A novel player in inflammation and cancer: The deubiquitinase CYLD controls HCC development
Authors: Jeremie Gautheron, Tom Luedde
Journal: Journal of Hepatology (2012)

Liver specific deletion of CYLDexon7/8 induces severe biliary damage, fibrosis and increases hepatocarcinogenesis in miceJournal of HepatologyVol. 57Issue 5PreviewCYLD is a tumor suppressor gene that is mutated in familial cylindromatosis, an autosomal dominant predisposition to tumors of skin appendages. Reduced CYLD expression has been observed in other tumor entities, including hepatocellular carcinoma. In the present study, we analyzed the role of CYLD in liver homeostasis and hepatocarcinogenesis in vivo. Full-Text PDF During the past decades, hepatocellular carcinoma (HCC) became a major health burden and currently represents the third leading cause of cancer-related mortality worldwide [[1]Sherman M. Hepatocellular carcinoma: epidemiology, surveillance, and diagnosis.Semin Liver Dis. 2010; 30: 3-16Crossref PubMed Scopus (331) Google Scholar]. While a continuous decline in cirrhosis-related mortality was observed over the past decades, mortality rates for HCC increased until the end of the last millennium. In fact, the incidence of HCC still nearly equals its mortality rate [[2]Cabibbo G. Craxi A. Epidemiology, risk factors and surveillance of hepatocellular carcinoma.Eur Rev Med Pharmacol Sci. 2010; 14: 352-355PubMed Google Scholar]. The introduction of the multi tyrosine kinase inhibitor sorafenib represented a major breakthrough for the therapy of HCC [[3]de Lope C.R. Tremosini S. Forner A. Reig M. Bruix J. Management of HCC.J Hepatol. 2012; 56: S75-S87Abstract Full Text PDF PubMed Scopus (503) Google Scholar]. However, despite extensive efforts, other chemotherapeutic agents and also alternative tyrosine kinase inhibitors did not show a significant benefit for HCC patients and it is currently unclear which therapeutic strategies beyond sorafenib will be available in the future [[4]Villanueva A. Llovet J.M. Targeted therapies for hepatocellular carcinoma.Gastroenterology. 2011; 140: 1410-1426Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar]. It is well established that in most instances HCC arises in a setting of chronic hepatic inflammation, but the knowledge on this unique association between inflammation and cancer has not yet translated into preventive or therapeutic concepts against HCC in patients with chronic liver disease [[5]Vucur M. Roderburg C. Bettermann K. Tacke F. Heikenwalder M. Trautwein C. et al.Mouse models of hepatocarcinogenesis: what can we learn for the prevention of human hepatocellular carcinoma?.Oncotarget. 2010; 1: 373-378Crossref PubMed Scopus (46) Google Scholar], highlighting the need for a better understanding of the function of inflammatory signaling pathways in hepatocarcinogenesis. Among the cytokines that can induce inflammation, tumour necrosis factor (TNF) withholds a privileged role in the chronically inflamed liver [[6]Tacke F. Luedde T. Trautwein C. Inflammatory pathways in liver homeostasis and liver injury.Clin Rev Allergy Immunol. 2009; 36: 4-12Crossref PubMed Scopus (309) Google Scholar]. After its binding to TNF receptors 1 (TNFR1) or 2 (TNFR2), several distinct protein complexes can be assembled, leading to the activation of different signaling pathways, such as the pro -survival and -inflammatory NF-κB pathway, the stress-related Jun-(N)-terminal kinase (JNK) and p38MAPK pathways or the pro-apoptotic caspase cascade [[7]Luedde T. Schwabe R.F. NF-kappaB in the liver-linking injury, fibrosis and hepatocellular carcinoma.Nat Rev Gastroenterol Hepatol. 2011; 8: 108-118Crossref PubMed Scopus (917) Google Scholar]. In addition to phosphorylation events, the activation status of these respective signaling pathways is tightly controlled by ubiquitination, which involves the attachment of one or several ubiquitin molecules to a substrate [[8]Karin M. Gallagher E. TNFR signaling: ubiquitin-conjugated TRAFfic signals control stop-and-go for MAPK signaling complexes.Immunol Rev. 2009; 228: 225-240Crossref PubMed Scopus (150) Google Scholar]. Until recently, ubiquitination was mainly considered to play a role in controlling the half-life of proteins, but it soon became apparent that ubiquitination also regulates signaling events by acting as modulator of the enzymatic activity or docking of regulatory molecules [[8]Karin M. Gallagher E. TNFR signaling: ubiquitin-conjugated TRAFfic signals control stop-and-go for MAPK signaling complexes.Immunol Rev. 2009; 228: 225-240Crossref PubMed Scopus (150) Google Scholar]. To add an additional level of complexity, ubiquitination is a dynamic process that can be counterbalanced by deubiquitinase enzymes, including the molecule CYLD. CYLD was initially discovered in patients with familial cylindromatosis, a rare inherited cancer, characterized by the formation of benign tumours in hairy parts of the body [[9]Bignell G.R. Warren W. Seal S. Takahashi M. Rapley E. Barfoot R. et al.Identification of the familial cylindromatosis tumour-suppressor gene.Nat Genet. 2000; 25: 160-165Crossref PubMed Scopus (591) Google Scholar]. Later, the group of Gilles Courtois could show that CYLD is an essential modifier of NF-κB and the ubiquitination state of the NF-κB-activating molecule NEMO [[10]Kovalenko A. Chable-Bessia C. Cantarella G. Israel A. Wallach D. Courtois G. The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination.Nature. 2003; 424: 801-805Crossref PubMed Scopus (857) Google Scholar]. In addition to NF-κB, various studies performed mainly in cell culture revealed that CYLD deubiquitinates multiple other members of the TNF-signaling cascade, including TGF-β-activated kinase-1 (TAK1) and the TRAF family of ligases [[11]Courtois G. Tumor suppressor CYLD: negative regulation of NF-kappaB signaling and more.Cell Mol Life Sci. 2008; 65: 1123-1132Crossref PubMed Scopus (66) Google Scholar] (Fig. 1). Interestingly, expression of CYLD was shown to be dysregulated in several human cancers, including lung and colon cancer and multiple myeloma [[12]Massoumi R. CYLD: a deubiquitination enzyme with multiple roles in cancer.Future Oncol. 2011; 7: 285-297Crossref PubMed Scopus (89) Google Scholar]. In the paper from the group of Henning Schulze-Bergkamen published in the present issue of the Journal of Hepatology [[13]Urbanik T. Boger R.J. Longerich T. Becker K. Ehrenberg K.R. Hovelmeyer N. et al.Liver specific deletion of CYLDexon7/8 induces severe biliary damage, fibrosis and increases hepatocarcinogenesis in mice.J Hepatol. 2012; 57: 995-1003Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar], the authors examined the role of CYLD in liver homeostasis and the development of HCC by applying a conditional, cre/loxP-based knockout approach of CYLD in parenchymal liver cells. Specifically, they did not ablate the whole CYLD protein but only exons 7/8, resulting in overexpression of a naturally occurring, shorter splicing version of CYLD (s-CYLD) that preserves its catalytic activity but lacks the 3rd CAP domain of the protein responsible for binding of e.g., NEMO and TRAF2 [10Kovalenko A. Chable-Bessia C. Cantarella G. Israel A. Wallach D. Courtois G. The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination.Nature. 2003; 424: 801-805Crossref PubMed Scopus (857) Google Scholar, 14Trompouki E. Hatzivassiliou E. Tsichritzis T. Farmer H. Ashworth A. Mosialos G. CYLD is a deubiquitinating enzyme that negatively regulates NF-kappaB activation by TNFR family members.Nature. 2003; 424: 793-796Crossref PubMed Scopus (808) Google Scholar] (CYLDxAlbCre). Expression of this mutant form in CYLDxAlbCre animals resulted in a spontaneous phenotype characterized by prominent biliary fibrosis and ductular reaction, reflecting a chronic inflammatory response with immune cell infiltration and overexpression of inflammatory cytokines and chemokines such as TNF, IL-6, and MCP-1. However, in contrast to other genetic models of chronic hepatic inflammation [15Bettermann K. Vucur M. Haybaeck J. Koppe C. Janssen J. Heymann F. et al.TAK1 suppresses a NEMO-dependent but NF-kappaB-independent pathway to liver cancer.Cancer Cell. 2010; 17: 481-496Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 16Luedde T. Beraza N. Kotsikoris V. van Loo G. Nenci A. De Vos R. et al.Deletion of NEMO/IKKgamma in liver parenchymal cells causes steatohepatitis and hepatocellular carcinoma.Cancer Cell. 2007; 11: 119-132Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar], CYLDxAlbCre mice did not develop spontaneous HCCs, but were more susceptible to liver tumours induced by the chemical carcinogen diethylnitrosamine (DEN). Interestingly, some liver tumours in CYLDxAlbCre mice showed histological characteristics of cholangiocellular carcinomas (CCC). On the one hand, this might be explained by the fact that the albumin-Cre line used in this paper is not specific for hepatocytes but mediates deletion in a parenchymal hepatic precursor cell compartment [[17]Xu X. Kobayashi S. Qiao W. Li C. Xiao C. Radaeva S. et al.Induction of intrahepatic cholangiocellular carcinoma by liver-specific disruption of Smad4 and Pten in mice.J Clin Invest. 2006; 116: 1843-1852Crossref PubMed Scopus (170) Google Scholar]. On the other hand, the detection of CCCs in CYLDxAlbCre mice might support recently published findings by the group of Holger Willenbring showing that CCC can directly evolve from hepatocytes by a pathway involving NOTCH and AKT signalling [[18]Fan B. Malato Y. Calvisi D.F. Naqvi S. Razumilava N. Ribback S. et al.Cholangiocarcinomas can originate from hepatocytes in mice.J Clin Invest. 2012; Google Scholar]. Finally, the authors demonstrated that their spontaneous phenotype was associated with a subtle increase in NF-κB signaling reflected by upregulation of NF-κB target genes like survivin and GADD45β. The study makes an important contribution to the field of inflammation and hepatocarcinogenesis, since it clearly demonstrates a novel, interesting function of CYLD in HCC development. However, like every good study, it leaves unanswered questions on top of the novel findings presented. While the authors demonstrated increased NF-κB activity in CYLDxAlbCre animals, it is presently not clear if NF-κB is really the main downstream target of CYLD mediating its anti-carcinogenic function, given the multiple other interaction partners of CYLD within the TNF pathway. Of note, it was previously demonstrated that blockage of NF-κB results in increased hepatic tumourigenesis in the DEN-HCC-model [[19]Maeda S. Kamata H. Luo J.L. Leffert H. Karin M. IKKbeta couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis.Cell. 2005; 121: 977-990Abstract Full Text Full Text PDF PubMed Scopus (982) Google Scholar]. Therefore, it is unclear if the opposite situation in CYLDxAlbCre mice – overactivation of NF-κB – really functionally explains increased tumourigenesis in these animals. In contrast, the phenotype in CYLDxAlbCre animals might be a consequence not of the absence of full-length-CYLD, but of the increased intracellular amount of the short-CYLD-version, putatively lacking binding sites to certain factors like NEMO and TRAF2 in exons 7/8, but still retaining the deubiquitinase-activity encoded from exon 9 on. Important complementary information to the present study might come from a recent paper published in the journal Cancer Cell by the group of Iannis Talianidis [[20]Nikolaou K. Tsagaratou A. Eftychi C. Kollias G. Mosialos G. Talianidis I. Inactivation of the deubiquitinase CYLD in hepatocytes causes apoptosis, inflammation, fibrosis, and cancer.Cancer Cell. 2012; 21: 738-750Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar]. In contrast to the present approach, this group performed a conditional knockout of exon 9 of CYLD, resulting in expression of a deubiquitinase-deficient form of CYLD similar to oncogenic mutations described in humans [[21]Trompouki E. Tsagaratou A. Kosmidis S.K. Dolle P. Qian J. Kontoyiannis D.L. et al.Truncation of the catalytic domain of the cylindromatosis tumor suppressor impairs lung maturation.Neoplasia. 2009; 11: 469-476Abstract Full Text PDF PubMed Scopus (38) Google Scholar]. Interestingly, expression of this mutant in liver cells resulted in spontaneous development of hepatic fibrosis and liver tumours. On a functional level, certain features of the phenotype seen in that CYLD mutant mouse were mediated through the kinase TAK1, since combined deletions of TAK1 and CYLD led to less fibrosis and periportal cell death than those observed in CYLD single mutants. However, TAK1/CYLD combined mutant animals still showed cancer development and other features of TAK1 single mutant animals [[15]Bettermann K. Vucur M. Haybaeck J. Koppe C. Janssen J. Heymann F. et al.TAK1 suppresses a NEMO-dependent but NF-kappaB-independent pathway to liver cancer.Cancer Cell. 2010; 17: 481-496Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar], demonstrating that neither the absence nor the over-activation of TAK1 is beneficial for liver cells. Nevertheless, given that absence of the deubiquitinase-activity of CYLD results in a much more dramatic phenotype than the exon 7/8 deletion presented in the current paper, it would be of high interest to identify the interaction partners of CYLD that still bind to the short form (s-CYLD) and prevent cell death and spontaneous cancer in CYLDxAlbCre animals. In the light of these two interesting studies and the elegant approach of partial deletion of certain exons in the Cyld gene, it is possible that future studies taking similar approaches rather than a complete genetic knockout might lead to novel important functional knowledge of key players in inflammation and cancer, since they are probably closer to mutations observed in human cancer and also more relevant to pharmacological applications. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.