miR-130b regulates B cell proliferation via CYLD-mediated NF-κB signaling.

Previous studies have revealed that B cell activation is regulated by various microRNAs(miRNAs). However, the role of microRNA-130b regulating B cell activation and apoptosis is still unclear. In the present study, we first found that the expression of miR-130b was the lowest in Pro/Pre-B cells and the highest in immature B cells. Besides, the expression of miR-130b decreased after activation in B cells. Through the immuno-phenotypic analysis of miR-130b transgenic and knockout mice, we found that miR-130b mainly promoted the proliferation of B cells and inhibited B cell apoptosis. Furthermore, we identified that Cyld, a tumor suppressor gene was the target gene of miR-130b in B cells. Besides, the Cyld-mediated NF-κB signaling was increased in miR-130b overexpressed B cells, which further explains the enhanced proliferation of B cells. In conclusion, we propose that miR-130b promotes B cell proliferation via Cyld-mediated NF-κB signaling, which provides a new theoretical basis for the molecular regulation of B cell activation.

The Met1-Linked Ubiquitin Machinery: Emerging Themes of (De)regulation.

The linear ubiquitin chain assembly complex, LUBAC, is the only known mammalian ubiquitin ligase that makes methionine 1 (Met1)-linked polyubiquitin (also referred to as linear ubiquitin). A decade after LUBAC was discovered as a cellular activity of unknown function, there are now many lines of evidence connecting Met1-linked polyubiquitin to NF-κB signaling, cell death, inflammation, immunity, and cancer. We now know that Met1-linked polyubiquitin has potent signaling functions and that its deregulation is connected to disease. Indeed, mutations and deficiencies in several factors involved in conjugation and deconjugation of Met1-linked polyubiquitin have been implicated in immune-related disorders. Here, we discuss current knowledge and recent insights into the role and regulation of Met1-linked polyubiquitin, with an emphasis on the mechanisms controlling the function of LUBAC.

Influenza A virus infection activates caspase-8 to enhance innate antiviral immunity by cleaving CYLD and blocking TAK1 and RIG-I deubiquitination.

Caspase-8, an aspartate-specific cysteine protease that primarily functions as an initiator caspase to induce apoptosis, can downregulate innate immunity in part by cleaving RIPK1 and IRF3. However, patients with caspase-8 mutations or deficiency develop immunodeficiency and are prone to viral infections. The molecular mechanism underlying this controversy remains unknown. Whether caspase-8 enhances or suppresses antiviral responses against influenza A virus (IAV) infection remains to be determined. Here, we report that caspase-8 is readily activated in A549 and NL20 cells infected with the H5N1, H5N6, and H1N1 subtypes of IAV. Surprisingly, caspase-8 deficiency and two caspase-8 inhibitors, Z-VAD and Z-IETD, do not enhance but rather downregulate antiviral innate immunity, as evidenced by decreased TBK1, IRF3, IκBα, and p65 phosphorylation, decreased IL-6, IFN-ß, MX1, and ISG15 gene expression; and decreased IFN-ß production but increased virus replication. Mechanistically, caspase-8 cleaves and inactivates CYLD, a tumor suppressor that functions as a deubiquitinase. Caspase-8 inhibition suppresses CYLD cleavage, RIG-I and TAK1 ubiquitination, and innate immune signaling. In contrast, CYLD deficiency enhances IAV-induced RIG-I and TAK1 ubiquitination and innate antiviral immunity. Neither caspase-3 deficiency nor treatment with its inhibitor Z-DEVD affects CYLD cleavage or antiviral innate immunity. Our study provides evidence that caspase-8 activation in two human airway epithelial cell lines does not silence but rather enhances innate immunity by inactivating CYLD.

SPATA2 regulates the activation of RIPK1 by modulating linear ubiquitination.

Stimulation of cells with TNFα leads to the formation of the TNF-R1 signaling complex (TNF-RSC) to mediate downstream cellular fate decision. Activation of the TNF-RSC is modulated by different types of ubiquitination and may lead to cell death, including apoptosis and necroptosis, in both RIPK1-dependent and RIPK1-independent manners. Spata2 (spermatogenesis-associated 2) is an adaptor protein recruited into the TNF-RSC to modulate the interaction between the linear ubiquitin chain assembly complex (LUBAC) and the deubiquitinase CYLD (cylindromatosis). However, the mechanism by which Spata2 regulates the activation of RIPK1 is unclear. Here, we report that Spata2-deficient cells show resistance to RIPK1-dependent apoptosis and necroptosis and are also partially protected against RIPK1-independent apoptosis. Spata2 deficiency promotes M1 ubiquitination of RIPK1 to inhibit RIPK1 kinase activity. Furthermore, we provide biochemical evidence for the USP domain of CYLD and the PUB domain of the SPATA2 complex preferentially deubiquitinating the M1 ubiquitin chain in vitro. Spata2 deficiency also promotes the activation of MKK4 and JNK and cytokine production independently of RIPK1 kinase activity. Spata2 deficiency sensitizes mice to systemic inflammatory response syndrome (SIRS) induced by TNFα, which can be suppressed by RIPK1 inhibitor Nec-1s. Thus, Spata2 can regulate inflammatory response and cell death in both RIPK1-dependent and RIPK1-independent manners.

The TRIF-RIPK1-Caspase-8 signalling in the regulation of TLR4-driven gene expression.

The inflammatory response is tightly regulated to eliminate invading pathogens and avoid excessive production of inflammatory mediators and tissue damage. Caspase-8 is a cysteine protease that is involved in programmed cell death. Here we show the TRIF-RIPK1-Caspase-8 is required for LPS-induced CYLD degradation in macrophages. TRIF functions in the upstream of RIPK1. The homotypic interaction motif of TRIF and the death domain of RIPK1 are essential for Caspase-8 activation. Caspase-8 cleaves CYLD and the D235A mutant is resistant to the protease activity of Caspase-8. TRIF and RIPK1 serve as substrates of Capase-8 in vitro. cFLIP interacts with Caspase-8 to modulate its protease activity on CYLD and cell death. Deficiency in TRIF, Caspase-8 or CYLD can lead to a decrease or increase in the expression of genes encoding inflammatory cytokines. Together, the TRIF-Caspase-8 and CYLD play opposite roles in the regulation of TLR4 signalling.

PLK4 deubiquitination by Spata2-CYLD suppresses NEK7-mediated NLRP3 inflammasome activation at the centrosome.

The innate immune sensor NLRP3 assembles an inflammasome complex with NEK7 and ASC to activate caspase-1 and drive the maturation of proinflammatory cytokines IL-1ß and IL-18. NLRP3 inflammasome activity must be tightly controlled, as its over-activation is involved in the pathogenesis of inflammatory diseases. Here, we show that NLRP3 inflammasome activation is suppressed by a centrosomal protein Spata2. Spata2 deficiency enhances NLRP3 inflammasome activity both in the macrophages and in an animal model of peritonitis. Mechanistically, Spata2 recruits the deubiquitinase CYLD to the centrosome for deubiquitination of polo-like kinase 4 (PLK4), the master regulator of centrosome duplication. Deubiquitination of PLK4 facilitates its binding to and phosphorylation of NEK7 at Ser204. NEK7 phosphorylation in turn attenuates NEK7 and NLRP3 interaction, which is required for NLRP3 inflammasome activation. Pharmacological or shRNA-mediated inhibition of PLK4, or mutation of the NEK7 Ser204 phosphorylation site, augments NEK7 interaction with NLRP3 and causes increased NLRP3 inflammasome activation. Our study unravels a novel centrosomal regulatory pathway of inflammasome activation and may provide new therapeutic targets for the treatment of NLRP3-associated inflammatory diseases.

Mettl1 knockdown alleviates cardiac I/R injury in mice by inactivating the Mettl1-CYLD-P53 positive feedback loop.

The N7-methylguanosine (m7G) methyltransferase Mettl1 has been recently implicated in cardiac repair and fibrosis. In this study we investigated the role of Mettl1 in mouse cardiomyocytes injury and the underlying mechanisms. Cardiac ischemia/reperfusion (I/R) I/R model was established in mice by ligation of the left anterior descending coronary artery (LAD) for 45 min followed by reperfusion for 24 h. We showed the mRNA and protein levels of Mettl1 were significantly upregulated in mouse I/R hearts and H2O2-treated neonatal mouse cardiomyocytes (NMCMs). Mettl1 knockdown markedly ameliorated cardiac I/R injury, evidenced by decreased infarct size, apoptosis, and improved cardiac function. Overexpression of Mettl1 triggered cardiomyocytes apoptosis in vivo and in vitro. By performing RNA sequencing combined with m7G methylated RNA sequencing in Mettl1-overexpressing mouse hearts, we revealed that Mettl1 catalyzed m7G modification of the deubiquitinase cylindromatosis (CYLD) mRNA to increase the expression of CYLD, which enhanced the stability of P53 via abrogating its ubiquitination degradation. Vice versa, P53 served as a transcriptional factor to positively regulate Mettl1 expression during I/R injury. Knockdown of CYLD mitigated cardiomyocytes apoptosis induced by Mettl1 overexpression or oxidative stress. From the available drug-targets databases and literature, we identified 4 small molecule inhibitors of m7G modification. Sinefungin, one of the Mettl1 inhibitors exerted profound protection against cardiac I/R injury in vivo and in vitro. Collectively, this study has identified Mettl1 as a key regulator of cardiomyocyte apoptosis, and targeting the Mettl1-CYLD-P53 positive feedback circuit may represent a novel therapeutic avenue for alleviating cardiac I/R injury.

The K48-K63 Branched Ubiquitin Chain Regulates NF-κB Signaling.

Polyubiquitin chains of different topologies regulate diverse cellular processes. K48- and K63-linked chains, the two most abundant chain types, regulate proteolytic and signaling pathways, respectively. Although recent studies reported important roles for heterogeneous chains, the functions of branched ubiquitin chains remain unclear. Here, we show that the ubiquitin chain branched at K48 and K63 regulates nuclear factor κB (NF-κB) signaling. A mass-spectrometry-based quantification strategy revealed that K48-K63 branched ubiquitin linkages are abundant in cells. In response to interleukin-1ß, the E3 ubiquitin ligase HUWE1 generates K48 branches on K63 chains formed by TRAF6, yielding K48-K63 branched chains. The K48-K63 branched linkage permits recognition by TAB2 but protects K63 linkages from CYLD-mediated deubiquitylation, thereby amplifying NF-κB signals. These results reveal a previously unappreciated cooperation between K48 and K63 linkages that generates a unique coding signal: ubiquitin chain branching differentially controls readout of the ubiquitin code by specific reader and eraser proteins to activate NF-κB signaling.

The K63 deubiquitinase CYLD modulates autism-like behaviors and hippocampal plasticity by regulating autophagy and mTOR signaling.

Nondegradative ubiquitin chains attached to specific targets via Lysine 63 (K63) residues have emerged to play a fundamental role in synaptic function. The K63-specific deubiquitinase CYLD has been widely studied in immune cells and lately also in neurons. To better understand if CYLD plays a role in brain and synapse homeostasis, we analyzed the behavioral profile of CYLD-deficient mice. We found that the loss of CYLD results in major autism-like phenotypes including impaired social communication, increased repetitive behavior, and cognitive dysfunction. Furthermore, the absence of CYLD leads to a reduction in hippocampal network excitability, long-term potentiation, and pyramidal neuron spine numbers. By providing evidence that CYLD can modulate mechanistic target of rapamycin (mTOR) signaling and autophagy at the synapse, we propose that synaptic K63-linked ubiquitination processes could be fundamental in understanding the pathomechanisms underlying autism spectrum disorder.

CYLD inhibits osteoclastogenesis to ameliorate alveolar bone loss in mice with periodontitis.

Periodontitis is a chronic immune inflammatory disease that can lead to the destruction and loss of the tooth-supporting apparatus. During this process, the balance between bone absorption mediated by osteoclasts and bone formation mediated by osteoblasts is damaged. Consistent with previous studies, we observed that depletion of cylindromatosis (CYLD) resulted in an osteoporotic bone phenotype. However, the effect of CYLD deficiency on periodontitis is undetermined. Here, we investigated whether CYLD affects periodontal tissue homeostasis in experimental periodontitis in Cyld knockout (KO) mice, and we explored the underlying mechanisms. Interestingly, we discovered significant alveolar bone density loss and severely reduced alveolar bone height in Cyld KO mice with experimentally induced periodontitis. We observed increased osteoclast number and activity in both the femurs and alveolar bones, accompanied by the downregulation of osteogenesis genes and upregulation of osteoclastogenesis genes of alveolar bones in ligatured Cyld KO mice. Taken together, our findings demonstrate that the deletion of CYLD in mice plays a vital role in the pathogenesis of periodontal bone loss and suggest that CYLD might exert an ameliorative effect on periodontal inflammatory responses.

Brooke-Spiegler syndrome: radiotherapy as the last resort?

PURPOSE: To describe the case of successful radiotherapeutic treatment of a woman suffering from Brooke-Spiegler syndrome who had multiple disfiguring cylindromas on the entire scalp and further tumors on the trunk. METHODS: After decades of treatment with conventional therapies including surgery and topically applied salicylic acid, the 73-year-old woman agreed to undergo radiotherapeutic treatment. She received 60 Gy to the scalp and 36 Gy to painful nodules in the lumbar spine region. RESULTS: Over a follow-up period of 14 and 11 years, respectively, the scalp nodules almost completely regressed, while the lumbar nodules became painless and considerably smaller. Apart from alopecia, no late adverse effects of treatment remain. CONCLUSION: This case should remind us of the potential role that radiotherapy could play in treating Brooke-Spiegler syndrome. The required dose for treatment of such extensive disease is still a matter of debate due to the scarcity of radiotherapeutic experience. This case demonstrates that for scalp tumors, 30â€¯× 2 Gy can result in long-term tumor control, while other dose prescriptions may be adequate for tumors in other locations.

Multiple brain regions are involved in reaction to acute restraint stress in CYLD-knockout mice.

The lysine 63 deubiquitinase cylindromatosis (CYLD) is expressed at high levels in the brain and is considered to be involved in anxious and depressive behavior, cognitive inflexibility, and autism disorders. Previous research was limited in some brain regions, including the hippocampus, striatum, and amygdala. To better understand whether CYLD plays a role in adaptation to stress and which brain regions are involved, we analyzed the behavior of CYLD-knockout mice in the elevated plus maze (EPM) and light-dark box test (LDT) after acute restraint stress (ARS) and mapped their c-Fos immunoreactivity in brain sections. Here we report that CYLD deficiency leads to an unexpected reaction to ARS in mice, and is accompanied by significant neuronal activation of brain regions including the medial prefrontal cortex (mPFC), dorsal striatum (DS), nucleus accumbens (NAc), and basal lateral amygdala (BLA), but not ventral hippocampus (vHPC). Our findings show that CYLD participates in ARS-induced anxious behavior and that this involves multiple brain regions.

Deubiquitinase cylindromatosis (CYLD) regulates antibacterial immunity and apoptosis in Chinese mitten crab (Eriocheir sinensis).

Ubiquitination and deubiquitination of target proteins is an important mechanism for cells to rapidly respond to changes in the external environment. The deubiquitinase, cylindromatosis (CYLD), is a tumor suppressor protein. CYLD from Drosophila melanogaster participates in the antimicrobial immune response. In vertebrates, CYLD also regulates bacterial-induced apoptosis. However, whether CYLD can regulate the bacterial-induced innate immune response in crustaceans is unknown. In the present study, we reported the identification and cloning of CYLD in Chinese mitten crab, Eriocheir sinensis. Quantitative real-time reverse transcription polymerase chain reaction analysis showed that EsCYLD was widely expressed in all the examined tissues and was upregulated in the hemolymph after Vibrio parahaemolyticus challenge. Knockdown of EsCYLD in hemocytes promoted the cytoplasm-to-nucleus translocation of transcription factor Relish under V. parahaemolyticus stimulation and increased the expression of corresponding antimicrobial peptides. In vivo, silencing of EsCYLD promoted the removal of bacteria from the crabs and enhanced their survival. In addition, interfering with EsCYLD expression inhibited apoptosis of crab hemocytes caused by V. parahaemolyticus stimulation. In summary, our findings revealed that EsCYLD negatively regulates the nuclear translocation of Relish to affect the expression of corresponding antimicrobial peptides and regulates the apoptosis of crab hemocytes, thus indirectly participating in the innate immunity of E. sinensis.

FOSL1 promotes proneural-to-mesenchymal transition of glioblastoma stem cells via UBC9/CYLD/NF-κB axis.

Proneural (PN) to mesenchymal (MES) transition (PMT) is a crucial phenotypic shift in glioblastoma stem cells (GSCs). However, the mechanisms driving this process remain poorly understood. Here, we report that Fos-like antigen 1 (FOSL1), a component of AP1 transcription factor complexes, is a key player in regulating PMT. FOSL1 is predominantly expressed in the MES subtype, but not PN subtype, of GSCs. Knocking down FOSL1 expression in MES GSCs leads to the loss of MES features and tumor-initiating ability, whereas ectopic expression of FOSL1 in PN GSCs is able to induce PMT and maintain MES features. Moreover, FOSL1 facilitates ionizing radiation (IR)-induced PMT and radioresistance of PN GSCs. Inhibition of FOSL1 enhances the anti-tumor effects of IR by preventing IR-induced PMT. Mechanistically, we find that FOSL1 promotes UBC9-dependent CYLD SUMOylation, thereby inducing K63-linked polyubiquitination of major nuclear factor κB (NF-κB) intermediaries and subsequent NF-κB activation, which results in PMT induction in GSCs. Our study underscores the importance of FOSL1 in the regulation of PMT and suggests that therapeutic targeting of FOSL1 holds promise to attenuate molecular subtype switching in patients with glioblastomas.

The Potential of Cylindromatosis (CYLD) as a Therapeutic Target in Oxidative Stress-Associated Pathologies: A Comprehensive Evaluation.

Oxidative stress (OS) arises as a consequence of an imbalance between the formation of reactive oxygen species (ROS) and the capacity of antioxidant defense mechanisms to neutralize them. Excessive ROS production can lead to the damage of critical biomolecules, such as lipids, proteins, and DNA, ultimately contributing to the onset and progression of a multitude of diseases, including atherosclerosis, chronic obstructive pulmonary disease, Alzheimer's disease, and cancer. Cylindromatosis (CYLD), initially identified as a gene linked to familial cylindromatosis, has a well-established and increasingly well-characterized function in tumor inhibition and anti-inflammatory processes. Nevertheless, burgeoning evidence suggests that CYLD, as a conserved deubiquitination enzyme, also plays a pivotal role in various key signaling pathways and is implicated in the pathogenesis of numerous diseases driven by oxidative stress. In this review, we systematically examine the current research on the function and pathogenesis of CYLD in diseases instigated by oxidative stress. Therapeutic interventions targeting CYLD may hold significant promise for the treatment and management of oxidative stress-induced human diseases.

MiR-182-5p Is Upregulated in Hepatic Tissues from a Diet-Induced NAFLD/NASH/HCC C57BL/6J Mouse Model and Modulates Cyld and Foxo1 Expression.

Non-alcoholic fatty liver disease (NAFLD) is considered a relevant liver chronic disease. Variable percentages of NAFLD cases progress from steatosis to steatohepatitis (NASH), cirrhosis and, eventually, hepatocellular carcinoma (HCC). In this study, we aimed to deepen our understanding of expression levels and functional relationships between miR-182-5p and Cyld-Foxo1 in hepatic tissues from C57BL/6J mouse models of diet-induced NAFL/NASH/HCC progression. A miR-182-5p increase was detected early in livers as NAFLD damage progressed, and in tumors compared to peritumor normal tissues. An in vitro assay on HepG2 cells confirmed Cyld and Foxo1, both tumor-suppressor, as miR-182-5p target genes. According to miR-182-5p expression, decreased protein levels were observed in tumors compared to peritumor tissues. Analysis of miR-182-5p, Cyld and Foxo1 expression levels, based on datasets from human HCC samples, showed results consistent with those from our mouse models, and also highlighted the ability of miR-182-5p to distinguish between normal and tumor tissues (AUC 0.83). Overall, this study shows, for the first time, miR-182-5p overexpression and Cyld-Foxo1 downregulation in hepatic tissues and tumors from a diet-induced NAFLD/HCC mouse model. These data were confirmed by the analysis of datasets from human HCC samples, highlighting miR-182-5p diagnostic accuracy and demonstrating the need for further studies to assess its potential role as a biomarker or therapeutic target.

Biocytin-Labeling in Whole-Cell Recording: Electrophysiological and Morphological Properties of Pyramidal Neurons in CYLD-Deficient Mice.

Biocytin, a chemical compound that is an amide formed from the vitamin biotin and the amino acid L-lysine, has been used as a histological dye to stain nerve cells. Electrophysiological activity and morphology are two key characteristics of neurons, but revealing both the electrophysiological and morphological properties of the same neuron is challenging. This article introduces a detailed and easy-to-operate procedure for single-cell labeling in combination with whole-cell patch-clamp recording. Using a recording electrode filled with a biocytin-containing internal solution, we demonstrate the electrophysiological and morphological characteristics of pyramidal (PNs), medial spiny (MSNs) and parvalbumin neurons (PVs) in brain slices, where the electrophysiological and morphological properties of the same individual cell are elucidated. We first introduce a protocol for whole-cell patch-clamp recording in various neurons, coupled with the intracellular diffusion of biocytin delivered by the glass capillary of the recording electrode, followed by a post hoc procedure to reveal the architecture and morphology of biocytin-labeled neurons. An analysis of action potentials (APs) and neuronal morphology, including the dendritic length, number of intersections, and spine density of biocytin-labeled neurons, were performed using ClampFit and Fiji Image (ImageJ), respectively. Next, to take advantage of the techniques introduced above, we uncovered defects in the APs and the dendritic spines of PNs in the primary motor cortex (M1) of deubiquitinase cylindromatosis (CYLD) knock-out (Cyld-/-) mice. In summary, this article provides a detailed methodology for revealing the morphology as well as the electrophysiological activity of a single neuron that will have many applications in neurobiology.

Associations of A20, CYLD, Cezanne and JAK2 Genes and Immunophenotype with Psoriasis Susceptibility.

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.

IKK-mediated CYLD phosphorylation and cellular redox activity.

Oxidative stress is important in the development of obesity-related nephropathy (ORN). A causal relationship between IKK and ORN via CYLD-mediated inhibition of NRF2 has been described. However, contradictory explanations about the functioning of the mechanisms that will be effective in the pathogenesis require clarification.

Potential use of EGFR-targeted molecular therapies for tumor suppressor CYLD-negative and poor prognosis oral squamous cell carcinoma with chemoresistance.

BACKGROUND: Tumor suppressor CYLD dysfunction by loss of its expression, triggers malignant transformation, especially drug resistance and tumor invasion/metastasis. Although loss of CYLD expression is significantly associated with poor prognosis in a large variety of tumors, no clinically-effective treatment for CYLD-negative cancer patients is available. METHODS: We focused on oral squamous cell carcinoma (OSCC), and sought to develop novel therapeutic agents for CYLD-negative cancer patients with poor prognosis. CYLD-knockdown OSCC cells by using CYLD-specific siRNA, were used to elucidate and determine the efficacy of novel drug candidates by evaluating cell viability and epithelial-mesenchymal transition (EMT)-like change. Therapeutic effects of candidate drug on cell line-derived xenograft (CDX) model and usefulness of CYLD as a novel biomarker using patient-derived xenograft (PDX) model were further investigated. RESULTS: CYLD-knockdown OSCC cells were resistant for all currently-available cytotoxic chemotherapeutic agents for OSCC, such as, cisplatin, 5-FU, carboplatin, docetaxel, and paclitaxel. By using comprehensive proteome analysis approach, we identified epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, played key roles in CYLD-knockdown OSCC cells. Indeed, cell survival rate in the cisplatin-resistant CYLD-knockdown OSCC cells was markedly inhibited by treatment with clinically available EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib. In addition, gefitinib was significantly effective for not only cell survival, but also EMT-like changes through inhibiting transforming growth factor-ß (TGF-ß) signaling in CYLD-knockdown OSCC cells. Thereby, overall survival of CYLD-knockdown CDX models was significantly prolonged by gefitinib treatment. Moreover, we found that CYLD expression was significantly associated with gefitinib response by using PDX models. CONCLUSIONS: Our results first revealed that EGFR-targeted molecular therapies, such as EGFR-TKIs, could have potential to be novel therapeutic agents for the CYLD-negative OSCC patients with poor prognosis.