Fuquan Jiang

Regulation of Nur77 expression by β-catenin and its mitogenic effect in colon cancer cells

The orphan nuclear receptor Nur77 is an immediate‐early response gene whose expression is rapidly induced by various extracellular stimuli. The aims of this study were to study the role of Nur77 expression in the growth and survival of colon cancer cells and the mechanism by which Nur77 expression was regulated. We showed that levels of Nur77 were elevated in a majority of human colon tumors (9/12) compared to their nontumorous tissues and that Nur77 expression could be strongly induced by different colonic carcinogens including deoxycholic acid (DCA). DCA‐induced Nur77 expression resulted in up‐regulation of antiapoptotic BRE and angiogenic VEGF, and it enhanced the growth, colony formation, and migration of colon cancer cells. In studying the mechanism by which Nur77 was regulated in colon cancer cells, we found that β‐catenin was involved in induction of Nur77 expression through its activation of the tran‐scriptional activity of AP‐1 (c‐Fos/c‐Jun) that bound to and transactivated the Nur77 promoter. Together, our results demonstrate that Nur77 acts to promote the growth and survival of colon cancer cells and serves as an important mediator of the Wnt/β‐catenin and AP‐1 signaling pathways.—Wu, H., Lin, Y., Li, W., Sun, Z., Gao, W., Zhang, H., Xie, L., Jiang, F., Qin, B., Yan, T., Chen, L., Zhao, Y., Cao, X., Wu, Y., Lin, B., Zhou, H., Wong, A.S.‐T., Zhang, X.‐K., Zeng, J.‐Z. Regulation of Nur77 expression by β‐catenin and its mitogenic effect in colon cancer cells. FASEB J. 25, 192–205 (2011). www.fasebj.org

NSAID Sulindac and Its Analog Bind RXRα and Inhibit RXRα-Dependent AKT Signaling

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their anticancer effects through cyclooxygenase-2 (COX-2)-dependent and independent mechanisms. Here, we report that Sulindac, an NSAID, induces apoptosis by binding to retinoid X receptor-alpha (RXRalpha). We identified an N-terminally truncated RXRalpha (tRXRalpha) in several cancer cell lines and primary tumors, which interacted with the p85alpha subunit of phosphatidylinositol-3-OH kinase (PI3K). Tumor necrosis factor-alpha (TNFalpha) promoted tRXRalpha interaction with the p85alpha, activating PI3K/AKT signaling. When combined with TNFalpha, Sulindac inhibited TNFalpha-induced tRXRalpha/p85alpha interaction, leading to activation of the death receptor-mediated apoptotic pathway. We designed and synthesized a Sulindac analog K-80003, which has increased affinity to RXRalpha but lacks COX inhibitory activity. K-80003 displayed enhanced efficacy in inhibiting tRXRalpha-dependent AKT activation and tRXRalpha tumor growth in animals.

Inhibition of β-catenin signaling by nongenomic action of orphan nuclear receptor Nur77.

Dysregulation of β-catenin turnover due to mutations of its regulatory proteins including adenomatous polyposis coli (APC) and p53 is implicated in the pathogenesis of cancer. Thus, intensive effort is being made to search for alternative approaches to reduce abnormally activated β-catenin in cancer cells. Nur77, an orphan member of the nuclear receptor superfamily, has a role in the growth and apoptosis of cancer cells. Here, we reported that Nur77 could inhibit transcriptional activity of β-catenin by inducing β-catenin degradation via proteasomal degradation pathway that is glycogen synthase kinase 3β and Siah-1 independent. Nur77 induction of β-catenin degradation required both the N-terminal region of Nur77, which was involved in Nur77 ubiquitination, and the C-terminal region, which was responsible for β-catenin binding. Nur77/ΔDBD, a Nur77 mutant lacking its DNA-binding domain, resided in the cytoplasm, interacted with β-catenin, and induced β-catenin degradation, demonstrating that Nur77-mediated β-catenin degradation was independent of its DNA binding and transactivation, and might occur in the cytoplasm. In addition, we reported our identification of two digitalis-like compounds (DLCs), H-9 and ATE-i2-b4, which potently induced Nur77 expression and β-catenin degradation in SW620 colon cancer cells expressing mutant APC protein in vitro and in animals. DLC-induced Nur77 protein was mainly found in the cytoplasm, and inhibition of Nur77 nuclear export by the CRM1-dependent nuclear export inhibitor leptomycin B or Jun N-terminal kinase inhibitor prevented the effect of DLC on inducing β-catenin degradation. Together, our results demonstrate that β-catenin can be degraded by cytoplasmic Nur77 through their interaction and identify H-9 and ATE-i2-b4 as potent activators of the Nur77-mediated pathway for β-catenin degradation.

Celastrol-Induced Nur77 Interaction with TRAF2 Alleviates Inflammation by Promoting Mitochondrial Ubiquitination and Autophagy

Mitochondria play an integral role in cell death, autophagy, immunity, and inflammation. We previously showed that Nur77, an orphan nuclear receptor, induces apoptosis by targeting mitochondria. Here, we report that celastrol, a potent anti-inflammatory pentacyclic triterpene, binds Nur77 to inhibit inflammation and induce autophagy in a Nur77-dependent manner. Celastrol promotes Nur77 translocation from the nucleus to mitochondria, where it interacts with tumor necrosis factor receptor-associated factor 2 (TRAF2), a scaffold protein and E3 ubiquitin ligase important for inflammatory signaling. The interaction is mediated by an LxxLL motif in TRAF2 and results not only in the inhibition of TRAF2 ubiquitination but also in Lys63-linked Nur77 ubiquitination. Under inflammatory conditions, ubiquitinated Nur77 resides at mitochondria, rendering them sensitive to autophagy, an event involving Nur77 interaction with p62/SQSTM1. Together, our results identify Nur77 as a critical intracellular target for celastrol and unravel a mechanism of Nur77-dependent clearance of inflamed mitochondria to alleviate inflammation.

Targeting Truncated Retinoid X Receptor-α by CF31 Induces TNF-α–Dependent Apoptosis

A truncated version of retinoid X receptor-α, tRXR-α, promotes cancer cell survival by activating the phosphoinositide 3-kinase (PI3K)/AKT pathway. However, targeting the tRXR-α-mediated survival pathway for cancer treatment remains to be explored. We report here our identification of a new natural product molecule, CF31, a xanthone isolated from Cratoxylum formosum ssp. pruniflorum, and the biologic evaluation of its regulation of the tRXR-α-mediated PI3K/AKT pathway. CF31 binds RXR-α and its binding results in inhibition of RXR-α transactivation. Through RXR-α mutational analysis and computational studies, we show that Arg316 of RXR-α, known to form salt bridges with certain RXR-α ligands, such as 9-cis-retinoic acid (9-cis-RA), is not required for the antagonist effect of CF31, showing a distinct binding mode. Evaluation of several CF31 analogs suggests that the antagonist effect is mainly attributed to an interference with Leu451 of helix H12 in RXR-α. CF31 is a potent inhibitor of AKT activation in various cancer cell lines. When combined with TNF-α, it suppresses TNF-α activation of AKT by inhibiting TNF-α-induced tRXR-α interaction with the p85α regulatory subunit of PI3K. CF31 inhibition of TNF-α activation of AKT also results in TNF-α-dependent activation of caspase-8 and apoptosis. Together, our results show that CF31 is an effective converter of TNF-α signaling from survival to death by targeting tRXR-α in a unique mode and suggest that identification of a natural product that targets an RXR-mediated cell survival pathway that regulates PI3K/AKT may offer a new therapeutic strategy to kill cancer cells.

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

Antagonist Effect of Triptolide on AKT Activation by Truncated Retinoid X Receptor-alpha

Background Retinoid X receptor-alpha (RXRα) is a key member of the nuclear receptor superfamily. We recently demonstrated that proteolytic cleavage of RXRα resulted in production of a truncated product, tRXRα, which promotes cancer cell survival by activating phosphatidylinositol-3-OH kinase (PI3K)/AKT pathway. However, how the tRXRα-mediated signaling pathway in cancer cells is regulated remains elusive. Methodology/Principal Findings We screened a natural product library for tRXRα targeting leads and identified that triptolide, an active component isolated from traditional Chinese herb Trypterygium wilfordii Hook F, could modulate tRXRα-mediated cancer cell survival pathway in vitro and in animals. Our results reveal that triptolide strongly induces cancer cell apoptosis dependent on intracellular tRXRα expression levels, demonstrating that tRXRα serves as an important intracellular target of triptolide. We show that triptolide selectively induces tRXRα degradation and inhibits tRXRα-dependent AKT activity without affecting the full-length RXRα. Interestingly, such effects of triptolide are due to its activation of p38. Although triptolide also activates Erk1/2 and MAPK pathways, the effects of triptolide on tRXRα degradation and AKT activity are only reversed by p38 siRNA and p38 inhibitor. In addition, the p38 inhibitor potently inhibits tRXRα interaction with p85α leading to AKT inactivation. Our results demonstrate an interesting novel signaling interplay between p38 and AKT through tRXRα mediation. We finally show that targeting tRXRα by triptolide strongly activates TNFα death signaling and enhances the anticancer activity of other chemotherapies Conclusions/Significance Our results identify triptolide as a new xenobiotic regulator of the tRXRα-dependent survival pathway and provide new insight into the mechanism by which triptolide acts to induce apoptosis of cancer cells. Triptolide represents one of the most promising therapeutic leads of natural products of traditional Chinese medicine with unfortunate side-effects. Our findings will offer new strategies to develop improved triptolide analogs for cancer therapy.

Sulindac-Derived RXRα Modulators Inhibit Cancer Cell Growth by Binding to a Novel Site

Retinoid X receptor-alpha (RXRα), an intriguing and unique drug target, can serve as an intracellular target mediating the anticancer effects of certain nonsteroidal anti-inflammatory drugs (NSAIDs), including sulindac. We report the synthesis and characterization of two sulindac analogs, K-8008 and K-8012, which exert improved anticancer activities over sulindac in a RXRα-dependent manner. The analogs inhibit the interaction of the N-terminally truncated RXRα (tRXRα) with the p85α subunit of PI3K, leading to suppression of AKT activation and induction of apoptosis. Crystal structures of the RXRα ligand-binding domain (LBD) with K-8008 or K-8012 reveal that both compounds bind to tetrameric RXRα LBD at a site different from the classical ligand-binding pocket. Thus, these results identify K-8008 and K-8012 as tRXRα modulators and define a binding mechanism for regulating the nongenomic action of tRXRα.

TBMS1 exerts its cytotoxicity in NCI-H460 lung cancer cells through nucleolar stress-induced p53/MDM2-dependent mechanism, a quantitative proteomics study

Tubeimoside-1 (TBMS1) exerts its anticancer effects by inducing G2/M arrest and apoptosis of cancer cells. However, the precise molecular mechanism of its anti-tumor effects has not been fully elucidated, especially the signaling pathways involved in the early stage of TBMS1 stimulation. In this study, we employed stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics approach and identified 439 proteins that exhibit significant differential expressions in NCI-H460 lung cancer cells upon exposure to TBMS1. Gene ontology and network analysis using DAVID and STRING on-line tools revealed that several nucleolar stress (ribosomal biogenesis) response proteins were differentially regulated by TBMS1. Functional validation demonstrated that TBMS1-induced NCI-H460 cell cytotoxicity involved nucleolar stress-induced p53/murine double minute clone 2 (MDM2), mTOR, and NF-κB signaling pathways.

Modulation of the Nur77-Bcl-2 apoptotic pathway by p38α MAPK

Orphan nuclear receptor Nur77 promotes apoptosis by targeting mitochondria through interaction with Bcl-2, an event that converts Bcl-2 from a survival to killer. However, how the Nur77-Bcl-2 apoptotic pathway is regulated remains largely unknown. In this study, we examined the regulation of the Nur77-Bcl-2 pathway by CCE9, a xanthone compound. Our results demonstrated that the apoptotic effect of CCE9 depended on its induction of Nur77 expression, cytoplasmic localization, and mitochondrial targeting. The activation of the Nur77-Bcl-2 pathway by CCE9 was associated with its activation of p38α MAPK. Inhibition of p38α MAPK activation by knocking down or knocking out p38α MAPK impaired the effect of CCE9 on inducing apoptosis and the expression and cytoplasmic localization of Nur77. In addition, CCE9 activation of p38α MAPK resulted in Bcl-2 phosphorylation and Bcl-2 interaction with Nur77, whereas inhibition of p38α MAPK activation or expression suppressed the interaction. Moreover, mutating Ser87 and Thr56 in the loop of Bcl-2, which are known to be phosphorylated by p38α MAPK, impaired the ability Bcl-2 to interact with Nur77. Together, our results reveal a profound role of p38α MAPK in regulating the Nur77-Bcl-2 apoptotic pathway through its modulation of Nur77 expression, Bcl-2 phosphorylation, and their interaction.Orphan nuclear receptor Nur77 promotes apoptosis by targeting mitochondria through interaction with Bcl-2, an event that converts Bcl-2 from a survival to killer. However, how the Nur77-Bcl-2 apoptotic pathway is regulated remains largely unknown. In this study, we examined the regulation of the Nur77-Bcl-2 pathway by CCE9, a xanthone compound. Our results demonstrated that the apoptotic effect of CCE9 depended on its induction of Nur77 expression, cytoplasmic localization, and mitochondrial targeting. The activation of the Nur77-Bcl-2 pathway by CCE9 was associated with its activation of p38α MAPK. Inhibition of p38α MAPK activation by knocking down or knocking out p38α MAPK impaired the effect of CCE9 on inducing apoptosis and the expression and cytoplasmic localization of Nur77. In addition, CCE9 activation of p38α MAPK resulted in Bcl-2 phosphorylation and Bcl-2 interaction with Nur77, whereas inhibition of p38α MAPK activation or expression suppressed the interaction. Moreover, mutating Ser87 and Thr56 in the loop of Bcl-2, which are known to be phosphorylated by p38α MAPK, impaired the ability Bcl-2 to interact with Nur77. Together, our results reveal a profound role of p38α MAPK in regulating the Nur77-Bcl-2 apoptotic pathway through its modulation of Nur77 expression, Bcl-2 phosphorylation, and their interaction.