The role of the tumor suppressor CYLD for the proliferation and invasion of hepatocellular carcinoma cells

Background and Aims: The deubiquitination enzyme CYLD has originally been identified as a gene mutated in familial cylindromatosis, an autosomal-dominant predisposition to multiple tumors of the skin appendages.[1] CYLD is an important regulator of cell proliferation, survival and invasion and is downregulated in a variety of cancers, including hepatocellular carcinoma.[2] CYLD acts as a negative regulator of NFκB-signaling and prevents the nuclear translocation of Bcl–3, a coactivator of NFκB subunits.[3] The aim of our study is to examine the effects of CYLD knockdown on the proliferation and invasion of HCC cells. Methods: HCC cells (Huh7 and Hep3B) were treated with siRNA specifically targeting CYLD mRNA and resulting in a knockdown of CYLD expression. We examined the levels of different cell cycle and adhesion markers on both mRNA level via quantitative realtime-PCR and protein level via Western blot analysis. Furthermore, we investigated the invasive potential of HCC cells in transwell assays after knockdown of CYLD. Results: After siRNA-mediated CYLD knockdown we observed that invasion of HCC cells through a Matrigel layer was elevated after 48h. A possible explanation could be the increased expression of N-Cadherin which we observed after CYLD siRNA transfection in both cell lines. Our analysis also revealed an increase in Bcl–3 and ß-Catenin expression, both potent enhancers of proliferation and transcriptional coactivators of Cyclin D1, which we also found upregulated. Consistent with these findings, we observed a downregulation of p21, a CDK-inhibitor and negative regulator of G1/S-progression, in Huh7 cells following CYLD knockdown. Conclusion: The deubiquitination enzyme CYLD affects a variety of pathways crucial for the regulation of proliferation, cell cycle progression and invasion. Knockdown of CYLD promotes cell cycle progression and the invasive potential of HCC cells.

Literatur: 1. Bignell, G.R., et al., Identification of the familial cylindromatosis tumour-suppressor gene. Nat Genet, 2000. 25(2): p. 160-5. 2. Urbanik, T., et al., Down-regulation of CYLD as a trigger for NF-kappaB activation and a mechanism of apoptotic resistance in hepatocellular carcinoma cells. Int J Oncol, 2011. 38(1): p. 121-31. 3. Massoumi, R., et al., Cyld inhibits tumor cell proliferation by blocking Bcl-3-dependent NF-kappaB signaling. Cell, 2006. 125(4): p. 665-77.

CYLD - hepatocellular carcinoma