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

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 JNK

CYLD controls c-MYC expression through the JNK-dependent signaling pathway in hepatocellular carcinoma

Posttranslational modification of different proteins via direct ubiquitin attachment is vital for mediating various cellular processes. Cylindromatosis (CYLD), a deubiquitination enzyme, is able to cleave the polyubiquitin chains from the substrate and to regulate different signaling pathways. Loss, or reduced expression, of CYLD is observed in different types of human cancer, such as hepatocellular carcinoma (HCC). However, the molecular mechanism by which CYLD affects cancerogenesis has to date not been unveiled. The aim of the present study was to examine how CYLD regulates cellular functions and signaling pathways during hepatocancerogenesis. We found that mice lacking CYLD were highly susceptible to chemically induced liver cancer. The mechanism behind proved to be an elevated proliferation rate of hepatocytes, owing to sustained c-Jun N-terminal kinase 1 (JNK1)-mediated signaling via ubiquitination of TNF receptor-associated factor 2 and expression of c-MYC. Overexpression of wild-type CYLD in HCC cell lines prevented cell proliferation, without affecting apoptosis, adhesion and migration. A combined immunohistochemical and tissue microarray analysis of 81 human HCC tissues revealed that CYLD expression is negatively correlated with expression of proliferation markers Ki-67 and c-MYC. To conclude, we found that downregulation of CYLD induces tumor cell proliferation, consequently contributing to the aggressive growth of HCC. Our findings suggest that CYLD holds potential to serve as a marker for HCC progression, and its link to c-MYC via JNK1 may provide the foundation for new therapeutic strategies for HCC patients

Serum response factor controls CYLD expression via MAPK signaling pathway.

Tumor suppressor gene CYLD is a deubiquitinating enzyme which negatively regulates various signaling pathways by removing the lysine 63-linked polyubiquitin chains from several specific substrates. Loss of CYLD in different types of tumors leads to either cell survival or proliferation. In this study we demonstrate that lack of CYLD expression in CYLD-/- MEFs increases proliferation rate of these cells compared to CYLD+/+ in a serum concentration dependent manner without affecting cell survival. The reduced proliferation rate in CYLD+/+ in the presence of serum was due to the binding of serum response factor (SRF) to the serum response element identified in the CYLD promoter for the up-regulation of CYLD levels. The serum regulated recruitment of SRF to the CYLD promoter was dependent on p38 mitogen-activated protein kinase (MAPK) activity. Elimination of SRF by siRNA or inhibition of p38 MAPK reduced the expression level of CYLD and increased cell proliferation. These results show that SRF acts as a positive regulator of CYLD expression, which in turn reduces the mitogenic activation of serum for aberrant proliferation of MEF cells

Expression of Id proteins is regulated by the Bcl-3 proto-oncogene in prostate cancer.

B-cell leukemia 3 (Bcl-3) is a member of the inhibitor of κB family, which regulates a wide range of biological processes by functioning as a transcriptional activator or as a repressor of target genes. As high levels of Bcl-3 expression and activation have been detected in different types of human cancer, Bcl-3 has been labeled a proto-oncogene. Our study uncovered a markedly upregulated Bcl-3 expression in human prostate cancer (PCa), where inflammatory cell infiltration was observed. Elevated Bcl-3 expression in PCa was dependent on the proinflammatory cytokine interleukin-6-mediated STAT3 activation. Microarray analyses, using Bcl-3 knockdown in PCa cells, identified the inhibitor of DNA-binding (Id) family of helix-loop-helix proteins as potential Bcl-3-regulated genes. Bcl-3 knockdown reduced the abundance of Id-1 and Id-2 proteins and boosted PCa cells to be more receptive to undergoing apoptosis following treatment with anticancer drug. Our data imply that inactivation of Bcl-3 may lead to sensitization of cancer cells to chemotherapeutic drug-induced apoptosis, thus suggesting a potential therapeutic strategy in PCa treatment.Oncogene advance online publication, 14 May 2012; doi:10.1038/onc.2012.175

Recruitment of SRF to the promoter of CYLD induced by serum.

<p>(<b>A</b>). Location of two serum response elements identified at CYLD promoter. (<b>B</b>). Lysates from CYLD+/+ MEFs were examined by ChIP assay using an anti- SRF (H-300, Santa Cruz) and a PCR primer pair corresponding to the promoter of the CYLD gene (270 bp). Immunoprecipitation (ChIP) using specific antibodies; IgG: IP using negative control rabbit immunoglobulin; Input: 10% of the cell lysate used for the IP is shown. (<b>C</b>). Reporter assays revealing inducible CYLD promoter (-1297 to -1) activity in MEF cells; (p1194SRE), whereas mutation of the consensus SRF binding site (p1194ΔSRE) led to reduced promoter activity. (<b>D</b>). Western blot analysis of SRF and tubulin expression in the presence of 10% FCS for 48 hours (control) or cells incubated in the absence of serum over a period of 24–72 hours (0% FBS). (<b>E</b>). Western blot analysis of SRF, CYLD and tubulin expression in scrambled siRNA control transfected cells or cells transfected with the SRF siRNA nucleotides. (<b>F</b>). Western blot analysis of SRF, CYLD and tubulin expression in cells transiently transfected with the SRF siRNA nucleotides after serum withdrawal (24 hours) and re-addition of FCS over a period of 24–72 hours. Control cells are transiently transfected with the scramble siRNA nucleotides after re-addition of FCS for 48 hours. (<b>G</b>). Cell counting of CYLD+/+ MEFs transiently transfected with scramble or SRF siRNA nucleotides in the presence of 10% FCS over period of 24–48 hours.</p

Serum concentration dependent proliferation of CYLD+/+ and CYLD−/− primary MEFs.

<p>(<b>A–D</b>). Measurement of the growth rate of MEFs by cell counting after serum withdrawal (24 hours) followed by re-addition of FCS: 0, 1, 5 and 10% over a period of 24–96 hours. (<b>E</b>). Analysis of the levels of cyclin D1 and tubulin in primary CYLD+/+ compared to CYLD −/− MEFs in the absence (0%) or presence of 10% FCS for 48 hours. (<b>F</b>). Confocal plane of Bcl-3 (red) and DAPI (blue) in CYLD+/+ (upper) and CYLD−/− (lower) MEFs after serum withdrawal (24 hours) followed by re-addition of 10% FCS for 24 hours. (<b>G–J</b>). Measurement of the growth rate of MEFs by cell counting treated with serum free for 24 hours before re-addition of 1% FCS together with EGF (100 ng/ml), LPA (1 µM), TPA (100 nM) and or TNF-α (100 ng/ml) over a period of 24–96 hours.</p

Effects of p38MAPK inhibition to serum mediated CYLD expression.

<p>(<b>A–C</b>). Western blot analysis of CYLD and tubulin expression in serum starved WT MEFs (24 hours) and readdition of 10% FCS for 24 hours in the absence or presence of SB-203580 (500 nM), PD 98058 (25 µM), UO126 (20 µM), SP600125 (20 µM) or solvent (DMSO). (<b>D</b>). Western blot analysis of active and total p38MAPK in WT MEFs in the absence (24 hours) or readdition of 10% FCS for 30, 60 or 120 minutes. (<b>E</b>). Lysates from CYLD+/+ MEFs were examined by ChIP assay in the absence or presence of solvent (DMSO) or SB203580 (500 nM for 1 hour). Cells were serum starved for 24 hours and one hour before readdition of serum, SB203580 (500 nM for 1 hour) or solvent (DMSO) was added to the cell culture. Cell lysates were chromatin immunoprecipitated using anti-SRF antibody and a PCR primer pair corresponding to the promoter of the CYLD gene was used. Immunoprecipitation (ChIP) using specific antibodies; IgG: IP using negative control rabbit immunoglobulin; Input: 10% of the cell lysate used for the IP is shown. (F). Lysates from CYLD+/+ MEFs were examined by ChIP assay using anti-pSRF or anti- pELK1 and a PCR primer pair corresponding to the promoter of the CYLD gene (270 bp). Immunoprecipitation (ChIP) using specific antibodies; IgG: IP using negative control rabbit immunoglobulin; Input: 10% of the cell lysate used for the IP is shown. (<b>G</b>). Cell counting of CYLD+/+ MEFs in serum starved WT MEFs (24 hours) before re-addition of 10% FCS for 48 hours in the absence or presence of solvent (DMSO) or SB203580 (500 nM for 1 hour).</p

Effects of TNF-α mediated apoptosis in CYLD +/+ and CYLD−/− MEFs.

<p>(<b>A</b>). The apoptotic cells were analyzed by detecting the gradual degradation of internucleosomal DNA with the DNA binding fluorescent dye propidium iodide in WT and CYLD-KO MEF cells in the absence or presence of 10% FCS over a period of 24–96 hours. (<b>B</b>). The apoptotic cells were analyzed by detecting the gradual degradation of internucleosomal DNA with the DNA binding fluorescent dye propidium iodide in WT and CYLD-KO MEF cells treated with TNF-α (100 ng/ml) for 12 hours, cyclohexamide (10 µg/ml) for 12 hours or a combination of TNF-α and cyclohexamide (100 ng/ml respective 10 µg/ml) for 12 hours.</p

CYLD gene expression is regulated at the transcriptional level by serum.

<p>(<b>A</b>). Analysis of the levels of CYLD, cyclin D1 and tubulin in primary CYLD+/+ MEFs in 10% FCS (control) or serum deprived cells over a period of 24–72 hours. (<b>B–C</b>). Analysis of the levels of CYLD and tubulin in primary CYLD+/+ MEFs in 10% FCS (control) or serum deprived cells for 24 hours (0%) or re-addition of FCS (10%) to the cells over a period of 24–72 hours. (<b>D</b>). Analysis of the levels of CYLD and tubulin in primary CYLD+/+ MEFs in 10% FCS over a period of 0.5–24 hours. (<b>E</b>). CYLD gene expression by using qRT-PCR upon withdrawal (0% FCS) or re-addition of serum to the cell cultures for 1 or 4 hours. (<b>F</b>). Analysis of the levels of CYLD and tubulin in primary CYLD+/+ MEFs after serum deprivation 24 hours before re-addition of 10% FCS (control) or 10% FCS together with 0.5–1.0 µg/ml actinomycin D for 12 hours.</p