Liver specific deletion of CYLDexon7/8 induces severe biliary damage, fibrosis and increases hepatocarcinogenesis in mice.

BACKGROUND & AIMS: CYLD 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. METHODS: Mice with liver-specific deletion of CYLDexon7/8 (CYLD(FF)xAlbCre) were generated. Liver tissues were histologically analyzed and oval cell activation was investigated. Hepatocarcinogenesis was induced by diethylnitrosamine/phenobarbital (DEN/PB). Microarray expression profiling of livers was performed in untreated as well as DEN/PB-treated mice. NF-κB signaling was assessed by ELISA, quantitative real-time PCR, and Western blotting. RESULTS: CYLD(FF)xAlbCre hepatocytes and cholangiocytes did not express full-length CYLD (FL-CYLD) protein but showed increased expression of the naturally occurring short-CYLD splice variant (s-CYLD). CYLD(FF)xAlbCre mice exhibited a prominent biliary phenotype with ductular reaction and biliary-type fibrosis. In addition, CYLD(FF)xAlbCre mice showed a significantly increased sensitivity towards DEN/PB-induced hepatocarcinogenesis. Moreover, we could observe the development of cholangiocellular carcinoma, in line with enhanced oval cell activity. NF-κB-signaling was increased in livers of CYLD(FF)xAlbCre mice and likely contributed to the inflammatory and fibrotic response. CONCLUSIONS: The deletion of exon7/8 of the CYLD gene activates oval cells, leads to a biliary phenotype, and increases the susceptibility towards carcinogenesis in the liver. Thus, our study presents a novel model of biliary damage and liver fibrosis, followed by cancer development.

Down-regulation of CYLD as a trigger for NF-κB activation and a mechanism of apoptotic resistance in hepatocellular carcinoma cells.

The cylindromatosis gene (CYLD) was identified as a tumor suppressor gene, which is mutated in familial cylindromatosis (Brooke-Spiegler syndrome), an autosomal-dominant predisposition to multiple tumors of the skin appendages. CYLD is a deubiquitinating enzyme acting as a negative regulator of the nuclear factor κB (NF-κB) signaling pathway by removing lysine-63-linked polyubiquitin chains from NF-κB activating proteins. In order to investigate the role of CYLD in apoptotic signaling in human hepatocellular carcinoma (HCC) cells, we first studied the expression levels of CYLD in HCC tissues. CYLD expression was lower in HCC both at protein and mRNA levels compared to the surrounding non-malignant tissue. In order to further study the role of CYLD in the apoptotic sensitivity of HCC cells, CYLD was specifically down-regulated in HCC cell lines via RNA interference. The specific down-regulation of CYLD resulted in increased resistance towards treatment with doxorubicin, 5-fluorouracil and cisplatin. In addition, the down-regulation of CYLD in HCC cells decreased the sensitivity towards tumor necrosis factor-α-induced apoptosis. The CYLD knockdown also led to the degradation of the NF-κB inhibitor, IκB-α, resulting in enhanced NF-κB activity in HCC cells. Finally, we found that CYLD expression was triggered by the multikinase inhibitor, sorafenib, by the inhibition of Raf-1, as well as by the blockage of the pro-survival kinases, MEK (U0126) and the epidermal growth factor receptor (AG1478). In summary, we show that CYLD is down-regulated in human HCC and is involved in the apoptotic resistance of HCC cells. Our data identify the reconstitution of CYLD expression as an attractive approach for overcoming resistance to treatment in HCC.

TRAIL-induced apoptosis of hepatocellular carcinoma cells is augmented by targeted therapies.

AIM: To analyze the effect of chemotherapeutic drugs and specific kinase inhibitors, in combination with the death receptor ligand tumor necrosis factor-related apoptosis inducing ligand (TRAIL), on overcoming TRAIL resistance in hepatocellular carcinoma (HCC) and to study the efficacy of agonistic TRAIL antibodies, as well as the commitment of antiapoptotic BCL-2 proteins, in TRAIL-induced apoptosis. METHODS: Surface expression of TRAIL receptors (TRAIL-R1-4) and expression levels of the antiapoptotic BCL-2 proteins MCL-1 and BCL-x(L) were analyzed by flow cytometry and Western blotting, respectively. Knock-down of MCL-1 and BCL-x(L) was performed by transfecting specific small interfering RNAs. HCC cells were treated with kinase inhibitors and chemotherapeutic drugs. Apoptosis induction and cell viability were analyzed via flow cytometry and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS: TRAIL-R1 and -R2 were profoundly expressed on the HCC cell lines Huh7 and Hep-G2. However, treatment of Huh7 and Hep-G2 with TRAIL and agonistic antibodies only induced minor apoptosis rates. Apoptosis resistance towards TRAIL could be considerably reduced by adding the chemotherapeutic drugs 5-fluorouracil and doxorubicin as well as the kinase inhibitors LY294002 [inhibition of phosphoinositol-3-kinase (PI3K)], AG1478 (epidermal growth factor receptor kinase), PD98059 (MEK1), rapamycin (mammalian target of rapamycin) and the multi-kinase inhibitor Sorafenib. Furthermore, the antiapoptotic BCL-2 proteins MCL-1 and BCL-x(L) play a major role in TRAIL resistance: knock-down by RNA interference increased TRAIL-induced apoptosis of HCC cells. Additionally, knock-down of MCL-1 and BCL-x(L) led to a significant sensitization of HCC cells towards inhibition of both c-Jun N-terminal kinase and PI3K. CONCLUSION: Our data identify the blockage of survival kinases, combination with chemotherapeutic drugs and targeting of antiapoptotic BCL-2 proteins as promising ways to overcome TRAIL resistance in HCC.