Nur77, an immediate-early response gene, participates in a wide range of biological functions. Its human homologue, NUR77, is known by several names and has the HGNC-approved gene symbol NR4A1. However, the role of Nur77 in inflammatory bowel disease (IBD) and its underlying mechanisms remain elusive. Here, using public data from the International Inflammatory Bowel Disease Genetics Consortium (IIBDGC) on the most recent genome-wide association studies (GWAS) for ulcerative colitis (UC) and Crohn's disease (CD), we found that genetic variants of the NUR77 gene are associated with increased risk for both UC and CD. Accordingly, Nur77 expression was significantly reduced in colon tissues from patients with UC or CD and mice treated with DSS. Nur77 deficiency increased the susceptibility of mice to DSS-induced experimental colitis and prevented intestinal recovery, whereas treatment with cytosporone B (Csn-B), an agonist for Nur77, significantly attenuated excessive inflammatory response in the DSS-induced colitis mouse model. Mechanistically, NUR77 acts as a negative regulator of TLR-IL-1R signalling by interacting with TRAF6. This interaction prevented auto-ubiquitination and oligomerization of TRAF6 and subsequently inhibited NF-κB activation and pro-inflammatory cytokine production. Taken together, our GWAS-based analysis and in vitro and in vivo studies have demonstrated that Nur77 is an important regulator of TRAF6/TLR-IL-1R-initiated inflammatory signalling, and loss of Nur77 may contribute to the development of IBD, suggesting Nur77 as a potential target for the prevention and treatment of IBD.

Ubiquitin specific protease 4 (USP4) is a deubiquitinating enzyme with key roles in the regulation of p53 and TGFb signaling, suggesting its importance in tumorigenesis. However, the mechanisms and regulatory roles of USP4 in cancer, including colorectal cancer, remain largely elusive. Here, we present the first evidence that USP4 regulates the growth, invasion, and metastasis of colorectal cancer. USP4 expression was significantly elevated in colorectal cancer tissues and was significantly associated with tumor size, differentiation, distant metastasis, and poor survival. Knockdown of USP4 diminished colorectal cancer cell growth, colony formation, migration, and invasion in vitro and metastasis in vivo. Importantly, we found that phosphatase of regenerating liver-3 (PRL-3) is indispensable for USP4-mediated oncogenic activity in colorectal cancer.Mechanistically, we observed that USP4 interacted with and stabilized PRL-3 via deubiquitination. This resulted in activation of Akt and reduction of E-cadherin, critical regulators of cancer cell growth and metastasis. Examination of clinical samples confirmed that USP4 expression positively correlates with PRL-3 protein expression, but not mRNA transcript levels. Taken together, our results demonstrate that aberrant expression of USP4 contributes to the development and progression of colorectal cancer and reveal a critical mechanism underlying USP4-mediated oncogenic activity. These observations suggest that the potential of harnessing proteolytic degradation processes for therapeutic manipulation may offer a much-needed new approach for improving colorectal cancer treatment strategies. Cancer Res; 76(1); 83-95. Ó2015 AACR.

Nur77, an orphan member of the nuclear receptor superfamily, has been implicated in tumorigenesis. However, its contributions to colorectal cancer (CRC) invasion and metastasis are largely under characterized. Here, we present the first evidence that the invasion and metastasis of CRC is regulated by Nur77. High expression of Nur77 was observed in clinical CRC tissues, and this elevated expression was significantly associated with advanced tumor, lymph nodes, distant metastasis stage (P = 0.003), lymph node metastasis (P = 0.001) and poor survival (P = 0.03). Overexpression of Nur77 in CRC cells enhanced cell invasion in vitro, whereas knockdown of Nur77 diminished cell invasion and metastasis both in vitro and in vivo. In studying the possible mechanism by which overexpression of Nur77 contributes to CRC invasion and metastasis, we observed that the nuclear protein Nur77 promoted the expression of matrix metalloproteinase (MMP)-9, a novel downstream target of Nur77, and subsequently decreased the expression of E-cadherin. Examination of clinical samples further showed that Nur77 expression is positively correlated with MMP-9, whereas negatively correlated with E-cadherin. Interestingly, Nur77-mediated CRC invasion via MMP-9 and E-cadherin could be mimicked by some metastasis-inducible factors including hypoxia and prostaglandin E2. Collectively, our study demonstrated that Nur77 could promote the invasion and metastasis of CRC cells through regulation of MMP-9/E-cadherin signaling. These observations provide a possible new strategy for potentially treating or preventing the metastasis of CRC through targeting of Nur77.

Liver metastasis is the main cause of death in patients with colorectal cancer (CRC). Here, we searched for CRC metastasis‐associated circular RNA in a mouse model of liver metastasis of CRC by using RNA (transcriptome)‐sequencing. We identified a novel and conserved circular RNA, circ‐NSD2, functioning as a promoter of CRC metastasis. Circ‐NSD2 expression was elevated in CRC tissues and was markedly increased in advanced stages or metastatic tumours of CRC patients. Gain‐of‐function and loss‐of‐function experiments demonstrated that circ‐NSD2 promoted migration and metastasis of CRC in vitro and in vivo. Mechanistically, circ‐NSD2 acted as a sponge for the tumour suppressor miR‐199b‐5p and activated DDR1 (discoidin domain receptor tyrosine kinase 1) and JAG1 (Jagged 1) genes, which synergistically helped with cell–matrix interaction, migration and metastasis of CRC cells. Taken together, our findings highlight a novel oncogenic function of circ‐NSD2 and uncover a key mechanism for the circ‐NSD2/miR‐199b‐5p/DDR1/JAG1 axis in CRC metastasis, which may serve as a prognostic factor and therapeutic target for antimetastatic therapy in CRC patients. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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The oncogene c‐Myc is aberrantly expressed and plays a key role in malignant transformation and progression of hepatocellular carcinoma (HCC). Here, we report that c‐Myc is significantly up‐regulated by tumor necrosis factor receptor–associated factor 6 (TRAF6), an E3 ubiquitin ligase, in hepatocarcinogenesis. High TRAF6 expression in clinical HCC samples correlates with poor prognosis, and the loss of one copy of the Traf6 gene in Traf6+/– mice significantly impairs liver tumorigenesis. Mechanistically, TRAF6 first interacts with and ubiquitinates histone deacetylase 3 (HDAC3) with K63‐linked ubiquitin chains, which leads to the dissociation of HDAC3 from the c‐Myc promoter and subsequent acetylation of histone H3 at K9, thereby epigenetically enhancing the mRNA expression of c‐Myc. Second, the K63‐linked ubiquitination of HDAC3 impairs the HDAC3 interaction with c‐Myc and promotes c‐Myc protein acetylation, which thereby enhances c‐Myc protein stability by inhibiting carboxyl terminus of heat shock cognate 70‐kDa–interacting protein–mediated c‐Myc ubiquitination and degradation. Importantly, TRAF6/HDAC3/c‐Myc signaling is also primed in hepatitis B virus–transgenic mice, unveiling a critical role for a mechanism in inflammation–cancer transition. In clinical specimens, TRAF6 positively correlates with c‐Myc at both the mRNA and protein levels, and high TRAF6 and c‐Myc expression is associated with an unfavorable prognosis, suggesting that TRAF6 collaborates with c‐Myc to promote human hepatocarcinogenesis. Consistently, curbing c‐Myc expression by inhibition of TRAF6 activity with a TRAF6 inhibitor peptide or the silencing of c‐Myc by small interfering RNA significantly suppressed tumor growth in mice. Conclusion: These findings demonstrate the oncogenic potential of TRAF6 during hepatocarcinogenesis by modulating TRAF6/HDAC3/c‐Myc signaling, with potential implications for HCC therapy.

Background & Aims The circadian clock is crucial for physiological homeostasis including gut homeostasis. Disorder of the circadian clock may contribute to many diseases including inflammatory bowel disease (IBD). However, the role and the mechanisms of circadian clock involvement in IBD still are unclear. Methods Disorder of the circadian clock including chronic social jet lag and circadian clock gene deficiency mice ( Bmal1 -/- , and Per1 -/- Per2 -/- ) were established. Dextran sulfate sodium (DSS) and/or azoxymethane were used to induce mouse models of colitis and its associated colorectal cancer. Flow cytometry, immunohistochemistry, immunofluorescence, Western blot, and reverse-transcription quantitative polymerase chain reaction were used to analyze the characteristics of immune cells and their related molecules. Results Mice with disorders of the circadian clock including chronic social jet lag and circadian clock gene deficiency were susceptible to colitis. Functionally, regulatory B (Breg) cells highly expressing Programmed cell death 1 ligand 1 (PDL1) in intestinal intraepithelial lymphocytes (IELs) helped to alleviate the severity of colitis after DSS treatment and was dysregulated in DSS-treated Bmal1 -/- mice. Notably, interleukin 33 in the intestinal microenvironment was key for Bmal1-regulated PDL1 + Breg cells and interleukin 33 was a target of Bmal1 transcriptionally. Dysregulated PDL1 + B cells induced cell death of activated CD4 + T cells in DSS-treated Bmal1 -/- mice. Consequently, circadian clock disorder was characterized as decreased numbers of Breg + PDL1 + cells in IELs and dysfunction of CD4 + T cells promoted colitis-associated colorectal cancer (CRC) in mice. In clinical samples from CRC patients, low expression of Bmal1 gene in paracancerous tissues and center area of tumor was associated closely with a poorer prognosis of CRC patients. Conclusions Our study uncovers the importance of the circadian clock regulating PDL1 + Breg + cells of IELs in IBD and IBD-associated CRC.

Background In the central nervous system (CNS), connexin 43 (Cx43) is mainly expressed in astrocytes and regulates astrocytic network homeostasis. Similar to Cx43 overexpression, abnormal excessive opening of Cx43 hemichannels (Cx43Hcs) on reactive astrocytes aggravates the inflammatory response and cell death in CNS pathologies. However, the role of excessive Cx43Hc opening in intracerebral hemorrhage (ICH) injury is not clear. Methods Hemin stimulation in primary cells and collagenase IV injection in C57BL/6J (B6) mice were used as ICH models in vitro and in vivo. After ICH injury, the Cx43 mimetic peptide Gap19 was used for treatment. Ethidium bromide (EtBr) uptake assays were used to measure the opening of Cx43Hcs. Western blotting and immunofluorescence were used to measure protein expression. qRT-PCR and ELISA were used to determine the levels of cytokines. Coimmunoprecipitation (Co-IP) and the Duolink in situ proximity ligation assay (PLA) were applied to measure the association between proteins. Results In this study, Cx43 expression upregulation and excessive Cx43Hc opening was observed in mice after ICH injury. Delayed treatment with Gap19 significantly alleviated hematoma volume and neurological deficits after ICH injury. In addition, Gap19 decreased inflammatory cytokine levels in the tissue surrounding the hematoma and decreased reactive astrogliosis after ICH injury in vitro and in vivo. Intriguingly, Cx43 transcriptional activity and expression in astrocytes were significantly increased after hemin stimulation in culture. However, Gap19 treatment downregulated astrocytic Cx43 expression through the ubiquitin-proteasome pathway without affecting Cx43 transcription. Additionally, our data showed that Gap19 increased Yes-associated protein (YAP) nuclear translocation. This subsequently upregulated SOCS1 and SOCS3 expression and then inhibited the TLR4-NFκB and JAK2-STAT3 pathways in hemin-stimulated astrocytes. Finally, the YAP inhibitor, verteporfin (VP), reversed the anti-inflammatory effect of Gap19 in vitro and almost completely blocked its protective effects in vivo after ICH injury. Conclusions This study provides new insight into potential treatment strategies for ICH injury involving astroglial Cx43 and Cx43Hcs. Suppression of abnormal astroglial Cx43 expression and Cx43Hc opening by Gap19 has anti-inflammatory and neuroprotective effects after ICH injury.