Cylindromatosis Knockdown Research Articles

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.

Wnt/β‑catenin signaling is a novel therapeutic target for tumor suppressor CYLD‑silenced glioblastoma cells.

Tumor suppressor cylindromatosis (CYLD) dysfunction by its downregulation is significantly associated with poor prognosis in patients with glioblastoma (GBM), the most aggressive and malignant type of glioma. However, no effective treatment is currently available for patients with CYLD‑downregulated GBM. The aim of the present study was to identify the crucial cell signaling pathways and novel therapeutic targets for CYLD downregulation in GBM cells. CYLD knockdown in GBM cells induced GBM malignant characteristics, such as proliferation, metastasis, and GBM stem‑like cell (GSC) formation. Comprehensive proteomic analysis and RNA sequencing data from the tissues of patients with GBM revealed that Wnt/β‑catenin signaling was significantly activated by CYLD knockdown in patients with GBM. Furthermore, a Wnt/β‑catenin signaling inhibitor suppressed all CYLD knockdown‑induced malignant characteristics of GBM. Taken together, the results of the present study revealed that Wnt/β‑catenin signaling is responsible for CYLD silencing‑induced GBM malignancy; therefore, targeting Wnt/β‑catenin may be effective for the treatment of CYLD‑negative patients with GBM with poor prognosis.

YTHDC2 Retards Cell Proliferation and Triggers Apoptosis in Papillary Thyroid Cancer by Regulating CYLD-Mediated Inactivation of Akt Signaling.

N6-Methyladenosine (m6A) mRNA methylation modification is regarded as an important mechanism involved in diverse physiological processes. YT521-B homology (YTH) domain family members are associated with the tumorigenesis of several cancers. However, the role of YTHDC2 in papillary thyroid cancer (PTC) progression remains unknown. Results showed that YTHDC1, YTHDF1, YTHDF2, and YTHDF3 showed no observable difference in thyroid cancer samples. YTHDC2 was significantly downregulated in thyroid cancer samples and cells. YTHDC2 inhibited cell proliferation in PTC cells. YTHDC2 elicited apoptosis in PTC cells, as demonstrated by the elevated expression of pro-apoptotic factors cl-caspase-3/caspase-3 and Bcl-2-associated (Bax), and the reduced anti-apoptotic B cell lymphoma-2 (Bcl-2) expression. There was a positive correlation between YTHDC2 and cylindromatosis (CYLD) expression based on GEPIA database. YTHDC2 increased CYLD expression in PTC cells. CYLD knockdown abolished the effects of YTHDC2 on PTC cell proliferation and apoptosis. Additionally, YTHDC2 inactivated the protein kinase B (Akt) pathway by increasing CYLD in PTC cells. Overall, YTHDC2 inhibited cell proliferation and induced apoptosis in PTC cells by regulating CYLD-mediated inactivation of Akt pathway.

Down-regulated cylindromatosis enhances NF-κB activation and aggravates inflammation in HBV-ACLF patients

ABSTRACT The pathogenesis of liver in patients with hepatitis B virus-associated acute chronic liver failure (HBV-ACLF) remains largely unknown. We aimed to elucidate the molecular mechanism underlying the pathogenesis of liver in HBV-ACLF patients by using multiple approaches including transcriptome analysis. We performed transcriptomic sequencing analysis on the liver of HBV-ACLF patients (n = 6), chronic hepatitis B (n = 6), liver cirrhosis (n = 6) and normal control (n = 5), then explored the potential pathogenesis mechanism in liver specimen from another 48 subjects and further validated the molecular and cellular mechanisms using THP-1 cells. RNA-sequencing data analysis indicated that, among the genes up-regulated in HBV-ACLF, genes related to inflammatory response and chemotaxis accounted for a large proportion of the total DEGs. A number of key chemokines (CCL2, CCL5, CCL20, CXCL5, CXCL6, CXCL8) and NF-ĸB pathway were identified to be robust in the liver samples from HBV-ACLF patients. Interestingly, cylindromatosis (CYLD) was found to be downregulated in the liver of HBV-ACLF patients, in line with the well-established role of CYLD in regulating most of the chemokines and pro-inflammatory cytokines (CCL2, CCL5, CCL20, CXCL5, CXCL6, CXCL8, IL-6, IL-1β) via inhibition of NF-ĸB. Indeed, the knockdown of CYLD resulted in sustained activation of NF-ĸB in macrophages and enhanced chemokines and inflammatory cytokines production, which in turn enhanced chemotactic migration of neutrophil, monocyte, T lymphocytes, and NK cell. In conclusions, down-regulated CYLD aggravated inflammatory cell chemotaxis through enhancing NF-κB activation in HBV-ACLF patients, thereby participating in the pathogenesis of HBV-ACLF injury.

Deubiquitinase CYLD acts as a negative regulator of dopamine neuron survival in Parkinson's disease.

Mutations in PINK1 and parkin highlight the mitochondrial axis of Parkinson's disease (PD) pathogenesis. PINK1/parkin regulation of the transcriptional repressor PARIS bears direct relevance to dopamine neuron survival through augmentation of PGC-1α-dependent mitochondrial biogenesis. Notably, knockout of PARIS attenuates dopaminergic neurodegeneration in mouse models, indicating that interventions that prevent dopaminergic accumulation of PARIS could have therapeutic potential in PD. To this end, we have identified the deubiquitinase cylindromatosis (CYLD) to be a regulator of PARIS protein stability and proteasomal degradation via the PINK1/parkin pathway. Knockdown of CYLD in multiple models of PINK1 or parkin inactivation attenuates PARIS accumulation by modulating its ubiquitination levels and relieving its repressive effect on PGC-1α to promote mitochondrial biogenesis. Together, our studies identify CYLD as a negative regulator of dopamine neuron survival, and inhibition of CYLD may potentially be beneficial in PD by lowering PARIS levels and promoting mitochondrial biogenesis.

I\u03baB kinase promotes Nrf2 ubiquitination and degradation by phosphorylating cylindromatosis, aggravating oxidative stress injury in obesity-related nephropathy

BackgroundObesity-related nephropathy (ORN) has become one of the leading causes of end-stage renal disease and has tripled over the past decade. Previous studies have demonstrated that decreased reactive oxygen species production may contribute to improving ORN by ameliorating oxidative stress injury. Here, IκB kinase (IKK) was hypothesized to inactivate the deubiquitination activity of cylindromatosis (CYLD) by activating the phosphorylation of CYLD, thus promoting the ubiquitination of NF-E2-related factor 2 (Nrf2) and further aggravating oxidative stress injury of the kidney in ORN. This study was aimed to confirm this hypothesis.MethodsHaematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Oil Red O staining were performed to assess histopathology. Dihydroethidium (DHE) staining and MDA, SOD, CAT, and GSH-PX assessments were performed to measure reactive oxygen species (ROS) production. Immunohistochemical (IHC) staining, qRT–PCR and/or western blotting were performed to assess the expression of related genes. JC-1 assays were used to measure the mitochondrial membrane potential (ΔΨm) of treated HK-2 cells. Co-immunoprecipitation experiments (Co-IP) were used to analyse the interaction between CYLD and Nrf2 in ORN.ResultsORN in vivo and in vitro models were successfully constructed, and oxidative stress injury was detected in the model tissues and cells. Compared with the control groups, the phosphorylation level of CYLD increased while Nrf2 levels decreased in ORN model cells. An IKK inhibitor reduced lipid deposition, ROS production, CYLD phosphorylation levels and ΔΨm in vitro, which were reversed by knockdown of CYLD. Nrf2 directly bound to CYLD and was ubiquitinated in ORN cells. The proteasome inhibitor MG132 activated the Nrf2/ARE signalling pathway, thereby reversing the promoting effect of CYLD knockdown on oxidative stress.ConclusionIKK inactivates the deubiquitination activity of CYLD by activating the phosphorylation of CYLD, thus promoting the ubiquitination of Nrf2 and further aggravating oxidative stress injury of the kidney in ORN. This observation provided a feasible basis for the treatment of kidney damage caused by ORN.

Knockdown of microRNA-130b improves doxorubicin sensitivity in bladder urothelial carcinoma by negatively regulating cylindromatosis expression.

IntroductionChemotherapeutic resistance reduces the sensitivity of bladder urothelial carcinoma (BUC) to chemotherapeutic drugs and contributes a barrier leading to treatment failure. The purpose of this research project is to investigate the regulatory effects of miR-130b on chemotherapeutic drug resistance of BUC and its mechanism.Material and methodsThe relative expression of miRNA-130b and cylindromatosis (CYLD) was examined using real-time quantitative PCR. The cell proliferation and doxorubicin sensitivity were detected with the enhanced CCK-8 assay. The specific combination of miR-130b and CYLD was verified with the luciferase reporter gene assay. Protein expression was detected by Western blot.ResultsOur study found that miR-130b was up-regulated in doxorubicin-insensitive BUC tissues and cell lines, and its high expression was negatively related to doxorubicin sensitivity in BUC. The miR-130b knockdown reduced the IC50 of doxorubicin and improved doxorubicin sensitivity of J82/Dox and T24/Dox cells. For the regulation mechanism analysis of miR-130b, bioinformatics analysis software was used to predict the potential targets of miR-130b, including the CYLD gene. The following luciferase activities assay, quantitative real time-PCR and western blot identified the CYLD gene as a target of miR-130b. Knockdown of CYLD reversed miR-130b’s regulatory roles in doxorubicin sensitivity in J82/Dox and T24/Dox cells.ConclusionsHigh expression of miR-130b is negatively related to doxorubicin sensitivity in BUC, and knockdown of miR-130b improves doxorubicin sensitivity in BUC by negatively regulating CYLD expression. Our findings will provide guidance for the clinical chemotherapy of BUC.

The inhibitory effect of the deubiquitinase cylindromatosis (CYLD) on inflammatory responses in human gingival fibroblasts.

Experiments were performed to evaluate CYLD expression in human gingival tissue samples and to examine the effects of CYLD on inflammatory responses in lipopolysaccharide (LPS)- or TNF-α-stimulated human gingival fibroblasts (HGFs). Immunohistochemistry for CYLD and p65 expression was performed with healthy and inflamed gingival tissue samples. siRNA was used to knock down the expression of CYLD in HGFs. Upon LPS or TNF-α stimulation, NF-κB activation was detected in control and CYLD-knockdown HGFs. RT-PCR was applied to determine gene expression. Western blot analyses were employed to assess protein expression. Immunofluorescence staining was carried out to evaluate the nuclear translocation of p65. Immunohistochemical staining showed the expression of CYLD in human gingival tissues. In addition, CYLD protein expression was reduced in inflamed gingival tissue samples compared with healthy tissue samples. CYLD knockdown greatly enhanced the mRNA expression of proinflammatory cytokines in LPS- or TNF-α-stimulated HGFs. Furthermore, knocking down CYLD expression increased LPS-stimulated NF-κB activation in HGFs. Unexpectedly, CYLD knockdown did not affect TNF-α-induced NF-κB activation. Our results suggest that CYLD participates in periodontal inflammatory responses by negatively regulating LPS-induced NF-κB signalling.

Cylindromatosis Is Required for Survival of a Subset of Melanoma Cells.

The deubiquitinase cylindromatosis (CYLD) functions as a tumor suppressor inhibiting cell proliferation in many cancer types including melanoma. Here we present evidence that a proportion of melanoma cells are nonetheless addicted to CYLD for survival. The expression levels of CYLD varied widely in melanoma cell lines and melanomas in vivo, with a subset of melanoma cell lines and melanomas displaying even higher levels of CYLD than melanocyte lines and nevi, respectively. Strikingly, although short hairpin RNA (shRNA) knockdown of CYLD promoted, as anticipated, cell proliferation in some melanoma cell lines, it reduced cell viability in a fraction of melanoma cell lines with relatively high levels of CYLD expression and did not impinge on survival and proliferation in a third type of melanoma cell lines. The decrease in cell viability caused by CYLD knockdown was due to induction of apoptosis, as it was associated with activation of the caspase cascade and was abolished by treatment with a general caspase inhibitor. Mechanistic investigations demonstrated that induction of apoptosis by CYLD knockdown was caused by upregulation of receptor-interacting protein kinase 1 (RIPK1) that was associated with elevated K63-linked polyubiquitination of the protein, indicating that CYLD is critical for controlling RIPK1 expression in these cells. Of note, microRNA (miR) profiling showed that miR-99b-3p that was predicted to target the 3′-untranslated region (3′-UTR) of the CYLD mRNA was reduced in melanoma cell lines with high levels of CYLD compared with melanocyte lines. Further functional studies confirmed that the reduction in miR-99b-3p expression was responsible for the increased expression of CYLD in a highly cell line-specific manner. Taken together, these results reveal an unexpected role of CYLD in promoting survival of a subset of melanoma cells and uncover the heterogeneity of CYLD expression and its biological significance in melanoma.

Downregulation of CYLD promotes IFN-γ mediated PD-L1 expression in thymic epithelial tumors

ObjectivesRecent genomic studies suggest the biological significance of the cylindromatosis (CYLD) gene in thymic epithelial tumors (TETs). CYLD is a crucial regulator of immune response, and we previously reported that CYLD mutation is associated with high PD-L1 expression in thymic carcinoma. Therefore, we wanted to explore the role and mechanism of CYLD in regulating PD-L1 expression in TETs. Materials and methodsThe role of CYLD in PD-L1 expression was assessed by knockdown of CYLD in TET cells upon stimulation with interferon gamma (IFN-γ), tumor necrosis factor-α (TNF-α) or polyinosinic-polycytidylic acid (poly I:C). The molecular mechanism was investigated through analysis of downstream molecules in the STAT1/IRF1 pathway. Moreover, the clinical correlation between low CYLD and high PD-L1 expression, and the clinical impact of CYLD expression were evaluated in tissue microarrays of 105 TET cases. ResultsCYLD knockdown significantly enhanced the expression of PD-L1 in presence of IFN-γ stimulation in most TET cell lines. However, this phenomenon was not observed in presence of TNF-α stimulation. CYLD knockdown upregulated IFN-γ mediated activation of the STAT1/IRF1 axis, which in turn induced PD-L1 expression. Interestingly, we found a significant association between low CYLD expression and ≥ 50 % PD-L1 expression (p = 0.001). In addition, the average proportion of tumor cells exhibiting PD-L1 staining was significantly higher in the low CYLD expression group (24.7 %) than in the high CYLD expression group (5.2 %) (p = 0.005). There was no correlation between CYLD expression and the frequency of pre-existing paraneoplastic auto-immune diseases. In advanced stages (III/IV), the low CYLD expressing group had numerically worse survival than the high CYLD group (log-rank p = 0.089). ConclusionsOur findings provide insight into the mechanism of regulation of PD-L1 expression by CYLD in TET cells. Tumors with low CYLD expression could be potential targets for PD-1/PD-L1 inhibitors.

BTK Inhibitors Induce ABC-DLBCL Cell Apoptosis By Inhibiting CYLD Phosphorylation

BTK inhibitors induce ABC-DLBCL cell apoptosis by inhibiting CYLD phosphorylation Xin Xu1, Zhigang Zhu1, Feng Liu1 , Weijie Zhong1,Huabao Xiong2, Tiefeng Feng3, and Qingshan Li3 1Geriatrics Department, GuangZhou First People's Hospital, GuangDong, 510180, People's Republic of China. 2Precision Immunology Institute, Mount Sinai School of Medicine, New York, NY 10029, U.S.A. 3Hemotology Department, GuangZhou First People's Hospital, GuangDong, 510180, People's Republic of China. Activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL) is a common aggressive diffuse large B cell lymphoma (DLBCL). Even though CD20 antibody (Rituximab) combination therapy improved the total prognosis of DLBCL, there are still many relapsed and refractory ABC-DLBCL cases. The new treatment options are urgently needed. Pathogenesis of ABC-DLBCL is different from germinal center B cell-like diffuse large B cell lymphoma (GCB-DLBCL), which is related to activation of B cell antigen receptor(BCR) signaling. The Bruton tyrosine kinase(BTK) plays a central role in BCR signaling. Inhibition of BTK function will affect BCR signaling activity, which is involved in maintaining the malignant phenotype in B-cell lymphoma. Now in clinic, BTK inhibitors like Ibrutinib or Acalabrutinib are used extensively in chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma(MCL) as first line therapy or second line therapy. But in DLBCL its role is unclear now.Some DLBCL clinic trials got the better PFS and OS in BTK inhibitor combination therapy group(combined with Rituximab or RCHOP) but some got the negative results. Whether BTK inhibitors can definitely improve ABC-DLBCL survival and the mechanism how BTK inhibitors inducing cell death are needed further investigations. CYLD (cylindromatosis) is a tumor suppressor, its activity is inhibited by phosphorylation, which is related to apoptosis regulation pathway NF-kB in many kinds of tumors.In this study, we found that CYLD to be constitutively phosphorylated in ABC-DLBCL cell line models (HBL-1 and OCI-Ly10) as well as primary samples from patients with non-GCB DLBCL. We subsequently examined the effect of BTK inhibitors Ibrutinib (PCI-32765) and Acalabrutinib (ACP-196) on CYLD phosphorylation. We found that BTK inhibitors Ibrutinib and Acalabrutinib both induced ABC-DLBCL apoptosis by down-regulating CYLD phosphorylation in vivo and in vitro, even in the Rituximab resistant ABC-DLBCL cell line.Combination of BTK inhibitors and Rituximab would enhance this effect. Knockdown of CYLD in ABC-DLBCL cells reduced the level of cell death induced by BTK inhibitors, which showed that this cell death induced by BTK inhibitors was CYLD dependent. Through this study, we conclude that BTK inhibitors induce CYLD inactivation by regulating CYLD phosphorylation is clinical beneficial in ABC-DLBCL therapy, especially the relapsed or refractory ABC-DLBCL cases after Rituximab therapy. Disclosures No relevant conflicts of interest to declare.

Loss of Tumor Suppressor CYLD Expression Triggers Cisplatin Resistance in Oral Squamous Cell Carcinoma.

Cisplatin is one of the most effective chemotherapeutic agents commonly used for several malignancies including oral squamous cell carcinoma (OSCC). Although cisplatin resistance is a major obstacle to effective treatment and is associated with poor prognosis of OSCC patients, the molecular mechanisms by which it develops are largely unknown. Cylindromatosis (CYLD), a deubiquitinating enzyme, acts as a tumor suppressor in several malignancies. Our previous studies have shown that loss of CYLD expression in OSCC tissues is significantly associated with poor prognosis of OSCC patients. Here, we focused on CYLD expression in OSCC cells and determined whether loss of CYLD expression is involved in cisplatin resistance in OSCC and elucidated its molecular mechanism. In this study, to assess the effect of CYLD down-regulation on cisplatin resistance in human OSCC cell lines (SAS), we knocked-down the CYLD expression by using CYLD-specific siRNA. In cisplatin treatment, cell survival rates in CYLD knockdown SAS cells were significantly increased, indicating that CYLD down-regulation caused cisplatin resistance to SAS cells. Our results suggested that cisplatin resistance caused by CYLD down-regulation was associated with the mechanism through which both the reduction of intracellular cisplatin accumulation and the suppression of cisplatin-induced apoptosis via the NF-κB hyperactivation. Moreover, the combination of cisplatin and bortezomib treatment exhibited significant anti-tumor effects on cisplatin resistance caused by CYLD down-regulation in SAS cells. These findings suggest the possibility that loss of CYLD expression may cause cisplatin resistance in OSCC patients through NF-κB hyperactivation and may be associated with poor prognosis in OSCC patients.

Abstract 5150: Loss of CYLD promotes cell invasion via ALK5 stabilization in oral squamous cell carcinoma

Abstract Oral squamous cell carcinoma (OSCC) has a very poor prognosis because of its highly invasive nature, and the 5-year survival rate has not changed appreciably for the past 30 years. Although cylindromatosis (CYLD), a deubiquitinating enzyme, is thought to be a potent tumor suppressor, its biological and clinical significance in OSCC is largely unknown. This study aimed to clarify the roles of CYLD in OSCC progression. Our immunohistochemical analyses revealed significantly reduced CYLD expression in invasive areas in OSCC tissues, whereas CYLD expression was conserved in normal epithelium and carcinoma in situ. Furthermore, downregulation of CYLD by siRNA led to acquisition of mesenchymal features and increased migratory and invasive properties in OSCC cells and HaCaT keratinocytes. It is interesting that CYLD knockdown promoted TGF-β signaling by inducing stabilization of TGF-β receptor I (ALK5) in a cell autonomous fashion. In addition, the response to exogenous TGF-β stimulation was enhanced by CYLD downregulation. The invasive phenotypes induced by CYLD knockdown were completely blocked by an ALK5 inhibitor. Also, lower CYLD expression was significantly associated with the clinical features of deep invasion, poor overall survival, and increased Smad3 phosphorylation, which is an indicator of activation of TGF-β signaling in invasive OSCC. These findings suggest that downregulation of CYLD promotes invasion with mesenchymal transition via ALK5 stabilization in OSCC cells. Citation Format: Satoru Shinriki, Hirofumi Jono, Manabu Maeshiro, Takuya Nakamura, Jianying Guo, Jian-Dong Li, Ryoji Yoshida, Masanori Shinohara, Hideki Nakayama, Hirotaka Matsui, Yukio Ando. Loss of CYLD promotes cell invasion via ALK5 stabilization in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5150.

Loss of CYLD promotes cell invasion via ALK5 stabilization in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) has a very poor prognosis because of its highly invasive nature, and the 5-year survival rate has not changed appreciably for the past 30 years. Although cylindromatosis (CYLD), a deubiquitinating enzyme, is thought to be a potent tumour suppressor, its biological and clinical significance in OSCC is largely unknown. This study aimed to clarify the roles of CYLD in OSCC progression. Our immunohistochemical analyses revealed significantly reduced CYLD expression in invasive areas in OSCC tissues, whereas CYLD expression was conserved in normal epithelium and carcinoma in situ. Furthermore, downregulation of CYLD by siRNA led to the acquisition of mesenchymal features and increased migratory and invasive properties in OSCC cells and HaCaT keratinocytes. It is interesting that CYLD knockdown promoted transforming growth factor-β (TGF-β) signalling by inducing stabilization of TGF-β receptor I (ALK5) in a cell autonomous fashion. In addition, the response to exogenous TGF-β stimulation was enhanced by CYLD downregulation. The invasive phenotypes induced by CYLD knockdown were completely blocked by an ALK5 inhibitor. In addition, lower expression of CYLD was significantly associated with the clinical features of deep invasion and poor overall survival, and also with increased phosphorylation of Smad3, which is an indicator of activation of TGF-β signalling in invasive OSCC. These findings suggest that downregulation of CYLD promotes invasion with mesenchymal transition via ALK5 stabilization in OSCC cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MiR-767 promoted cell proliferation in human melanoma by suppressing CYLD expression

MicroRNAs (miRNAs) have emerged as critical regulators for cancer development and progression of human melanoma. However, the potential molecular mechanism of miR-767 in human melanoma has not been intensively investigated. In this present study, we confirmed that miR-767 was frequently up-regulated in human melanoma tissues and cell lines. Ectopic expression of miR-767 promoted cell proliferation in human melanoma cell lines A375 and WM35, whereas miR-767-in reversed the function. Bioinformatics analysis revealed that cylindromatosis (CYLD) was hypothesized to be a possible target gene of miR-767, and this was confirmed by luciferase activity assay. Knockdown of CYLD counteracted the proliferation arrest by miR-767-in in melanoma cells A375 and WM35. In conclusion, our study indicated that miR-767 acted as a role of tumor promoter by targeting CYLD in human melanoma, and might serve as a prognostic or therapeutic target for human melanoma.

Blocking Notch signal in myeloid cells alleviates hepatic ischemia reperfusion injury by repressing the activation of NF-κB through CYLD.

Ischemia-reperfusion (I/R) is a major reason of hepatocyte injury during liver surgery and transplantation. Myeloid cells including macrophages and neutrophils play important roles in sustained tissue inflammation and damage, but the mechanisms regulating myeloid cells activity have been elusive. In this study, we investigate the role of Notch signaling in myeloid cells during hepatic I/R injury by using a mouse model of myeloid specific conditional knockout of RBP-J. Myeloid-specific RBP-J deletion alleviated hepatic I/R injury. RBP-J deletion in myeloid cells decreased hepatocytes apoptosis after hepatic I/R injury. Furthermore, myeloid-specific RBP-J deletion led to attenuated inflammation response in liver after I/R injury. Consistently, Notch blockade reduced the production of inflammatory cytokines by macrophages in vitro. We also found that blocking Notch signaling reduced NF-κB activation and increased cylindromatosis (CYLD) expression and knockdown of CYLD rescued reduction of inflammatory cytokines induced by Notch blockade in macrophages during I/R injury in vitro. On the other hand, activation of Notch signaling in macrophages led to increased inflammatory cytokine production and NF-κB activation and decreased CYLD expression in vitro. These data suggest that activation of Notch signaling in myeloid cells aggravates I/R injury, by enhancing the inflammation response by NF-κB through down regulation of CYLD.

MiR-186 suppressed CYLD expression and promoted cell proliferation in human melanoma.

Previous studies have shown that microRNA-186 (miR-186) is overexpressed in various human cancers and is associated with the regulation of the carcinogenic processes. However, the underlying mechanisms of this microRNA in melanoma remain largely unknown. In the present study, the overexpression of miR-186 was identified in melanoma tissues and melanoma cells compared to the expression of miR-186 in the matched tumor adjacent tissues and normal human epidermal melanocytes. Overexpression of miR-186 promoted the proliferation and anchorage-independent growth of melanoma cells, whereas inhibition of miR-186 reduced this effect. Bioinformatics analysis also revealed cylindromatosis (CYLD), a putative tumor suppressor, to be a potential target of miR-186. Luciferase reporter assays showed that miR-186 directly targeted the 3′-untranslated regions of CYLD messenger RNA. Additional experiments showed that overexpression of miR-186 promoted the proliferation of melanoma cells, which was consistent with the inhibitory effects induced by knockdown of CYLD. In summary, the present study indicated that miRNA-186 plays a crucial role in melanoma growth and its oncogenic effect is mediated chiefly through the direct suppression of CYLD expression.

CYLD Promotes TNF-α-Induced Cell Necrosis Mediated by RIP-1 in Human Lung Cancer Cells.

Lung cancer is one of the most common cancers in the world. Cylindromatosis (CYLD) is a deubiquitination enzyme and contributes to the degradation of ubiquitin chains on RIP1. The aim of the present study is to investigate the levels of CYLD in lung cancer patients and explore the molecular mechanism of CYLD in the lung cancer pathogenesis. The levels of CYLD were detected in human lung cancer tissues and the paired paracarcinoma tissues by real-time PCR and western blotting analysis. The proliferation of human lung cancer cells was determined by MTT assay. Cell apoptosis and necrosis were determined by FACS assay. The results demonstrated that low levels of CYLD were detected in clinical lung carcinoma specimens. Three pairs of siRNA were used to knock down the endogenous CYLD in lung cancer cells. Knockdown of CYLD promoted cell proliferation of lung cancer cells. Otherwise overexpression of CYLD induced TNF-α-induced cell death in A549 cells and H460 cells. Moreover, CYLD-overexpressed lung cancer cells were treated with 10 μM of z-VAD-fmk for 12 hours and the result revealed that TNF-α-induced cell necrosis was significantly enhanced. Additionally, TNF-α-induced cell necrosis in CYLD-overexpressed H460 cells was mediated by receptor-interacting protein 1 (RIP-1) kinase. Our findings suggested that CYLD was a potential target for the therapy of human lung cancers.

Myeloid-specific disruption of recombination signal binding protein Jκ ameliorates hepatic fibrosis by attenuating inflammation through cylindromatosis in mice.

Macrophages play multidimensional roles in hepatic fibrosis, but their control has not been fully understood. The Notch pathway mediated by recombination signal binding protein Jκ (RBP-J), the transcription factor transactivated by signals from four mammalian Notch receptors, is implicated in macrophage activation and plasticity. In this study, by using mouse hepatic fibrosis models, we show that myeloid-specific disruption of RBP-J resulted in attenuated fibrosis. The activation of hepatic stellate cells and production of profibrotic factors including platelet-derived growth factor (PDGF)-B and transforming growth factor beta1 (TGF-β1) reduced significantly in myeloid-specific RBP-J deficient mice. The infiltration of inflammatory cells and production of proinflammatory factors were reduced in liver of myeloid-specific RBP-J-deficient mice during fibrosis. In RBP-J-deficient macrophages, the nuclear factor kappa B (NF-κB) activation was remarkably attenuated as compared with the control. This could be attributed to the up-regulation of cylindromatosis (CYLD), a negative regulator of NF-κB, in Notch signal-compromised macrophages, because the knockdown of CYLD in RBP-J-deficient macrophages or overexpression of p65 in RBP-J knockdown cells both restored NF-κB activation and the production of proinflammatory and/or profibrotic factors by macrophages. In human hepatic fibrosis biopsies, stronger Notch activation is correlated with more severe fibrosis, which is accompanied by a lower level of CYLD but irrespective of etiological reasons. RBP-J-mediated Notch signaling is required for macrophages to promote hepatic fibrosis by up-regulation of NF-κB activation through CYLD.

Abstract 4314: Loss of CYLD promotes cell migration through stabilized ALK5 in human oral squamous cell carcinoma cells.

Abstract Five-year survival of patients with oral squamous cell carcinoma (OSCC) has not changed apparently for the past 30 years. Although cylindromatosis (CYLD) is thought as a potent tumor suppressor, its biological significances in malignancies are largely unknown. The aim of this study was to clarify the role of CYLD in OSCC progression. We investigated expression of CYLD in OSCC including intraepithelial neoplasia (IEN; n = 48) and invasive carcinomas (n = 133) tissues and normal oral mucosal tissues (n = 35) by immunohistochemistry. In addition, effects of CYLD knockdown by siRNA or its overexpression on OSCC progression using OSCC cell lines. Our immunohistochemical analyses revealed that CYLD expression was significantly reduced at invasive lesions in OSCC tissues whereas it was conserved in normal epithelium and IEN. In addition, lower CYLD expression was associated with poor overall survival in invasive OSCC. Accordingly, CYLD knockdown led to acquisition of mesenchymal state and increased migratory activity in OSCC cells. Interestingly, loss of CYLD enhanced TGFβR1 (ALK5) protein expression by promoting ALK5 stability while overexpression of wild-type CYLD downregulated ALK5. Furthermore, EMT-like changes induced by CYLD knockdown were completely blocked by a TGFβR1 kinase inhibitor in OSCC cells. These findings suggest that downregulation of CYLD promotes invasion through EMT-like changes via ALK5 stabilization in OSCC cells. Further studies for these mechanisms probably provide new insights into the TGFβ signal transduction in cancer and development of a novel therapy in OSCC. Citation Format: Satoru Shinriki, Hirofumi Jono, Jian-Dong Li, Takuya Nakamura, Mitsuhiro Hayashi, Jianying Guo, Masanori Shinohara, Yukio Ando. Loss of CYLD promotes cell migration through stabilized ALK5 in human oral squamous cell carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4314. doi:10.1158/1538-7445.AM2013-4314