CYLD Expression Research Articles

Grading Challenges and Prognostic Insights in Chromophobe Renal Cell Carcinoma: A Retrospective Study of 72 Patients.

Chromophobe RCC (ChRCC) carries the best prognosis among all RCC subtypes, yet it lacks a proper grading system. Various systems have been suggested in the past, causing much controversy, and Avulova et al. recently proposed a promising four-tier grading system that takes into consideration tumor necrosis. Dysregulation of the mammalian target of the rapamycin (mTOR) pathway plays a key role in ChRCC pathogenesis, highlighting its molecular complexity. The present retrospective study aimed to evaluate the prognostic factors associated with a more aggressive ChRCC phenotype. Materials and Methods: Seventy-two patients diagnosed with ChRCC between 2004 and 2017 were included in our study. Pathology reports and tissue blocks were reviewed, and immunohistochemistry (IHC) was performed in order to assess the expressions of CYLD (tumor-suppressor gene) and mTOR, among other markers. Univariate analysis was performed, and OS was assessed using the Kaplan-Meier method. Results: In our study, 74% of patients were male, with a mean age of 60 years, and the mean tumor size was 63 mm (±44). The majority (54%) were followed for more than 10 years at intervals ranging between 44 and 222 months. The risk of death was significantly higher for patients that were classified as Grade 4 in the Avulova system (HR: 5.83; 95% CI, 1.37-24.7; p: = 0.017). As far as the IHC is concerned, mTOR expression was associated with an HR of 8.57 (95% CI, 1.91-38.5; p = 0.005), and CYLD expression was associated with an HR of 17.3 (95% CI, 1.57-192; p = 0.02). Conclusions: In our study, the Avulova grading system seems to be positively correlated with OS in patients diagnosed with ChRCC. Furthermore, an elevated mTOR expression also shows a negative correlation with OS, whereas an elevated CYLD expression does not seem to exert a protective role. However, because only a small proportion (4.2%) of our patients died due to ChRCC, despite the long follow-up period, the results must be interpreted with caution. Further research is needed to validate our findings.

Characterization of necroptosis activators in colorectal cancer.

3522 Background: Necroptosis is a pro-inflammatory form of programmed cell death triggered by cellular stresses and extrinsic cytokines. It is a potential therapeutic target for immunotherapy (IO) in colorectal cancer (CRC). Here, we present comprehensive clinical and molecular characterization of key necroptosis regulators in CRC. Methods: 24,257 samples of CRC tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq), NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES), and PD-L1 expression (22C3 clone; TPS ≥1%) were analyzed. Expression of activators in the necroptosis pathway ( RIPK3, MLKL, CYLD, and ZBP1) was categorized as either high (H) or low (L) using median expression as the cutoff. CRCs with high expression of all four activators were considered to have high propensity for necroptosis activity (NA-H, N=4595) compared to tumors with low expression in all activators (NA-L, N = 4946). RNA deconvolution analysis with QuantiSEQ estimated immune cell infiltration in the tumor microenvironment (TME). Differences in overall survival (OS) were analyzed from insurance claims data and calculated from treatment initiation using Kaplan-Meier estimates. Statistical significance was set as a P-value adjusted for multiple comparisons ( q<0.05). Results: NA-H was associated with increased PD-L1 positivity (4.0% vs 2.3%), immune cell infiltration, and T-cell inflamed score (all q<0.001). TP53 mutations were more frequent in NA-H, while SMAD2, SMAD4, KRAS, and NRAS mutations were associated with NA-L (all q<0.01). NA-H was associated with activation of KRAS signaling, allograft rejection, inflammatory/interferon gamma response, and pancreatic beta cell function pathways (all q<0.05). NA-H showed significantly improved OS in FOLFOX/FOLFIRI (F/F) (NA-L: 34.3 vs NA-H: 39.6 months [mo], P < 0.001; HR = 0.83, 95% CI 0.77 – 0.90) and those treated with IO (15.3 vs 22.4 mo, P = 0.018; HR = 0.77, 95% CI 0.62– 0.96) treated CRC patients. The benefit remained in TP53-mutated CRC treated with F/F (34.6 vs 41.0 mo, P < 0.001; HR = 0.82, 95% CI 0.74-0.90) and IO (10.0 vs 21.8 mo, P = 0.027; HR = 0.68, 95% CI 0.48-0.96) but not in the TP53-wild-type subgroup. Improved OS was observed with increased expression of CYLD and ZBP1(Table). The benefit remained in TP53-mutated CRC but not in the TP53 wildtype subgroup. Conclusions: This is the largest molecular and clinical characterization of necroptotic genes in CRC. Our data shows that activation of the necroptosis pathway is associated with immune cell infiltration/pathway activation, PD-L1 expression, and improved OS on F/F and IO. This benefit is more pronounced in TP53-mutated CRC, suggesting possible novel therapeutic targets.[Table: see text]

CYLD regulates cell ferroptosis through Hippo/YAP signaling in prostate cancer progression

Prostate cancer (PCa) is one of the most common malignancy in men. However, the molecular mechanism of its pathogenesis has not yet been elucidated. In this study, we demonstrated that CYLD, a novel deubiquitinating enzyme, impeded PCa development and progression via tumor suppression. First, we found that CYLD was downregulated in PCa tissues, and its expression was inversely correlated with pathological grade and clinical stage. Moreover, we discovered that CYLD inhibited tumor cell proliferation and enhanced the sensitivity to cell ferroptosis in PCa in vitro and in vivo, respectively. Mechanistically, we demonstrated that CYLD suppressed the ubiquitination of YAP protein, then promoted ACSL4 and TFRC mRNA transcription. Then, we demonstrated that CYLD could enhance the sensitivity of PCa xenografts to ferroptosis in vivo. Furthermore, we discovered for the first time that there was a positive correlation between CYLD expression and ACSL4 or TFRC expression in human PCa specimens. The results of this study suggested that CYLD acted as a tumor suppressor gene in PCa and promoted cell ferroptosis through Hippo/YAP signaling.

Cancer-associated fibroblasts-derived CXCL12 enhances immune escape of bladder cancer through inhibiting P62-mediated autophagic degradation of PDL1

BackgroundCancer-associated fibroblasts (CAFs), the predominant stromal cell of tumor microenvironment (TME), play an important role in tumor progression and immunoregulation by remodeling extracellular matrix (ECM) and secreting cytokines. However, little is known about the details of the underlying mechanism in bladder cancer.MethodsBioinformatics analysis was performed to analyze the prognostic value of CAFs and CXCL12 using GEO, TCGA and SRA databases. The effects of CXCL12 on bladder cancer progression were investigated through in vitro and in vivo assays. The biological mechanism of the effect of CXCL12 on PDL1 were investigated using western blotting, immunoprecipitation, RT-PCR, immunofluorescence, mass spectrometry, protein stability, and flow cytometry.ResultsThe results demonstrated that CAFs-derived CXCL12 promoted cancer cell migration and invasion and upregulated PDL1. Mechanistically, upon binding to its specific receptor, CXCL12 activated the downstream JAK2/STAT3 pathway and rapidly up-regulated the expression of deubiquitinase CYLD. CYLD deubiquitinated P62 causing P62 accumulation, which in turn inhibited the autophagic degradation of PDL1. In vivo experiments demonstrated that blocking CXCL12 inhibited tumor growth, reduced tumor PDL1 expression and increased immune cell infiltration.ConclusionsThis study revealed a novel mechanism for the role of CXCL12 in P62-mediated PDL1 autophagic regulation. Combined application of CXCL12 receptor blocker and PD1/PDL1 blocker can more effectively inhibit PDL1 expression and enhance antitumor immune response. Targeting CAFs-derived CXCL12 may provide an effective strategy for immunotherapy in bladder cancer.

The Deubiquitinase CYLD Acts As an Oncogene in a Cellular Model of Chronic Lymphocytic Leukemia

The cylindromatosis protein (CYLD) is a functional deubiquitinase that regulates critical signaling pathways, e.g. NF-κB and Wnt, thus modulating several cellular functions. CYLD acts as a tumor suppressor gene in solid tumors, and is also involved in the pathogenesis of hematological malignancies, including B cell lymphomas, in as yet unclear ways. In chronic lymphocytic leukemia (CLL), preliminary evidence suggests that reduced expression of CYLD correlates with a worse clinical prognosis, which appears to be in line with its postulated role as a tumor suppressor. Here we sought to gain insight into the function of CYLD in CLL through genetic engineering, molecular characterization and bioenergetic profiling. To this end, we used CRISPR/Cas9 technology and a CYLD-targeting or an unrelated gRNA to generate stable CYLD-knockout ( CYLDko) and control ( CYLDwt) MEC1 cells. Phenotypic characterization of CYLDkoversus CYLDwt MEC1 cells by flow cytometry showed (i) significantly reduced viability, assessed by Annexin V (fold difference, FD=1.2, p<0.05); (ii) lower cell proliferation rate, assessed by Ki67 expression (FD=1.4, p<0.05); (iii) increased apoptosis, determined by measuring active caspase 3 expression levels (FD=6.1, p<0.01); and, (iv) reduced expression of CD86 (FD=4.3, p<0.001) and CD40 (FD=4.1, p=0.05). Western blotting analysis of CYLDko versus CYLDwtMEC1 cells revealed down-regulation of the NF-κB pathway evidenced by diminished expression of ΙΚΚβ (FD=2.4, p<0.01), phospho-ΙκΒα (FD=3.4, p<0.01) and phospho-p105 (FD=2.5, p=0.2); and, the Wnt pathway evidenced by reduced β-catenin levels (FD=2, p<0.05). Transcriptome profiling by RNA-seq gave concordant results, in documenting increased apoptosis and decreased NF-κΒ signaling in CYLDkoversus CYLDwtMEC1 cells. The former also showed downregulation of calcium, BcR and PI3K/AKT/mTOR signaling pathways and, in contrast, upregulation of the glutathione and KEAP1-NFE2L2 pathways, which contribute to antioxidant defense and nutrient metabolism, as well as, regulation of redox balance and cellular metabolism, respectively. To evaluate the impact of CYLD deletion on CLL bioenergetics, we assessed ATP production rate, as a marker of active cellular metabolism, using the Seahorse XF Real-Time ATP Rate assay. CYLDkoMEC1 cells exhibited impaired ATP production reflected in decreased OCR (oxygen consumption rate) and ECAR (extracellular acidification rate), that are proportional to OXPHOS (oxidative phosphorylation) and glycolysis, respectively. Regarding the metabolic phenotype, CYLDwtMEC1 cells displayed a shift towards mitochondrial OXPHOS, whereas, in CYLDkoMEC1 cells ATP production was mostly based on glycolysis. High-performance liquid chromatography was performed using culture supernatants obtained at sequential time points to quantify glucose uptake and lactate secretion rates. The analysis revealed that CYLDwt MEC1 cells primarily directed glucose carbon toward biomass biosynthetic pathways, as reflected in both higher cell number and proliferation rate achieved, while CYLD deletion redirected carbon flux enhancing lactate formation. In both cases, once glucose was consumed (day 4), the secreted lactate was re-used, yet this was more pronounced in CYLDko MEC1 cells. Finally, we explored whether CYLD knockout might impact MEC1 sensitivity to targeted agents i.e. the BTK inhibitor ibrutinib and the BCL2 inhibitor venetoclax. We found that CYLDkoMEC1 cells presented increased apoptosis compared to their CYLDwtcounterparts when cultured in the presence of either drug. Moreover, treatment with ibrutinib or venetoclax led to reduced ATP production rates in both CYLDkoand CYLDwtMEC1 cells, albeit the reduction was more pronounced in the former (FD=4.5 for ibrutinib; FD=10 for venetoclax compared to the respective CYLDwt MEC1 treated cells). Taken together, we demonstrate for the first time that CYLD can also act as an oncogene, at least in the context of CLL and in particular in the MEC1 cell line model of CLL, since its elimination leads to (i) lower proliferation and increased apoptosis rates coupled with diminished signaling capacity; (ii) metabolic rewiring toward enhanced lactate formation; and, (iii) augmented sensitivity to CLL therapeutic agents. It remains to be elucidated under which condition this could also occur in the patients with CLL or other B lymphoproliferative disorders.

Up-regulated lncRNA CYLD as a ceRNA of miR-2383 facilitates bovine viral diarrhea virus replication by promoting CYLD expression to counteract RIG-I-mediated type-I IFN production

Bovine viral diarrhea virus (BVDV) is one of the most important pathogens of cattle, causing numerous economic losses to the cattle industry. To date, many potential mechanisms of BVDV evading or subverting innate immunity are still unknown. In this study, an lnc-CYLD/miR-2383/CYLD axis involved in BVDV-host interactions was screened from RNA-seq-based co-expression networks analysis of long noncoding RNAs, microRNAs and mRNAs in BVDV-infected bovine cells, and underlying mechanisms of lnc-CYLD/miR-2383/CYLD axis regulating BVDV replication were explored. Results showed that BVDV-induced up-regulation of the lnc-CYLD competed for binding to the miR-2383, and then promoted CYLD expression, thereby inhibiting RIG-I-mediated type-I interferon (IFN) production, which was subsequently confirmed by treatment with lnc-CYLD overexpression and miR-2383 inhibitor. However, miR-2383 transfection and small interfering RNA-mediated lnc-CYLD knockdown inhibited CYLD expression and enhanced RIG-I-mediated type-I IFN production, inhibiting BVDV replication. In addition, interaction relationship between lnc-CYLD and miR-2383, and colocalization relationship of lnc-CYLD, miR-2383 and CYLD were confirmed by dual-luciferase assay and in situ hybridization assay. Conclusively, up-regulation of the lnc-CYLD as a competing endogenous RNA binds to the miR-2383 to reduce inhibitory effect of the miR-2383 on the CYLD expression, playing an important role in counteracting type-I IFN-dependent antiviral immunity to facilitate BVDV replication.

A 3-gene signature comprising CDH4, STAT4 and EBV-encoded LMP1 for early diagnosis and predicting disease progression of nasopharyngeal carcinoma

PurposeNasopharyngeal carcinoma is highly metastatic but difficult to detect in its early stages. It is critical to develop a simple and highly efficient molecular diagnostic method for early detection of NPC in clinical biopsies.MethodsThe transcriptomic data of primary NPC cell strains were used as a discovery tool. Linear regression approach was used to define signatures distinctive between early and late stage of NPC. Expressions of candidates were validated with an independent set of biopsies (n = 39). Leave-one-out cross-validation technique was employed to estimate the prediction accuracy on stage classification. The clinical relevance of marker genes was verified using NPC bulk RNA sequencing data and IHC analysis.ResultsThree genes comprising CDH4, STAT4, and CYLD were found to have a significant differentiating power to separate NPC from normal nasopharyngeal samples and predicting disease malignancy. IHC analyses showed stronger CDH4, STAT4, and CYLD immunoreactivity in adjacent basal epithelium compared with that in tumor cells (p < 0.001). EBV-encoded LMP1 was exclusively expressed in NPC tumors. Using an independent set of biopsies, we showed that a model combining CDH4, STAT4, and LMP1 had a 92.86% of diagnostic accuracy, whereas a combination of STAT4 and LMP1 had a 70.59% accuracy for predicting advanced disease. Mechanistic studies suggested that promoter methylation, loss of DNA allele, and LMP1 contributed to the suppressive expression of CDH4, CYLD, and STAT4, respectively.ConclusionA model combining CDH4 and STAT4 and LMP1 was proposed to be a feasible model for diagnosing NPC and predicting late stage of NPC.

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.

Novel pharmacotherapy targeting NF-κB signal for triple-negative breast cancer patients with loss of CYLD expression

Novel pharmacotherapy targeting NF-κB signal for triple-negative breast cancer patients with loss of CYLD expression

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

BackgroundTumor 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.MethodsWe 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.ResultsCYLD-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-β (TGF-β) 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.ConclusionsOur 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.

Effects of Vinblastine and Vincristine on the function of chronic myeloid leukemic cells through expression of A20 and CYLD.

Chronic myelogenous leukemia (CML) is characterised by the translocation of regions of the BCR and ABL genes, leading to the fusion gene BCR-ABL forming the Philadelphia (Ph) chromosome. Vinblastine (Vinb) and Vincristine (Vinc) are Vinca alkaloids and frequently used in combination chemotherapy in leukemias and lymphomas. Deubiquitinating enzyme (DUB) genes such as A20, Otubain 1 and CYLD are known as inhibitors of functional activation of immune cells mediated through the NF-κB/STAT pathway. Little is known about the regulatory role of Vinb/Vinc on the function of CML cells and the contribution of the DUBs to those effects. In the end, the gene expression profile was determined by quantitative RT-PCR, physiological properties of CML cells by flow cytometry and cytokine production by ELISA. As a result, inactivated expression of the DUBs A20, CYLD, Otubain 1 and Cezanne and enhanced activation of CD11b+ and CD4T cells were observed in CML patients. Importantly, Vinc enhanced the expression of A20 and CYLD and inhibited the proliferation and survival of CML (K562) cells. The effects were abolished in the presence of A20 siRNA, while cell proliferation only depended on the presence of CYLD. In conclusion, the up-regulation of A20 by Vinc could involve inhibitory effects on the proliferation and survival of K562 cells. The events might contribute to the anticancer effect of Vinc on A20-sensitive CML cells.

RNF213-associated urticarial lesions with hypercytokinemia

Urticarial lesions are observed in both cutaneous and systemic disorders. Familial forms of urticarial syndromes are rare and can be encountered in systemic autoinflammatory diseases. We sought to investigate a large family with dominantly inherited chronic urticarial lesions associated with hypercytokinemia. We performed a genetic linkage analysis in 14 patients from a 5-generation family, as well as whole-exome sequencing, cytokine profiling, and transcriptomic analyses on samples from 2 patients. The identified candidate protein was studied after invitro expression of the corresponding normal and mutated recombinant proteins. An unsupervised proteomic approach was used to unveil the associated protein network. The disease phenotype of the most affected family members is characterized by chronic urticarial flares associated with extremely high plasma levels of proinflammatory (IL-1β, IL-6, and TNF-α) and anti-inflammatory (IL-10 and IL-1 receptor antagonist [IL-1RA]) cytokines, with no secondary organ dysfunction, no susceptibility to infections, no fever, and normal C-reactive protein levels. Monocyte transcriptomic analyses identified an immunotolerant profile in the most affected patient. The affected family members carried a loss-of-function mutation in RNF213 that encodes mysterin, a protein with a poorly known physiologic role. We identified the deubiquitinase CYLD, a major regulator of inflammation, as an RNF213 partner and showed that CYLD expression is inhibited by wild-type but not mutant RNF213. We identified a new entity characterized by chronic urticarial lesions associated with a clinically blunted hypercytokinemia. This disease, which is due to loss of function of RNF213, reveals mysterin's key role in the complex molecular network of innate immunity.

Melatonin Increases the Sensitivity of Osteosarcoma Cells to Chemotherapy Drug Cisplatin.

Chemotherapy, which is one of the common treatments for osteosarcoma (OS), has many side effects and in some cases has low effectiveness due to chemoresistance, hence it is vital to study new therapies for OS. In this regard, we combined melatonin with cisplatin and evaluate their effect on MG63 OS cells. Since melatonin has anti-cancer properties, we hypothesized that its combination with cisplatin could increase the effectiveness of cisplatin. Firstly, MTT assay was used to evaluate the cell viability and cytotoxicity of cisplatin on MG63 cells and the results showed that melatonin in combination with cisplatin increases the sensitivity of MG63 cells to cisplatin. In addition, qRT-PCR results showed that the expressions of miR-181 and P53, CYLD, CBX7 and BCL2 genes change in MG63 cells after treatment with the combination of cisplatin and melatonin, so that the expression of P53, CYLD and CBX7 increased and the expression of BCL2 and miR-181b decreases significantly. Furthermore, analysis of Annexin V/FITC assay data revealed that the rate of apoptosis in MG63 OS cell line remarkably promoted after treated with cisplatin and melatonin combination. As a result, our findings show that melatonin in combination with cisplatin increases the effectiveness of cisplatin in osteosarcoma cells and this study provides a new therapeutic approach for OS.

TRPA1 promotes melanosome phagocytosis in keratinocytes via PAR-2/CYLD axis

BackgroundKeratinocytes 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 in human epidermal melanocytes; however, the mechanism remains unknown. ObjectiveThis study aimed to investigate the roles and mechanism(s) of action of TRPA1 in keratinocytes. MethodsThe correlation between TRPA1 expression levels and the ability of keratinocytes to phagocytize melanosomes was examined using melanin silver staining. TRPA1 depleted human epidermal keratinocytes 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. ResultsTRPA1 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. ConclusionsOur 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.

Quercetin and Methotrexate in Combination have Anticancer Activity in Osteosarcoma Cells and Repress Oncogenic MicroRNA-223

Osteosarcoma (OS) is one of the most common bone neoplasms in adolescents. Notable short- and long-term toxic effects of OS chemotherapy regimens have been reported. Hence, new chemotherapeutic agents with the ability to potentiate OS chemotherapy drugs and protect non-tumorous tissues are required. Saos-2 cells were treated with Methotrexate (MTX) and Quercetin (Que) (a polyphenolic flavonoid with anti-tumor effects) alone and in combination. MTT assay was performed to investigate the cytotoxicity of the drugs. Moreover, apoptosis-involved genes, including miR-223, p53, BCL-2, CBX7, and CYLD expression were analyzed via qRT-PCR. Annexin V-FITC/PI kit was employed to assess the apoptosis rate. The MTT results showed that Que increases MTX cytotoxicity on OS cells. The measured IC50s are 142.3 µM for QUE and 13.7 ng/ml for MTX. A decline in MTX IC50 value was observed from 13.7 ng/ml to 8.45 ng/ml in the presence of Que. Moreover, the mRNA expression outcomes indicated that the combination therapy significantly up-regulates the tumor suppressor genes, such as p53, CBX7, and CYLD, and declines anti-apoptotic genes BCL-2 and miR-223, which can lead to proliferation inhibition and apoptosis inducement. Furthermore, the apoptosis rate increased significantly from 6.03% in the control group to 38.35% in Saos-2 cells that were treated with the combination of MTX and Que. Que, with the potential to boost the anticancer activity of MTX on Saos-2 cancer cells through proliferation inhibition and apoptosis induction, is a good candidate for combination therapy.

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

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

CYLD deficiency enhances metabolic reprogramming and tumor progression in nasopharyngeal carcinoma via PFKFB3

Aberrant cancer metabolism contributes to cell proliferation and tumor progression. However, the contribution of enhanced glycolysis, observed during cancer metabolism, to the pathogenesis and progression of nasopharyngeal carcinoma (NPC) remains unclear. CYLD, an NF-κB inhibitor, is frequently deficient in NPC. Here, we investigated the role of CYLD in the metabolic reprogramming of NPC and found that restoration of CYLD expression suppressed glycolysis in NPC cells. Mechanistic dissection showed that CYLD stabilized p53 and facilitated its nuclear translocation, thereby enhancing p53 activity by removing K63-linked and K48-linked ubiquitin chains of p53, which can bind to the PFKFB3 promoter and inhibit its transcription. Additionally, CYLD interacted with FZR1 to promote APC/C-FZR1 E3 ligase activity, which further ubiquitinated and degraded PFKFB3 via the 26S proteasomal system. Furthermore, clinical tissue array analysis indicated that low expression of CYLD was correlated with high expression of PFKFB3 and poor prognosis among patients with NPC. In conclusion, CYLD suppressed PFKFB3 expression via two factors, namely, p53 and FZR1, to inhibit glycolysis and delay tumor growth and progression in NPC. CYLD is a biomarker indicating poor prognosis of patients with NPC.

Transforming Growth Factor-β Activated Kinase 1 (Tak1) Is Activated in Hepatocellular Carcinoma, Mediates Tumor Progression, and Predicts Unfavorable Outcome.

Simple SummaryChronic inflammation is known to drive cancer initiation and progression in the liver and other organs. In different genetic mouse models, the role of the pro-inflammatory kinase Tak1 in liver cancer development has been controversial so far. To clarify the role of Tak1 in human hepatocellular carcinoma (HCC), we investigated the expression of Tak1 in a large and clinicopathologically well-characterized patient cohort with HCC. In human livers and HCCs, Tak1 is predominantly present in its isoform Tak1A localizing to the cell nucleus. Tak1 is upregulated in HCCs of the diethylnitrosamine mouse model as well as in human HCCs, independent of etiology, and is further induced in distant metastases. Overexpression of the isoform Tak1A in the HCC cell line Huh7 resulted in increased tumor cell migration, whereas overexpression of full-length Tak1 had no significant effect. In human HCCs, high nuclear Tak1 expression is associated with vascular invasion and short overall survival.Although knowledge on inflammatory signaling pathways driving cancer initiation and progression has been increasing, molecular mechanisms in hepatocarcinogenesis are still far from being completely understood. Hepatocyte-specific deletion of the MAPKKK Tak1 in mice recapitulates important steps of hepatocellular carcinoma (HCC) development, including the occurrence of cell death, steatohepatitis, dysplastic nodules, and HCCs. However, overactivation of Tak1 in mice upon deletion of its deubiquitinase Cyld also results in steatohepatitis and HCC development. To investigate Tak1 and Cyld in human HCCs, we created a tissue microarray to analyze their expression by immunohistochemistry in a large and well-characterized cohort of 871 HCCs of 561 patients. In the human liver and HCC, Tak1 is predominantly present as its isoform Tak1A and predominantly localizes to cell nuclei. Tak1 is upregulated in diethylnitrosamine-induced mouse HCCs as well as in human HCCs independent of etiology and is further induced in distant metastases. A high nuclear Tak1 expression is associated with short survival and vascular invasion. When we overexpressed Tak1A in Huh7 cells, we observed increased tumor cell migration, whereas overexpression of full-length Tak1 had no significant effect. A combined score of low Cyld and high Tak1 expression was an independent prognostic marker in a multivariate Cox regression model.

TRIM15 and CYLD regulate ERK activation via lysine-63-linked polyubiquitination.

The extracellular signal-regulated kinases ERK1 and ERK2 represent the foremost mitogenic pathway in mammalian cells, and their dysregulation drives tumorigenesis and confers therapeutic resistance. ERK1/2 are known to be activated by MAPK/ERK kinase (MEK)-mediated phosphorylation. Here we show that ERK1/2 are also modified by Lys63-linked polyubiquitin chains. We identify the tripartite motif-containing protein TRIM15 as a ubiquitin ligase, and the tumor suppressor CYLD as a deubiquitinase, for ERKs. TRIM15 and CYLD regulate ERK ubiquitination at defined lysine residues via mutually exclusive interactions as well as opposing activities. K63-linked polyubiquitination enhances ERK interaction with and activation by MEK. Down-regulation of TRIM15 inhibits growth of both drug-responsive and -resistant melanomas. Moreover, high TRIM15 expression and low CYLD expression are associated with poor prognosis of melanoma patients. These findings define a role of Lys63-linked polyubiquitination in the ERK signaling pathway and suggest a potential target for cancer therapy.

MiR-130-CYLD Axis Is Involved in the Necroptosis and Inflammation Induced by Selenium Deficiency in Pig Cerebellum.

Selenium (Se) is an essential trace element in creatures which deficiency can cause necroptosis and inflammation of multiple tissues. MicroRNAs (miRNAs) have been identified to participate multiple biological processes by regulating the expression of target genes. In the present study, the Se-deficient pig cerebellar model was established and conducted by light microscopy, qRT-PCR, and Western blot. Morphological observation exhibited necrosis-like lesions and inflammatory infiltration in the cerebellum of the Se-deficient group. Quantitative analysis result showed that Se deficiency significantly suppressed miR-130 expression, which in turn disinhibited the expression of CYLD. Meanwhile, in comparison to the control group, the expression levels of TNF-α pathway genes (TNF-α, TNFR1, and NF-κB p65) and necroptosis-related genes (RIPK1, RIPK3, and MLKL) in Se deficiency group were obviously increased (P < 0.05). Moreover, Se deficiency induced the occurrence of inflammation by upregulating the expression of inflammatory cytokines (IL-1β, IL-2, IL-8, IL-18, IFN-γ, COX-2, PTGEs, and NLRP3). In conclusion, we proved Se deficiency could induce the deregulation of miR-130-CYLD axis to cause RIPK3-dependent necroptosis and inflammation in pig cerebellum.