCP than HCCB. Messenger RNA levels of these genes were up-regulated in samples in which these genes were not methylated; protein levels increased only in HCCB because of CKS1- and SKP2-dependent ubiquitination of these proteins in HCCP. The level of SKP2 expression correlated with rate of HCC cell proliferation and level of microvascularization of samples and was inversely correlated with apoptosis and survival. In HCCB, SKP2 activity was balanced by degradation by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-CDH1 and up-regulation of SKP2 suppressor histidine triad nucleotide binding protein 1 (HINT1). In HCCP, however, SKP2 was not degraded because of down-regulation of the phosphatase CDC14B, CDK2-dependent serine phosphorylation (which inhibits interaction between CDH1 and SKP2), and HINT1 inactivation. In HCC cells, small interfering RNA knockdown of SKP2 reduced proliferation and ubiquitination of the cell cycle regulators, whereas SKP2 increased proliferation and reduced expression of cell cycle regulators. Conclusions Ubiquitination and proteasome degradation of P21WAF1, P27KIP1, P57KIP2, P130, RASSF1A, and FOXO1 and mechanisms that prevent degradation of SKP2 by APC/C-CDH1 contribute to HCC progression. CKS1-SKP2 ligase might be developed as a therapeutic target or diagnostic marker.