The expression of tumor suppressor CYLD in specific neoplasias

The tumor suppressor CYLD is a deubiquitinating enzyme that acts as a negativeregulator of several signaling pathways including NF-kappa-B and MAPK activationpathways, by affecting the function of important mediators. CYLD regulates severalcellular processes such as immune responses, inflammation, cell survival,proliferation and differentiation. The tumor suppressor function of CYLD seems to becell-type specific and it is important to delineate its involvement in the homeostasis ofspecific tissues in order to understand and exploit its role in oncogenesis . In thisreview, we summarize existing data on the expression and alterations of CYLD incertain types of lung and colorectal neoplasias

The Tumor Suppressor CYLD Inhibits Mammary Epithelial to Mesenchymal Transition by the Coordinated Inhibition of YAP/TAZ and TGFβ Signaling

Downregulation of the cylindromatosis (CYLD) tumor suppressor has been associated with breast cancer development and progression. Here, we report a critical role for CYLD in maintaining the phenotype of mammary epithelial cells in vitro and in vivo. CYLD downregulation or inactivation induced an epithelial to mesenchymal transition of mammary epithelial cells that was dependent on the concomitant activation of the transcription factors Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) and transforming growth factor beta (TGF�)signaling. CYLD inactivation enhanced the nuclear localization of YAP/TAZ and the phosphorylation of Small Mothers Against Decapentaplegic (SMAD)2/3 proteins in confluent cell culture conditions. Consistent with these findings were the hyperplastic alterations of CYLD-deficient mouse mammary epithelia, which were associated with enhanced nuclear expression of the YAP/TAZ transcription factors. Furthermore, in human breast cancer samples, downregulation of CYLD expression correlates with enhanced YAP/TAZ-regulated target gene expression. Our results identify CYLD as a critical regulator of a signaling node that prevents the coordinated activation of YAP/TAZ and the TGF� pathway in mammary epithelial cells, in order to maintain their phenotypic identity and homeostasis. Consequently, they provide a novel conceptual framework that supports and explains a causal implication of deficient CYLD expression in aggressive human breast cancers

Induction of Apoptosis by Rewiring the Signal Transduction of Epstein-Barr Virus Oncoprotein LMP1 toward Caspase Activation

The Epstein-Barr virus latent membrane protein 1 (LMP1) is an oncoprotein which mimics activated tumor necrosis factor receptor family members. Here we demonstrate the principle that an inducible association of the LMP1 cytoplasmic carboxyl terminus with caspase-8 by a heterodimerizing agent causes apoptosis. This process depends on the catalytic activity of caspase-8 and the ability of LMP1 to oligomerize constitutively at the plasma membrane. Our data indicate that chemical inducers of the association of the LMP1 carboxyl terminus with caspase-8 can kill LMP1-expressing cells selectively. Such compounds could be used as chemotherapeutic agents for LMP1-associated malignancies

Functional specificity of two hormone response elements present on the human apoA-II promoter that bind retinoid X receptor alpha/thyroid receptor beta heterodimers for retinoids and thyroids: synergistic interactions between thyroid receptor beta and upstream stimulatory factor 2a.

DNA binding and mutagenesis in vitro established that the -67/-55 region of the apoA-II (apolipoprotein A-II) promoter contains a thyroid HRE (hormone response element), which strongly binds RXRalpha (retinoid X receptor alpha)/T(3)Rbeta (thyroid receptor beta) heterodimers and weakly T(3)Rbeta homodimers, but does not bind other homo- or heterodimers of RXRalpha or orphan nuclear receptors. Transactivation was abolished by point mutations in the thyroid HRE. In co-transfection experiments of HEK-293 (human embryonic kidney 293) cells, the -911/+29 human apoA-II promoter was transactivated strongly by RXRalpha/T(3)Rbeta heterodimers in the presence of RA (9- cis retinoic acid) or T(3) (tri-iodothyronine). Homopolymeric promoters containing either three copies of the -73/-40 (element AIIAB) or four copies of the -738/-712 (element AIIJ) apoA-II promoter could be transactivated by RXRalpha/T(3)Rbeta heterodimers in COS-7 cells only in the presence of T(3) or RA respectively. RXRalpha/T(3)Rbeta heterodimers and USF2a (upstream stimulatory factor 2a) synergistically transactivated the -911/+29 apoA-II promoter in the presence of T(3). USF2a also enhanced the activity of a GAL4-T(3)Rbeta fusion protein in the presence of T(3) and suppressed the activity of a GAL4-RXRalpha fusion protein in the presence of RA. These findings suggest a functional specificity of the two HREs of the apoA-II promoter for retinoids and thyroids, which is modulated by synergistic or antagonistic interactions between RXRalpha/T(3)Rbeta heterodimers and the ubiquitous transcription factor USF2a

Characterization of tumor suppressor CYLD expression in clear cell renal cell carcinoma

Cyld is a tumor suppressor gene that has attracted particular interest recently, due to its involvement in many types of neo-plasia, including head and neck squamous cell carcinoma, multiple myeloma, melanoma, hepatocellular carcinoma and colon cancer. Cyld encodes a predominantly cytoplasmic protein (CYLD), which is a deubiquitinating enzyme that regulates primarily cell survival and cell division pathways. Studies on the molecular function of CYLD have shown that it can modulate NF-κΒ, JNK, p38, TGF-beta, Wnt and Notch signaling. The present study aimed to investigate whether CYLD expression can be correlated with the development of clear cell renal cell carcinoma (ccRCC). Towards this goal, immunohistochemistry and Real Time PCR experiments were performed in order to analyze CYLD expression in clear cell renal cell carcinoma and matched normal tissue specimens. In addition, a clonogenic assay was performed to analyze the effect of CYLD wt and a catalytically inactive mutant CYLD on the growth of human embryonic kidney cells. The results of the present study show that CYLD is downregulated at protein and mRNA level in patients with ccRCC. This is further corroborated by the results of a clonogenic assay, which showed a deubiquitinating activity-dependent growth inhibitory role of CYLD in human embryonic kidney cells. Our results support the notion that CYLD can have a tumor suppressing role, at least in a subset of clear cell renal cell carcinoma, suggesting that it can be incorporated in the future in the development of targeted therapeutic approaches

CeCYLD can inhibit the activation of AP1.

<p>HEK 293T cells (2x10⁵ cells per well in 12-well plates) were cotransfected with a AP1-dependent luciferase reporter plasmid (7xAP1), a β-galactosidase expression plasmid for transfection efficiency normalization (pGKβgal), and 0.1μg, 1μg and 0.3μg of plasmids expressing HsCYLD, CeCYLD and CeCYLDC774S respectively. After 18h, the cells were harvested and lysed in luciferase lysis buffer (Promega). The lysates were assayed for luciferase and β-galactosidase activities. (A) Values are shown as the mean +/- standard error of relative luciferase activity from five independent experiments. The values that were compared statistically are indicated by horizontal lines. (B) Representative immunoblot showing the expression levels of transfected proteins and β-actin. *p< 0.05, **p<0.01.</p

CeCyld inhibits TNF-induced IL8 expression.

<p>HeLa cells (10⁵ cells per well in 12-well plates) were transfected with 0.1μg, 1.4μg and 0.4μg of plasmids expressing HsCYLD, CeCYLD and CeCYLDC774S respectively. After 24h, each transfected cell population was split equally in two new wells and 24 h later, half of each transfected cell population was left untreated or treated with TNFα (40ng/ml, 1h). The cells were then harvested and from each cell population RNA and total protein was extracted. The overexpressed FLAG-tagged proteins were immuno-precipitated and the products of immunoprecipitation were used to assess the expression levels of each protein by immuoblotting. (A) Values are shown as the mean +/- standard error of relative IL8 expression induction from four independent experiments of RT-PCR. The values that were compared statistically are indicated by horizontal lines. (B) Immunoblot showing the expression levels of immuno-precipitated proteins with anti-FLAG antibody (IP:αFLAG) along with the immunoglobulin heavy chains (Ig-HC) and β-actin in the whole cell lysate (WCL). *p< 0.05, **p<0.01 ***p<0.001.</p

Functional analysis of the <i>C</i>. <i>elegans cyld-1</i> gene reveals extensive similarity with its human homolog

<div><p>The human cylindromatosis tumor suppressor (HsCyld) has attracted extensive attention due to its association with the development of multiple types of cancer. HsCyld encodes a deubiquitinating enzyme (HsCYLD) with a broad range of functions that include the regulation of several cell growth, differentiation and death pathways. HsCyld is an evolutionarily conserved gene. Homologs of HsCyld have been identified in simple model organisms such as <i>Drosophila melanogaster</i> and <i>Caenorhabditis elegans</i> (C. <i>elegans</i>) which offer extensive possibilities for functional analyses. In the present report we have investigated and compared the functional properties of HsCYLD and its <i>C</i>. <i>elegans</i> homolog (CeCYLD). As expected from the mammalian CYLD expression pattern, the CeCyld promoter is active in multiple tissues with certain gastrointestinal epithelia and neuronal cells showing the most prominent activity. CeCYLD is a functional deubiquitinating enzyme with similar specificity to HsCYLD towards K63- and M1-linked polyubiquiting chains. CeCYLD was capable of suppressing the TRAF2-mediated activation of NF-kappaB and AP1 similarly to HsCYLD. Finally, CeCYLD could suppress the induction of TNF-dependent gene expression in mammalian cells similarly to HsCYLD. Our results demonstrate extensively overlapping functions between the HsCYLD and CeCYLD, which establish the <i>C</i>. <i>elegans</i> protein as a valuable model for the elucidation of the complex activity of the human tumor suppressor protein.</p></div