Jing-Ru Wang

Ubiquitin-Specific Protease 4-Mediated Deubiquitination and Stabilization of PRL-3 Is Required for Potentiating Colorectal Oncogenesis

Ubiquitin specific protease 4 (USP4) is a deubiquitinating enzyme with key roles in the regulation of p53 and TGFβ 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.

Orphan nuclear receptor Nur77 promotes colorectal cancer invasion and metastasis by regulating MMP-9 and E-cadherin

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.

NUR77 exerts a protective effect against inflammatory bowel disease by negatively regulating the TRAF6/TLR-IL-1R signalling axis.

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 Crohns 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. Copyright

RARγ Downregulation Contributes to Colorectal Tumorigenesis and Metastasis by Derepressing the Hippo–Yap Pathway

The Hippo-Yap pathway conveys oncogenic signals, but its regulation during cancer development is not well understood. Here, we identify the nuclear receptor RARγ as a regulator of the Hippo-Yap pathway in colorectal tumorigenesis and metastasis. RARγ is downregulated in human colorectal cancer tissues, where its expression correlates inversely with tumor size, TNM stage, and distant metastasis. Functional studies established that silencing of RARγ drove colorectal cancer cell growth, invasion, and metastatic properties both in vitro and in vivo Mechanistically, RARγ controlled Hippo-Yap signaling to inhibit colorectal cancer development, acting to promote phosphorylation and binding of Lats1 to its transcriptional coactivator Yap and thereby inactivating Yap target gene expression. In clinical specimens, RARγ expression correlated with overall survival outcomes and expression of critical Hippo-Yap pathway effector molecules in colorectal cancer patients. Collectively, our results defined RARγ as tumor suppressor in colorectal cancer that acts by restricting oncogenic signaling by the Hippo-Yap pathway, with potential implications for new approaches to colorectal cancer therapy. Cancer Res; 76(13); 3813-25. ©2016 AACR.

Nur77-mediated TRAF6 signalling protects against LPS-induced sepsis in mice

BackgroundNur77, a key member of the NR4A receptor subfamily, is involved in the regulation of inflammation and immunity. However, the in vivo regulatory roles of Nur77 in sepsis and the mechanisms involved remains largely elusive. In this study, we used Nur77-deficient (Nur77−/−) mice and investigated the function of Nur77 in sepsis.FindingsCompared to wild-type (Nur77+/+) mice, Nur77−/− mice are more susceptible to LPS-induced sepsis and acute liver inflammation. Mechanistically, we observed that Nur77 can interact with TRAF6, a crucial adaptor molecule in the Toll-like receptor-interleukin 1 receptor (TLR-IL-1R) signalling pathway, in in vivo mouse model of sepsis. The interaction may affect TRAF6 auto-ubiquitination, thereby inhibiting NF-κB activation and pro-inflammatory cytokines production.ConclusionsThese in vivo observations reveals an important protective role for Nur77 in LPS-induced sepsis through its regulation to TRAF6 signalling, and highlights the potential clinical application of Nur77 as a molecular target in prevention and/or treatment of sepsis.

Molecular cloning, organization, expression and 3D structural analysis of the MHC class Ia gene in the whitespotted bamboo shark (Chiloscyllium plagiosum)

Cartilaginous fishes are the oldest jawed vertebrates, from which the major histocompatibility complex (MHC) derived approximately 500 MYA; however, full-length genomic sequences for MHC genes in these species remain undescribed. This lack of basic information about MHC organization in cartilaginous fish is hindering investigations into the relationship between MHC polymorphism and disease, and leaves a large gap in our understanding of shark MHC evolution. Here, we obtained a complete 4887 bp genomic DNA of chplUAA (designated as chplUAA) from the whitespotted bamboo shark (Chiloscyllium plagiosum) using long PCR. The full-length cDNA sequence was 1385 bp, with a 1029 bp open reading frame (ORF) encoding 343 amino acids. Six unique sequences (chplUAA*01-06) were detected from 51 sequences from three samples. No more than two sequences were found in each individual, suggesting that only one UAA locus was amplified in each sample. Phylogenetic analysis supports monophyly of all available shark classical class Ia sequences.

Dicer suppresses cytoskeleton remodeling and tumorigenesis of colorectal epithelium by miR-324-5p mediated suppression of HMGXB3 and WASF-2

Emerging evidence indicates that microRNAs, a class of small and well-conserved noncoding RNAs, participate in many physiological and pathological processes. RNase III endonuclease DICER is one of the key enzymes for microRNA biogenesis. Here, we found that DICER was downregulated in tumor samples of colorectal cancer (CRC) patients at both mRNA and protein levels. Importantly, intestinal epithelial cell (IEC)-specific deletion of Dicer mice got more tumors after azoxymethane and dextran sulfate sodium (DSS) administration. Interestingly, IEC-specific deletion of Dicer led to severe chronic inflammation and epithelium layer remodeling in mice with or without DSS administration. Microarray analysis of 3 paired Dicer deletion CRC cell lines showed that miR-324-5p was one of the most significantly decreased miRNAs. In the intestinal epithelium of IEC-specific deletion of Dicer mice, miR-324-5p was also found to be markedly reduced. Mechanistically, miR-324-5p directly bound to the 3′untranslated regions (3′UTRs) of HMG-box containing 3 (HMGXB3) and WAS protein family member 2 (WASF-2), two key proteins participated in cell motility and cytoskeleton remodeling, to suppress their expressions. Intraperitoneal injection of miR-324-5p AgomiR (an agonist of miR-324-5p) curtailed chronic inflammation and cytoskeleton remodeling of colorectal epithelium and restored intestinal barrier function in IEC-specific deletion of Dicer mice induced by DSS. Therefore, our study reveals a key role of a DICER/miR-324-5p/HMGXB3/WASF-2 axis in tumorigenesis of CRC by regulation of cytoskeleton remodeling and maintaining integrity of intestinal barriers.

Erbin exerts a protective effect against inflammatory bowel disease by suppressing autophagic cell death

The pathogenesis and key functional molecules involved in inflammatory bowel disease (IBD) including Crohns disease (CD) and ulcerative colitis (UC) remain unclear. Here, we reported that Erbin, a protein required for the polarity of epithelial cells, is conserved across species and highly expressed in the intestinal mucosa in mice and zebrafish. Pathologically, Erbin expression in the intestinal mucosa was significantly decreased in DSS induced acute colitis mice, IL-10 deficient mice and clinical biopsy specimens from patients with ulcerative colitis. Moreover, Erbin deficient mice are more susceptible to experimental colitis, exhibiting more severe intestinal barrier disruption, with increased histological scores and excessive production of proinflammatory cytokines. Mechanistically, Erbin deficiency or knockdown significantly exacerbated activation of autophagic program and autophagic cell death in vivo and in vitro. And, inhibition of autophagy by Chloroquine attenuates excessive inflammatory response in the DSS-induced colitis mouse model of Erbin deletion. Generally, our study uncovers a crucial role of Erbin in autophagic cell death and IBD, giving rise to a new strategy for IBD therapy by inhibiting excessive activation of autophagy and autophagic cell death.

Nur77 deficiency in mice accelerates tumor invasion and metastasis by facilitating TNFα secretion and lowering CSF-1R expression

Nur77, an orphan member of the nuclear receptor superfamily, plays critical roles in inflammation and immunity. However, the role of Nur77 in tumor microenvironment remains elusive. Results showed that deletion of Nur77 strikingly enhanced tumor metastasis compared to WT mice. Additionally, compared to the conditioned media derived from Nur77+/+ peritoneal macrophages (CM1), the conditioned media derived from Nur77-/- peritoneal macrophages (CM2) significantly promoted the EMT of cancer cells, and greatly enhanced the migratory and invasive abilities of cancer cells. Moreover, studies using TNF-α blocking antibody demonstrated that pro-inflammatory cytokine TNF-α was indispensable in supporting CM2-induced EMT to drive cancer cells migration and invasion. Furthermore, we found that Nur77 promoted the expression of CSF-1R, a novel downstream target gene of Nur77, and subsequently enhanced the migration of inflammatory cells. Notably, infiltration of inflammatory cells in the tumors of Nur77-/- mice was markedly abrogated compared to Nur77+/+ mice. Collectively, these results revealed that host Nur77 expression was pivotal in antitumor immune response, and in inhibiting tumor metastasis.

Orphan nuclear receptor Nur77 inhibits poly (I:C)-triggered acute liver inflammation by inducing the ubiquitin-editing enzyme A20

Inflammation is a key contributor to various types of acute and chronic liver disease. We recently reported that lack of Nur77, an orphan nuclear receptor, contributes to the pathogenesis of inflammatory diseases including inflammatory bowel disease and sepsis. However, whether Nur77 plays a critical role in liver inflammation remains to be fully understood. Employing in vivo acute liver inflammation model in wild-type (Nur77+/+) and Nur77-/- mice, we here found that Nur77 deficiency dramatically increased the production of pro-inflammatory cytokines and accelerated liver injury induced by Poly (I:C)/D-GalN in Nur77-/- mice. Mechanistically, Nur77 acts as a negative regulator of NF-κB signaling by inducing the expression of ubiquitin-editing enzyme A20, a novel target gene of Nur77. Notably, in inflammatory cells, overexpression of A20 enhanced, whereas knockdown of A20 by siRNA approach impaired, the inhibitory effect of Nur77 on Poly (I:C)-triggered inflammation. Collectively, our data suggest that the orphan nuclear receptor Nur77 plays a protective role in Poly (I:C)-triggered liver inflammation by inducing A20, thus making it a promising target for the prevention and treatment of liver inflammation.Inflammation is a key contributor to various types of acute and chronic liver disease. We recently reported that lack of Nur77, an orphan nuclear receptor, contributes to the pathogenesis of inflammatory diseases including inflammatory bowel disease and sepsis. However, whether Nur77 plays a critical role in liver inflammation remains to be fully understood. Employing in vivo acute liver inflammation model in wild-type (Nur77+/+) and Nur77-/- mice, we here found that Nur77 deficiency dramatically increased the production of pro-inflammatory cytokines and accelerated liver injury induced by poly (I:C)/D-GalN in Nur77-/- mice. Mechanistically, Nur77 acts as a negative regulator of NF-κB signaling by inducing the expression of ubiquitin-editing enzyme A20, a novel target gene of Nur77. Notably, in inflammatory cells, overexpression of A20 enhanced, whereas knockdown of A20 by siRNA approach impaired, the inhibitory effect of Nur77 on poly (I:C)-triggered inflammation. Collectively, our data suggest that the orphan nuclear receptor Nur77 plays a protective role in poly (I:C)-triggered liver inflammation by inducing A20, thus making it a promising target for the prevention and treatment of liver inflammation.