No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Cyld Inhibits Tumor Cell Proliferation by Blocking Bcl-3-Dependent NF-??B Signaling
Abstract
Mutations in the CYLD gene cause tumors of hair-follicle keratinocytes. The CYLD gene encodes a deubiquitinase that removes lysine 63-linked ubiquitin chains from TRAF2 and inhibits p65/p50 NF-kappaB activation. Here we show that mice lacking Cyld are highly susceptible to chemically induced skin tumors. Cyld-/- tumors and keratinocytes treated with 12-O-tetradecanoylphorbol-13 acetate (TPA) or UV light are hyperproliferative and have elevated cyclin D1 levels. The cyclin D1 elevation is caused not by increased p65/p50 action but rather by increased nuclear activity of Bcl-3-associated NF-kappaB p50 and p52. In Cyld+/+ keratinocytes, TPA or UV light triggers the translocation of Cyld from the cytoplasm to the perinuclear region, where Cyld binds and deubiquitinates Bcl-3, thereby preventing nuclear accumulation of Bcl-3 and p50/Bcl-3- or p52/Bcl-3-dependent proliferation. These data indicate that, depending on the external signals, Cyld can negatively regulate different NF-kappaB pathways; inactivation of TRAF2 controls survival and inflammation, while inhibition of Bcl-3 controls proliferation and tumor growth.
... Bcl-3 was originally identified as a gene involved in the recurring chromosomal translocation t (14;19), which is found in patients with chronic lymphocytic leukemia [14]. Since the discovery that Bcl-3 is an oncogene involved in leukemia, other studies have demonstrated the oncogenic function of Bcl-3 in solid tumors originating from breast [15][16][17], liver [18], prostate [19], colon [20][21][22][23], cylindromatosis [24], basal cell carcinoma [25], squamous cell carcinoma [26], and nasopharyngeal carcinoma [27]. In transgenic animals, sustained Bcl-3 nuclear translocation leads to the development of skin papilloma [24] or squamous cell carcinoma [26] by augmentation of p50/p52-related NF-κB binding in mouse keratinocytes. ...
... Since the discovery that Bcl-3 is an oncogene involved in leukemia, other studies have demonstrated the oncogenic function of Bcl-3 in solid tumors originating from breast [15][16][17], liver [18], prostate [19], colon [20][21][22][23], cylindromatosis [24], basal cell carcinoma [25], squamous cell carcinoma [26], and nasopharyngeal carcinoma [27]. In transgenic animals, sustained Bcl-3 nuclear translocation leads to the development of skin papilloma [24] or squamous cell carcinoma [26] by augmentation of p50/p52-related NF-κB binding in mouse keratinocytes. It has also been shown that p53 decreases the expression of Bcl-3, which changes the ratio of p52/Bcl-3 to p52/ HDAC complexes on the cyclin D1 promoter, thus inhibiting cyclin D1 expression [28]. ...
... Cell Line authentication has been performed for all the cell lines presented in this study. Immortalized keratinocytes were cultured and transfected as described previously [24]. BCL3ANT was dissolved in DMSO at different concentrations and stored at -20 °C until use. ...
Molecular targeted therapy using a drug that suppresses the growth and spread of cancer cells via inhibition of a specific protein is a foundation of precision medicine and treatment. High expression of the proto-oncogene Bcl-3 promotes the proliferation and metastasis of cancer cells originating from tissues such as the colon, prostate, breast, and skin. The development of novel drugs targeting Bcl-3 alone or in combination with other therapies can cure these patients or prolong their survival. As a proof of concept, in the present study, we focused on metastatic melanoma as a model system. High-throughput screening and in vitro experiments identified BCL3ANT as a lead molecule that could interfere with Bcl-3-mediated cyclin D1 expression and cell proliferation and migration in melanoma. In experimental animal models of melanoma, it was demonstrated that the use of a Bcl-3 inhibitor can influence the survival of melanoma cells. Since there are no other inhibitors against Bcl-3 in the clinical pipeline for cancer treatment, this presents a unique opportunity to develop a highly specific drug against malignant melanoma to meet an urgent clinical need.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12885-023-11663-y.
... Deletion of OTULIN in mice leads to embryonic lethality due to increased TNFR1 and RIPK1 mediated cell death, presumably due to the resulting inactivation of LUBAC, and aberrant angiogenesis (Heger et al., 2018;Rivkin et al., 2013). Perturbations of CYLD functions are associated with tumorigenesis of certain cancer types as well as increased NF-κB activation (Massoumi et al., 2006;Zhang et al., 2006). Therefore, better understanding of the ubiquitination machinery and associated factors is important as it is an attractive target for therapeutic intervention against immune-related disorders and cancer development. ...
... CYLD deficiency or mutations also promote the development and progression of other cancer types such as melanoma, myeloma, kidney cancer and various carcinomas (colon, uterine cervix, hepatocellular) (Sun, 2010). The tumor suppressor function of CYLD was further supported by animal models in which skin or colon tumors were chemically induced upon CYLD loss (Massoumi et al., 2006;Zhang et al., 2006). ...
... Of note, the independent generation of CYLD-deficient mice resulted in different phenotypes, probably arising from compensatory mechanisms by redundant proteins or dominant-negative effects of truncated CYLD variants (Lork et al., 2017). Deletion of the N-terminus containing the start codon induces a mild phenotype with slighly elevated B and T cell responsiveness (Massoumi et al., 2006;Zhang et al., 2006;. This phenotype is more pronounced in CYLD-mice lacking exons 7 and 8 (Hövelmeyer et al., 2007). ...
Pro-inflammatory signaling mediated by immune and cytokine receptors is crucial for the induction of adequate immune responses to eliminate invading pathogens. Tight regulation of these pathways is important to prevent pathologies or the onset of autoimmune or chronic inflammatory diseases. Upon immune receptor engagement, non-degradative ubiquitylation is an essential posttranslational modification (PTM), which allows the recruitment of kinases to activate pro-survival/inflammatory NF-κB and MAPK signaling. It is mediated mainly by E3 ubiquitin ligases such as cIAP1/2 and the linear ubiquitin assembly complex (LUBAC). Deubiquitinases (DUBs) like CYLD and OTULIN play critical roles for the regulation of pro-inflammatory signaling by removing K63- and M1-linked ubiquitin chains from receptor complexes, e.g., the tumor necrosis factor (TNF) receptor I (TNFR1). The adaptor protein SPATA2, which bridges the interaction of CYLD with LUBAC, mediates recruitment of CYLD to the TNFR1 signaling complex (TNFR1-SC). Whether SPATA2 exhibits other functions independently of CYLD is not known so far. Therefore, the aim of this study was to identify if SPATA2 and CYLD can function independently of each other.
It could be shown that homozygous deletion of SPATA2 and CYLD in mice resulted in perinatal lethality of the majority of double knockout (Cyld-/-Spata2-/-) offspring. Surviving Cyld-/-Spata2-/- animals were smaller than littermates, had a kinked tail and elevated marginal zone B cell numbers, indicating unrestricted NF-κB activation in the absence of both proteins. Combined loss of SPATA2 and CYLD in murine embryonic fibroblasts (MEF) and bone marrow-derived macrophages (BMDM) induced elevated pro-inflammatory gene expression and increased NF-κB and MAPK activation. Increased TNFR1-SC ubiquitylation in Cyld-/-Spata2-/- MEF in comparison to Cyld-/- or Spata2-/- cells. TNF-induced cell death was reduced in Cyld-/-Spata2-/- MEF as compared to single knockout cells, which was reversed after reconstitution of SPATA2 or CYLD. In addition to the PUB interacting motif (PIM), the zinc finger region (ZnF) of SPATA2 was identified as novel motif required for HOIP interaction. Furthermore, it was seen that the PIM and ZnF domain of SPATA2 are necessary to attenuate pro-inflammatory signaling in the absence of CYLD. SPATA2 counteracts LUBAC activity by displacing OTULIN from HOIP, thereby promoting auto-ubiquitylation of LUBAC. In consequence, increased pro-inflammatory signaling upon combined loss of SPATA2 and CYLD depends on the presence of OTULIN. As another finding, BMDM) generated from Cyld-/-Spata2-/- animals exhibited stronger priming and induction of the NLRP1b inflammasome. Additional deletion of caspase-1 resulted in increased survival of Cyld-/-Spata2-/- mice. Furthermore, it could be seen that SPATA2 is a crucial regulator of intestinal inflammation as combined loss of SPATA2 and CYLD led to villus blunting, increased pro-inflammatory cytokine expression in intestinal epithelial cells and developmental defects of intestinal organoids. Thus, this study revealed that SPATA2 and CYLD have mutually independent functions for regulating inflammatory responses and cell death induction.
... It is relevant that the in vivo role of CYLD as a tumor suppressor of NMSC has only been analyzed through the study of transgenic mice that either contain a functionally inactive CYLD (by expression of mutated forms of CYLD that act as dominant negative) [11,12] or that are deficient in CYLD [32]. However, the in vivo role of wild type CYLD in the development and progression of NMSC has not yet been fully characterized. ...
... The different lines of transgenic mice were viable, and no relevant histological differences were detected in the skin of any of them with respect to the skin histology of Control mice ( Figure S1); therefore, we generically refer to them as K5-CYLDwt mice (most of the experiments were performed in line 1). [11,12] or that are deficient in CYLD [32]. However, the in vivo role of wild type CYLD in the development and progression of NMSC has not yet been fully characterized. ...
... Our data suggesting the role of CYLD as a tumor suppressor of skin squamous cell tumors in vivo are in agreement with those of Cyld-/mice, in which other authors found that mice lacking the Cyld gene exhibited increased susceptibility to skin cancer development in chemical carcinogenesis experiments [32]; they also agree with our recent results showing that transgenic mice lacking the deubiquitinase function of CYLD in keratinocytes, K5-CYLD C/S mice, spontaneously develop skin tumors [11]. ...
Cylindromatosis (CYLD) is a deubiquitinase (DUB) enzyme that was initially characterized as a tumor suppressor of adnexal skin tumors in patients with CYLD syndrome. Later, it was also shown that the expression of functionally inactive mutated forms of CYLD promoted tumor development and progression of non-melanoma skin cancer (NMSC). However, the ability of wild-type CYLD to inhibit skin tumorigenesis in vivo in immunocompetent mice has not been proved. Herein, we generated transgenic mice that express the wild type form of CYLD under the control of the keratin 5 (K5) promoter (K5-CYLDwt mice) and analyzed the skin properties of these transgenic mice by WB and immunohistochemistry, studied the survival and proliferating characteristics of primary keratinocytes, and performed chemical skin carcinogenesis experiments. As a result, we found a reduced activation of the nuclear factor kappa B (NF-κB) pathway in the skin of K5-CYLDwt mice in response to tumor necrosis factor-α (TNF-α); accordingly, when subjected to insults, K5-CYLDwt keratinocytes are prone to apoptosis and are protected from excessive hyperproliferation. Skin carcinogenesis assays showed inhibition of tumor development in K5-CYLDwt mice. As a mechanism of this tumor suppressor activity, we found that a moderate increase in CYLD expression levels reduced NF-κB activation, which favored the differentiation of tumor epidermal cells and inhibited its proliferation; moreover, it decreased tumor angiogenesis and inflammation. Altogether, our results suggest that increased levels of CYLD may be useful for anti-skin cancer therapy.
... CYLD is mainly a cytoplasmic protein that belongs to the ubiquitin-specific protease (USP) family [2] and is abundant in the brain [3], skeletal muscle [4], and immune cells [5]. CYLD processes larger substrate molecule by cleaving lysin63-linked ubiquitin chains from that molecule [6,7] and thereby involved in corresponding cellular events, namely: cell cycle control [8], cellular differentiation [9], oncogenesis [10], cellular proliferation [11,12], and apoptosis [13]. Mutation in CYLD can give rise to the constant activation and deregulation of cell survival proteins associated with tumorigenesis [10]. ...
... By deubiquitinating TRAF2/TRAF6, NF-κB essential modifier (NEMO), CYLD, acts as a key regulator in the typical p65/NF-κB pathway [19,20]. CYLD also contributes greatly by preventing the Bcl3 from being localized in the nucleus and thus controls tumor development and proliferation [11]. Therefore, any mutation disrupting the deubiquitinating (DUB) activity of CYLD may lead to oncogenic function gain, as DUB activity is fundamental for CYLD as a tumor suppressor [21,22]. ...
Tumor suppressor cylindromatosis protein (CYLD) regulates NF-κB and JNK signaling pathways by cleaving K63-linked poly-ubiquitin chain from its substrate molecules and thus preventing the progression of tumorigenesis and metastasis of the cancer cells. Mutations in CYLD can cause aberrant structure and abnormal functionality leading to tumor formation. In this study, we utilized several computational tools such as PANTHER, PROVEAN, PredictSNP, PolyPhen-2, PhD-SNP, PON-P2, and SIFT to find out deleterious nsSNPs. We also highlighted the damaging impact of those deleterious nsSNPs on the structure and function of the CYLD utilizing ConSurf, I-Mutant, SDM, Phyre2, HOPE, Swiss-PdbViewer, and Mutation 3D. We shortlisted 18 high-risk nsSNPs from a total of 446 nsSNPs recorded in the NCBI database. Based on the conservation profile, stability status, and structural impact analysis, we finalized 13 nsSNPs. Molecular docking analysis and molecular dynamic simulation concluded the study with the findings of two significant nsSNPs (R830K, H827R) which have a remarkable impact on binding affinity, RMSD, RMSF, radius of gyration, and hydrogen bond formation during CYLD-ubiquitin interaction. The principal component analysis compared native and two mutants R830K and H827R of CYLD that signify structural and energy profile fluctuations during molecular dynamic (MD) simulation. Finally, the protein–protein interaction network showed CYLD interacts with 20 proteins involved in several biological pathways that mutations can impair. Considering all these in silico analyses, our study recommended conducting large-scale association studies of nsSNPs of CYLD with cancer as well as designing precise medications against diseases associated with these polymorphisms.
... Early studies of the oncogenic role of BCL3 naturally focused on NF-kB mediated pathways in cancer, most notably the transcriptional control of cyclin D1 and p53 to regulate cell cycle [98][99][100][101][102][103], and cell viability (including p53 mediated apoptosis) [58,[104][105][106]. More recently, as other regulatory mechanisms have come to light, it has become clear that BCL3 impacts several of the 'acquired capabilities' and 'enabling characteristics' outlined as the key Hallmarks of Cancer [107]. ...
... BCL3 is a heavily modified protein which regulates its stability, subcellular localisation and activity and impacts on its oncogenicity [29,31,52,100,146,147]. Whether these modifications which include phosphorylation and ubiquitination (reviewed by [16,17]) could be harnessed directly or through targeting of BCL3 upstream modifiers, as suggested for the cancer related deubiquitination enzyme CYLD [148], remains to be determined. ...
In the early 1990’s a group of unrelated genes were identified from the sites of recurring translocations in B-cell lymphomas. Despite sharing the nomenclature ‘Bcl’, and an association with blood-borne cancer, these genes have unrelated functions. Of these genes, BCL2 is best known as a key cancer target involved in the regulation of caspases and other cell viability mechanisms. BCL3 on the other hand was originally identified as a non-canonical regulator of NF-kB transcription factor pathways – a signaling mechanism associated with important cell outcomes including many of the hallmarks of cancer. Most of the early investigations into BCL3 function have since focused on its role in NF-kB mediated cell proliferation, inflammation/immunity and cancer. However, recent evidence is coming to light that this protein directly interacts with and modulates a number of other signaling pathways including DNA damage repair, WNT/β-catenin, AKT, TGFβ/SMAD3 and STAT3 – all of which have key roles in cancer development, metastatic progression and treatment of solid tumours. Here we review the direct evidence demonstrating BCL3’s central role in a transcriptional network of signaling pathways that modulate cancer biology and treatment response in a range of solid tumour types and propose common mechanisms of action of BCL3 which may be exploited in the future to target its oncogenic effects for patient benefit.
... However, it is unclear if there is aberrant NF-κB activation in SPATA2-deficient mouse intestines. Mouse macrophages, fibroblasts and keratinocytes lacking SPATA2 or CYLD exhibit, at best, a modest enhancement in TNF-induced activation of MAPKs or NF-κB [97,98,102,[108][109][110]. ...
... Although SPATA2-deficient mice and several different strains of CYLD-deficient mice are viable [102,[108][109][110][111], mice expressing inactive CYLD, owing to truncation of the C-terminal USP domain, die soon after birth [112]. Whether this lethality reflects a gain-of-function of the mutant CYLD scaffold remains unclear. ...
Apoptosis, pyroptosis, and necroptosis are distinct forms of programmed cell death that eliminate infected, damaged, or obsolete cells. Many proteins that regulate or are a part of the cell death machinery undergo ubiquitination, a post-translational modification made by ubiquitin ligases that modulates protein abundance, localization, and/or activity. For example, some ubiquitin chains target proteins for degradation, while others function as scaffolds for the assembly of signaling complexes. Deubiquitinases (DUBs) are the proteases that counteract ubiquitin ligases by cleaving ubiquitin from their protein substrates. Here, we review the DUBs that have been found to suppress or promote apoptosis, pyroptosis, or necroptosis.
... The deubiquitinase CYLD lysine 63 deubiquitinase (CYLD) was first described in cylindromatosis (1) and its tumor suppressive properties have been investigated in various types of cancer, such as pancreas, breast and liver cancer (2)(3)(4). In malignant melanoma, the expression of CYLD is downregulated by elevated expression of the transcription factor SNAIL1, which results in increased proliferative and migratory potential of melanoma cells (5,6). Recently, our group generated a novel transgenic (Tg) melanoma mouse model, Tg(Grm1) Cyld, showing enhanced tumor development and growth in Cyld-knockdown (Cyld -/-) mice compared with Cyld-wild type (Cyld +/+ ) mice (7). ...
... Human primary melanoma cell line Mel Juso [provided by dr Judith Johnson (Ludwig Maximilian University of Munich, Munich, Germany), stably transfected with control green fluorescent protein [human (h)CYLD -], CYLD (hCYLD + ) or CYLD C/S (catalytically inactive mutant of cYLd) vector (5,6) were cultivated (8% cO 2 at 37˚C) in RPMI-1640 (Sigma-Aldrich; Merck KGaA) with NaHcO 3 and the aforementioned supplements. For cell counting the light microscope AE2000 from Motic Incorporation Ltd. was used (x4 or x20 magnification). ...
The tumor suppressive role of CYLD lysine 63 deubiquitinase (CYLD) is known in melanoma. To the best of our knowledge, however, the precise mechanism underlying the tumor suppressive function of CYLD has yet to be clarified. In the present study, a novel melanoma mouse model was generated, which revealed accelerated tumor growth in Cyld‑knockout (Cyld‑/‑) compared with Cyld‑wild‑type (Cyld+/+) mice. To determine the underlying molecular mechanism, mutation analysis of primary tumor‑derived cell lines from Cyld+/+ and Cyld‑/‑ mice was performed using RNA sequencing data. Variant calling revealed no common mutations in Cyld‑/‑ compared with Cyld+/+ cells. Thus, the epigenetic processes influencing development and progression of melanoma were investigated. Initial analysis of expression pattern of known hypermethylated genes in melanoma (suppressor of cytokine signalling, methylthioadenosine phosphorylase, cadherin 1) in the presence or absence of 5'‑Aza‑deoxyctidine treatment revealed that CYLD does not play a key role in DNA methylation. Chromatin accessibility and histone H3 modification assay uncovered a role of CYLD in the formation of chromatin structure. Subsequent inhibitor experiments confirmed the effect of CYLD on H3K9me2 level associated with heterochromatin. Furthermore, enhanced H3K9 dimethylation in Cyld‑/‑ melanoma cells was associated with upregulation of euchromatic histone lysine methyltransferase 2 (EHMT2). Moreover, the specific inhibitor of EHMT2, CM272, resulted in decreased proliferation and relaxation of compact chromatin in Cyld‑deficient melanoma cells. These results reveal a novel role of CYLD in histone methylation and chromatin packaging.
... This translocation and nuclear activity depends on ubiquitinylation [15] and phosphorylation by AKT, ERK, or IKK1/2 [16]. ...
... BCL3 protein abundance is regulated by an auto-regulatory loop via NF-kB [12]. Furthermore, the amount of BCL3 in the cytosol is determined by ubiquitinylation, which regulates its ongoing degradation [15]. In this localization, BCL3 has inhibitory functions on the NF-κB transcription factor, whereas upon activation of cells, BCL3 can be phosphorylated and located to the nucleus where it acts as a transcriptional coactivator. ...
Patients with estrogen receptor positive breast cancer are usually receiving an anti-estrogen therapy by either aromatase inhibitors or selective estrogen receptor mediators such as tamoxifen. Nevertheless, acquired resistance to tamoxifen under treatment frequently hampers therapy. One proposed explanation for this phenomenon is the interaction of the tumor cells with cells of the tumor microenvironment via the Insulin-like growth factor RNA binding protein 5/B-cell lymphoma 3 (IGFBP5/ BCL3) axis. Here we investigated whether a high expression of BCL3 either cytoplasmic or nuclear is associated with the occurrence of a relapse under anti-estrogen therapy in patients. Formaldehyde-fixed, paraffin-embedded samples of 180 breast cancer patients were analyzed for BCL3 expression by immunohistochemistry. An immunoreactive score (IRS) was calculated from staining intensity in cytoplasm and nucleus as well as the percentage of positive tumor cells. These scores were correlated with clinico-pathological parameters using cross-tabulation analysis and patients' relapse free and overall survival by Kaplan-Meier analysis and Cox regression. A tamoxifen-adapted MCF-7 derived cell line was investigated for BCL3 localization by immunofluorescence. The cytosolic BCL3-IRS significantly correlated with the proliferation marker Ki-67, and with the occurrence of a relapse under tamoxifen treatment. Nuclear score correlated only with tamoxifen-relapse. In survival analysis, both scores were highly significant prognostic factors for relapse free, but not for overall survival. This was especially obvious for estrogen receptor positive and HER2/NEU negative cases as well as lobular breast cancer. Tamoxifen-treated, but not aromatase-treated patients had a poor survival when BCL3 scores were high. A tamoxifen adapted cell line exhibited a reduced expression and mainly nuclear localization of BCL3, compared to the parental estrogen receptor positive cell-line MCF-7. Altogether, these data strongly support a function of BCL3 in tamoxifen resistance and its potential use as a predictive biomarker for tamoxifen resistance.
... Mice. Cyld À/À mice were generated with a targeting construct in which the ATG-containing exon 4 of the Cyld gene was disrupted with a lacZ reporter and a neomycin gene (13). Both Cyld +/À and Cyld À/À are fertile, have a normal life span, and do not spontaneously develop tumors (13). ...
... Cyld À/À mice were generated with a targeting construct in which the ATG-containing exon 4 of the Cyld gene was disrupted with a lacZ reporter and a neomycin gene (13). Both Cyld +/À and Cyld À/À are fertile, have a normal life span, and do not spontaneously develop tumors (13). ...
Significance
The deubiquitinating enzyme CYLD specifically removes Lysine 63 (K63)–linked polyubiquitin chains from several substrates. Although it is highly expressed in the brain, its role in the central nervous system is far from being understood. A thorough behavioral, physiological, and morphological analysis revealed that CYLD-deficient mice exhibit major autism-like behaviors accompanied by deficits in the structure and function of hippocampal synapses. In addition, we show that CYLD is a modulator of mechanistic target of rapamycin (mTOR) signaling, synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits, and autophagy in the hippocampus. We thereby introduce CYLD and K63-linked polyubiquitination processes at the synapse as molecular targets for both a better understanding of the pathomechanisms underlying autism spectrum disorder and the development of novel autism therapies.
... Patients with high CYLD expression and multiple myeloma (van Andel et al., 2017), oral squamous cell carcinoma (Shinriki et al., 2018) or breast cancer (Hayashi et al., 2014) have a better prognosis. Several substrates of CYLD have been identified in recent studies, including DVL (Tauriello et al., 2010), TRAF (Brummelkamp et al., 2003), Bcl-3 (Massoumi et al., 2006), IKK (Chu et al., 2006), and p53 (Fernandez-Majada et al., 2016). Some of these substrates act as CSCs factors, which can activate and upregulate the SRSs. ...
... The downregulation of CYLD promotes the accumulation of β-catenin in the nucleus through ubiquitination of DVL at Lys-63 (Tauriello et al., 2010). In addition, CYLD deubiquitinates TRAF and Bcl-3 proteins directly to downregulate NF-kB activity, inhibiting oncogenic transformation in keratinocytes (Brummelkamp et al., 2003;Massoumi et al., 2006). In salivary gland tumors, CYLD has a negative regulatory effect on NF-kB activity after TNF-α stimulation (Fukuda et al., 2008). ...
Cancer stem cells (CSCs) are sparks for igniting tumor recurrence and the instigators of low response to immunotherapy and drug resistance. As one of the important components of tumor microenvironment, the tumor associated immune microenvironment (TAIM) is driving force for the heterogeneity, plasticity and evolution of CSCs. CSCs create the inhibitory TAIM (ITAIM) mainly through four stemness-related signals (SRSs), including Notch-nuclear factor-κB axis, Hedgehog, Wnt and signal transducer and activator of transcription. Ubiquitination and deubiquitination in proteins related to the specific stemness of the CSCs have a profound impact on the regulation of ITAIM. In regulating the balance between ubiquitination and deubiquitination, it is crucial for deubiquitinating enzymes (DUBs) to cleave ubiquitin chains from substrates. Ubiquitin-specific peptidases (USPs) comprise the largest family of DUBs. Growing evidence suggests that they play novel functions in contribution of ITAIM, including regulating tumor immunogenicity, activating stem cell factors, upregulating the SRSs, stabilizing anti-inflammatory receptors, and regulating anti-inflammatory cytokines. These overactive or abnormal signaling may dampen antitumor immune responses. The inhibition of USPs could play a regulatory role in SRSs and reversing ITAIM, and also have great potential in improving immune killing ability against tumor cells, including CSCs. In this review, we focus on the USPs involved in CSCs signaling pathways and regulating ITAIM, which are promising therapeutic targets in antitumor therapy.
... Par exemple, malgré le blocage au stade pré-B1 des cellules B des modèles invalidant le gène Btk (Khan et al. 1995;Kerner et al. 1995), il a été démontré une réduction de la prolifération en réponse à l'activation du récepteur CD40, soulignant la coopération de ces 2 voies de signalisation (Khan et al. 1995;Kerner et al. 1995). Parmi les études d'invalidation ubiquitaire d'autres inhibiteurs de la voie NF-κB, le modèle ciblant la désubiquitinase CYLD présente une augmentation du nombre de ses cellules B1, B2-CG et B2-ZM (Massoumi et al. 2006;Jin et al. 2007) alors que le modèle invalidant A20 semble ne présenter qu'une augmentation des cellules du CG (E. G. Lee et al. 2000;Tavares et al. 2010;Chu et al. 2011;Hövelmeyer et al. 2011). ...
... Le modèle d'invalidation ubiquitaire Cyld -/présente une tumeur des kératinocytes et des follicules pileux avec dans certains cas une hyperplasie sévère des cellules B (Massoumi et al. 2006 (Knittel et al. 2016). ...
Les lymphomes ont causé 557 730 décès dans le monde en 2018 et sont la 5ème cause de mortalité due à un cancer en France. Les recherches fondamentales et appliquées dans ce domaine sont donc cruciales pour améliorer les diagnostics et les thérapies. Les voies de signalisation cellulaire du facteur de transcription NF-κB, jouant un rôle central dans la survie et la prolifération de la cellule, sont fréquemment dérégulées et impliquées dans l’émergence des lymphomes non Hodgkinien des cellules B (LNHB). Les études menées au laboratoire sur ces trois années concernaient le rôle de MYD88 et de NF-κB dans le contexte de l’émergence des lymphomes liés à la cellule B. La mutation L265P de la protéine adaptatrice MYD88 (MYD88L265P), exacerbant la signalisation des « toll like receptor » (TLR), récepteurs impliqués dans la réponse immunitaire innée de la cellule B entraîne une activation constitutive de NF-κB. La protéine mutée MYD88L265P est retrouvée dans de nombreux cas de LNHB allant des lymphomes lymphoplasmocytaires (LLP) indolents aux lymphomes agressifs diffus à grandes cellules B (DLBCL). La macroglobulinémie de Waldenström (MW) présente dans environ 90% des cas, cette mutation MYD88L265P, faisant d’elle un évènement primaire dans l’émergence de ce LLP. Pour pouvoir étudier les conséquences d’une telle mutation sur la transformation de la cellule B, nous avons réalisé une étude in vivo, via un modèle murin exprimant MYD88L252P (orthologue murin de la mutation MYD88L265P chez l’humain) seulement des cellules B grâce à l’expression de la CRE-recombinase via le promoteur du gène Cd19. En effet, l’étude de ce modèle développant un lymphome présentant des similarités avec la MW, nous a permis de visualiser son pouvoir transformant sur la cellule B, mais aussi le comportement de la cellule dans le microenvironnement d’un organisme entier. Nous avons pu confirmer l’aptitude de MYD88L252P à former un clone lymphoplasmocytaire, ainsi que la présence d’un pic d’IgM monoclonal dans le sérum et une invasion de plasmocytes à IgM dans la moelle osseuse, qui sont des caractéristiques de la MW.L’échappement immunitaire est une composante majeure dans l’émergence des LNHB. Dans le cas des lymphomes liés au virus d’Epstein Barr (EBV), les cellules B immortalisées par ce virus sont aussi aptes à moduler la réponse immunitaire qui leur est adressée. Concernant ce dernier phénomène, nous avons décrit le profil immunorégulateur in vitro de cellules B infectées par l’EBV en latence III. La protéine LMP1, produite lors de la latence II et III, active essentiellement la voie canonique de NF-κB, et permet la surexpression des protéines immunomodulatrice PD-L1, IL-10, TGF- β1 et IL35 (EBI3 et IL12A). Nous avons aussi mis en évidence, l’expansion des cellules T régulatrices (Treg) conventionnelles et non conventionnelles dépendant de l’axe PD-1/PD-L1. Dans de nombreux cancers, la protéine de surface PD-L1 est surexprimée impactant fortement le microenvironnement des cellules tumorales. Ainsi, dans le cas des DLBCL, cette surexpression est associée à un mauvais pronostic. Du fait de son importance dans la progression de la tumeur, les tests thérapeutiques avec des anticorps anti-PD-L1 peuvent se révéler être très efficaces. Pour cela, nous avons réalisé des tests précliniques in vivo ciblant PD-L1 directement, et ex vivo ciblant les voies permettant son expression dans le modèle murin L.CD40 (lmp1/cd40) développant un lymphome indolent se rapprochant du lymphome de la zone marginale (LZM) dans lequel nous avons montré que les tumeurs B surexprimaient la protéine PD-L1. Nous avons pu démontrer les effets bénéfiques de ce traitement avec une réduction de la taille de la rate in vivo, une diminution de l’expression du gène PD-L1 ex vivo et une réactivation des cellules T. Ce travail ouvre de nouvelles perspectives de recherche sur l’échappement à la réponse immune dans les LNHB associés à la mutation activatrice de MYD88.
... The NF-κB signaling pathway regulates cell proliferation in a variety of cell types [35,36]. Here, we determined the role of A1CF in two renal cancer cell lines (OSRC-2 and 786-O). ...
Apobec-1 complementation factor (A1CF) functions as an RNA-binding cofactor for APO-BEC1-mediated C-to-U conversion during RNA editing and as a hepatocyte-specific regulator in the alternative pre-mRNA splicing of metabolic enzymes. Its role in RNA editing has not been clearly established. Western blot, co-immunoprecipitation (Co-IP), immunofluorescence (IF), methyl thiazolyl tetrazolium (MTT), and 5-ethynyl-2′-deoxyuridine (EdU) assays were used to examine the role of A1CF beyond RNA editing in renal carcinoma cells. We demonstrated that A1CF interacts with NKRF, independent of RNA and DNA, without affecting its expression or nuclear translocation; however, it modulates p65(Ser536) phosphorylation and IFN-β levels. Truncation of A1CF or deletion on NKRF revealed that the RRM1 domain of A1CF and the p65 binding motif of NKRF are required for their interaction. Deletion of RRM1 on A1CF abrogates NKRF binding, and the decrease in IFN-β expression and p65(Ser536) phosphorylation was induced by A1CF. Moreover, full-length A1CF, but not an RRM1 deletion mutant, promoted cell proliferation in renal carcinoma cells. Perturbation of A1CF levels in renal carcinoma cells altered anchorage-independent growth and tumor progression in nude mice. Moreover, p65(Ser536) phosphorylation and IFN-β expression were lower, but ki67 was higher in A1CF-overexpressing tumor tissues of a xenograft mouse model. Notably, primary and metastatic samples from renal cancer patients exhibited high A1CF expression, low p65(Ser536) phosphorylation, and decreased IFN-β levels in renal carcinoma tissues compared with the corresponding paracancerous tissues. Our results indicate that A1CF-decreased p65(Ser536) phosphorylation and IFN-β levels may be caused by A1CF competitive binding to the p65-combined site on NKRF and demonstrate the direct binding of A1CF independent of RNA or DNA in signal pathway regulation and tumor promotion in renal carcinoma cells.
... Next, CYLD is prominently expressed in the epidermis; however, CYLD −/− mice do not exhibit skin abnormalities [34]. Recently, two SNPs (rs4785452 and rs12925755) in the CYLD gene have been reported to link to autoimmune diseases and psoriasis [20]. ...
Background and Objectives: Psoriasis is an immune-mediated chronic inflammatory skin disorder and commonly associated with highly noticeable erythematous, thickened and scaly plaques. Deubiquitinase genes, such as tumor necrosis factor-alpha protein 3 (TNFAIP3, A20), the cylindromatosis (CYLD) and Cezanne, function as negative regulators of inflammatory response through the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathways. In this study, polymorphisms and expressions of A20, CYLD and Cezanne genes as well as immunophenotype in psoriatic patients were determined. Materials and Methods: In total, 82 patients with psoriasis and 147 healthy individuals with well-characterized clinical profiles were enrolled. Gene polymorphisms were determined by direct DNA sequencing, gene expression profile by quantitative real time-polymerase chain reaction (PCR), immunophenotype by flow cytometry, and the secretion of cytokines and cancer antigen (CA) 125 by enzyme-linked Immunosorbent assay (ELISA). Results: The inactivation of A20, CYLD and Cezanne and increased levels of TNF-α, IFN-γ and CA 125 was observed in psoriatic patients. Importantly, patients with low A20 expression had significant elevations of triglyceride and total cholesterol concentrations and higher numbers of CD13+CD117− and CD19+CD23+ (activated B) cells than those with high A20 expression. Genetic analysis indicated that all rs4495487 SNPs in the JAK2 gene, rs200878487 SNPs in the A20 gene and four SNPs (c.1584-375, c.1584-374, rs1230581026 and p.W433R) in the Cezanne gene were associated with significant risks, while the rs10974947 variant in the JAK2 gene was at reduced risk of psoriasis. Moreover, in the Cezanne gene, p.W433R was predicted to be probably damaging by the Polyphen-2 prediction tool and an AA/CC haplotype was associated with a high risk of psoriasis. In addition, patients with higher CA 125 levels than the clinical cutoff 35 U/mL showed increased levels of IFN-γ than those with normal CA 125 levels. Conclusions: A20 expression was associated with lipid metabolism and the recruitment of CD13+ CD117− and activated B cells into circulation in psoriatic patients. Besides this, the deleterious effect of the p.W433R variant in the Cezanne gene may contribute to the risk of psoriasis.
... Complementary work in cultured keratinocytes and mouse epidermal models with wildtype and deubiquitinase-defective CYLD shows influence of the protein over keratinocyte differentiation, response to cytokine exposure, and activation of NF-κB and other transcription factors [109,[112][113][114][115]. HaCaT keratinocytes stably transfected to over-express CYLD have increased levels of the early differentiation markers keratin 1 and 10 [114]. ...
The overactivity of keratinocyte cytoplasmic signaling contributes to several cutaneous inflammatory and immune pathologies. An important emerging complement to proteins responsible for this overactivity is signal repression brought about by several proteins and protein complexes with the native role of limiting inflammation. The signaling repression by these proteins distinguishes them from transmembrane receptors, kinases, and inflammasomes, which drive inflammation. For these proteins, defects or deficiencies, whether naturally arising or in experimentally engineered skin inflammation models, have clearly linked them to maintaining keratinocytes in a non-activated state or returning cells to a post-inflamed state after a signaling event. Thus, together, these proteins help to resolve acute inflammatory responses or limit the development of chronic cutaneous inflammatory disease. We present here an integrated set of demonstrated or potentially inflammation-repressive proteins or protein complexes (linear ubiquitin chain assembly complex [LUBAC], cylindromatosis lysine 63 deubiquitinase [CYLD], tumor necrosis factor alpha-induced protein 3-interacting protein 1 [TNIP1], A20, and OTULIN) for a comprehensive view of cytoplasmic signaling highlighting protein players repressing inflammation as the needed counterpoints to signal activators and amplifiers. Ebb and flow of players on both sides of this inflammation equation would be of physiological advantage to allow acute response to damage or pathogens and yet guard against chronic inflammatory disease. Further investigation of the players responsible for repressing cytoplasmic signaling would be foundational to developing new chemical-entity pharmacologics to stabilize or enhance their function when clinical intervention is needed to restore balance.
... The atypical IκB proteins IκBζ, IκBδ (aka IκBNS), IκBη, and BCL3 control the transcriptional activity of NF-κB in the nucleus [18,19,25,29]. IκBδ is a repressive regulator [19,30,31], whereas IκBη is an activating regulator [20], and IκBζ and BCL3 act as both repressors and activators of NF-κB-driven gene transcription [25,32,33]. The NF-κB precursor proteins p105/NF-κB1 and p100/NF-κB2 also share sequence similarities with Iκb factors, thereby allowing them to bind and retain preformed NF-κB/Rel proteins in the cytoplasm [14,21]. ...
NF-κB signalling is largely controlled by the family of ‘inhibitors of NF-κB’ (IκB). The relevant databases indicate that the genome of rainbow trout contains multiple gene copies coding for iκbα (nfkbia), iκbε (nfkbie), iκbδ (nkfbid), iκbζ (nfkbiz), and bcl3, but it lacks iκbβ (nfkbib) and iκbη (ankrd42). Strikingly, three nfkbia paralogs are apparently present in salmonid fish, two of which share a high sequence identity, while the third putative nfkbia gene is significantly less like its two paralogs. This particular nfkbia gene product, iκbα, clusters with the human IκBβ in a phylogenetic analysis, while the other two iκbα proteins from trout associate with their human IκBα counterpart. The transcript concentrations were significantly higher for the structurally more closely related nfkbia paralogs than for the structurally less similar paralog, suggesting that iκbβ probably has not been lost from the salmonid genomes but has been incorrectly designated as iκbα. In the present study, two gene variants coding for iκbα (nfkbia) and iκbε (nfkbie) were prominently expressed in the immune tissues and, particularly, in a cell fraction enriched with granulocytes, monocytes/macrophages, and dendritic cells from the head kidney of rainbow trout. Stimulation of salmonid CHSE-214 cells with zymosan significantly upregulated the iκbα-encoding gene while elevating the copy numbers of the inflammatory markers interleukin-1-beta and interleukin-8. Overexpression of iκbα and iκbε in CHSE-214 cells dose-dependently quenched both the basal and stimulated activity of an NF-κB promoter suggesting their involvement in immune-regulatory processes. This study provides the first functional data on iκbε—versus the well-researched iκbα factor—in a non-mammalian model species.
... Cyld À/À mice have been described. 33 Spata2 À/À mice were obtained from the KOMP Repository (UC Davis). Cyld À/À Spata2 À/À mice were generated by crossing Cyld À/À to Spata2 À/À mice, and Cyld À/À Spata2 +/À mice subsequently derived from this breeding were intercrossed. ...
SPATA2 mediates the recruitment of CYLD to immune receptor complexes by bridging the interaction of CYLD with the linear ubiquitylation assembly complex (LUBAC) component HOIP. Whether SPATA2 exhibits functions independently of CYLD is unclear. Here, we show that, while Cyld−/− and Spata2−/− mice are viable, double mutants exhibit highly penetrant perinatal lethality, indicating independent functions of SPATA2 and CYLD. Cyld−/−Spata2−/− fibroblasts show increased M1-linked TNFR1-SC ubiquitylation and, similar to Cyld−/−Spata2−/− macrophages and intestinal epithelial cells, elevated pro-inflammatory gene expression compared with Cyld−/− or Spata2−/− cells. We show that SPATA2 competes with OTULIN for binding to HOIP via its PUB-interacting motif (PIM) and its zinc finger domain, thereby promoting autoubiquitylation of LUBAC. Consistently, increased pro-inflammatory signaling in Cyld−/−Spata2−/− cells depends on the presence of OTULIN. Our data therefore indicate that SPATA2 counteracts, independently of CYLD, the deubiquitylation of LUBAC by OTULIN and thereby attenuates LUBAC activity and pro-inflammatory signaling.
... Importantly, the tumor suppressive function of CYLD has been demonstrated in vivo using genetically engineered mouse (GEMs) model. Cyld-deficient mice are highly susceptible to chemical-induced skin tumors, chemical-induced colitis, and colon-tumorigenesis (Massoumi et al., 2006;Reiley et al., 2006Reiley et al., , 2007Zhang et al., 2006). ...
... CYLD also negatively regulates other processes, such as TGFβ signaling via deubiquitinating its substrate Akt/PKB [8]. Mice deficient in CYLD display modest immune phenotypes, as they do not develop tumors spontaneously but show increased susceptibility to tumorigenesis and other immune deficits when challenged [9][10][11]. ...
The lysine-63 deubiquitinase cylindromatosis (CYLD) is long recognized as a tumor suppressor in immunity and inflammation, and its loss-of-function mutations lead to familial cylindromatosis. However, recent studies reveal that CYLD is enriched in mammalian brain postsynaptic densities, and a gain-of-function mutation causes frontotemporal dementia (FTD), suggesting critical roles at excitatory synapses. Here we report that CYLD drives synapse elimination and weakening by acting on the Akt-mTOR-autophagy axis. Mice lacking CYLD display abnormal sociability, anxiety- and depression-like behaviors, and cognitive inflexibility. These behavioral impairments are accompanied by excessive synapse numbers, increased postsynaptic efficacy, augmented synaptic summation, and impaired NMDA receptor-dependent hippocampal long-term depression (LTD). Exogenous expression of CYLD results in removal of established dendritic spines from mature neurons in a deubiquitinase activity-dependent manner. In search of underlying molecular mechanisms, we find that CYLD knockout mice display marked overactivation of Akt and mTOR and reduced autophagic flux, and conversely, CYLD overexpression potently suppresses Akt and mTOR activity and promotes autophagy. Consequently, abrogating the Akt-mTOR-autophagy signaling pathway abolishes CYLD-induced spine loss, whereas enhancing autophagy in vivo by the mTOR inhibitor rapamycin rescues the synaptic pruning and LTD deficits in mutant mice. Our findings establish CYLD, via Akt-mTOR signaling, as a synaptic autophagy activator that exerts critical modulations on synapse maintenance, function, and plasticity.
... Bcl3 is involved in a wide range of biological processes, such as cell survival [39], proliferation [40,41], immune response [42,43], inflammation [44], and stemness [22,45,46]. Weighted gene coexpression network analysis following PHx indicated that Bcl3 might play a key role in the proliferation stage [47]. ...
Recent reports have demonstrated that Sox9+HNF4α+ hepatocytes are involved in liver regeneration after chronic liver injury; however, little is known about the origin of Sox9+HNF4α+ hepatocytes and the regulatory mechanism. Employing a combination of chimeric lineage tracing, immunofluorescence, and immunohistochemistry, we demonstrate that Sox9+HNF4α+ hepatocytes, generated by transition from mature hepatocytes, play an important role in the initial phase after partial hepatectomy (PHx). Additionally, knocking down the expression of Sox9 suppresses hepatocyte proliferation and blocks the recovery of lost hepatic tissue. In vitro and in vivo assays demonstrated that Bcl3, activated by LPS, promotes hepatocyte conversion and liver regeneration. Mechanistically, Bcl3 forms a complex with and deubiquitinates YAP1 and further induces YAP1 to translocate into the nucleus, resulting in Sox9 upregulation and mature hepatocyte conversion. We demonstrate that Bcl3 promotes Sox9+HNF4α+ hepatocytes to participate in liver regeneration, and might therefore be a potential target for enhancing regeneration after liver injury.
... TNF receptor associated factor 2) sowie Bcl-3 (engl. B-cell leukemia/lymphoma 3) identifiziert werden (Massoumi et al. 2006;Saito et al. 2004;Kovalenko et al. 2003). ...
Die Deubiquitinase CYLD beeinflusst ein weites Spektrum an physiologischen Prozessen, welche von der Immunreaktion und Entzündung bis hin zur Zell-Zyklus-Progression oder auch der Osteoklastogenese reichen. Darüber hinaus konnte der Verlust von CYLD in diversen Tumorentitäten, wie dem malignen Melanom, bereits mit der Onkogenese in Verbindung gebracht werden. Die bisherigen Daten bezüglich des Tumorsuppressors CYLD und der Entstehung und Progression des malignen Melanoms beschränkt sich zumeist auf dessen Einfluss auf die Proliferation und Migration/Invasion. Die genauen Mechanismen der tumorsuppressiven Funktion von CYLD sind jedoch noch unklar. Aufgrund dessen wurde das Tg(Grm1)Cyld-/- Melanommausmodell entwickelt, welches ein beschleunigtes Tumorwachstum der Cyld-knockout Mäuse im Vergleich zu den Cyld-wildtyp Mäusen aufweist. Die vorliegende Arbeit beschäftigte sich mit der Aufklärung des zugrundeliegenden Mechanismus unter Nutzung der generierten Tg(Grm1)Cyld+/+ und Tg(Grm1)Cyld-/- Melanomzellen des Mausmodells. Mittels Transkriptom-Sequenzierung muriner Gewebeproben des Tg(Grm1)Cyld Melanommodells und in vitro Luciferase-Reportergen-Analysen muriner Zelllinien konnte der Einfluss von CYLD auf Tumor-assoziierte Signalwege, welche die Prozesse der Proliferation, Migration und Angiogenese nachweislich beeinflussen, gezeigt werden. Zudem gaben in vitro Analysen Aufschluss über den detaillierten Mechanismus der regulatorischen Rolle von CYLD im Prozess der Lymphangiogenese. Diese deuten darauf hin, dass CYLD einen supprimierenden Einfluss auf verschiedene Lymphangiogenese-assoziierte Faktoren, wie LYVE1, VEGF-C, PDPN oder auch MDK besitzt und dass ein Verlust von CYLD die Bildung von neuen Lymphgefäßen und der damit einhergehenden frühzeitigen Metastasierung des Melanoms begünstigt. Um potentielle genomische Aberrationen, welche zum beschleunigten Tumorwachstum der Cyld-knockout Mäuse beitragen, zu identifizieren, wurden Next Generation Sequencing-Analysen von murinen Primärtumorzelllinien durchgeführt. Der Vergleich von Tg(Grm1)Cyld+/+ und Tg(Grm1)Cyld-/- Melanomzellen ergaben keinen Unterschied hinsichtlich chromosomaler Veränderungen. Zudem konnten nur wenige Mutationen detektiert werden, welche das signifikant frühere Tumor-Onset nicht erklären können. Da neben genomischen Veränderungen epigenetische Prozesse für die Entwicklung und Progression des malignen Melanoms von Bedeutung sind, wurden diese anschließend betrachtet. Anhand von in vitro Analysen konnte eine CYLD-abhängige Modulation der Chromatinstruktur festgestellt werden. Die gewonnenen Daten lassen vermuten, dass bei CYLD-Defizienz die verstärkte Expression der Methyltransferase EHMT2 zur Erhöhung der Di-Methylierung von H3K9, der spezifischen Methylierungsstelle von EHMT2, führt. Dies fördert eine kompaktere Heterochromatinstruktur und führt zur Induktion der Proliferation von Melanomzellen. Zudem bewirkte die Behandlung von CYLD-defizienten Melanomzellen mit dem EHMT2 Inhibitor CM272 eine Auflockerung der Chromatinstruktur, sowie eine Suppression der Proliferation, was eine vielsprechende Strategie für die Melanombehandlung darstellen könnte. Die Relevanz des Mausmodells und der daraus generierten Zelllinien für die Untersuchung der molekularen Mechanismen des Melanoms zeigt sich durch die Bestätigung der gewonnenen Daten in einem etablierten humanen System. In dieser Dissertation konnte somit die Relevanz von CYLD in entscheidenden Prozessen, welche für der Entstehung und Progression des malignen Melanoms verantwortlich sind, aufgezeigt werden. Zum einen wurde die Rolle von CYLD in Tumor-assoziierten Signalwegen aufgezeigt und der molekulare Mechanismus der Lymphangiogenese näher analysiert. Des Weiteren konnte zum ersten Mal eine Auswirkung des Tumorsuppressors CYLD auf die Chromatin-Zugänglichkeit und der damit assoziierten Histon Methylierungen, insbesondere H3K9me2, nachgewiesen werden.
... Inactive Bcl-3 is localized in the cytoplasm in certain cell types, although it primarily located in the nucleus (50, 51), Bcl-3 in the cytoplasm requires K63-linked polyubiquitination for translocation to the nucleus. The de-ubiquitinase CYLD is demonstrated to control the localization of Bcl-3 in keratinocytes by removing these polyubiquitin chains, decreasing the amount Bcl-3 in nuclear and gene transcription mediated by Bcl-3 (52). ...
The NF-κB transcription factor family controls the transcription of many genes and regulates a number of pivotal biological processes. Its activity is regulated by the IκB family of proteins. Bcl-3 is an atypical member of the IκB protein family that regulates the activity of nuclear factor NF-κB. It can promote or inhibit the expression of NF-κB target genes according to the received cell type and stimulation, impacting various cell functions, such as proliferation and differentiation, induction of apoptosis and immune response. Bcl-3 is also regarded as an environment-dependent cell response regulator that has dual roles in the development of B cells and the differentiation, survival and proliferation of Th cells. Moreover, it also showed a contradictory role in inflammation. At present, in addition to the work aimed at studying the molecular mechanism of Bcl-3, an increasing number of studies have focused on the effects of Bcl-3 on inflammation, immunity and malignant tumors in vivo. In this review, we focus on the latest progress of Bcl-3 in the regulation of the NF-κB pathway and its extensive physiological role in inflammation and immune cells, which may help to provide new ideas and targets for the early diagnosis or targeted treatment of various inflammatory diseases, immunodeficiency diseases and malignant tumors.
... CYLD is known as a tumor suppressor protein in DUB [45], and this function of CYLD has also been studied using mouse models. CYLD knockout mice are not naturally tumor-causing but are more susceptible to chemically induced skin tumors than wild type mice [46]. (59) OTU (21) JAMM (14) UCH (4) MJD (4) MINDY (5) ...
Although damaged cells can be repaired, cells that are considered unlikely to be repaired are eliminated through apoptosis, a type of predicted cell death found in multicellular organisms. Apoptosis is a structured cell death involving alterations to the cell morphology and internal biochemical changes. This process involves the expansion and cracking of cells, changes in cell membranes, nuclear fragmentation, chromatin condensation, and chromosome cleavage, culminating in the damaged cells being eaten and processed by other cells. The ubiquitin–proteasome system (UPS) is a major cellular pathway that regulates the protein levels through proteasomal degradation. This review proposes that apoptotic proteins are regulated through the UPS and describes a unique direction for cancer treatment by controlling proteasomal degradation of apoptotic proteins, and small molecules targeted to enzymes associated with UPS.
... CYLD also negatively regulates other processes, such as TGF signaling via deubiquitinating its substrate Akt/PKB [9]. Mice deficient in CYLD display modest immune phenotypes as they do not develop tumors spontaneously but show increased susceptibility to tumorigenesis and other immune deficits when challenged [10][11][12]. ...
The lysine-63 deubiquitinase cylindromatosis (CYLD) is long recognized as a tumor suppressor in immunity and inflammation and its loss-of-function mutations lead to familial cylindromatosis. However, recent studies reveal that CYLD is enriched in mammalian brain postsynaptic densities, and a gain-of-function mutation causes frontotemporal dementia (FTD), suggesting critical roles at excitatory synapses. Here we report that CYLD drives synapse elimination and weakening by acting on the Akt-mTOR-autophagy axis. Mice lacking CYLD display abnormal sociability, anxiety- and depression-like behaviors, and cognitive inflexibility. These behavioral impairments are accompanied by excessive synapse numbers, increased postsynaptic efficacy, augmented synaptic summation, and impaired NMDA receptor-dependent hippocampal long-term depression (LTD). Exogenous expression of CYLD results in removal of established dendritic spines from mature neurons in a deubiquitinase activity-dependent manner. In search of underlying molecular mechanisms, we find that CYLD knockout mice display marked overactivation of Akt and mTOR and reduced autophagic flux and, conversely, CYLD overexpression potently suppresses Akt and mTOR activity and promotes autophagy. Consequently, abrogating the Akt-mTOR-autophagy signaling pathway abolishes CYLD-induced spine loss, whereas enhancing autophagy in vivo by the mTOR inhibitor rapamycin rescues the synaptic pruning and LTD deficits in mutant mice. Our findings establish CYLD, via Akt-mTOR signaling, as a synaptic autophagy activator that exerts critical modulations on synapse maintenance, function, and plasticity.
... Ablation of Bcl-3 significantly reduced the amount of CYLD rather than A20 or cIAPs in TNF-induced Complex I (Fig. 7A) and II (Fig. 7B) formation, consequentially resulting in decreased binding of RIP1 and FADD in Complex II (Fig. 7B). As Bcl-3 has been reported to bind to the USP domain harboring the DUB activity of CYLD [32][33][34], we hypothesize that Bcl-3 may regulate RIP1 deubiquitination in a CYLD-dependent manner. We examined the interaction between Bcl-3 and CYLD as reported in 293 T cells by co-IP assay (Fig. 7C). ...
Tumor necrosis factor-α (TNF) is described as a main regulator of cell survival and apoptosis in multiple types of cells, including hepatocytes. Dysregulation in TNF-induced apoptosis is associated with many autoimmune diseases and various liver diseases. Here, we demonstrated a crucial role of Bcl-3, an IκB family member, in regulating TNF-induced hepatic cell death. Specifically, we found that the presence of Bcl-3 promoted TNF-induced cell death in the liver, while Bcl-3 deficiency protected mice against TNF/D-GalN induced hepatoxicity and lethality. Consistently, Bcl-3-depleted hepatic cells exhibited decreased sensitivity to TNF-induced apoptosis when stimulated with TNF/CHX. Mechanistically, the in vitro results showed that Bcl-3 interacted with the deubiquitinase CYLD to synergistically switch the ubiquitination status of RIP1 and facilitate the formation of death-inducing Complex II. This complex further resulted in activation of the caspase cascade to induce apoptosis. By revealing this novel role of Bcl-3 in regulating TNF-induced hepatic cell death, this study provides a potential therapeutic target for liver diseases caused by TNF-related apoptosis.
... Consistently, KO mice for Cyld have T cell developmental defects and exhibit fewer mature CD4 + and CD8 + single positive thymocytes and peripheral T cells (188). Mice lacking Cyld are highly sensitive to skin tumor development (189). In B cells, the consequences of Cyld deficiency are controversial. ...
The transcription factor NF-κB regulates multiple aspects of innate and adaptive immune functions and serves as a pivotal mediator of inflammatory response. In the first part of this review, we discuss the NF-κB inducers, signaling pathways, and regulators involved in immune homeostasis as well as detail the importance of post-translational regulation by ubiquitination in NF-κB function. We also indicate the stages of central and peripheral tolerance where NF-κB plays a fundamental role. With respect to central tolerance, we detail how NF-κB regulates medullary thymic epithelial cell (mTEC) development, homeostasis, and function. Moreover, we elaborate on its role in the migration of double-positive (DP) thymocytes from the thymic cortex to the medulla. With respect to peripheral tolerance, we outline how NF-κB contributes to the inactivation and destruction of autoreactive T and B lymphocytes as well as the differentiation of CD4⁺-T cell subsets that are implicated in immune tolerance. In the latter half of the review, we describe the contribution of NF-κB to the pathogenesis of autoimmunity and autoinflammation. The recent discovery of mutations involving components of the pathway has both deepened our understanding of autoimmune disease and informed new therapeutic approaches to treat these illnesses.
... p65, RELB, and c-REL have a DNAbinding domain (REL homology domain), and a transactivation domain through which they activate gene transcription. In contrast, p50 and p52 only have a DNA-binding domain and lack a transactivation domain, so p50 and p52 are only capable of promoting gene transcription if they form a heterodimer with p65, RELB, and c-REL; also, it was reported that in certain circumstances, such as described in the case of cyld-deficient keratinocytes, BCL3 accumulates in the nucleus and activates the NF-κB subunits p50 and p52 [14], although BCL3 works more commonly by inhibiting the DNA binding and transactivation of NF-κB heterodimer p50-p65 [15]. ...
Ageing is a complex process, induced by multifaceted interaction of genetic, epigenetic, and environmental factors. It is manifested by a decline in the physiological functions of organisms and associated to the development of age-related chronic diseases and cancer development. It is considered that ageing follows a strictly-regulated program, in which some signaling pathways critically contribute to the establishment and maintenance of the aged state. Chronic inflammation is a major mechanism that promotes the biological ageing process and comorbidity, with the transcription factor NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) as a crucial mediator of inflammatory responses. This, together with the finding that the activation or inhibition of NF-κB can induce or reverse respectively the main features of aged organisms, has brought it under consideration as a key transcription factor that acts as a driver of ageing. In this review, we focused on the data obtained entirely through the generation of knockout and transgenic mouse models of either protein involved in the NF-κB signaling pathway that have provided relevant information about the intricate processes or molecular mechanisms that control ageing. We have reviewed the relationship of NF-κB and premature ageing; the development of cancer associated with ageing and the implication of NF-κB activation in the development of age-related diseases, some of which greatly increase the risk of developing cancer.
... Homozygous knockout of CYLD rendered mice more susceptible to papilloma development than wild-type (WT) littermates following Te Li and Yiyan Wang are these authors contributed equally to this work treatment with the chemical carcinogens dimethylbenz[a] anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). 27 In another study, CYLD knockout mice developed chronic colonic inflammation and colon tumors following treatment with dextran sulfate sodium. 28 Furthermore, abnormal expression of or mutations in CYLD have been identified in several diseases including lung fibrosis, 29 osteolytic lesions, 30 and cardiovascular disease. ...
Background
The cylindromatosis (CYLD) tumor suppressor is a microtubule‐associated deubiquitinase that plays a critical role in the regulation of cell signaling and contributes to a variety of physiological and pathological processes. However, the functions of CYLD in zebrafish are less well known, particularly with regard to their development and physiology. In this context, we investigated the loss of function of CYLD in zebrafish via transcription activator‐like effector nuclease (TALEN)‐based gene deletion.
Methods
Semi‐quantitative RT‐PCR was used to quantify CYLD mRNA expression in zebrafish embryos at various developmental stages. We also performed whole‐mount in situ hybridization to further assess the dynamic expression and distribution of CYLD in the entire zebrafish embryos at different stages. In addition, we deleted CYLD in zebrafish with TALENs to investigate its potential impact on embryonic development.
Results
The expression of CYLD mRNA varied during early embryonic development. The CYLD mRNA localized to the brain and notochord of developing zebrafish embryos. Homozygous deletion of CYLD resulted in embryonic death before 8 h post‐fertilization.
Conclusions
CYLD appears to play an important role in central nervous system development in zebrafish. Although severe embryonic death restricted analysis of homozygous mutants, further research into the role of CYLD in central nervous system development is warranted.
... NF-κB activity and cyclin D1 levels were upregulated in Cyld-KO mice. Furthermore, CYLD-deficient cancer cells are resistant to cell death induced by anticancer compounds [204][205][206]. CYLD preferentially cleaves K63-and M1linked polyubiquitin chains from substrates and cleaves K11-and K48-linked ubiquitin chains [207]. ...
The mechanisms and physiological implications of regulated cell death (RCD) have been extensively studied. Among the regulatory mechanisms of RCD, ubiquitination and deubiquitination enable post-translational regulation of signaling by modulating substrate degradation and signal transduction. Deubiquitinases (DUBs) are involved in diverse molecular pathways of RCD. Some DUBs modulate multiple modalities of RCD by regulating various substrates and are powerful regulators of cell fate. However, the therapeutic targeting of DUB is limited, as the physiological consequences of modulating DUBs cannot be predicted. In this review, the mechanisms of DUBs that regulate multiple types of RCD are summarized. This comprehensive summary aims to improve our understanding of the complex DUB/RCD regulatory axis comprising various molecular mechanisms for diverse physiological processes. Additionally, this review will enable the understanding of the advantages of therapeutic targeting of DUBs and developing strategies to overcome the side effects associated with the therapeutic applications of DUB modulators.
... Patients affected from CYLD truncations suffer from cylindromatosis, a disease characterized by the formation of multiple tumors in the skin [217]. Similarly, Cyld-deficient mice are more susceptible to colitis-induced tumorigenesis and chemically-induced skin tumors due to increased NFκB activation [218,219]. ...
The rapid and dynamic activation of the innate immune system is achieved through complex signaling networks regulated by post-translational modifications modulating the subcellular localization, activity, and abundance of signaling molecules. Many constitutively expressed signaling molecules are present in the cell in inactive forms, and become functionally activated once they are modified with ubiquitin, and, in turn, inactivated by removal of the same post-translational mark. Moreover, upon infection resolution a rapid remodeling of the proteome needs to occur, ensuring the removal of induced response proteins to prevent hyperactivation. This review discusses the current knowledge on the negative regulation of innate immune signaling pathways by deubiquitinating enzymes, and through degradative ubiquitination. It focusses on spatiotemporal regulation of deubiquitinase and E3 ligase activities, mechanisms for re-establishing proteostasis, and degradation through immune-specific feedback mechanisms vs. general protein quality control pathways.
... Upregulation of FUCA1 was found to suppress tumor growth and promote chemotherapy-induced cell death. 54,55 These results are consistent with previous findings. Our prediction model performed well in predicting overall survival in TCGA and in the Zhengzhou external validation cohort. ...
Background:
Immunotherapy has revolutionized the treatment of clear cell renal cell carcinoma (ccRCC). However, the therapy is constrained by drug resistance. Therefore, further characterization of immune infiltration in ccRCC is needed to improve its efficacy.
Methods:
Here, we adopted the CIBERSORT method to analyze the level of 22 immune cells, and analyzed the correlation of immune cells and clinical parameters in ccRCC in The Cancer Genome Atlas. We used consensus clustering to cluster ccRCC and identified differently expressed genes (DEGs) between hot and cold tumors using the "Limma" package, and then performed enrichment analysis of DEGs. Finally, we constructed and validated a Cox regression model using the "survival", "glmnet", and "survivalROC" packages, implemented in R.
Results:
Regulatory T cells upregulated in tumor tissue increased during tumor progression, and correlated with poor overall survival in ccRCC. Consensus clustering identified four clusters of ccRCC. To elucidate the underlying mechanisms of immune cell infiltration, we subdivided these four clusters into two major types, immune hot and cold, and identified DEGs between them. The results revealed different transcription profiles in the two tumor types, with hot tumors being enriched in immune-related signaling, whereas cold tumors were enriched in extracellular matrix remodeling and the phosphatidylinositol 3-kinase-AKT (PI3K/AKT) pathway. We further identified hub genes and prognostic-related genes from the DEGs, and constructed a Cox regression model for predicting the overall survival of patients with ccRCC. The areas under the receiver operating characteristics curve for the risk model for the training, testing, and external Zhengzhou validation cohorts were 0.834, 0.733, and 0.812, respectively. Notably, gene sets in the prediction model could also predict the overall survival of patients receiving immunotherapy.
Conclusion:
These findings provide a comprehensive characterization of immune infiltration in ccRCC, while the constructed model can be used effectively to predict the overall survival of ccRCC patients.
Ubiquitination is an essential regulator of most, if not all, signalling pathways, and defects in cellular signalling are central to cancer initiation, progression and, eventually, metastasis. The attachment of ubiquitin signals by E3 ubiquitin ligases is directly opposed by the action of approximately 100 deubiquitinating enzymes (DUBs) in humans. Together, DUBs and E3 ligases coordinate ubiquitin signalling by providing selectivity for different substrates and/or ubiquitin signals. The balance between ubiquitination and deubiquitination is exquisitely controlled to ensure properly coordinated proteostasis and response to cellular stimuli and stressors. Not surprisingly, then, DUBs have been associated with all hallmarks of cancer. These relationships are often complex and multifaceted, highlighted by the implication of multiple DUBs in certain hallmarks and by the impact of individual DUBs on multiple cancer-associated pathways, sometimes with contrasting cancer-promoting and cancer-inhibiting activities, depending on context and tumour type. Although it is still understudied, the ever-growing knowledge of DUB function in cancer physiology will eventually identify DUBs that warrant specific inhibition or activation, both of which are now feasible. An integrated appreciation of the physiological consequences of DUB modulation in relevant cancer models will eventually lead to the identification of patient populations that will most likely benefit from DUB-targeted therapies.
Deubiquitylating enzymes (DUBs) maintain relative homeostasis of the cellular ubiquitome by removing the post‐translational modification ubiquitin moiety from substrates. Numerous DUBs have been demonstrated specificity for cleaving a certain type of ubiquitin linkage or positions within ubiquitin chains. Moreover, several DUBs perform functions through specific protein–protein interactions in a catalytically independent manner, which further expands the versatility and complexity of DUBs’ functions. Dysregulation of DUBs disrupts the dynamic equilibrium of ubiquitome and causes various diseases, especially cancer and immune disorders. This review summarizes the Janus‐faced roles of DUBs in cancer including proteasomal degradation, DNA repair, apoptosis, and tumor metastasis, as well as in immunity involving innate immune receptor signaling and inflammatory and autoimmune disorders. The prospects and challenges for the clinical development of DUB inhibitors are further discussed. The review provides a comprehensive understanding of the multi‐faced roles of DUBs in cancer and immunity.
Intense ultraviolet (UV) exposure can cause phototoxic reactions, such as skin inflammation, resulting in injury. UV is a direct cause of DNA damage, but the mechanisms underlying transcriptional regulation within cells after DNA damage are unclear. The bioinformatics analysis of transcriptome sequencing data from UV-irradiated and non-UV-irradiated skin showed that transcription-related proteins, such as HSF4 and COIL, mediate cellular response to UV irradiation. HSF4 and COIL can form a complex under UV irradiation, and the preference for binding target genes changed because of the presence of a large number of R-loops in cells under UV irradiation and the ability of COIL to recognize R-loops. The regulation of target genes was altered by the HSF4-COIL complex, and the expression of inflammation and ageing-related genes, such as Atg7, Tfpi, and Lims1, was enhanced. A drug screen was performed for the recognition sites of COIL and R-loop. N6-(2-hydroxyethyl)-adenosine can competitively bind COIL and inhibit the binding of COIL to the R-loop. Thus, the activation of downstream inflammation-related genes and inflammatory skin injury was inhibited.
CYLD lysine 63 deubiquitinase (CYLD) is a ubiquitin hydrolase with important roles in immunity and cancer. Complete CYLD ablation, truncation and expression of alternate isoforms, including short CYLD, drive distinct phenotypes and offer insights into CYLD function in inflammation, cell death, cell cycle progression and cell transformation. Research in diverse model systems has shown that these are mediated via CYLD regulation of cellular pathways including the NF-κB, Wnt and TGF-β pathways. Recent biochemical advances and models have offered new insights into the regulation and function of CYLD. In addition, recent discoveries of gain-of-function germline pathogenic CYLD variants in patients with a neurodegenerative phenotype contrast with the more widely known loss-of-function mutations seen in patients with CYLD cutaneous syndrome and with sporadic cancers. Here, we provide a current review of mechanistic insights into CYLD function gained from CYLD animal models, as well as an update on the role of CYLD in human disease.
Background and study aim:
There is currently a lack of sensitive biomarkers for the diagnosis of hepatocellular carcinoma (HCC). Low expression of cylindromatosis (CYLD), a tumor suppressor gene that encodes a deubiquitinase, is associated with the development of HCC. The present study, therefore, aimed to determine the clinical utility of measuring CYLD expression in the early diagnosis of HCC.
Patients and methods:
The present study comprised 257 patients from the Affiliated Hospital of Qingdao University including 90 patients with HCC, 41 patients with liver cirrhosis (LC), 46 patients with hepatitis B (HB), and 80 healthy controls. qPCR was used to measure the amounts of CYLD mRNA in stored blood samples. The sensitivity and specificity of CYLD mRNA in diagnosing HCC was analyzed using receiver operator characteristic (ROC) curves. We also obtained HCC data from the Oncomine database to further verify our results.
Results:
The relative levels of CYLD mRNA in peripheral blood from patients with HCC (median, 0.060; interquartile range [IQR], 0.019-0.260) was significantly lower than in blood from patients with LC (median, 3.732; IQR, 0.648-14.573), HB (median, 0.419; IQR, 0.255-1.809) and healthy controls (median, 1.262; IQR, 0.279-3.537; P < 0.05). CYLD mRNA levels in peripheral blood were significantly higher in patients with LC compared to healthy controls and patients with HB. Oncomine data demonstrated that CYLD mRNA expression levels in HCC tissues were significantly lower than in normal liver tissues. ROC analysis demonstrated that the combined use of peripheral blood levels of CYLD and AFP had the greatest diagnostic accuracy for HCC (area under the curve (AUC), 0.897; 95 % confidence interval [CI], 0.853-0.942). CYLD had utility as a supplementary marker to AFP for diagnosing HCC.
Conclusion:
Circulating levels of CYLD mRNA are significantly decreased in patients with HCC, indicating CYLD may have utility as a biomarker of HCC. Combined measurement of CYLD mRNA and AFP protein had the greatest diagnostic accuracy.
More than 400 single gene defects have been identified as inborn errors of immunity, including many arising from genes encoding proteins that affect NF-κB activity. We summarise the skin phenotypes in this subset of disorders and provide an overview of pathogenic mechanisms. NF-κB acts cell-intrinsically in basal epithelial cells during differentiation of skin appendages, influences keratinocyte proliferation and survival, and both responses to and amplification of inflammation, particularly TNF. Skin phenotypes include ectodermal dysplasia, reduction and hyperproliferation of keratinocytes, and aberrant recruitment of inflammatory cells, which often occur in combination. Phenotypes conferred by these rare monogenic syndromes often resemble those observed with more common defects. This includes oral and perineal ulceration and pustular skin disease as occurs with Behcet's disease, hyperkeratosis with microabscess formation similar to psoriasis, and atopic dermatitis. Thus, these genotype-phenotype relations provide diagnostic clues for this subset of IEIs, and also provide insights into mechanisms of more common forms of skin disease.
Bladder carcinoma (BC) is one of the most commonly diagnosed malignant cancers worldwide. Kelch-like protein 21 (KLHL21) has been shown to be involved in a number of human tumors. The study aimed to investigate the effects and mechanism of KLHL21 on BC progression. We found that KLHL21 expression was significantly decreased in human BC tissues and cell lines compared with the paired normal samples, and patients with lower KLHL21 expression exhibited poorer overall survival. In vitro studies then showed that KLHL21 over-expression significantly reduced the proliferation, migration and invasion in BC cells, while KLHL21 knockdown markedly accelerated the proliferative, migratory and invasive properties of BC cells. Animal studies confirmed that KLHL21 exhibited anti-tumor function in the xenograft mouse models, as indicated by the reduced tumor growth rates, and mice with KLHL21 knockdown showed the opposite tumor growth profile. Additionally, we found that KLHL21 negatively mediated the nuclear factor-κB (NF-κB) signaling activation, as well as its down-streaming molecules involved in the biological regulation of cell survival, death and migratory processes. Mechanistically, cylindromatosis (CYLD) expression levels were significantly up-regulated in BC cells over-expressing KLHL21, but were down-regulated upon KLHL21 knockdown. We further uncovered that KLHL21 directly interacted with CYLD in BC cells. Of note, we found that KLHL21 mainly in cytoplasm could restrain CYLD degradation by prohibiting its ubiquitination in BC cells. More importantly, our in vitro experiments displayed that KLHL21-inhibited progression and NF-κB/p65 activation in BC cells were completely abolished by CYLD deletion, revealing that CYLD expression was required for KLHL21 to perform its anti-tumor function in BC. Collectively, all these findings uncovered that KLHL21/CYLD axis may be a promising therapeutic target for BC treatment.
The transcription factor NF-κB plays a pivotal role in the adaptive immune response. Pathogenic variants in NFKB1 are the most common genetic etiology of common variable immunodeficiency (CVID). Patients frequently present with impaired terminal B cell differentiation, autoimmunity, and hyperinflammatory immune dysregulation. NF-κB signaling and target gene expression are expected to be dysregulated in NFKB1 -mutated patients. Here, we performed a multi-omics characterization of B cells from a cohort of clinically affected and unaffected NFKB1 mutation carriers. Our analysis identified specific epigenetic dysregulation and gene expression differences on B cells from NFKB1 -mutated patients. We observed an aberrant expression of negative regulators of NF-κB signaling in NFKB1 mutation carriers, which may be a key factor for the autoinflammatory phenotype of these patients. Moreover, our analysis points towards a dysregulation of XBP1 and BCL3 , key players of B cell activation and proliferation at different stages of B cell differentiation. The reduced expression of negative regulators of the NF-κB network is likely to be one of several mechanisms responsible for the aberrant NF-κB signaling, which impairs the maintenance of a normal humoral immune response. In summary, our findings highlight epigenetic and gene expression changes in B cells associated with NFKB1 mutations. Our data give insight into future therapeutic opportunities for patients with NFKB1 (haplo)insufficiency.
BackgroundHDAC6, a structurally and functionally distinct member of the HDAC family, is an integral part of multiple cellular functions such as cell proliferation, apoptosis, senescence, DNA damage and genomic stability, all of which when deregulated contribute to carcinogenesis. Among several HDAC family members known so far, HDAC6 holds a unique position. It differs from the other HDAC family members not only in terms of its subcellular localization, but also in terms of its substrate repertoire and hence cellular functions. Recent findings have considerably expanded the research related to the substrate pool, biological functions and regulation of HDAC6. Studies in HDAC6 knockout mice highlighted the importance of HDAC6 as a cell survival player in stressful situations, making it an important anticancer target. There is ample evidence stressing the importance of HDAC6 as an anti-cancer synergistic partner of many chemotherapeutic drugs. HDAC6 inhibitors have been found to enhance the effectiveness of conventional chemotherapeutic drugs such as DNA damaging agents, proteasome inhibitors and microtubule inhibitors, thereby highlighting the importance of combination therapies involving HDAC6 inhibitors and other anti-cancer agents.Conclusions
Here, we present a review on HDAC6 with emphasis on its role as a critical regulator of specific physiological cellular pathways which when deregulated contribute to tumorigenesis, thereby highlighting the importance of HDAC6 inhibitors as important anticancer agents alone and in combination with other chemotherapeutic drugs. We also discuss the synergistic anticancer effect of combination therapies of HDAC6 inhibitors with conventional chemotherapeutic drugs.
Oxidative stress, which can cause DNA damage, can both activate TNF-R1 directly in the absence of TNF stimulation and phosphorylate c-Abl, thus promoting its cytoplasmic translocation. Persistent cytoplasmic localization of c-Abl has been associated with cellular transformation. c-Abl phosphorylates OTULIN at tyrosine 56, thereby disrupting its relationship with LUBAC. OTULIN-released LUBAC interacts with SPATA2 and is recruited to the TNF-R1sc, facilitating SPATA2-CYLD interaction. All these interactions are required for the activation of IKKβ to stimulate NF-κB transcriptional activity following genotoxic stress. IKKβ also induces the critical phosphorylation of CYLD at serine 568 to increase its deubiquitinating (DUB) activity required for the termination of signaling cascades. Contrary to the widespread belief that CYLD is an absolute tumor suppressor, CYLD initiates and terminates NF-κB activity by alternately using its oncoprotein and tumor suppressor activities, respectively. If IKKβ fails to achieve the DUB activity-inducing phosphorylation at serine 568, CYLD would operate in a sustained mode of oncogenic activity. The resulting dysregulated NF-κB activation and other accompanying pathologies will disrupt cellular homeostasis in favor of transformation.
Ubiquitination is a critical type of protein post-translational modification playing an essential role in many cellular processes. To date, more than eight types of ubiquitination exist, all of which are involved in distinct cellular processes based on their structural differences. Studies have indicated that activation of the ubiquitination pathway is tightly connected with inflammation-related diseases as well as cancer, especially in the non-proteolytic canonical pathway, highlighting the vital roles of ubiquitination in metabolic programming. Studies relating degradable ubiquitination through lys48 or lys11-linked pathways to cellular signaling have been well-characterized. However, emerging evidence shows that non-degradable ubiquitination (linked to lys6, lys27, lys29, lys33, lys63, and Met1) remains to be defined. In this review, we summarize the non-proteolytic ubiquitination involved in tumorigenesis and related signaling pathways, with the aim of providing a reference for future exploration of ubiquitination and the potential targets for cancer therapies.
Background
Keratinocytes are recipients of melanosomes. Although the chemical basis of melanogenesis is well documented, the molecular mechanism of melanosome transfer must be elucidated. TRPA1 is a member of the transient receptor potential A subfamily. Previous studies have shown that inhibition of TRPA1 activity reduces melanin synthesis inhuman primary melanocytes (HEMs); however, the mechanism remains unknown.
Objective
This study aimed to investigate the roles and mechanism(s) of action of TRPA1 in keratinocytes.
Methods
The correlation between TRPA1 expression levels and the ability of keratinocytes to phagocytize melanosomes was examined using melanin silver staining. TRPA1 depleted human primary keratinocytes (HEKs) and keratinocyte cell lines (HaCaT) were established using adenovirus-expressing shRNAs against TRPA1. The effects of TRPA1 on keratinocytes and HaCaT cells were determined using cell-based analyses, including light stimulation, calcium imaging, melanin phagocytosis, immunoblotting, and co-immunoprecipitation assays. The degree of epidermal pigmentation was determined in a guinea pig model.
Results
TRPA1 mediated the phagocytic activity of keratinocytes. TRPA1 knockdown markedly suppressed melanosome transport to keratinocytes. Mechanistically, TRPA1 was required for PAR-2-induced melanosome phagocytosis in keratinocytes. Furthermore, TRPA1 activation indirectly stabilized microtubules by promoting the competitive binding of CYLD and acetylated α-tubulin. In addition, bortezomib (PS-341), a proteasome inhibitor, increased TRPA1 and CYLD expression and promoted phagocytic activity both in vitro and in vivo.
Conclusions
Our findings firstly suggest that TRPA1 promotes melanosome transport in keratinocytes and reveal that TRPA1 is a regulator of PAR-2 activation and microtubule stability via the PAR-2/CYLD axis.
Receptor interacting protein 1 (RIP1) kinase is a critical regulator of inflammation and cell death signaling, and plays a crucial role in maintaining immune responses and proper tissue homeostasis. Mounting evidence argues for the importance of RIP1 post-translational modifications in control of its function. Ubiquitination by E3 ligases, such as inhibitors of apoptosis (IAP) proteins and LUBAC, as well as the reversal of these modifications by deubiquitinating enzymes, such as A20 and CYLD, can greatly influence RIP1 mediated signaling. In addition, cleavage by caspase-8, RIP1 autophosphorylation, and phosphorylation by a number of signaling kinases can greatly impact cellular fate. Disruption of the tightly regulated RIP1 modifications can lead to signaling disbalance in TNF and/or TLR controlled and other inflammatory pathways, and result in severe human pathologies. This review will focus on RIP1 and its many modifications with an emphasis on ubiquitination, phosphorylation, and cleavage, and their functional impact on the RIP1's role in signaling pathways.
Significance
The mechanisms underlying inflammatory disorders are poorly understood. In this study, we show that inappropriate cell death may cause uncontrolled inflammation. We found that CYLD, an enzyme that removes K63-linked polyubiquitination, is normally inhibited. But in the cpdm mouse strain that has a loss-of-function in the Sharpin gene, the brake on CYLD is no longer present. When CYLD is no longer inhibited, it turns on death signaling in cells exposed to the cytokine TNF, and the ensuing inappropriate cell death causes skin inflammation and other immune disorders in the cpdm mouse. Removing CYLD from the cpdm mouse prevents cell death and reverses the inflammation. We conclude that excessive CYLD activity leads to inappropriate cell death and inflammation.
The NADPH oxidase (Nox) family of enzymes is solely dedicated in the generation of reactive oxygen species (ROS). ROS generated by Nox are involved in multiple signaling cascades and a myriad of pathophysiological conditions including cancer. As such, ROS seem to have both detrimental and beneficial roles in a number of cellular functions, including cell signaling, growth, apoptosis and proliferation. Regulatory mechanisms are required to control the activity of Nox enzymes in order to maintain ROS balance within the cell. Here, we performed genome-wide screening for deubiquitinating enzymes (DUBs) regulating Nox organizer 1 (NoxO1) protein expression using a CRISPR/Cas9-mediated DUB-knockout library. We identified cylindromatosis (CYLD) as a binding partner regulating NoxO1 protein expression. We demonstrated that the overexpression of CYLD promotes ubiquitination of NoxO1 protein and reduces the NoxO1 protein half-life. The destabilization of NoxO1 protein by CYLD suppressed excessive ROS generation. Additionally, CRISPR/Cas9-mediated knockout of CYLD in PC-3 cells promoted cell proliferation, migration, colony formation and invasion in vitro. In xenografted mice, injection of CYLD-depleted cells consistently led to tumor development with increased weight and volume. Taken together, these results indicate that CYLD acts as a destabilizer of NoxO1 protein and could be a potential tumor suppressor target for cancer therapeutics.
Significance
Resistance to endocrine therapy is a major obstacle to managing breast cancer. Here, we introduce that tripartite motif protein TRIM47 enhances endocrine therapy resistance and facilitates breast cancer tumorigenesis. We showed that TRIM47 immunoreactivity scores in breast cancer specimens were positively associated with shorter disease-free survival of the patients with postoperative endocrine therapy. Overexpression of TRIM47 in breast cancer cells activated NF-κB signaling, which is partly due to the stabilization of PKC-ε and PKD3 by interacting with TRIM47. As an underlying mechanism, we demonstrated TRIM47-dependent modification of PKC-ε by atypical lysine 27–linked polyubiquitination. Our study implies that TRIM47 and its associated kinases can be potential diagnostic and therapeutic targets for patients with breast cancer refractory to endocrine therapy.
Due to the complexity of psoriasis, people have been searching for better treatment methods, and probiotics therapy is gaining acceptance. In this study, tumor necrosis factor-α (TNF-α) was used to stimulate abnormal proliferation of HaCaT cells, and the inhibitory effects of potential probiotics supernatants on hyperproliferation were compared. Among 102 strains, two with better suppressive effects, Bifidobacterium animalis CCFM1148 and Lactobacillus paracasei CCFM1147, were selected, whereas the other two were selected as control strains. Based on RNA-seq results, CCFM1148 and CCFM1147 inhibited TNF-α-induced activation of the nuclear factor kappa-B (NF-κB) signaling pathway and altered the cellular response to interleukin (IL)-8, while the two control strains did not. It was speculated that the inhibitory effect of strains was through the inhibiting of the activation of the NF-κB and the release of proinflammatory cytokines, and subsequent experiments confirmed this. Collectively, these findings suggest that CCFM1148 and CCFM1147 have potential to ameliorate psoriasis.
To clarify the genetic aberrations involved in the development and progression of hepatitis C virus-associated hepatocellular carcinoma (HCV-HCC), we investigated DNA copy number aberrations (DCNAs) in 19 surgically resected HCCs by conventional CGH and array CGH. Conventional CGH revealed that increases of DNA copy number were frequent at 1q (79% of the cases), 8q (37%), 6p (32%), and 10p (32%) and that decreases were frequent at 17p (79%), 16q (58%), 4q (53%), 13q (42%), 10q (37%), 1p (32%), and 8p (32%). In general, genes that showed DCNAs by array CGH were usually located in chromosomal regions with DCNAs detected by conventional CGH analysis. Increases in copy numbers of the LAMC2, TGFB2, and AKT3 genes (located on 1q) and decreases in copy numbers of FGR/SRC2 and CYLD (located on 1p and 16q, respectively) were observed in more than 30% of tumors, including small, well-differentiated carcinomas. These findings suggest that these genes are associated with the development of HCV-HCC. Increases of MOS, MYC, EXT1, and PTK2 (located on 8q) were detected exclusively in moderately and poorly differentiated tumors, suggesting that these alterations contribute to tumor progression. In conclusion, chromosomal and array CGH technologies allow identification of genes involved in the development and progression of HCV-HCC.Keywords: hepatocellular carcinoma, hepatitis C virus, carcinogenesis, comparative genomic hybridization, array CGH
Familial cylindromatosis is an autosomal dominant genetic predisposition to multiple tumours of the skin appendages. The susceptibility gene (CYLD) has previously been localized to chromosome 16q and has the genetic attributes of a tumour-suppressor gene (recessive oncogene). Here we have identified CYLD by detecting germline mutations in 21 cylindromatosis families and somatic mutations in 1 sporadic and 5 familial cylindromas. All mutations predict truncation or absence of the encoded protein. CYLD encodes three cytoskeletal-associated-protein-glycine-conserved (CAP-GLY) domains, which are found in proteins that coordinate the attachment of organelles to microtubules. CYLD also has sequence homology to the catalytic domain of ubiquitin carboxy-terminal hydrolases (UCH).
BCL3 is a candidate proto-oncogene involved in the recurring translocation t(14;19) found in some patients with chronic lymphocytic leukemia. BCL3 protein acts as an I kappa B in that it can specifically inhibit the DNA binding of NF-kappa B factors. Here, we demonstrate that BCL3 is predominantly a nuclear protein and provide evidence that its N terminus is necessary to direct the protein into the nucleus. In contrast to I kappa B alpha (MAD3), BCL3 does not cause NF-kappa B p50 to be retained in the cytoplasm; instead, in cotransfection assays, it alters the subnuclear localization of p50. The two proteins colocalize, suggesting that they interact in vivo. Further immunofluorescence experiments showed that a mutant p50, lacking a nuclear localization signal and restricted to the cytoplasm, is brought into the nucleus in the presence of BCL3. Correspondingly, a wild-type p50 directs into the nucleus a truncated BCL3, which, when transfected alone, is found in the cytoplasm. We tested whether BCL3 could overcome the cytoplasmic retention of p50 by I kappa B alpha. Results from triple cotransfection experiments with BCL3, I kappa B alpha, and p50 implied that BCL3 can successfully compete with I kappa B alpha and bring p50 into the nucleus; thus, localization of NF-kappa B factors may be affected by differential expression of I kappa B proteins. These novel properties of BCL3 protein further establish BCL3 as a distinctive member of the I kappa B family.
The product of the putative proto-oncogene bcl-3 is an I kappa B-like molecule with novel binding properties specific for a subset of the rel family of transcriptional regulators. In vitro, Bcl-3 protein specifically inhibited the DNA binding of both the homodimeric NF-kappa B p50 subunit and a closely related homolog, p52 (previously p49), to immunoglobulin kappa NF-kappa B DNA motifs. Bcl-3 could catalyze the removal of these proteins from DNA. At concentrations that significantly inhibited DNA binding by homodimeric p50, Bcl-3 did not inhibit binding of reconstituted heterodimeric NF-kappa B (p50:p65), a DNA-binding homodimeric form of p65, or homodimers of c-Rel. Phosphatase treatment of Bcl-3 partially inactivated its inhibitory properties, implicating a role for phosphorylation in the regulation of Bcl-3 activity. Bcl-3, like p50, localizes to the cell nucleus. In cells cotransduced with Bcl-3 and p50, both molecules could be found in the nucleus of the same cells. Interestingly, coexpression of Bcl-3 with a p50 mutant deleted for its nuclear-localizing signal resulted in the relocalization of Bcl-3 to the cytoplasm, showing that the proteins interact in the cell. These properties contrast Bcl-3 to classically defined I kappa B, which maintains heterodimeric NF-kappa B p50:p65 in the cytoplasm through specific interactions with the p65 subunit. Bcl-3 appears to be a nuclear, I kappa B-related molecule that regulates the activity of homodimeric nuclear p50 and its homolog p52.
The candidate oncoprotein BCL-3 has been shown to function as a transcriptional co-activator for homodimers of NF-kappaB p50 and p50B. We expressed BCL-3 ectopically in pro-B cell lines and found that these cells exhibited a dramatic increase in nuclear kappaB motif binding activity of p50 homodimers containing BCL-3 in the complex. Co-transfection and in vitro reconstitution experiments revealed that the complex of p50 with its precursor p105 (p50-p105), which was shown to accumulate in the cytoplasm of the pro-B cell lines, is required for induction of DNA binding of p50 homodimers by BCL-3. However, we could see no in vivo or in vitro evidence of a BCL-3-induced increase in proteolytic processing. Instead, BCL-3-mediated reorganization of NFKB1 subunits was demonstrated in vitro. Immunofluorescence staining clearly demonstrated that the transition from cytoplasmic p50-p105 to nuclear p50 homodimers was induced by BCL-3 expression. Thus BCL-3 has versatile functions: cytoplasmic activation of p50 homodimers, their nuclear translocation and, as previously shown, modulation of the transcriptional machinery in the nucleus.
Nuclear factor kappa B (NF-κB) has been implicated in the regulation of cell proliferation, transformation, and tumor development. We provide evidence for a direct link between NF-κB activity and cell cycle regulation. NF-κB was found to stimulate transcription of cyclin D1, a key regulator of G
1
checkpoint control. Two NF-κB binding sites in the human cyclin D1 promoter conferred activation by NF-κB as well as by growth factors. Both levels and kinetics of cyclin D1 expression during G
1
phase were controlled by NF-κB. Moreover, inhibition of NF-κB caused a pronounced reduction of serum-induced cyclin D1-associated kinase activity and resulted in delayed phosphorylation of the retinoblastoma protein. Furthermore, NF-κB promotes G
1
-to-S-phase transition in mouse embryonal fibroblasts and in T47D mammary carcinoma cells. Impaired cell cycle progression of T47D cells expressing an NF-κB superrepressor (IκBαΔN) could be rescued by ectopic expression of cyclin D1. Thus, NF-κB contributes to cell cycle progression, and one of its targets might be cyclin D1.
Modulation of the balance between pro- and antiangiogenic factors holds great promise for the treatment of a broad spectrum of human disease ranging from ischemic heart disease to cancer. This requires both the identification of angiogenic regulators and their efficient delivery to target organs. Here, we demonstrate the use of a noncatalytic fragment of matrix metalloproteinase 2 (termed PEX) delivered by lentiviral vectors in different angiogenesis models. Transduction of human endothelial cells with PEX virus suppressed endothelial invasion and formation of capillary-like structures without affecting chemotaxis in vitro. Lentiviral delivery of PEX blocked basic fibroblast growth factor-induced matrix metalloproteinase 2 activation and angiogenesis on chicken chorioallantoic membranes. PEX expression also inhibited tumor-induced angiogenesis and tumor growth in a nude mouse model. Thus, our study shows that lentiviral vectors can deliver sufficient quantities of antiangiogenic substances to achieve therapeutic effects in vivo.
In an often rapidly changing environment, cells must adapt by monitoring and reacting quickly to extracellular stimuli detected by membrane-bound receptors and proteins. Reversible phosphorylation of intracellular regulatory proteins has emerged as a crucial mechanism effecting the transmission and modulation of such signals and is determined by the relative activities of protein kinases and phosphatases within the cell. These are often arranged into complex signaling networks that may function independently or be subject to cross-regulation. Recently, genetic and biochemical analyses have identified the universally conserved mitogen-activated protein (MAP) kinase cascade as one of the most ubiquitous signal transduction systems. This pathway is activated after a variety of cellular stimuli and regulates numerous physiological processes, particularly the cell division cycle. Progression through the cell cycle is critically dependent on the presence of environmental growth factors and stress stimuli, and failure to correctly integrate such signals into the cell cycle machinery can lead to the accumulation of genetic damage and genomic instability characteristic of cancer cells. Here we focus on the MAP kinase cascade and discuss the molecular mechanisms by which these extensively studied signaling pathways influence cell growth and proliferation.
NF-kappa B is a transcription factor whose nuclear residence is controlled by I kappa B family members. In the NF-kappa B-I kappa B autoregulatory loop, activated (nuclear) Rel A.NF-kappa B1 induces the resynthesis of I kappa B alpha recapturing nuclear Rel A back into the cytoplasm within 1 h of stimulation. In contrast, NF-kappa B1 subunits redistribute more slowly into the cytoplasm (from 6 to 12 h). Here we examine the role of inducible cytoplasmic BCL-3 expression in terminating nuclear NF-kappa B1. Although BCL-3 is a nuclear protein in B lymphocytes, surprisingly, BCL-3 is primarily a cytoplasmic protein in HepG2 cells. Cytoplasmic BCL-3 abundance is induced 6-12 h after tumor necrosis factor-alpha stimulation where it complexes with NF-kappa B1 homodimers. Moreover, BCL-3 mRNA and protein expression are induced by NF-kappa B-activating agents. Two observations are interpreted to indicate that bcl-3 is transactivated by NF-kappa B/Rel A: 1) expression of a dominant negative NF-kappa B inhibitor blocks tumor necrosis factor-alpha-induced BCL-3 expression and 2) expression of constitutively active Rel A is sufficient to induce BCL-3 expression. In gene transfer studies, we identify two high affinity NF-kappa B-binding sites, kappa B1 (located at -872 to -861 nucleotides) and kappa B2 (-106 to -96 nucleotides), and although both bind with high affinity to Rel A, only kappa B2 is required for NF-kappa B-dependent induction of the native BCL-3 promoter. Down-regulation of BCL-3 induction results in prolonged, enhanced NF-kappa B1 binding and increased NF-kappa B-dependent transcription. Together, these data suggest the presence of an NF-kappa B-BCL-3 autoregulatory loop important in terminating NF-kappa B1 action and that individual NF-kappa B isoforms are actively terminated through coordinate induction of inhibitory I kappa B molecules to restore cellular homeostasis.
Bcl-3 is a distinctive member of the IκB family of NF-κB inhibitors because it can function to coactivate transcription. A
potential involvement of Bcl-3 in oncogenesis is highlighted by the fact that it was cloned due to its location at a breakpoint
junction in some cases of human B-cell chronic lymphocytic leukemia and that it is highly expressed in human breast tumor
tissue. To analyze the effects of Bcl-3 dysregulation in breast epithelial cells, we created stable immortalized human breast
epithelial cell lines either expressing Bcl-3 or carrying the corresponding vector control plasmid. Analysis of the Bcl-3-expressing
cells suggests that these cells have a shortened G1 phase of the cell cycle as well as a significant increase in hyperphosphorylation of the retinoblastoma protein. Additionally,
the cyclin D1 gene was found to be highly expressed in these cells. Upon further analysis, Bcl-3, acting as a coactivator
with NF-κB p52 homodimers, was demonstrated to directly activate the cyclin D1 promoter through an NF-κB binding site. Therefore,
our results demonstrate that dysregulated expression of Bcl-3 potentiates the G1 transition of the cell cycle by stimulating the transcription of the cyclin D1 gene in human breast epithelial cells.
Anaplastic large cell lymphoma (ALCL) with t(2;5)(p23;q35) and Hodgkin disease (HD) share many cellular features, including expression of CD30. We compared gene expression profiles of 4 ALCL (Karpas 299, SU-DHL-1, DEL, SR-786) and 3 HD cell lines and found that BCL3, which encodes a nuclear protein belonging to the I kappa B family of inhibitors of nuclear factor-kappa B (NF-kappa B) transcriptional factors, was expressed at higher levels in ALCL than HD. Northern and Western blotting analyses confirmed the high-level expression of BCL3 in ALCL at both mRNA and protein levels. We established a real-time reverse transcriptase-mediated polymerase chain reaction assay to measure the BCL3 mRNA level and found a predominant level of BCL3 expression in t(2;5)(+) ALCL; the levels of cell lines and clinical materials were comparable to or higher than that of a B-cell chronic lymphocytic leukemia carrying t(14;19)(q32;q13). Southern blotting and fluorescence in situ hybridization disclosed that the BCL3 gene copies were amplified in SU-DHL-1, whereas Karpas 299 carried 4 BCL3 gene loci. The BCL3 gene contains 2 cytosine-guanine dinucleotide (CpG) islands, and the intragenic 3' CpG was entirely demethylated in SU-DHL-1 and DEL. In contrast to HD, in which NF-kappa B was constitutively activated, ALCL cells consistently showed (p50)(2) homodimer binding activity on electrophoretic mobility shift assay. It is suggested that the high-level nuclear Bcl-3 sequesters the (p50)(2) homodimer to the nucleus, which may account for the contradictory effect of CD30 stimulation on ALCL and HD. We propose that BCL3 is overexpressed by genetic and epigenetic modifications, potentially contributing to the development of t(2;5)(+) ALCL.
NF-kappaB transcription factors have key roles in inflammation, immune response, oncogenesis and protection against apoptosis. In most cells, these factors are kept inactive in the cytoplasm through association with IkappaB inhibitors. After stimulation by various reagents, IkappaB is phosphorylated by the IkappaB kinase (IKK) complex and degraded by the proteasome, allowing NF-kappaB to translocate to the nucleus and activate its target genes. Here we report that CYLD, a tumour suppressor that is mutated in familial cylindromatosis, interacts with NEMO, the regulatory subunit of IKK. CYLD also interacts directly with tumour-necrosis factor receptor (TNFR)-associated factor 2 (TRAF2), an adaptor molecule involved in signalling by members of the family of TNF/nerve growth factor receptors. CYLD has deubiquitinating activity that is directed towards non-K48-linked polyubiquitin chains, and negatively modulates TRAF-mediated activation of IKK, strengthening the notion that ubiquitination is involved in IKK activation by TRAFs and suggesting that CYLD functions in this process. Truncations of CYLD found in cylindromatosis result in reduced enzymatic activity, indicating a link between impaired deubiquitination of CYLD substrates and human pathophysiology.
Protein modification by the conjugation of ubiquitin moieties--ubiquitination--plays a major part in many biological processes, including cell cycle and apoptosis. The enzymes that mediate ubiquitin-conjugation have been well-studied, but much less is known about the ubiquitin-specific proteases that mediate de-ubiquitination of cellular substrates. To study this gene family, we designed a collection of RNA interference vectors to suppress 50 human de-ubiquitinating enzymes, and used these vectors to identify de-ubiquitinating enzymes in cancer-relevant pathways. We report here that inhibition of one of these enzymes, the familial cylindromatosis tumour suppressor gene (CYLD), having no known function, enhances activation of the transcription factor NF-kappaB. We show that CYLD binds to the NEMO (also known as IKKgamma) component of the IkappaB kinase (IKK) complex, and appears to regulate its activity through de-ubiquitination of TRAF2, as TRAF2 ubiquitination can be modulated by CYLD. Inhibition of CYLD increases resistance to apoptosis, suggesting a mechanism through which loss of CYLD contributes to oncogenesis. We show that this effect can be relieved by aspirin derivatives that inhibit NF-kappaB activity, which suggests a therapeutic intervention strategy to restore growth control in patients suffering from familial cylindromatosis.
Cylindromas are benign adnexal skin tumors caused by germline mutations in the CYLD gene. In most cases the second wild-type allele is lost in tumor tissue, suggesting that CYLD functions as tumor suppressor. CYLD is a protein of 956 amino acids harboring a functional deubiquitinating domain at the COOH-terminal end. To shed more light on the function of CYLD, we have performed a yeast two hybrid screen using an HaCaT cDNA library that identified the RING finger protein TRIP (TRAF-interacting protein) as interactor with full-length CYLD. Mapping of the interacting domains revealed that the central domain of CYLD binds to the COOH-terminal end of TRIP. Far Western analysis and coimmunoprecipitations in mammalian cells confirmed that full-length CYLD binds to the COOH-terminal domain of TRIP. Because TRIP is an inhibitor of nuclear factor (NF)-kappaB activation by tumor necrosis factor (TNF), the effect of CYLD on NF-kappaB activation was investigated in HeLa cells. The results established that CYLD down-regulates NF-kappaB activation by TNF-alpha. The inhibition by CYLD depends on the presence of the central domain interacting with TRIP and its deubiquitinating activity. These findings indicate that cylindromas arise through constitutive NF-kappaB activation leading to hyperproliferation and tumor growth.
Ubiquitin is a small polypeptide that is conjugated to proteins and commonly serves as a degradation signal. The attachment of ubiquitin (Ub) to a substrate proceeds through a multi-enzyme cascade involving an activating enzyme (E1), a conjugating enzyme (E2), and a protein ligase (E3). We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11. We now show that the import mechanism for UbcM2 and two other human class III E2s (UbcH6 and UBE2E2) uniquely requires the covalent attachment of Ub to the active site cysteine of these enzymes. This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes. Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.
EBV latent infection is associated with the development of lymphoid and epithelial malignancies such as nasopharyngeal carcinoma (NPC). The EBV latent membrane protein 1 (LMP1) acts as a constitutively active tumor necrosis factor receptor and activates cellular signaling pathways such as c-Jun-NH(2)-terminal kinase, cdc42, Akt, and nuclear factor (NF)-kappaB. In epithelial cells, two regions of LMP1 induce specific forms of NF-kappaB. COOH-terminal activating region 2 only activates p52/p65 dimers, whereas COOH-terminal activating region 1 activates p50/p50, p50/p52, and p52/p65 dimers and also uniquely up-regulates the epidermal growth factor receptor (EGFR) at the mRNA level. Deregulation of specific NF-kappaB members is associated with the development of many cancers. In this study, the status of NF-kappaB activation was investigated in NPC to determine which NF-kappaB dimers may contribute to the development of NPC. Electrophoretic mobility shift assay, immunoblot, ELISA, and immunohistochemistry data demonstrate that in NPC, NF-kappaB p50 homodimers are specifically activated, and this activation is not dependent on LMP1 expression. Coimmunoprecipitation assays indicate that homodimers are bound to the transcriptional coactivator Bcl-3, and chromatin immunoprecipitation indicates that this complex is bound to NF-kappaB consensus motifs within the egfr promoter in NPC. The discrete yet striking NF-kappaB p50 activation in NPC suggests that p50/p50 homodimers may be important factors in the development of NPC and may contribute to oncogenesis through transcriptional up-regulation of target genes through their interaction with Bcl-3.
BCL3 is a candidate proto-oncogene involved in the recurring translocation t(14;19) found in some patients with chronic lymphocytic leukemia. BCL3 protein acts as an I kappa B in that it can specifically inhibit the DNA binding of NF-kappa B factors. Here, we demonstrate that BCL3 is predominantly a nuclear protein and provide evidence that its N terminus is necessary to direct the protein into the nucleus. In contrast to I kappa B alpha (MAD3), BCL3 does not cause NF-kappa B p50 to be retained in the cytoplasm; instead, in cotransfection assays, it alters the subnuclear localization of p50. The two proteins colocalize, suggesting that they interact in vivo. Further immunofluorescence experiments showed that a mutant p50, lacking a nuclear localization signal and restricted to the cytoplasm, is brought into the nucleus in the presence of BCL3. Correspondingly, a wild-type p50 directs into the nucleus a truncated BCL3, which, when transfected alone, is found in the cytoplasm. We tested whether BCL3 could overcome the cytoplasmic retention of p50 by I kappa B alpha. Results from triple cotransfection experiments with BCL3, I kappa B alpha, and p50 implied that BCL3 can successfully compete with I kappa B alpha and bring p50 into the nucleus; thus, localization of NF-kappa B factors may be affected by differential expression of I kappa B proteins. These novel properties of BCL3 protein further establish BCL3 as a distinctive member of the I kappa B family.
Bcl-3 is a proto-oncogene involved in the chromosomal translocation t(14;19) found in some patients with chronic lymphocytic leukemia. It shares structural similarities with and is a member of the IκB family of proteins. In this report, involvement of Bcl-3in hematopoietic growth factor-stimulated erythroid proliferation and differentiation was examined. In TF-1 cells, an erythroleukemia cell line, granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) greatly enhanced Bcl-3 expression at both the protein and mRNA levels in association with stimulation of proliferation. Bcl-3 protein was also highly expressed in early burst-forming unit-erythroid (BFU-E)–derived erythroid precursors (day 7) and decreased during maturation (days 10 and 14), suggesting that Bcl-3 is involved in normal erythroid proliferation. In these hematopoietic cells, Bcl-3 was hyperphosphorylated. GM-CSF and Epo modulated the subcellular localization of Bcl-3. Upon stimulation of TF-1 cells with GM-CSF or Epo, the nuclear translocation ofBcl-3 was dramatically enhanced. Overexpression of Bcl-3 in TF-1 cells by transient transfection along with the NF-κB factors p50 or p52 resulted in significant induction of an human immunodeficiency virus–type 1 (HIV-1) κB-TATA-luceriferase reporter plasmid, demonstrating that Bcl-3 has a positive role in transactivation of κB-containing genes in erythroid cells. Stimulation with GM-CSF enhanced c-myb mRNA expression in these cells. Bcl-3 in nuclear extracts of TF-1 cells bound to a κB enhancer in the c-mybpromoter together with NF-κB2/p52 and this binding activity was enhanced by GM-CSF stimulation. Furthermore, cotransfection of Bcl-3 with p52 or p50 in TF-1 cells resulted in significant activation of ac-myb κB-TATA-luceriferase reporter plasmid. These findings suggest that Bcl-3 may participate in the transcriptional regulation of certain κB-containing genes involved in hematopoiesis, includingc-myb.
© 1998 by The American Society of Hematology.
The product of the putative proto-oncogene bcl-3 is an I kappa B-like molecule with novel binding properties specific for a subset of the rel family of transcriptional regulators. In vitro, Bcl-3 protein specifically inhibited the DNA binding of both the homodimeric NF-kappa B p50 subunit and a closely related homolog, p52 (previously p49), to immunoglobulin kappa NF-kappa B DNA motifs. Bcl-3 could catalyze the removal of these proteins from DNA. At concentrations that significantly inhibited DNA binding by homodimeric p50, Bcl-3 did not inhibit binding of reconstituted heterodimeric NF-kappa B (p50:p65), a DNA-binding homodimeric form of p65, or homodimers of c-Rel. Phosphatase treatment of Bcl-3 partially inactivated its inhibitory properties, implicating a role for phosphorylation in the regulation of Bcl-3 activity. Bcl-3, like p50, localizes to the cell nucleus. In cells cotransduced with Bcl-3 and p50, both molecules could be found in the nucleus of the same cells. Interestingly, coexpression of Bcl-3 with a p50 mutant deleted for its nuclear-localizing signal resulted in the relocalization of Bcl-3 to the cytoplasm, showing that the proteins interact in the cell. These properties contrast Bcl-3 to classically defined I kappa B, which maintains heterodimeric NF-kappa B p50:p65 in the cytoplasm through specific interactions with the p65 subunit. Bcl-3 appears to be a nuclear, I kappa B-related molecule that regulates the activity of homodimeric nuclear p50 and its homolog p52.
We describe the morphology and comparative genomic hybridization findings in a tumor for which we propose the term “spiradenocylindroma” of the kidney. The tumor arose in the wall of a renal cyst in an otherwise healthy male patient who had a favorable clinical course after nephrectomy. Tumor cells formed either large nodules exhibiting a solid or trabecular architecture with conspicuous perivascular spaces or cylindromatous small tumor cell islands arranged in a jigsaw pattern. Focally, there were interspersed tubular structures and tumor cell rosettes with central deposits of periodic acid–Schiff-positive material. A minor tumor component showed epidermoid differentiation. The tumor cells were strongly positive for cytokeratins 5/6, high molecular weight cytokeratins 34βE12 and AE1/3, and E-cadherin, but only weakly positive for cytokeratins 7, 8, 18, 19, and epithelial membrane antigen. Focal reactivity for actin, vimentin, and S-100 protein or lysozyme and α 1 -antichymotrypsin within tubular and cylindromatous areas suggested myoepithelial and apocrine differentiation, respectively. By comparative genomic hybridization, the only abnormality was loss of the long arm of chromosome 16 and gain of genetic material on the short arm of chromosome 16, suggesting isochromosome i(16p). This finding is unique among renal neoplasms and implies loss of heterozygosity at 16q12–13 of the CYLD1 gene that is critically involved in the oncogenesis of familial cylindromatosis and some sporadic spiradenocylindromas. We conclude that somatic mutation of the CYLD1 gene outside the skin can have a role in the oncogenesis of tumors with cylindromatous features.
Members of the NF-κB/Rel transcription factor family have been shown recently to be required for cellular transformation by oncogenic Ras and by other oncoproteins and to suppress transformation-associated apoptosis. Furthermore, NF-κB has been shown to be activated by several oncoproteins including HER2/Neu, a receptor tyrosine kinase often expressed in human breast cancer. Human breast cancer cell lines, human breast tumors and normal adjacent tissue were analysed by gel mobility shift assay, immunoblotting of nuclear extracts and immunohistochemistry for activation of NF-κB. Furthermore, RNA levels for NF-κB-activated genes were analysed in order to determine if NF-κB is functionally active in human breast cancer. Our data indicate that the p65/RelA subunit of NF-κB is activated (i.e., nuclear) in breast cancer cell lines. However, breast tumors exhibit an absence or low level of nuclear p65/RelA but show activated c-Rel, p50 and p52 as compared to nontumorigenic adjacent tissue. Additionally, the IκB homolog Bcl-3, which functions to stimulate transcription with p50 or p52, was also activated in breast tumors. There was no apparent correlation between estrogen receptor status and levels of nuclear NF-κB complexes. Transcripts of NF-κB-regulated genes were found elevated in breast tumors, as compared to adjacent normal tissue, indicating functional NF-κB activity. These data suggest a potential role for a subset of NF-κB and IκB family proteins, particularly NF-κB/p52 and Bcl-3, in human breast cancer. Additionally, the activation of functional NF-κB in these tumors likely involves a signal transduction pathway distinct from that utilized by cytokines.
The regulation of the transcription factor NF-κB activity occurs at several levels including controlled cytoplasmic-nuclear shuttling and modulation of its transcriptional activity. A critical component in NF-κB regulation is the IκB kinase (IKK) complex. This review is focused on recent progress as well as unanswered questions regarding the regulation and function of NF-κB and IKK.
The t(14;19)(q32.3;q13.1) is a recurring translocation found in the neoplastic cells of some patients with chronic lymphocytic leukemia (CLL). We have previously cloned the translocation breakpoint junction present in the leukemic cells from one such patient. In the present study, we have cloned and sequenced the breakpoint junction from a second patient. The breakpoint on chromosome 14 occurs within a switch region upstream of the immunoglobulin heavy chain C1 sequence. We detected a 2.1–2.3 kb transcript on Northern blots using a probe from chromosome 19 adjacent to this breakpoint. S1 nuclease protection experiments showed that transcription of the gene proceeds in a direction away from the breakpoint junction. This gene (for which we propose the name BCL3) may contribute to the malignant development of B-lymphocytes following the chromosome translocation. If so, it is the first protooncogene identified whose activation is principally associated with CLL.
Cellular proto-oncogenes can be activated by both point mutations and chromosomal translocations, suggesting that there may be a direct link between exposure to agents which damage DNA and genetic change leading to malignancy. Several groups have therefore analysed mutations found in cellular oncogenes of tumours induced by particular physical or chemical carcinogens. Here, we have analysed the molecular changes at different stages of carcinogenesis in mouse skin tumours induced by initiating and promoting agents. Over 90% of tumours, including premalignant papillomas, initiated with dimethylbenzanthracene (DMBA) have a specific A----T transversion at the second nucleotide of codon 61 of the Harvey-ras (Ha-ras) gene. The frequency of this mutation was dependent on the initiating agent used, but not on the promoter, suggesting that the mutation occurs at the time of initiation. The mutation was heterozygous in most papillomas tested, but was homozygous or amplified in some carcinomas. The development of further chromosomal changes at the c-Ha-ras gene locus is therefore a common feature of tumour progression.
The candidate proto-oncogene bcl-3 encodes a protein that shares structural features with I kappa B-alpha and other proteins that bind to members of the Rel protein family. Here, we show that in contrast to the inhibitory activity of I kappa B-alpha, the bcl-3 gene product superactivates NF-kappa B p50 homodimer-mediated gene expression both in vivo and in vitro. BCL-3 protein can, as well, selectively associate with p50 homodimers in the presence of DNA containing a kappa B motif. These results strongly suggest that BCL-3 can act as a transcriptional coactivator, acting through DNA-bound p50 homodimers.
Bcl-3 is an I kappa B-related protein with ankyrin repeat motifs. Its gene is located at a site of recurrent translocations in a subset of B cell chronic lymphocytic leukemias. Bcl-3 associates tightly with p50B (NFKB2, p52) homodimers in cells, and together these proteins form a ternary complex with DNA at kappa B sites. Such an association functionally leads to a novel and potent form of transactivation through the kappa B motif: the tethering of Bcl-3 to DNA via the p50B homodimers allows Bcl-3 to transactivate directly, while p50B homodimers alone cannot. Transactivation mediated by Bcl-3 requires two cooperating domains located amino- and carboxy-terminal to the ankyrin domain. Bcl-3 is localized to the nucleus, and a Bcl-3-p50B complex is detected in certain lymphoid cells. Our data reveal a novel role for Bcl-3, distinct from that of the inhibitor I kappa B. The results have implications for tumorigenesis.
Stratified epithelium contains a mitotically active basal layer of cells that cease proliferating, then migrate outwards and undergo terminal differentiation. The control of this process, which is abnormal in cutaneous neoplasia and inflammation, is not well understood. In normal epidermis, NF-kappaB proteins were found to exist in the cytoplasm of basal cells and then to localize in the nuclei of suprabasal cells, suggesting a role for NF-kappaB in the switch from proliferation to growth arrest and differentiation. Functional blockade of NF-kappaB by expressing dominant-negative NF-kappaB inhibitory proteins in transgenic murine and human epidermis produced hyperplastic epithelium in vivo. Consistent with this, application of a pharmacologic inhibitor of NF-kappaB to intact skin induced epidermal hyperplasia. In contrast, overexpression of active p50 and p65 NF-kappaB subunits in transgenic epithelium produced hypoplasia and growth inhibition. These data suggest that spatially restricted NF-kappaB activation occurs in stratified epithelium and indicate that NF-kappaB activation in this tissue, in contrast to its role in other settings, is important for cellular growth inhibition.
Bcl-3 is a proto-oncogene involved in the chromosomal translocation t(14;19) found in some patients with chronic lymphocytic leukemia. It shares structural similarities with and is a member of the IκB family of proteins. In this report, involvement of Bcl-3 in hematopoietic growth factor- stimulated erythroid proliferation and differentiation was examined. In TF-1 cells, an erythroleukemia cell line, granulocyte-macrophage colony- stimulating factor (GM-CSF) and erythropoietin (Epo) greatly enhanced Bcl-3 expression at both the protein and mRNA levels in association with stimulation of proliferation. Bcl-3 protein was also highly expressed in early burst-forming unit-erythroid (BFU-E)-derived erythroid precursors (day 7) and decreased during maturation (days 10 and 14), suggesting that Bcl-3 is involved in normal erythroid proliferation. In these hematopoietic cells, Bcl-3 was hyperphosphorylated. GM-CSF and Epo modulated the subcellular localization of Bcl-3. Upon stimulation of TF-1 cells with GM-CSF or Epo, the nuclear translocation of Bcl-3 was dramatically enhanced. Overexpression of Bcl-3 in TF-1 cells by transient transfection along with the NF-κB factors p50 or p52 resulted in significant induction of an human immunodeficiency virus-type 1 (HIV-1) κB-TATA-luceriferase reporter plasmid, demonstrating that Bcl-3 has a positive role in transactivation of κB-containing genes in erythroid cells. Stimulation with GM-CSF enhanced c-myb mRNA expression in these cells. Bcl-3 in nuclear extracts of TF-1 cells bound to a κB enhancer in the c-myb promoter together with NF-κB2/p52 and this binding activity was enhanced by GM-CSF stimulation. Furthermore, cotransfection of Bcl-3 with p52 or p50 in TF-1 cells resulted in significant activation of a c-myb κB-TATA- luceriferase reporter plasmid. These findings suggest that Bcl-3 may participate in the transcriptional regulation of certain κB-containing genes involved in hematopoiesis, including c-myb.
Many important transgenic mouse models of benign and neoplastic skin diseases have been generated through the use of promoters that target transgene expression to the different epidermal layers. However, more mechanistic studies of the specific effects of the transgenes on keratinocytes have been hampered by difficulties in culturing keratinocytes from adult mouse epidermis and by the low differentiation potential of many established mouse keratinocyte lines. We have used the Rheinwald & Green technique to cultivate primary adult keratinocytes and to generate keratinocyte lines from transgenic mice which have a sporadic psoriatic phenotype due to expression of human integrin subunits under the control of the involucrin promoter. We show that the transgenes are induced when keratinocytes are placed in suspension and that the transgenic integrins are capable of clustering in focal adhesions and mediating cell adhesion and spreading. We also show that suprabasal integrin expression has no direct effect on proliferation of cells in the underlying basal layer, ruling this out as a possible explanation for the epidermal hyperproliferation observed in the transgenic mice.
Accumulating evidence implicates the transcription factor NF-κB as a positive mediator of cell growth, but the molecular mechanism(s) involved in this process remains largely unknown. Here we use both a skeletal muscle differentiation model and normal diploid fibroblasts to gain insight into how NF-κB regulates cell growth and differentiation. Results obtained with the C2C12 myoblast cell line demonstrate that NF-κB functions as an inhibitor of myogenic differentiation. Myoblasts generated to lack NF-κB activity displayed defects in cellular proliferation and cell cycle exit upon differentiation. An analysis of cell cycle markers revealed that NF-κB activates cyclin D1 expression, and the results showed that this regulatory pathway is one mechanism by which NF-κB inhibits myogenesis. NF-κB regulation of cyclin D1 occurs at the transcriptional level and is mediated by direct binding of NF-κB to multiple sites in the cyclin D1 promoter. Using diploid fibroblasts, we demonstrate that NF-κB is required to induce cyclin D1 expression and pRb hyperphosphorylation and promote G
1
-to-S progression. Consistent with results obtained with the C2C12 differentiation model, we show that NF-κB also promotes cell growth in embryonic fibroblasts, correlating with its regulation of cyclin D1. These data therefore identify cyclin D1 as an important transcriptional target of NF-κB and reveal a mechanism to explain how NF-κB is involved in the early phases of the cell cycle to regulate cell growth and differentiation.
To elucidate the possible role of NF-kappaB in mouse skin carcinogenesis we studied the expression of p50 (NF-kappaB1), p52 (NF-kappaB2), p65 (RelA) and IkappaB-alpha inhibitor as well as kappaB-binding activity in adult SENCAR mouse skin, skin papillomas, and squamous cell carcinomas (SCC) generated by a two-stage carcinogenesis protocol. We found that in normal epidermis all of the above proteins were mostly expressed in the cytoplasm of basal cells. Western blot analysis revealed a dramatic increase of p50 and p52 expression in mouse skin tumors starting from the middle stage of promotion. We also found that the level of IkappaB-alpha protein in many late papillomas and SCC was lower than in normal epidermis. Results of EMSA showed an increase in kappaB-binding activity in mouse skin tumors and suggested that p50 is the major component of constitutive kappaB-binding complexes in normal epidermis and in tumors. It has been shown that nuclear IkappaB protein Bcl-3 is able to increase p50/p50 homodimer binding to the different kappaB sites in mouse thymocytes. Our finding on Bcl-3 overexpression in late papillomas and SCC could explain the selective increase of p50-related kappaB-binding in mouse skin tumors. Thus, our results strongly suggest the important role of p50 in skin carcinogenesis.
The NF-kappaB family of transcription factors plays a crucial role in the immune, inflammatory and apoptotic responses. These proteins are normally found in the cytoplasm, retained by interaction with an inhibitory molecule called IkappaB. Activation of the NF-kappaB signalling cascade results in phosphorylation and degradation of IkappaB, allowing nuclear translocation of the NF-kappaB complexes. The recent identification of a high-molecular-weight complex containing two kinases and a regulatory subunit has led to a flurry of new results that shed light on some of the most complex mechanisms contributing to the exquisite regulation of NF-kappaB activity.
Ubiquitylation ? the conjugation of proteins with a small protein
called ubiquitin ? touches upon all aspects of eukaryotic biology, and
its defective regulation is manifest in diseases that range from developmental
abnormalities and autoimmunity to neurodegenerative diseases and cancer. A
few years ago, we could only have dreamt of the complex arsenal of enzymes
dedicated to ubiquitylation. Why has nature come up with so many ways of doing
what seems to be such a simple job?
The cyclins are a family of proteins that are centrally involved in cell cycle regulation and which are structurally identified by conserved “cyclin box” regions. They are regulatory subunits of holoenzyme cyclin-dependent kinase (CDK) complexes controlling progression through cell cycle checkpoints by phosphorylating and inactivating target substrates. CDK activity is controlled by cyclin abundance and subcellular location and by the activity of two families of inhibitors, the cyclin-dependent kinase inhibitors (CKI). Many hormones and growth factors influence cell growth through signal transduction pathways that modify the activity of the cyclins. Dysregulated cyclin activity in transformed cells contributes to accelerated cell cycle progression and may arise because of dysregulated activity in pathways that control the abundance of a cyclin or because of loss-of-function mutations in inhibitory proteins.
The conjugation of ubiquitin to other cellular proteins regulates a broad range of eukaryotic cell functions. The high efficiency and exquisite selectivity of ubiquitination reactions reflect the properties of enzymes known as ubiquitin-protein ligases or E3s. An E3 recognizes its substrates based on the presence of a specific ubiquitination signal, and catalyzes the formation of an isopeptide bond between a substrate (or ubiquitin) lysine residue and the C terminus of ubiquitin. Although a great deal is known about the molecular basis of E3 specificity, much less is known about molecular mechanisms of catalysis by E3s. Recent findings reveal that all known E3s utilize one of just two catalytic domains--a HECT domain or a RING finger--and crystal structures have provided the first detailed views of an active site of each type. The new findings shed light on many aspects of E3 structure, function, and mechanism, but also emphasize that key features of E3 catalysis remain to be elucidated.
We describe the morphology and comparative genomic hybridization findings in a tumor for which we propose the term "spiradenocylindroma" of the kidney. The tumor arose in the wall of a renal cyst in an otherwise healthy male patient who had a favorable clinical course after nephrectomy. Tumor cells formed either large nodules exhibiting a solid or trabecular architecture with conspicuous perivascular spaces or cylindromatous small tumor cell islands arranged in a jigsaw pattern. Focally, there were interspersed tubular structures and tumor cell rosettes with central deposits of periodic acid-Schiff-positive material. A minor tumor component showed epidermoid differentiation. The tumor cells were strongly positive for cytokeratins 5/6, high molecular weight cytokeratins 34betaE12 and AE1/3, and E-cadherin, but only weakly positive for cytokeratins 7, 8, 18, 19, and epithelial membrane antigen. Focal reactivity for actin, vimentin, and S-100 protein or lysozyme and alpha 1 -antichymotrypsin within tubular and cylindromatous areas suggested myoepithelial and apocrine differentiation, respectively. By comparative genomic hybridization, the only abnormality was loss of the long arm of chromosome 16 and gain of genetic material on the short arm of chromosome 16, suggesting isochromosome i(16p). This finding is unique among renal neoplasms and implies loss of heterozygosity at 16q12-13 of the CYLD1 gene that is critically involved in the oncogenesis of familial cylindromatosis and some sporadic spiradenocylindromas. We conclude that somatic mutation of the CYLD1 gene outside the skin can have a role in the oncogenesis of tumors with cylindromatous features.
The regulation of the transcription factor NF-kappaB activity occurs at several levels including controlled cytoplasmic-nuclear shuttling and modulation of its transcriptional activity. A critical component in NF-kappaB regulation is the IkappaB kinase (IKK) complex. This review is focused on recent progress as well as unanswered questions regarding the regulation and function of NF-kappaB and IKK.
Nuclear factor of kappaB (NF-kappaB) is a sequence-specific transcription factor that is known to be involved in the inflammatory and innate immune responses. Although the importance of NF-KB in immunity is undisputed, recent evidence indicates that NF-kappaB and the signalling pathways that are involved in its activation are also important for tumour development. NF-kappaB should therefore receive as much attention from cancer researchers as it has already from immunologists.
The p53 and NF-κB transcription factor families are important, multifunctional regulators of the cellular response to stress.
Here we have investigated the regulatory mechanisms controlling p53-dependent cell cycle arrest and cross talk with NF-κB.
Upon induction of p53 in H1299 or U-2 OS cells, we observed specific repression of cyclin D1 promoter activity, correlating
with a decrease in cyclin D1 protein and mRNA levels. This repression was dependent on the proximal NF-κB binding site of
the cyclin D1 promoter, which has been shown to bind the p52 NF-κB subunit. p53 inhibited the expression of Bcl-3 protein,
a member of the IκB family that functions as a transcriptional coactivator for p52 NF-κB and also reduced p52/Bcl-3 complex
levels. Concomitant with this, p53 induced a significant increase in the association of p52 and histone deacetylase 1 (HDAC1).
Importantly, p53-mediated suppression of the cyclin D1 promoter was reversed by coexpression of Bcl-3 and inhibition of p52
or deacetylase activity. p53 therefore induces a transcriptional switch in which p52/Bcl-3 activator complexes are replaced
by p52/HDAC1 repressor complexes, resulting in active repression of cyclin D1 transcription. These results reveal a unique
mechanism by which p53 regulates NF-κB function and cell cycle progression.
Familial cylindromatosis is an autosomal dominant predisposition to tumours of skin appendages called cylindromas. Familial cylindromatosis is caused by mutations in a gene encoding the CYLD protein of previously unknown function. Here we show that CYLD is a deubiquitinating enzyme that negatively regulates activation of the transcription factor NF-kappaB by specific tumour-necrosis factor receptors (TNFRs). Loss of the deubiquitinating activity of CYLD correlates with tumorigenesis. CYLD inhibits activation of NF-kappaB by the TNFR family members CD40, XEDAR and EDAR in a manner that depends on the deubiquitinating activity of CYLD. Downregulation of CYLD by RNA-mediated interference augments both basal and CD40-mediated activation of NF-kappaB. The inhibition of NF-kappaB activation by CYLD is mediated, at least in part, by the deubiquitination and inactivation of TNFR-associated factor 2 (TRAF2) and, to a lesser extent, TRAF6. These results indicate that CYLD is a negative regulator of the cytokine-mediated activation of NF-kappaB that is required for appropriate cellular homeostasis of skin appendages.
Cultured primary human keratinocytes were screened for their expression of various members of the toll-like receptor (TLR) family. Keratinocytes were found to constitutively express TLR1, TLR2, TLR3, TLR5, and TLR9 but not TLR4, TLR6, TLR7, TLR8, or TLR10 as shown by polymerase chain reaction analysis. The expression of the crucial receptor for signaling of staphylococcal compounds TLR2 was also confirmed by immunohistochemistry, in contrast to TLR4, which showed a negative staining pattern. Next, we analyzed the activation of the proinflammatory nuclear transcription factor kappaB by Staphylococcus aureus strain 8325-4. Using nuclear extract gel shifts, RelA staining, and luciferase reporter transfection plasmids we found a clear induction of nuclear factor kappaB translocation by the bacteria. This translocation induced the transcription of nuclear factor kappaB controlled genes such as inducible nitric oxide synthetase, COX2, and interleukin-8. Transcription of these genes was followed by production of increased amounts of interleukin-8 protein and NO. Inhibition experiments using monoclonal antibodies and the specific platelet activating factor receptor inhibitor CV3988 showed that nuclear factor kappaB activation by S. aureus was TLR2 but not TLR4 or platelet activating factor receptor dependent. In line, the purified staphylococcal cell wall components lipoteichoic acid and peptidoglycan, known to signal through TLR2, also showed nuclear factor kappaB translocation in human keratinocytes, indicating a crucial role of the staphylococcal cell wall in the innate immune stimulation of human keratinocytes. These results help to explain the complex activation of human keratinocytes by S. aureus and its cell wall components in various inflammatory disorders of the skin.
Toll-like receptors (TLRs) are important pattern recognition molecules that activate the nuclear factor (NF)-kappaB pathway leading to the production of antimicrobial immune mediators. As keratinocytes represent the first barrier against exogenous pathogens in human skin, we investigated their complete functional TLR1-10 expression profile. First, reverse transcription-polymerase chain reaction (PCR) analysis revealed a very similar pattern of TLR mRNA expression when comparing freshly isolated human epidermis and cultured primary human keratinocytes. Thus, further experiments were carried out with primary keratinocytes in comparison with the spontaneously immortalized human keratinocyte cell line HaCaT. The quantitative expression of TLR1-10 mRNA in real-time PCR of primary human keratinocytes and HaCaT cells was analysed. Both cell types constitutively expressed TLR2, TLR3, TLR5, and to a lesser extent TLR10. TLR4 was only found in HaCaT cells, TLR1 to a higher degree in primary keratinocytes. In line with this, LPS induced mRNA expression of CD14 and TLR4 only in HaCaT cells. After stimulation with various TLR ligands, the NF-kappaB-activated chemokine interleukin-8 (IL-8) was measured. In primary keratinocytes and HaCaT cells the TLR3 ligand poly (I:C) was the most potent stimulator of IL-8 secretion. The TLR ligands peptidoglycan, Pam3Cys and flagellin which bind to TLR2, TLR1/TLR2 heterodimer, and TLR5, respectively, also induced IL-8 secretion, whereas no IL-8 was induced by LPS, R-848, loxoribine and cytosine guanine dinucleotide-containing oligodeoxynucleotide. A corresponding pattern was found in the RelA NF-kappaB translocation assay after ligand stimulation of primary keratinocytes. These studies provide substantial evidence for a functional TLR expression and signalling profile of normal human keratinocytes contributing to the antimicrobial defence barrier of human skin.
As its role in tumor progression emerges, the PI3K/PKB (Akt) pathway presents an appealing cancer therapeutic target. Recent studies have investigated the mechanisms underlying the tumor-promoting effects of this pathway. PKB triggers a network that positively regulates G1/S cell cycle progression through inactivation of GSK3-beta, leading to increased cyclin D1, and inhibition of Forkhead family transcription factors and the tumor suppressor tuberin (TSC2), leading to reduction of p27Kip1. The identification of p21Waf1/Cip1 and p27Kip1 as novel substrates of PKB provided new insights into mechanisms whereby hyperactivation of this lipid signaling pathway may lead to cell cycle deregulation in human cancers. The PI3K pathway may also play a key role in the G2/M transition and its constitutive activation may lead to defects in DNA damage checkpoint control.
We established 2 novel human cell lines (GCCOT-1, GCCRK) from glassy cell carcinoma. Both cell lines showed dual tendencies of glandular and squamous differentiation, and thus possess the characteristics resembling reserve cells, the putative origin of most carcinomas arising from the uterine cervix. HPV type 18 DNA including E6-E7, which is commonly found in cell types other than squamous cell carcinoma of uterine cervix, was detected in both cell lines. We analyzed gene copy number alterations of the 2 cell lines using conventional comparative genomic hybridization (CGH) coupled with array-based CGH. Among the putative oncogenes demonstrating copy number gain in both cell lines, FGR(SRC2) at 1p36.2-1 and LAMC2 at 1q25-31 have not been reported to show amplification in previous analyses of conventional cervical cell lines. These oncogenes are thus speculated to be directly associated with oncogenesis of glassy cell carcinoma. On the other hand, among the putative suppressor genes demonstrating copy number loss in both cell lines, the 9q region, ATM at 11q22.3, and CYLD at 16q12-13 have not been reported to show loss in conventional cervical cancer cell lines. These sites are speculated to be important as tumor suppressors directly associated with oncogenesis of glassy cell carcinoma. This study suggests for the first time that together with the presence of HPV type 18, alterations at the above sites are closely associated with oncogenesis of glassy cell carcinoma, a special type of carcinoma in the uterine cervix.
Selective activation of NF-kB subunits in human breast can-cer: potential roles for NF-kB2/p52 and for Bcl-3 The candidate proto-oncogene bcl-3 encodes a transcriptional coac-tivator that activates through NF-kB p50 homodimers Missing pieces in the NF-kB puzzle
- P C Cogswell
- D C Guttridge
- W K Funkhouser
- A S Baldwin
- Jr
- T Fujita
- G P Nolan
- H C Liou
- M L Scott
- D Andbaltimore
Cogswell, P.C., Guttridge, D.C., Funkhouser, W.K., and Baldwin, A.S., Jr. (2000). Selective activation of NF-kB subunits in human breast can-cer: potential roles for NF-kB2/p52 and for Bcl-3. Oncogene 19, 1123– 1131. Fujita, T., Nolan, G.P., Liou, H.C., Scott,M.L., andBaltimore,D.(1993). The candidate proto-oncogene bcl-3 encodes a transcriptional coac-tivator that activates through NF-kB p50 homodimers. Genes Dev. 7, 1354–1363. Ghosh, S., and Karin, M. (2002). Missing pieces in the NF-kB puzzle
B) Cyclin D1 staining (brown) in skin 72 hr after a single application of TPA. (C) Proliferation assay with isolated primary epidermal keratinocytes in the absence or presence of TNF-a (25 ng/ml) or TPA (100 nM). The proliferation rate after 48 hr is significantly higher in Cyld
- + Cyld
- Dmba Tpa
(A) Ki67 (brown) staining in Cyld +/+ and Cyld À/À tumors after application of DMBA/TPA. (B) Cyclin D1 staining (brown) in skin 72 hr after a single application of TPA. (C) Proliferation assay with isolated primary epidermal keratinocytes in the absence or presence of TNF-a (25 ng/ml) or TPA (100 nM). The proliferation rate after 48 hr is significantly higher in Cyld À/À compared to Cyld +/+ in the presence of TPA (n = 4; p < 0.05).
NF-kB in cancer: from innocent bystander to major culprit
- M Karin
- Y Cao
- F R Greten
- Z W Li
Karin, M., Cao, Y., Greten, F.R., and Li, Z.W. (2002). NF-kB in cancer:
from innocent bystander to major culprit. Nat. Rev. Cancer 2, 301-310.
Alterations in NF-kB function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-kB
- C S Seitz
- Q Lin
- H Deng
- P A Khavari
Seitz, C.S., Lin, Q., Deng, H., and Khavari, P.A. (1998). Alterations in
NF-kB function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-kB. Proc. Natl. Acad. Sci. USA 95, 2307-2312.