The Deubiquitinase USP17 Regulates the Stability and Nuclear Function of IL-33.

IL-33 is a new member of the IL-1 family cytokines, which is expressed by different types of immune cells and non-immune cells. IL-33 is constitutively expressed in the nucleus, where it can act as a transcriptional regulator. So far, no direct target for nuclear IL-33 has been identified, and the regulation of IL-33 nuclear function remains largely unclear. Here, we report that the transcription of type 2 inflammatory cytokine IL-13 is positively regulated by nuclear IL-33. IL-33 can directly bind to the conserved non-coding sequence (CNS) before the translation initiation site in the IL13 gene locus. Moreover, IL-33 nuclear function and stability are regulated by the enzyme ubiquitin-specific protease 17 (USP17) through deubiquitination of IL-33 both at the K48 and at the K63 sites. Our data suggest that IL13 gene transcription can be directly activated by nuclear IL-33, which is negatively regulated by the deubiquitinase USP17.

Identification of the E3 deubiquitinase ubiquitin-specific peptidase 21 (USP21) as a positive regulator of the transcription factor GATA3.

The expression of the transcription factor GATA3 in FOXP3(+) regulatory T (Treg) cells is crucial for their physiological function in limiting inflammatory responses. Although other studies have shown how T cell receptor (TcR) signals induce the up-regulation of GATA3 expression in Treg cells, the underlying mechanism that maintains GATA3 expression in Treg cells remains unclear. Here, we show how USP21 interacts with and stabilizes GATA3 by mediating its deubiquitination. In a T cell line model, we found that TcR stimulation promoted USP21 expression, which was further up-regulated in the presence of FOXP3. The USP21 mutant C221A reduced its capacity to stabilize GATA3 expression, and its knockdown led to the down-regulation of GATA3 protein expression in Treg cells. Furthermore, we found that FOXP3 could directly bind to the USP21 gene promoter and activated its transcription upon TcR stimulation. Finally, USP21, GATA3, and FOXP3 were found up-regulated in Treg cells that were isolated from asthmatic subjects. In summary, we have identified a USP21-mediated pathway that promotes GATA3 stabilization and expression at the post-translational level. We propose that this pathway forms an important signaling loop that stabilizes the expression of GATA3 in Treg cells.

CircMYC promotes proliferation, migration, invasion and inhibits apoptosis of small cell lung cancer by targeting miR-145/ Matrix Metallopeptidase 2 axis.

Circular RNAs (circRNAs) are involved in the carcinogenesis of lung cancer. Human MYC gene is highly expressed in melanoma, multiple myeloma, and nasopharyngeal carcinoma. We aimed to investigate the role of circMYC in small cell lung cancer (SCLC). The expression of cirMYC in SCLC tissues and cells were examined. Functional studies were performed to evaluate the roles of circMYC in SCLC cells. Luciferase reporter gene assay, RNA pull-down assay, and rescue experiments were performed to evaluate the regulatory relationship between circMYC and miR-145, and MiR-145 and MMP2 mRNA. We found that CirMYC was highly expressed in SCLC tissues and cells. Knockdown of circMYC could inhibit cell proliferation, migration, invasion, and induce apoptosis. CircMYC targeted miR-145 and miR-145 targeted MMP2 (Matrix Metallopeptidase 2) mRNA. Our data indicated that circMYC upregulates the expression of MMP-2 by inhibiting miR-145, which functions to promote the proliferation, migration, and invasion and inhibit the apoptosis of SCLC. These findings suggest that targeting circMYC/miR-145/MMP-2 could serve as a potential therapeutic strategy for SCLC treatment.

MicroRNA­506­3p reverses gefitinib resistance in non­small cell lung cancer by targeting Yes­associated protein 1.

Epidermal growth factor receptor­tyrosine kinase inhibitors, such as gefitinib, have been found to be clinically effective in the treatment of patients with non­small cell lung cancer (NSCLC). However, the therapeutic effect of gefitinib is often limited by the development of gefitinib resistance. MicroRNAs (miRNAs), a group of small non­coding RNAs, have been demonstrated to be frequently dysregulated in human malignancies. For instance, the downregulation of miR­506­3p has been reported in NSCLC patients. The aim of the present study was to determine the role and underlying molecular mechanism of miR­506­3p in the regulation of gefitinib sensitivity in NSCLC. A gefitinib­resistant PC­9 (PC­9GR) cell line was established, and reduced miR­506­3p expression was observed in PC­9GR cells as compared with that in parental cells. The results of cell cytotoxicity and cell apoptosis assays indicated that PC­9GR cells were more sensitive to gefitinib following the transfection with an miR­506­3p mimic, while transfection with an miR­506­3p antagonist reduced the sensitivity of PC­9GR cells to gefitinib. It was further revealed that Yes­associated protein 1 (YAP1) was directly suppressed by miR­506­3p in PC­9GR cells. The elevated sensitivity of PC­9GR cells to gefitinib following transfection with the miR­506­3p mimic was counteracted by the overexpression of YAP1. Furthermore, an inverse correlation between the miR­506­3p and YAP1 mRNA levels was detected in lung adenocarcinoma specimens. Collectively, the results of the present study suggested that the downregulation of miR­506­3p contributes to gefitinib resistance, and thus, the restoration of miR­506­3p may be a potential therapeutic approach for overcoming NSCLC gefitinib resistance.

Dynamics of Plasma EGFR T790M Mutation in Advanced NSCLC: A Multicenter Study.

BACKGROUND: Droplet digital polymerase chain reaction (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, a ddPCR assay for the epidermal growth factor receptor (EGFR) p.Thr790Met (T790M) mutation suitable for clinical use remains to be established with analytical and clinical validations. OBJECTIVE: We aimed to develop and validate a new ddPCR assay to quantify the T790M mutation in plasma for monitoring and predicting the progression of advanced non-small-cell lung cancer (NSCLC). METHODS: Specificity of the ddPCR assay was evaluated with genomic DNA samples from healthy individuals. The inter- and intraday variations of the assay were evaluated using mixtures of plasmid DNA containing wild-type EGFR and T790M mutation sequences. We assessed the clinical utility of the T790M assay in a multicenter prospective study in patients with advanced NSCLC receiving tyrosine kinase inhibitor (TKI) treatment by analyzing longitudinal plasma DNA samples. RESULTS: We set the criteria for a positive call when the following conditions were satisfied: (1) T790M mutation frequency > 0.098% (3 standard deviations above the background signal); (2) at least two positive droplets in duplicate ddPCR reactions. Among the 62 patients with advanced NSCLC exhibiting resistance to TKI treatment, 15 had one or more serial plasma samples that tested positive for T790M. T790M mutation was detected in the plasma as early as 205 days (median 95 days) before disease progression, determined by imaging analysis. Plasma T790M concentrations also correlated with intervention after disease progression. CONCLUSIONS: We developed a ddPCR assay to quantify the T790M mutation in plasma. Quantification of longitudinal plasma T790M mutation may allow noninvasive assessment of drug resistance and guide follow-up treatment in TKI-treated patients with NSCLC. TRIAL REGISTRATION: Clinical Trials.gov identifier: NCT02804100.

USP17-mediated deubiquitination and stabilization of HDAC2 in cigarette smoke extract-induced inflammation.

Histone deacetylase HDAC2 regulates genes transcription via removing the acetyl group from histones. Glucocorticoids, the most potent anti-inflammatory treatment available for inflammatory diseases, inhibit the expression of inflammatory genes by recruiting HDAC2 to activated genes. In the lungs of patients who smoke and have chronic obstructive pulmonary disease (COPD) or asthma, glucocorticoids are not effective enough to suppress airway inflammation, which is so called "glucocorticoid resistance", due to decreased HDAC2 level caused by cigarette smoke. We report that the ubiquitin-specific protease USP17 interacts with HDAC2. USP17 deubiquitinates and stabilizes the protein level of HDAC2. In cigarette smoke extract-exposed airway epithelial cells and macrophages, HDAC2 is excessively ubiquitinated and degraded in the proteasome attributed to low expression of USP17. Furthermore, over-expression of USP17 blocks the destruction of HDAC2 induced by cigarette smoke extract. These results provide a new insight into the mechanisms of glucocorticoid resistance in airway inflammatory disease. Small molecules which can specifically induce the expression of USP17 might be useful in reversing glucocorticoid resistance.

Efficacy of sublingual immunotherapy for allergic asthma: retrospective meta-analysis of randomized, double-blind and placebo-controlled trials.

BACKGROUND: Allergen-specific immunotherapy (SIT) is the only available curative choice with a disease-modifying effect against respiratory allergies. The efficacy of SIT via the sublingual route was demonstrated by a number of clinical trials. This meta-analysis was performed to investigate the clinical efficacy and safety of sublingual-specific immunotherapy (SLIT) for allergic asthma. METHODS: PubMed, EMBASE and the Cochrane Central Register of Controlled Trials were searched for randomized, double-blind and placebo-controlled (DBPC) trials evaluating the efficacy and safety of SLIT on allergic asthma. Subgroup analyses were performed according to age, type of allergen and duration of SLIT treatment. RESULTS: Sixteen randomized DBPC trials comprising 794 patients in total met the inclusion criteria. The results suggest that SLIT significantly reduces both symptom [standardized mean difference (SMD), -0.74; P=0.006] and medication scores (SMD, -0.78; P=0.02) compared with placebo. SLIT offers a better clinical response in mite sensitive asthmatics but without confirmed proof from subgroup analyses. Prolonged duration of treatment for more than 12 months brings no additive effects. Improvement in the skin prick test was also observed following immunotherapy. There was no consistent effect on forced expiratory volume in 1 s, serum levels of antigen-specific immunoglobulin G4 and immunoglobulin E in the treated group. The risk of adverse effects was relative risk 2.23 (P=0.01). CONCLUSIONS: SLIT is safe and clinically effective in reducing symptoms and medication use for allergic asthma. Our subgroup analyses failed to identify a disproportionate benefit of SLIT in any specific group of asthmatics, but some possible trends did emerge.

Deubiquitination and stabilization of IL-33 by USP21.

Interleukin-33 (IL-33) is a dual-function protein that acts both as a secreted cytokine and as a nuclear factor regulating gene transcription. It has been demonstrated that IL-33 exerts its nuclear function in promoting the transcription of NF-κB p65. Here, we show that USP21-mediated deubiquitination of IL-33 affects the transcription of p65. IL-33 can be post-translationally modified by ubiquitination. Ubiquitin-specific protease 21 (USP21) interacts with IL-33 and also localizes in nucleus. The protein stability of IL-33 is maintained by USP21 through deubiquitination. Furthermore, depletion of USP21 reduces IL-33 protein levels and IL-33-mediated NF-κB p65 promoter activity. Our findings reveal the role of ubiquitination modification in regulating the protein stability and the nuclear function of IL-33.

CD39/CD73 and the imbalance of Th17 cells and regulatory T cells in allergic asthma.

In the immune system, CD4+CD25+Foxp3+ regulatory T cells (Tregs) maintain self­tolerance and Th17 cells mediate inflammatory responses. CD39 is expressed on the surface of a subset of naturally occurring human Tregs that are important in constraining pathogenic Th17 cells. Additional studies have shown that Tregs differentiate into interleukin­17 (IL­17)+Foxp3+ T cells. Our previous study indicated an imbalance of Th17 and Tregs in allergic asthma; however, the underlying mechanisms remain poorly understood. Using quantitative PCR (qPCR), CD39 and CD73 mRNA levels in CD4+ T cells were investigated. Flow cytometry was used to analyze the proportion of IL­17+Foxp3+ T cells, and CD39 and CD73 expressed by CD4+ T cells and Tregs in the peripheral blood of the subjects. The results of the present study demonstrated an increased frequency of CD4+Foxp3+IL­17+ T cells in moderate to severe asthma. A deficiency in CD39 expressed on the surface of CD4+ T cells and Tregs was observed in asthma patients. The expression of CD39 and CD73 on the surface of CD4+ T cells and Tregs was negatively correlated with the number of Th17 cells. These results indicated that the plasticity of Tregs transforming to IL­17+Foxp3+CD4+ T cells, the reduced frequency of CD39+ Tregs and less effective suppression of IL­17 production by residual CD39+ Tregs leads to an imbalance of Th17 and Tregs in asthma. Therefore, enhanced CD39 activity is hypothesized to prevent the progression of asthma.