The role of the deubiquitinating enzyme CYLD and its substrate BCL-3 in solid tumors

University dissertation from Molecular tumor Pathology

Abstract: The tumor suppressor CYLD and the proto-oncogene BCL-3 are known to be deregulated in various cancer types. The molecular background of how these genes participate in carcinogenesis is not fully understood. CYLD is a deubiquitinating enzyme known to specifically target lysine 63 linked ubiquitin chains, which can negatively regulate the BCL-3, NF-κB and JNK signaling pathways, leading to a decrease of cell survival or proliferation. BCL-3 is an alternative IκB family member that is needed for activation (or repression) of target genes by homodimeric p50 and p52. The aim of this thesis was to further investigate the molecular mechanisms behind CYLD and BCL-3 regulation and how they might contribute to carcinogenesis. In particular, the role of BCL-3 in prostate cancer (PCa), and the role of CYLD in hepatocellular carcinoma (HCC) were studied. In PCa we found up-regulation of BCL-3 in human prostate cancers with abundant infiltration of inflammatory cells. Using PCa cell lines we found that interleukin-6 (IL-6) could activate STAT3 mediated up-regulation of BCL-3, which in turn could elevate Id-1 and Id-2 expression. Knockdown of BCL-3 increased the sensitivity for anticancer drug-induced apoptosis. PCa cells with reduced BCL-3 levels that were subcutaneously injected into NUDE mice formed smaller tumors due to a higher percentage of apoptotic cells. In other tissues Bcl-3 has been shown to regulate proliferation through expression of its target gene CYCLIN D1, a process that is negatively regulated by CYLD. We found that CYLD knockout MEF cells have significantly increased proliferation rates and increased levels of CYCLIND1 in a serum dependent manner when compared with wild type MEF cells. The reduced proliferation in wild type cells was mediated through up-regulation of CYLD by transcription factor serum response factor (SRF) in a p38 mitogen-activated protein kinase (p38MAPK) dependent manner. Knockdown of SRF by siRNA or inhibition of p38MAPK reduced the expression of CYLD and increased cell proliferation rate. These results suggest that SRF is a positive regulator of CYLD expression, which in turn reduces the mitogenic activation of wild type MEF cells. For further investigation of the molecular mechanisms of CYLD in cancer we performed a tissue microarray, comparing benign liver tissue with HCC. We found that CYLD is significantly down-regulated in human (HCC) and that CYLD expression was inversely correlated with the expression of proliferation marker Ki67. In vivo experiments showed that CYLD deficient mice were more susceptible to the chemical carcinogen DEN-induced HCC. Furthermore, HCC isolated from CYLD knockout mice had elevated cell proliferation compared to wild type mice. This effect was mediated via TRAF-2 ubiquitination, JNK activation and c-MYC expression. In correlation to this result, transient transfection of CYLD into a HCC cell line restricted cell proliferation and reduced the activation of JNK. Together these results suggest that CYLD down-regulation is a risk factor for development and progression of HCC mediated through activation of JNK1