Mounting evidence indicates that S-Phase Kinase-Associated Protein 2 (SKP2) is overexpressed in human hepatocellular carcinoma (HCC). However, the role of SKP2 in hepatocarcinogenesis remains poorly delineated. To elucidate the function(s) of SKP2 in HCC, we stably overexpressed the SKP2 gene in the mouse liver, either alone or in combination with activated forms of N-Ras (N-RasV12), AKT1 (myr-AKT1), or β-catenin (ΔN90-β-catenin) protooncogenes, via hydrodynamic gene delivery. We found that forced overexpression of SKP2, N-RasV12 or ΔN90-β-catenin alone as well as co-expression of SKP2 and ΔN90-β-catenin did not induce hepatic histological alterations. Overexpression of myr-AKT1 alone led to HCC development after long latency. In contrast, co-expression of SKP2 with N-RasV12 or myr-AKT1 resulted in early development of multiple hepatocellular tumors in all SKP2/N-RasV12 and SKP2/myr-AKT1 mice. At the molecular level, preneoplastic and neoplastic liver lesions from SKP2/N-RasV12 and SKP2/myr-AKT1 mice exhibited a strong induction of AKT/mTOR and Ras/MAPK pathways. Noticeably, the tumor suppressor proteins whose levels have been shown to be downregulated by SKP2-dependent degradation in various tumor types, including p27, p57, DUSP1, and Rassf1A were not decreased in liver lesions from SKP2/N-RasV12 and SKP2/myr-AKT1 mice. In human HCC specimens, nuclear translocation of SKP2 was associated with activation of the AKT/mTOR and Ras/MAPK pathways, but not with β-catenin mutations. In hepatoma cell lines, suppression of SKP2 activity by MLN4924 synergized with AKT and MAPK inhibitors to induce growth restraint. Altogether, the present data indicate that SKP2 cooperates with N-Ras or Akt proto-oncogenes to promote hepatocarcinogenesis in vivo.
MDM4 is a p53 binding protein that negatively regulates p53-dependent transcription. MDM4 was found overexpressed in a variety of human cancers and amplifications of the MDM4 gene locus were reported with high frequency in most of the tumors analyzed. We have previously demonstrated that MDM4 is upregulated in about 50% of human hepatocellular carcinomas (HCC), both at messenger RNA and protein level. Although we identified 1q32 gain as a mechanism leading to MDM4 overexpression in human HCCs, many liver tumors display a balanced MDM4 gene locus. Thus, additional mechanisms are responsible for MDM4 aberrant expression in the presence of an intact MDM4 locus. The transcriptional regulation of MDM4 remains largely elusive. Here, we show that activation of the Ras/ERK signaling pathway is involved in the transcriptional upregulation of MDM4 in human HCCs thereby supporting its protumorigenic function.
PIK3CA, encoding the catalytic subunit p110α of class I phosphoinositide-3-kinase (PI3K), is mutated or overexpressed in many tumors, including hepatocellular carcinoma (HCC), where it is supposed to act as an oncogene. Here, we developed new mouse models to investigate the oncogenic effects of gain-of-function PIK3CA mutations and identified downstream PI3K signaling components in the mouse liver.
Der Wnt/Beta-Catenin- und der Yes-associated protein (YAP)-Signalweg regulieren beide das Organwachstum in der Embryogenese. Ihre aberrante Aktivierung wird unabhängig voneinander in Lebertumoren beobachtet. Aktivierende Mutationen oder Deletionen im Beta-Catenin-Gen können sowohl zu Hepatozellulären Karzinomen (HCC) als auch Hepatoblastomen (HB) führen. Die pathogenetische Bedeutung von YAP, dem wesentlichen transkriptionellen Mediator des Hippo-Kinase-Signalwegs, ist hingegen weniger gut verstanden und über ein mögliches Zusammenspiel dieser beiden onkogenen Signalwege für die Lebertumorentstehung ist kaum etwas bekannt. In einem Kollektiv humaner HB fanden wir eine Ko-Expression von Beta-Catenin and YAP1 in 79% der Fälle, hingegen in keinem der Kontrolltumoren (HCC oder cholangiozelluläre Karzinome). Die Bedeutung einer Ko-Aktivität dieser Onkogene für die Lebertumorentstehung haben wir daraufhin in vivo untersucht: Die Ko-Injektion von aktiviertem Beta-Catenin (ΔN90-β-catenin) und aktiviertem YAP (YapS127A; mit intaktem TEAD-Bindungsvermögen) in Mäuse durch hydrodynamischen Gentransfer mit nachfolgender sleeping beauty Transposase-mediierter genomischer Integration der Genkonstrukte in reife Hepatozyten, führte zu einer raschen Entstehung von Lebertumoren, von denen einige histomorphologisch HB des Menschen ähnelten. Nach 11 Wochen ergab sich eine 100%-ige tumorbedingte Mortalität der Tiere. Immunhistochemisch und durch Immunoblotting zeigte sich neben der kombinierten Überexpression des injizierten Beta-Catenins und von YAP1 auch eine starke Aktivierung derer Zielstrukturen wie Axin-2, Cyclin-D1, CTGF, und Jag1. In vitro führte die Suppression von YAP1 zu einem starken Aktivitätsverlust des Beta-Catenin-Signals in HB-Zellen (TOPflash Reporter Aktivitäts-Assay). Zudem war die kombinierte Inhibition von Beta-Catenin and YAP1 deutlich effektiver wachstumshemmend für die Tumorzellen als ein Inhibitor alleine. Wir schlussfolgern daher, dass der Synergismus von YAP and Beta-Catenin für die Entstehung und den Progress von HB bei der Maus und beim Menschen von besonderer Bedeutung ist.
Background: Cumulating evidence underlines the crucial role of aberrant lipid biosyntesis in human hepatocellular carcinoma (HCC). Here, we investigated the oncogenic potential of fatty acid synthase (FASN), the master regulator of de novo lipogenesis, in the mouse liver.
The Mouse Double Minute 4 protein (MDM 4) is a p53-negative regulator, which inhibits its transcriptional activity. As previously demonstrated, MDM 4 mRNA and protein levels are upregulated in human hepatocellular carcinoma (HCC) as consequence of both copy number alterations as well as a post-transcriptional mechanism involving the AKT/mTOR axis. However, many human HCCs showed high MDM 4 mRNA levels with a balanced MDM 4 gene locus, leading us to hypothesize that aberrant transcriptional mechanism might be involved in the upregulation of MDM 4 in human HCC.
Aims: The Hippo-pathway regulates organ size and homeostasis. Studies revealed that deletion of Hippo-pathway kinases (e.g. Mst1/2) or overexpression of the transcriptional co-activator YAP lead to liver cancer; however, the underlying molecular mechanism have not identified so far. We therefore aimed to define possible effector mechanisms mediating YAP-dependent oncogenic activity in liver cancer. Methods: SiRNA-knockdown, expression profiling, and functional assays were performed using HCC cell lines. Gene expression and protein interaction were analyzed by immunoblotting or RT-PCR and co-immunoprecipitation, respectively. YAP transgenic (YAPLAP) and Mst1/2 knock-out mice as well as human HCCs were examined by IHC, immunoblotting, and RT-PCR. Results: Expression profiling after YAP inhibition in HCC cells and of YAPLAP liver tissues identified the Notch-pathway ligand Jag1 as potential target gene for the Hippo pathway. Expression of cleaved Notch receptor (NICD) and Notch target gene Hes1 were reduced in HCC cells after YAP inhibition. SiRNA-knockdown of Jag1 but not of Jag2 as well as inhibition of Notch signalling by γ-secretase phenocopied the biological effects detected after YAP inhibition (e.g., decreased viability). Overexpression of YAP induced tumor cell proliferation, whereas additional Jag1 inhibition partly compensated the effects. Expression of YAP/Jag1 and YAP/Hes1 significantly correlated in human HCCs. YAPLAP-mice developed dysplastic foci with increased Jag1 expression and elevated proliferation. Furthermore, time-resolved analysis showed a simultaneous increase of Jag1 and Hes1 levels after doxycylin-dependent YAP expression in isolated primary hepatocytes. Accordingly, Mst1/2-/- mice-derived HCCs showed high YAP, Jag1, and Hes1 levels. Conclusion: These data demonstrate that activation of YAP induces Notch-pathway activity by Jag1 expression and that Notch-signalling partly facilitates the oncogenic effects of YAP in vitro and in vivo.
Große epidemiologische Studien haben gezeigt, dass Patienten mit einem Diabetes mellitus oder einem metabolischen Syndrom ein erhöhtes Risiko für die Entstehung eines hepatozellulären Karzinoms (HCC) besitzen. Wir untersuchen die mögliche karzinogene Wirkung einer dauerhaft erhöhten Insulin- und Glukosekonzentration auf Hepatozyten in einem Tiermodell, in dem diabetischen Ratten eine niedrige Anzahl von Pankreasinseln über die Pfortader in die Leber transplantiert wird. Die Hepatozyten im Abstromgebiet der Transplantate entwickeln daraufhin zunächst Präneoplasien, die sich langfristig zu HCC weiterentwickeln.
Background and aims: Administration of the N-Nitrosamine N-Nitrosomorpholine (NNM) in rats is a well examined model of chemically induced hepatocarcinogenesis and starts with the formation of focal pre-neoplastic lesions in the liver, i.e. clear cell foci (CCF) of altered hepatocytes, and induces the formation of hepatocellular adenomas (HCA) and carcinomas (HCC). Similar lesions are known in the rat model of insulin-induced hepatocarcinogenesis and also in the human liver, which reveal an activation of the AKT/mTOR and Ras/MAPK pathways and the lipogenic phenotype, as we described previously.
Introduction: AKT and Ras pathways are frequently concurrently hyperactivated in HCC. Previously, we have shown that co-expression of constitutively active forms of AKT (Myr-AKT) and activated Ras (N-RasV12) rapidly induces liver tumors in mice. mTORC1 is the main downstream effector of AKT, acting by phosphorylation of RPS-6 and 4E-BP1, and thus regulating cell metabolism, protein translation and other cellular processes. However, in which manner mTORC1 and its key downstream effectors RPS-6 and 4E-BP1/eIF4E do contribute to hepatocarcinogenesis remains unknown. Methods: Plasmids encoding Myr-AKT, RasV12, 4EBP1A4 (dominant negative form of 4E-BP1), or 4EBP1WT (wild type) were delivered into the mouse liver by hydrodynamic injection. The mTORC1 inhibitor Rapamycin or vehicle were intraperitoneally administrated into the mice. Western blotting and immunohistochemistry were performed to analyze the expression levels of proteins in the tissues. Results: To determine whether the mTORC1 downstream effector RPS6 is required for hepatocarcinogenesis in AKT/Ras mice, we treated AKT/Ras mice with rapamycin (AKT/RAS/Rapa) or vehicle (AKT/RAS/Veh) daily for 7 weeks. All of the AKT/RAS/Veh mice developed large HCCs and were required to be euthanized. By contrast, none of the AKT/RAS/Rapa mice showed HCC, but microscopically small regressive tumor-like nodules and preneoplastic hepatocellular foci were still visible. In addition, withdrawal of rapamycin treatment was followed by a relapse in HCC development. Biochemical analysis demonstrated that Rapamycin inhibited the phosphorylation of RPS6, but had no effect on phosphorylated/inactivated levels of 4E-BP1. To define the role of 4EBP1/eIF4E in AKT/Ras-driven hepatocarcinogenesis, we overexpressed 4EBP1A4 or 4EBP1WT along with AKT and Ras into the mouse liver. Seven weeks after hydrodynamic injection, AKT/Ras/4EBP1WT mice developed large HCC which were equivalent to those developed in AKT/RAS/Veh mice. However, only few very small hepatocellular adenomas (HCA) developed in the livers of AKT/Ras/4EBP1A4 mice. These lesions, together with preneoplastic hepatocytes, occupied 40–60% of the liver parenchyma. 20 weeks post injection, the AKT/Ras/4EBP1A4 mice eventually developed large liver tumors, including HCC. These results indicated that inhibition of 4E-BP1/eIF4E axis efficiently delayed the AKT/RAS induced liver tumor, yet was unable to completely block the tumorigenesis process. Finally, we co-injected AKT/Ras mice with 4EBP1A4 and treated the mice with Rapamycin. We found that simultaneously blocking p-RPS6 and p-4EBP1 completely inhibited AKT/Ras induced hepatic carcinogenesis. Conclusion: Our experiments demonstrate the critical role of mTORC1 in mediating activated AKT and Ras induced liver tumor development in vivo. The two major downstream effectors of mTORC1, RPS6 and 4E-BP1/eIF4E, are both required for tumorigenesis. Yet they are likely to have distinct roles within the neoplastic process.
