Ubiquitin Carboxyl-Terminal Hydrolase CYLD
CYLD is a deubiquitination enzyme that is responsible for cleaving polyubiquitin chains from multiple target proteins and for regulating downstream signaling pathways. Originally, CYLD gene was discovered through positional cloning from chromosome 16q. The germ-line mutations in CYLD gene were identified in patients suffering from familial cylindromatosis, which is a skin cancer disease (Biggs et al. 1995; Bignell et al. 2000). Both copies of the gene must be inactivated to cause cylindromatosis, which is usually an inherited mutation in one copy and loss of the second copy during postnatal life. CYLD consists of 956 amino acids, 20 exons, and it is scattered over 56 kb of genomic DNA located at chromosome 16q12–13 (Biggs et al. 1995; Bignell et al. 2000). Based on the structure and function studies, CYLD has been classified as a member of deubiquitination enzymes (DUBs). The function of DUBs in the cell is to remove single or multiple ubiquitin chains from specific substrates. In principle, the fate of the protein depends on the type of polyubiquitination. For example, polyubiquitination through Lys-48-linkage degrades the substrate by means of the mechanism of proteosome, whereas Lys-63-linked ubiquitin chains do not lead to proteasome-mediated degradation, but can have a variety of other consequences for protein function (Pickart 2001; Weissman 2001). CYLD is an Lys-63 and a linear ubiquitin-specific DUB that is unable to hydrolyze Lys-48-ubiquitin chains (Komander et al. 2008).
CYLD Functions as a Deubiquitination Enzyme
CYLD protein contains three cytoskeletal-associated protein-glycine-conserved (CAP-Gly) domains. The CAP-Gly domains of CYLD can function both as a scaffold and for the subcellular localization of CYLD (Massoumi 2010). The C-terminal part of CYLD encodes the ubiquitin carboxyl-terminal hydrolase (UCH) domain. Mutations of the UCH domain result in catalytically inactive CYLD, and animals lacking the UCH domain of CYLD die shortly after birth (Trompouki et al. 2009). Removal of ubiquitin chains from specific substrates by CYLD negatively regulates multiple signaling pathways, including the Nuclear Factor of Kappa Light Polypeptide Gene Enhancer in B-Cells (NF-кB), C-Jun N-Terminal Kinase (JNK), Wingless-Type MMTV Integration Site Family (Wnt), Notch, and B-Cell CLL/Lymphoma 3 (Bcl-3) (Massoumi 2010). TNF Receptor-Associated Factor 2 (TRAF2) and TNF Receptor-Associated Factor 6 (TRAF6) were the first identified CYLD substrates. CYLD-mediated removal of K63-linked polyubiquitin chains from these proteins inhibits the NF-kB signaling cascade (Massoumi 2010). Bcl-3 is another substrate for CYLD, in which deubiquitination of Bcl-3 by CYLD leads to reduced expression of cyclin D1, and this prevents the hyperproliferation of cancer cells (Massoumi et al. 2006). So far, more than 20 CYLD-specific substrates have been discovered in different cell types. A few examples of CYLD substrates include TNF Receptor-Associated Factor 7 (TRAF7), TRAF-Interacting Protein (TRIP), Transforming Growth Factor-Beta-Activated Kinase 1 (TAK1), NF-Kappa-B Essential Modifier (NEMO), Lymphocyte Cell-Specific Protein-Tyrosine Kinase (LCK), Receptor-Interacting Protein 1 (RIP1), Retinoic Acid-Inducible Gene (RIG), and Polo-Like Kinase 1 (PLK1) (Massoumi 2010). Regulation of downstream signaling by CYLD can also be achieved, independent of its deubiquitination activity. Two examples are the direct interaction between CYLD and HDAC6 or HDAC7 (Hellerbrand and Massoumi 2016). The direct interaction between CYLD and HDAC6 elevates the total levels of tubulin acetylation and facilitates the relocalization of CYLD from the cytoplasm to the perinuclear region in keratinocytes (Wickstrom et al. 2010). In the perinuclear region, CYLD associates with its substrate Bcl-3 and prevents the nuclear localization of Bcl-3 (Massoumi et al. 2006; Wickstrom et al. 2010). Regulation of cell death by CYLD is dependent on the NF-kB signaling pathway. CYLD by a direct interaction to NEMO after TNF stimuli removes Lys-63-linked polyubiquitin chains from TRAF2 or TRAF6, thereby leading the cell to apoptosis. In another similar mechanism, CYLD can interact directly with TRIP for downregulation of NF-kB activity after TNF stimulation. CYLD can also function as a negative regulator for the Jun N-terminal kinase (JNK) signaling pathway. CYLD knockdown was shown to increase both TRAF2 ubiquitination and JNK activation, further enhancing cell survival (Massoumi 2010).
CYLD and Cancer-Associated Pathways
In a tissue-microarray analysis from melanoma, an inverse correlation between Snail and CYLD expression was observed. Snail recruitment to the promoter of CYLD and repression of CYLD facilitate the proto-oncogene Bcl-3 to translocate into the nucleus and activate Cyclin D1 and N-cadherin. Upregulation of these two genes results in proliferation and invasion of melanoma cells (Massoumi et al. 2009). In basal cell carcinoma, CYLD expression is downregulated via sustained activation of the GLI1 signaling pathway. Inhibition of this pathway initiates the expression of CYLD and causes reduced cell proliferation, suggesting that GLI1-mediated suppression of CYLD affects basal cell carcinoma progression (Kuphal et al. 2011). CYLD-deficient mice are highly sensitive to chemically induced skin tumors by developing fast-growing skin papillomas. This phenotype is explained by the TPA or UV light-mediated Bcl-3 that is ubiquitinated through Lys-63-chains in the cytoplasm and its recruitment to the promoter of Cyclin D1, leading to an elevated proliferation rate of cells (Massoumi et al. 2006). β-catenin is another protein that was recently observed to be elevated in the nuclei of skin cancer cylindroma cells. Wnt-mediated, β-catenin nuclear translocation in cylindroma cells enhances the proliferation potential of the cells. In general, canonical Wnt/β-catenin signaling leads to recruitment of Dishevelled (Dvl) and Axin to the Frizzled/LRP6 coreceptor complex, which further causes activation and nuclear translocation of B-catenin. Downregulation of CYLD in this pathway promoted β-catenin accumulation in the nucleus through Lys63-ubiquitination of Dvl (Tauriello et al. 2010).
Cervical cancer is mainly caused by human papilloma virus infection, and it is the second most common cancer among women worldwide. Cervical adenocarcinoma is strongly associated with human papilloma virus type 18 infections. Analysis from two established glassy cell carcinomas arising from the uterine cervix, which contains human papilloma virus type 18 DNA, showed alterations in the CYLD gene copy number using conventional comparative genomic hybridization (CGH) coupled with array-based CGH. The alteration of CYLD gene expression is elucidated by a loss of the CYLD gene in both of these cervical cancer cell lines (Massoumi 2011).
Downregulation of CYLD mRNA in Crohn disease and ulcerative colitis has been shown to predispose patients with these diseases to develop colon carcinoma (Massoumi 2011). However, until now, the mechanism for this has not been discovered. CYLD knockout mice are also susceptible to induced colonic inflammation by using colitis-associated cancer. These knockout animals develop more severe colonic inflammation and exhibit a greater induction of colonic tumors than controls. The chronic inflammation that was studied by isolating macrophages displayed increased TRAF2 ubiquitination and JNK activity compared with wild-type mice (Zhang et al. 2006).
Hepatocellular carcinoma is the seventh most common cancer and the third leading cause of cancer-related death worldwide. CYLD mRNA expression is significantly downregulated in human hepatocellular carcinoma, and restoration of CYLD expression in human hepatocellular carcinoma cell lines reduces their proliferation rate via inhibition of the c-MYC promoter. In addition, overexpression of CYLD in hepatocellular carcinoma cell lines augmented the antitumoral effect of TNF-Related Apoptosis-Inducing Ligand (TRAIL). This effect was mediated by the direct binding of CYLD to Inhibitor of Kappa Light Polypeptide Gene Enhancer in B-Cells (IKK) in TRAIL-mediated NF-кB signaling and deubiquitination of TRAF2 by CYLD. Downregulation of CYLD in hepatocellular carcinoma can be explained by different micro RNAs that target CYLD-RNA, including MiR-362-5p, miR-526a, and miR-501-5p (Hellerbrand and Massoumi 2016). In animals, an injection with the chemical carcinogen diethylnitrosamine, which is an established experimental hepatocellular carcinoma model, leads to faster development of hepatocellular carcinoma in CYLD-knockout mice compared with wild-type mice due to the elevated cell proliferation. More precisely, elevated cell proliferation was explained by sustained ubiquitination of TRAF2, leading to JNK1-mediated promoter activation of Activator Protein 1 (AP-1) and the expression of cell cycle-regulated genes, including Cyclin D1 and c-MYC. Besides proliferation, hepatocellular carcinomas from CYLD exon 9-deleted mice show reactivation of oncofetal hepatic genes and the expression of cancer stem cells, which suggest that the tumors developing in CYLD-deficient animals are highly aggressive and have a poor prognosis (Hellerbrand and Massoumi 2016).
Multiple myeloma is caused by the infiltration of malignant plasma cells, and it is characterized by a distinctive combination of gains, losses, or nonrandom chromosomal translocations. The chromosomal rearrangements that affect the NF-кB p100/p52 locus at chromosomal region 10q24 have been associated with multiple myeloma. Large-scale screening of multiple myeloma patients demonstrated deletion at chromosomes 14q32, 11q22, and 16q12. In these regions, target genes such as TNF Receptor-Associated Factor 3 (TRAF3), Cellular Inhibitor of Apoptosis (cIAP1 and cIAP2), Caspase Recruitment Domain family, member 15 (CARD15), and CYLD are located. In another similar study focusing on multiple myeloma, the expression of CYLD mRNA was below the median, and biallelic loss of the CYLD locus in all samples could be detected. These studies found that genetic abnormalities and constitutive activation in the NF-кB pathway by loss of TRAF2, TRAF3, cIAP1/2, and CYLD are strongly associated with the development of multiple myeloma (Massoumi 2011).
Lung cancer is generally divided into small cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). NSCLCs are further characterized into three major types: squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. It has been shown that CYLD levels are frequently downregulated in NSCLC cell lines (Zhong et al. 2007). High-throughput gene expression microarray analysis combined with pharmacologic inhibition of histone deacetylation in NSCLC cell lines upregulated the levels of CYLD. Furthermore, overexpression of the full-length but not the catalytic inactive mutant of CYLD in these cells significantly reduces cell growth. This result suggests that CYLD acts as a tumor suppressor gene for the progression of NSCLC and that the deubiquitin activity of CYLD is important for this mechanism (Zhong et al. 2007).
It has previously been demonstrated that TNF induces serine phosphorylation of CYLD by IKKα and IKKβ. The consequence of this phosphorylation is that CYLD dissociates from the TRAF2/NEMO complex after such phosphorylation (Massoumi 2010). Since phosphorylation-defective mutant CYLD protein decreases the TNF-induced ubiquitination of TRAF2, it has been hypothesized that phosphorylation of CYLD serves as a mechanism for allowing the accumulation of ubiquitin-conjugated TRAF2. In addition to IKKα and IKKβ, serine 418 of CYLD is also phosphorylated by IKKε (Hutti et al. 2009). Notably, IKKε is a breast cancer oncogene that is amplified and overexpressed in more than 30% of breast tumors analyzed so far (Hutti et al. 2009). Mutation of serine 418 in CYLD reduced anchorage-independent growth in IKKε-transformed cells. This finding could define the potential mechanism by which IKKε induces tumorigenesis through inactivation of CYLD (Hutti et al. 2009).
Renal Cell Carcinoma
Renal cell carcinoma is the most common type of kidney cancer, and it accounts for about 85% of cancers arising from the kidney. Renal cell carcinoma usually develops in the tubules of the kidney when normal cells undergo a transformation, and due to the malignancy, the tumors invade the tissues of neighboring organs (Strobel et al. 2002). Spiradenocylindroma is the tumor arising in the wall of a renal cyst. This type of tumor is similar to cylindroma exhibiting small tumor cell islands that are arranged in a jigsaw pattern (Strobel et al. 2002). Genomic hybridization showed loss of the long arm of chromosome 16, suggesting that renal neoplasms occurs due to the loss of heterozygosity at 16q12–13 of the CYLD gene (Strobel et al. 2002). This study also concluded that CYLD mutation has an important role in the oncogenesis of tumors with cylindromatous features (Strobel et al. 2002).
Prostate cancer is a malignant tumor that consists of cells from the prostate gland, which can spread locally into the surrounding tissues. Two predominant epigenetic mechanisms are DNA methylation and histone modification, which are actively involved in regulating gene transcription (Kikuno et al. 2008). It is well established that DNA methylation can be reversed by methylation inhibitors 5-Aza-2′-Deoxycytidine. Besides acting as a tyrosine kinases inhibitor, genistein can regulate gene activity by modulating epigenetic events such as histone acetylation and DNA methylation. Treatment of prostate cancer cells with genistein could modulate histone H3-lysine 9 (H3-K9) methylation and deacetylation of the heterochromatic domains at the promoter of CYLD (Kikuno et al. 2008). This chromatin modulation was shown to increase the levels of CYLD in the cancer cells, which was further confirmed by using 5-Aza-2′-Deoxycytidine to verify the epigenetic events caused by genistein (Kikuno et al. 2008).
Salivary Gland Tumors
Analysis of CYLD gene locus using microsatellite markers identified loss of heterozygosity in most of the dermal and salivary gland dermal analog tumors. Treatment of human salivary gland tumor cells with TNF led to an increase in the levels of CYLD expression. TNF also caused a rapid accumulation of NF-кB into the nucleus. Knockdown of CYLD, however, showed elevated NF-кB activation after TNF stimulation, indicating that CYLD negatively regulates NF-kB activation after TNF stimulation in human salivary gland tumor cells (Massoumi 2011).
Head and Neck Cancer
The causes for most head and neck cancers are environmental and lifestyle risk factors, including UV light and certain strains of viruses, such as human papilloma virus. Under hypoxia conditions in human papilloma virus-positive head and neck cancers, CYLD level is undetectable (An et al. 2008). Reduced expression of CYLD is caused by human papilloma virus-mediated Lys-48-ubiquitination of CYLD. This effect was achieved by human papilloma virus-encoded E6 protein, which functions as part of an unidentified E3 ubiquitin ligase complex, and it did not involve the well-known E6-AP ubiquitin ligase activation (An et al. 2008). The inhibition of proteasome function by using MG132 rescued CYLD degradation. The E6-mediated downregulation of CYLD, however, was intensified under hypoxic conditions (An et al. 2008).
The human genome encodes a large number of putative ubiquitin ligases and deubiquitination enzymes. Evidence indicates that most of these enzymes regulate a limited number of proteins and pathways. It has also been established that an alteration in ubiquitin and the ubiquitin system has direct and indirect roles in the genesis of different types of tumor. CYLD is a deubiquitination enzyme that can cleave the lysine 63-linked polyubiquitin chains from target proteins and regulate cell survival or cell proliferation. Since loss of CYLD expression can be observed in different types of human cancer, it is now well established that CYLD acts as a tumor suppressor gene. Depending on the type of cancer or in vivo model system, CYLD can interfere with ubiquitin-mediated signaling pathways, including NF-kB, wnt, JNK, and p38MAPK, by removing ubiquitin chains from substrates such as TRAF2, TRAF6, TAK1, Dvl, and Bcl-3. The interruption of these signaling pathways can either promote apoptosis or reduce proliferation depending on the tissue-cell type. Besides its loss of function in human tumors by gene deletion or mutation, CYLD expression can be downregulated at the RNA level through transcriptional regulation or at the protein level through posttranslational modifications. A detailed understanding of the molecular and cellular function of CYLD and its target proteins in various pathways and tissues will likely contribute to effective medical therapies in the treatment of cancer.
- Strobel P, Zettl A, Ren Z, Starostik P, Riedmiller H, Storkel S, et al. Spiradenocylindroma of the kidney: clinical and genetic findings suggesting a role of somatic mutation of the CYLD1 gene in the oncogenesis of an unusual renal neoplasm. Am J Surg Pathol. 2002;26(1):119–24.PubMedCrossRefGoogle Scholar