Degradation Of c-Jun Research Articles

Kumujan B suppresses TNF-α-induced inflammatory response and alleviates experimental colitis in mice.

Treatments of inflammatory bowel disease (IBD) are diverse, but their efficacy is limited, and it is therefore urgent to find better therapies. Controlling mucosal inflammation is a must in IBD drug treatment. The occurrence of anti-tumor necrosis factor α (TNF-α) monoclonal antibodies has provided a safer and more efficacious therapy. However, this kind of treatment still faces failure in the form of loss of response. β-Carboline alkaloids own an anti-inflammatory pharmacological activity. While Kumujan B contains β-carboline, its biological activity remains unknown. In this study, we attempted to determine the anti-inflammatory effects of Kumujan B using both the TNF-α- induced in vitro inflammation and DSS-induced in vivo murine IBD models. Our data show that Kumujan B attenuated the expression of interleukin 1β (IL-1β) and interleukin 6 (IL-6) induced by TNF-α in mouse peritoneal macrophages. Kumujan B suppressed c-Jun N-terminal protein kinases (JNK) signaling, especially c-Jun, for anti-inflammatory response. Furthermore, Kumujan B promoted K11-linked ubiquitination and degradation of c-Jun through the proteasome pathway. In an in vivo study, Kumujan B inhibited the expression of IL-1β, IL-6, and TNF-α and improved the colon barrier function in dextran sulfate sodium salt (DSS)-induced experimental mice colitis. Kumujan B exhibited in vivo and in vitro anti-inflammatory effects, making it a potential therapeutic candidate for treating IBD.

ALK inhibitors downregulate the expression of death receptor 4 in ALK-mutant lung cancer cells via facilitating Fra-1 and c-Jun degradation and subsequent AP-1 suppression

The successful treatment of patients with advanced non–small cell lung cancer (NSCLC) harboring chromosomal rearrangements of anaplastic lymphoma kinase (ALK) with ALK tyrosine kinase inhibitors (ALK-TKIs) represents a promising targeted therapy. As a result, various ALK-TKIs have been rapidly developed, some of which are approved while some are being tested in clinical trials. Death receptor 4 (DR4; also called TNFRSF10A or TRAIL-R1) is a cell surface protein, which functions as a pro-apoptotic protein that transduces TRAIL death signaling to trigger apoptosis. DR4 expression is positively regulated by MEK/ERK signaling and thus can be downregulated by MEK/ERK inhibition. This study thus focused on determining the effects of AKL-TKIs on DR4 expression and the underlying mechanisms. Three tested ALK-TKIs including APG-2449, brigatinib and alectinib effectively and preferentially inhibited Akt/mTOR as well as MEK/ERK signaling and decreased cell survival in ALK-mutant (ALKm) NSCLC cells with induction of apoptosis. This was also true for DR4 downregulation, which occurred even at 2 h post treatment. These ALK-TKIs did not affect DR4 protein stability, rather decreased DR4 mRNA expression. In parallel, they promoted degradation and reduced the levels of Fra-1 and c-Jun, two critical components of AP-1, and suppressed AP-1 (Fra-1/c-Jun)-dependent transcription/expression of DR4. Hence, it appears that ALK-TKIs downregulate DR4 expression in ALKm NSCLC cells via facilitating Fra-1 and c-Jun degradation and subsequent AP-1 suppression. Our findings thus warrant further investigation of the biological significance of DR4 downregulation in ALK-targeted cancer therapy.

Abstract 4864: ALK inhibitors downregulate the expression of death receptor 4 in ALK-mutant NSCLC cells via facilitating Fra-1 and c-Jun degradation and subsequent AP-1 suppression

Abstract The successful treatment of patients with advanced non-small cell lung cancer (NSCLC) harboring chromosomal rearrangements of anaplastic lymphoma kinase (ALK) with ALK tyrosine kinase inhibitors (ALK-TKIs) represents a promising targeted therapy. As a result, various ALK-TKIs have been rapidly developed, some of which were already approved while some are being tested in clinical trials. Death receptor 4 (DR4; also called TNFRSF10A or TRAIL-R1) is a cell surface protein, which is supposed to function as a pro-apoptotic protein that transduces TRAIL death signal to trigger apoptosis. Its expression is positively regulated by MEK/ERK signaling and thus can be downregulated by MEK/ERK inhibition. This study thus focused on determining the effects of AKL inhibition on DR4 expression and the underlying mechanisms. ALK-TKIs such as brigatinib and alectinib effectively and preferentially inhibited AKT/mTOR as well as MEK/ERK signaling and decreased cell survival in ALK-mutant (ALKm) NSCLC cells with induction of apoptosis. This was also true to DR4 downregulation, which occurred even at 2 h post treatment. These ALK-TKIs did not affect DR4 protein stability, rather potently decreased DR4 mRNA expression. In parallel, they promoted degradation and decreased the levels of Fra-1 and c-Jun, two critical components of AP-1, and suppressed AP-1 (Fra-1/c-Jun)-dependent transcription/expression of DR4. Hence, it appears that ALK-TKIs downregulate DR4 expression in ALKm NSCLC cells via facilitating Fra-1 and c-Jun degradation and subsequent AP-1 suppression. Our findings thus warrant further investigation on elucidating the biological significance of DR4 downregulation in ALK-targeted cancer therapy. Citation Format: Wen Zhao, Danlei Yu, Yifan Zhai, Shi-Yong Sun. ALK inhibitors downregulate the expression of death receptor 4 in ALK-mutant NSCLC cells via facilitating Fra-1 and c-Jun degradation and subsequent AP-1 suppression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4864.

Anti-inflammatory activity of Kyungok-go on Lipopolysaccharide-Stimulated BV-2 Microglia Cells

Objectives: Kyungok-go (KOG) is a traditional multi-herbal medicine commonly used for enforcing weakened immunity for long time. Recently, there are several reports that KOG has anti-inflammatory and immuno-stimulatory activities in many experimental models. However, the protective effects of KOG on neuronal inflammation are still undiscovered. Thus, we investigated the neuro-protective activity of KOG on lipopolysaccharide (LPS)-stimulated mouse microglia cells. To find out KOG’s anti-neuroinflammatory effects on microglial cells, we examined the production of nitrite using griess assay, and mRNA expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and interleukin (IL)-1<i>β</i>, IL-6, tumor necrosis factor (TNF)-<i>α</i> using real time RT-PCR. In addition, to examine the regulating mechanisms of KOG, we investigated the protein expression of mitogen-activated protein kinases (MAPKs) and I<i>κ</i>-B<i>α</i> by western blot. KOG inhibited the elevation of nitrite, iNOS and COX-2 on LPS-stimulated BV2 cells. Also, KOG significantly inhibited the pro-inflammatory cytokines such as IL-1<i>β</i>, IL-6, and TNF-<i>α</i> on LPS-stimulated BV2 microglial cells. Moreover, KOG inhibited the activation of c-Jun N-terminal kinase (JNK), P38 and degradation of I<i>κ</i>-B<i>α</i> but not the activation of extracellular signal regulated kinase (ERK) on LPS-stimulated BV2 microglial cells. These results showed KOG has the anti-inflammatory effects through the inhibition on nitrite, iNOS, COX-2, IL-1<i>β</i>, IL-6, and TNF-<i>α</i> via the deactivation of JNK, p38 and nuclear factor (NF)-<i>κ</i>B on LPS-stimulated BV2 microglial cells. Thereby, KOG could offer the new and promising treatment for neurodegenerative disease related to neuroinflammation.

P38-dependent c-Jun degradation contributes to reduced PGE2 production in sodium orthovanadate-treated macrophages

In particular, the phenomenon of c-Jun degradation within the inflammatory response has not yet been fully analyzed. In order to verify this, we investigated LPS-stimulated murine macrophages pre-treated with sodium orthovanadate (SO) in order to uncover the regulatory mechanisms of the MAPKs which regulate c-Jun degradation within the inflammatory response. Through our study, we found that SO suppressed the production of prostaglandin E2 (PGE2) and the expression of COX-2 in LPS-stimulated RAW264.7 cells. Additionally, SO decreased total c-Jun levels, without altering the amount of mRNA, although the phospho-levels of p38, ERK, and JNK were strongly enhanced. Through the usage of selective MAPK inhibitors, and knockdown and overexpression strategies, p38 was revealed to be a major MAPK which regulates c-Jun degradation. Further analysis indicates that the phosphorylation of p38 is a determinant for c-Jun degradation, and is sufficient to induce ubiquitination-dependent c-Jun degradation, recovered through MG132 treatment. Therefore, our results suggest that the hyperphosphorylation of p38 by SO contributes to c-Jun degradation, which is linked to the suppression of PGE2 secretion in inflammatory responses; and thus, finding drugs to increase p38 activity could be a novel strategy for the development of anti-inflammatory drugs.

The E3 ligase COP1 promotes ERα signaling and suppresses EMT in breast cancer.

ERα signaling drives proliferation, survival and cancer initiation in the mammary gland. Therefore, it is critical to elucidate mechanisms by which ERα expression is regulated. We show that the tumor suppressor E3 ligase COP1 promotes the degradative polyubiquitination of the microtubule-associated protein HPIP. As such, COP1 negatively regulates estrogen-dependent AKT activation in breast cancer cells. However, COP1 also induces ERα expression and ERα-dependent gene transcription, at least through c-Jun degradation. COP1 and ERα levels are positively correlated in clinical cases of breast cancer. COP1 also supports the metabolic reprogramming by estrogens, including glycolysis. On the other hand, COP1 suppresses EMT in breast cancer cells. COP1 deficiency also contributes to Tamoxifen resistance, at least through protective autophagy. Therefore, COP1 acts as an oncogenic E3 ligase by promoting ERα signaling but also acts as a tumor suppressor candidate by preventing EMT, which reflects a dual role of COP1 in breast cancer.

Erk1/2 Inactivation-Induced c-Jun Degradation Is Regulated by Protein Phosphatases, UBE2d3, and the C-Terminus of c-Jun.

Constitutive photomorphogenic 1 (COP1) is the ubiquitin E3 ligase that mediates degradation of c-Jun protein upon Erk1/2 inactivation. It remains unknown how this protein degradation pathway is regulated. In this study, we investigated the roles of protein phosphatases, ubiquitin-conjugating E2 enzymes (UBE2), and an intrinsic motif of c-Jun in regulating this degradation pathway. By using pharmacological inhibitors and/or gene knockdown techniques, we identified protein phosphatase 1 (PP1) and PP2A as the phosphatases and UBE23d as the UBE2 promoting c-Jun degradation, triggered by Erk1/2 inactivation. In addition, we report that the C-terminus of c-Jun protein facilitates its degradation. The addition of a C-terminal tag or deletion of the last four amino acid residues from the C-terminus of c-Jun protects it from degradation under Erk1/2-inactivating conditions. Taken together, this study reveals that the Erk1/2 inactivation-triggered and COP1-mediated c-Jun degradation is extrinsically and intrinsically regulated, providing a new understanding of the mechanisms underlying this protein degradation pathway.

Lipopolysaccharide stimulates bovine endometrium explants through toll‑like receptor 4 signaling and PGE2 synthesis

Bovine endometrium infection with gram-negative bacteria commonly causes uterine diseases. Previous studies indicate that prostaglandin E2 (PGE2) is an inflammatory mediator in bacterial endometritis. However, the mechanism underlying lipopolysaccharide (LPS)-induced inflammatory response regulation in bovine endometrial explants remains elusive. In the present study, bovine explants were pre-treated with 15-hydroxyprostaglandin dehydrogenase (15-PGDH) inhibitors before LPS stimulation. PGE2 secretion, prostaglandin synthetase, pro-inflammatory factor, damage-associated molecular pattern (DAMP), and related signaling pathway factor levels were evaluated. Using 15-PGDH inhibitors pre-treatment, LPS-treated bovine endometrial explants exhibited augmentation of PGE2 and DAMP expression, and upregulation of various signaling pathway factors. Protein kinase A (PKA), extracellular-signal-regulated kinase, and c-Jun N-terminal kinase phosphorylation and degradation of nuclear transcription factor-κB (NF-κB) inhibitors were induced in the pre-treated endometrial explants. The mechanism underlying LPS-induced PGE2 accumulation acting as a pro-inflammatory mediator through toll-like receptor 4 signaling in bovine explants could involve the PKA, mitogen-activated protein kinase, and NF-κB pathways.

Endoplasmic Reticulum Stress-Mediated p62 Downregulation Inhibits Apoptosis via c-Jun Upregulation.

Cereblon (CRBN), a substrate receptor of cullin 4-RING E3 ligase (CRL4) regulates the ubiquitination and degradation of c-Jun, mediating the lipopolysaccharide-induced cellular response. However, the upstream signaling pathway that regulates this process is unknown. In this study, we describe how endoplasmic reticulum (ER) stress reversely regulates sequestosome-1 (p62)and c-Jun protein levels. Furthermore, our study reveals that expression of p62 attenuates c-Jun protein levels through the ubiquitin-proteasome system. Conversely, siRNA knockdown of p62 elevates c-Jun protein levels. Immunoprecipitation and immunoblotting experiments demonstrate that p62 interacts with c-Jun and CRBN to form a ternary protein complex. Moreover, we find that CRBN knockdown completely abolishes the inhibitory effect of p62 on c-Jun. Using brefeldin A as an inducer of ER stress, we demonstrate that the p62/c-Jun axis participates in the regulation of ER stress-induced apoptosis, and that CRBN is required for this regulation. In summary, we have identified an upstream signaling pathway, which regulates p62-mediated c-Jun degradation. Our findings elucidate the underlying molecular mechanism by which p62/c-Jun axis regulates the ER stress-induced apoptosis, and provide a new molecular connection between ER stress and apoptosis.

KDM5c Promotes Colon Cancer Cell Proliferation Through the FBXW7-c-Jun Regulatory Axis.

KDM5c is a histone demethylase that specifically demethylates trimethylated and dimethylated H3 Lys-4 to play a central role in transcriptional repression. C-Jun is a proto-oncogene and promotes cell proliferation when ectopically accumulated, but can be ubiquitinated by SCF (FBXW7), leading to its degradation. FBXW7 is an E3 ubiquitin ligase of c-Jun, and exhibits carcinostasis in colon cancer. Here, we report that overexpression of KDM5c in human colon cancer cells results in attenuated FBXW7 transcription and accumulated c-Jun protein, leading to increased proliferation of colon cancer cells. We show that overexpression of KDM5c can result in increased c-Jun protein levels and decreased ubiquitin levels, with no significant change in mRNA levels of c-Jun. KDM5c overexpression blocks the ubiquitin-proteasome proteolytic pathway of c-Jun by down-regulating the expression of FBXW7. KDM5c down-regulation of FBXW7 occurs by demethylation of H3K4me3 at TSS and downstream of the FBXW7 gene. And interaction of KDM5c with H3K4me3 downstream of FBXW7 gene may be followed by recruitment of DNMT3b to methylate the spatially close CpG island located near the FBXW7 TSS. This methylation represses FBXW7 gene expression, which can reduce c-Jun degradation via the ubiquitin-proteasome pathway. TCGA database analysis revealed high expression of KDM5c in colon cancer tissues. KDM5c expression in colon cancer was correlated with poor overall survival of patients in the first 7 years. Data from TCGA showed that high expression of KDM5c was correlated with high DNA methylation of the FBXW7 gene, but was not positively correlated with methylation of the Jun gene. These results suggest that KDM5c regulation of colon cell proliferation is mainly mediated by the KDM5c-FBXW7-c-Jun axis.

The splice 1 variant of HTLV-1 bZIP factor stabilizes c-Jun

HBZ is expressed by the complex retrovirus, Human T-cell Leukemia Virus type 1, and implicated in pathological effects associated with viral infection. From the nucleus, HBZ alters gene expression by interacting with a variety of transcriptional regulatory proteins, among which is c-Jun. Previously, one of the three HBZ variants, HBZUS, was reported to decrease c-Jun expression by promoting its degradation. Here we show that another variant, HBZS1, produces the opposite effect. In the presence of HBZS1, c-Jun expression increases due to its stabilization. Our data suggest that this effect requires the ability of HBZS1 to interact with c-Jun. We provide evidence that HBZS1 inhibits the proteosomal degradation of c-Jun initiated by the Cop1-containing ubiquitin ligase complex. HBZS1 is the most abundant variant in HTLV-1-infected T-cells, and our data indicate that levels of c-Jun expression in infected cells are consistent with effects of HBZS1.

Secalonic acid D induces cell apoptosis in both sensitive and ABCG2-overexpressing multidrug resistant cancer cells through upregulating c-Jun expression

Secalonic acid D (SAD) could inhibit cell growth in not only sensitive cells but also multidrug resistant (MDR) cells. However, the molecular mechanisms need to be elucidated. Here, we identified that SAD possessed potent cytotoxicity in 3 pairs of MDR and their parental sensitive cells including S1-MI-80 and S1, H460/MX20 and H460, MCF-7/ADR and MCF-7 cells. Furthermore, SAD induced cell G2/M phase arrest via the downregulation of cyclin B1 and the increase of CDC2 phosphorylation. Importantly, JNK pathway upregulated the expression of c-Jun in protein level and increased c-Jun phosphorylation induced by SAD, which was linked to cell apoptosis via c-Jun/Src/STAT3 pathway. To investigate the mechanisms of upregulation of c-Jun protein by SAD, the mRNA expression level and degradation of c-Jun were examined. We found that SAD did not alter the mRNA level of c-Jun but inhibited its proteasome-dependent degradation. Taken together, these results implicate that SAD induces cancer cell death through c-Jun/Src/STAT3 signaling axis by inhibiting the proteasome-dependent degradation of c-Jun in both sensitive cells and ATP-binding cassette transporter sub-family G member 2 (ABCG2)-mediated MDR cells.

BLM Potentiates c-Jun Degradation and Alters Its Function as an Oncogenic Transcription Factor

Mutations in BLM helicase predispose Bloom syndrome (BS) patients to a wide spectrum of cancers. We demonstrate that MIB1-ubiquitylated BLM in G1 phase functions as an adaptor protein by enhancing the binding of transcription factor c-Jun and its E3 ligase, Fbw7α. BLM enhances the K48/K63-linked ubiquitylation on c-Jun, thereby enhancing the rate of its subsequent degradation. Functionally defective Fbw7α mutants prevalent in multiple human cancers are reactivated by BLM. However, BS patient-derived BLM mutants cannot potentiate Fbw7α-dependent c-Jun degradation. The decrease in the levels of c-Jun in cells expressing BLM prevents effective c-Jun binding to 2,584 gene promoters. This causes decreases in the transcript and protein levels of c-Jun targets in BLM-expressing cells, resulting in attenuated c-Jun-dependent effects during neoplastic transformation. Thus, BLM carries out its function as a tumor suppressor by enhancing c-Jun turnover and thereby preventing its activity as a proto-oncogene.

Cereblon suppresses the lipopolysaccharide-induced inflammatory response by promoting the ubiquitination and degradation of c-Jun

Chronic inflammation is associated with multiple human disorders, such as rheumatoid arthritis, metabolic diseases, and neurodegenerative diseases. Therefore, alleviation of inflammation induced by environmental stimuli is important for disease prevention or treatment. Cereblon (CRBN) functions as a substrate receptor of the cullin-4 RING E3 ligase to mediate protein ubiquitination and degradation. Although it has been reported that CRBN reduces the inflammatory response through its nonenzymatic function, its role as a substrate receptor of the E3 ligase is not explored in mediating this process. Here we used a quantitative proteomics approach to find that the major component of the activator protein 1 (AP-1) complex, c-Jun, is significantly down-regulated upon CRBN expression. Biochemical approaches further discover that CRBN interacts and partially colocalizes with c-Jun and promotes the formation of Lys48-linked polyubiquitin chains on c-Jun, enhancing c-Jun degradation. We further reveal that CRBN attenuates the transcriptional activity of the AP-1 complex and reduces the mRNA expression and protein level of several pro-inflammatory cytokines. Moreover, flow cytometry analyses show that CRBN attenuates lipopolysaccharide-induced apoptosis in differentiated THP-1 cells. Through genetic manipulation and pharmacological inhibition, we uncover a new molecular mechanism by which CRBN regulates the inflammatory response and apoptosis induced by lipopolysaccharide. Our work and previous studies demonstrate that CRBN suppresses the inflammatory response by promoting or inhibiting the ubiquitination of two key molecules at different levels of the inflammatory cascade through its enzymatic function as a substrate receptor and its nonenzymatic function as a protein binding partner.

UBE2M Is a Stress-Inducible Dual E2 for Neddylation and Ubiquitylation that Promotes Targeted Degradation of UBE2F

UBE2M and UBE2F are two family members of neddylation E2 conjugating enzyme that, together with E3s, activate CRLs (Cullin-RING Ligases) by catalyzing cullin neddylation. However, whether and how two E2s cross-talk with each other are largely unknown. Here, we report that UBE2M is a stress-inducible gene subjected to cis-transactivation by HIF-1 and AP1, and MLN4924, a small molecule inhibitor of E1 NEDD8-activating enzyme (NAE), upregulates UBE2M via blocking degradation of HIF-1α and c-JUN. UBE2M is a dual E2 for targeted ubiquitylation and degradation of UBE2F, acting as a neddylation E2 to activate CUL3-Keap1 E3 under physiological conditions but as a ubiquitylation E2 for Parkin-DJ-1 E3 under stressed conditions. UBE2M-induced UBE2F degradation leads to CRL5 inactivation and subsequent NOXA accumulation to suppress the growth of lung cancer cells. Collectively, our study establishes a negative regulatory axis between two neddylation E2s with UBE2M ubiquitylating UBE2F, and two CRLs with CRL3 inactivating CRL5.

Activation and auto‐destruction of the ubiquitin ligase Itch

Ubiquitin ligases of the Nedd4 family are maintained inactive by intramolecular interactions (1). We reported that, like Nedd4, the closely related ubiquitin ligase Itch strongly binds to the endosomal protein LITAF through its WW domains, that recognize two PPXY motifs in the N‐terminal half of LITAF. Unlike Nedd4, Itch binding to LITAF brings the ligase to the lysosomal compartments. We thus postulated that LITAF overexpression might bring Itch to the lysosome, where it would access different substrates and target them for degradation (2). Concomitantly, LITAF binding to Itch may increase Itch ligase activity, as other PPXY motif proteins such as NDFIP and LRAD3 (3,4). Here, we set up to determine if Itch ubiquitin ligase activity is indeed up regulated by LITAF binding, and if Itch most common substrate degradation is increased in response to LITAF binding to Itch. We find that Itch, WWP1, WWP2 and Nedd4 all bind to LITAF, and that the four ligases autoubiquitylation is greatly increased upon LITAF binding, albeit to different levels. Both LITAF overexpression and addition of recombinant LITAF to crude cell lisates lead to rapid degradation of Itch, and, to a lesser extent, Nedd4. Itch substrates cJun and Endophilin degradation was also increased. In vitro ubiquitylation assays show that Itch autoubiquitylation is enhanced in the presence of LITAF, but substrate ubiquitylation was not significantly increased. In conclusion, LITAF binding strongly activate Itch autoubiquitylation, as well as that of other ligases of the Nedd4 family. The consequence of this activation is Itch proteasomal degradation, with little effect on its substrates ubiquitylation. LITAF is thus likely to be a negative regulator of Itch in cells where it is strongly expressed.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multivalent Interactions with Fbw7 and Pin1 Facilitate Recognition of c-Jun by the SCFFbw7 Ubiquitin Ligase

Many regulatory proteins, including the transcription factor c-Jun, are highly enriched in disordered protein regions that govern growth, division, survival, differentiation, and response to signals. The stability of c-Jun is controlled by poorly understood regulatory interactions of its disordered region with both the E3 ubiquitin ligase SCFFbw7 and prolyl cis-trans isomerase Pin1. We use nuclear magnetic resonance and fluorescence studies of c-Jun to demonstrate that multisite c-Jun phosphorylation is required for high-affinity interaction with Fbw7. We show that the Pin1 WW and PPIase domains interact in a dynamic complex with multiply phosphorylated c-Jun. Importantly, Pin1 isomerizes a pSer-Pro peptide bond at the c-Jun N terminus that affects binding to Fbw7 and thus modulates the ubiquitin-mediated degradation of c-Jun. Our findings support the general principle that multiple weak binding motifs within disordered regions can synergize to yield high-affinity interactions and provide rapidly evolvable means to build and fine-tune regulatory events.

MFHAS1 suppresses TLR4 signaling pathway via induction of PP2A C subunit cytoplasm translocation and inhibition of c-Jun dephosphorylation at Thr239

TLR4, an important Toll-like receptor in innate immunity, can be activated by LPS and induce proinflammatory cytokines to resist invasion of pathogenic microorganism, but excessive inflammation can trigger tissue injury. Many genes negatively regulate TLR4 signaling pathway. Recent studies found that malignant fibrous histiocytoma amplified sequence 1 (MFHAS1) suppressed the expression of cytokine IL6 in Raw264.7 cells stimulated by LPS, but the mechanisms remained unclear. This study investigated the role of MFHAS1 in TLR4 signaling pathway and the possible mechanisms implicated. The results indicated that the expression of MFHAS1 was significantly increased in cells stimulated with LPS. Up-regulation of MFHAS1 effectively suppressed inflammatory cytokine expression in cells exposed to LPS, whereas down-regulation of MFHAS1 markedly increased inflammatory cytokines expression. Co-immunoprecipitation, pull-down and immunofluorescence tests demonstrated that MFHAS1 combined with the B and C subunits of PP2A and induced cytoplasm translocation of the C subunit, leading to decrease dephosphorylation of c-Jun at Thr239 and increase degradation of c-Jun. Reduction of c-Jun protein results in decreased AP-1 activity, which is independent of inhibition of JNK or p38MAPK phosphorylation. Taken together, these results indicate that MFHAS1 suppresses TLR4 signaling pathway through induction of PP2A C subunit cytoplasm translocation and subsequent c-Jun degradation, leading finally to decrease AP-1 activity and cytokines expression.

Promoting tumorigenesis in nasopharyngeal carcinoma, NEDD8 serves as a potential theranostic target

Nasopharyngeal carcinoma (NPC), is one of the most common human malignancies in south China, it has the highest recurrence rate and treatment resistance. The underlying molecular mechanisms of NPC relapse and treatment tolerance are not fully understood. In this study, the effects of NEDD8 and NEDD8-activating enzyme inhibitor (MLN4924) on NPC were studied both in vitro and in vivo. Immunohistochemical staining of 197 NPC tissues revealed an elevated NEDD8 expression as an unfavorable independent factor in overall survival and disease-free survival rates. NEDD8 expression was positively correlated with a high risk of death and positivity of lymph node metastasis. Depleted NEDD8 expression by shRNA and inhibited by specific inhibitor MLN4924 dramatically suppressed cell proliferation, cell apoptosis, cell cycle arrest, while ectopic NEDD8 exhibited opposing effects. NEDD8 affected cancer stem cell phenotypes of NPC as assessed in vitro using the cell number of side population (SP) by flow cytometry analysis, colony formation assay, sphere formation assay, and tumor initiation ability in vivo. Downregulation of NEDD8 enhanced the susceptibility of NPC cells to cisplatin and radiation. Moreover, we found that MLN4924 suppressed c-Jun degradation in human NPC cells. Taken together, this report revealed that NEDD8 may act as a novel prognostic marker and MLN4924 may serve as a promising therapeutic target for patients with NPC.

FBXW7 expression is associated with prognosis and chemotherapeutic outcome in Chinese patients with gastric adenocarcinoma

BackgroundFBXW7, a component of the Skp-Cullin1-F-box, mediates target protein recognition. It is a tumor suppressor gene that plays a role in the regulation of cell cycle exit and reentry via c-Myc, c-Jun and Notch degradation. There are few studies, particularly involving a large patient cohort, that have evaluated FBXW7 during gastric cancer progression.MethodsOur study aimed to evaluate the value of FBXW7 as a clinical marker in gastric adenocarcinoma (GC) patients including a subset treated with postoperative chemotherapy. Quantitative reverse transcription PCR (qRT-PCR) assay was used to measure FBXW7 transcript levels in tumors paired with normal gastric tissue in 24 gastric adenocarcinoma patients. Subsequently, 546 additional GC samples were evaluated from patients that underwent radical gastrectomy, including 118 early stage cases(Stage I) and 428 advanced stage cases (Stages II or III). Amongst the advanced stage patient cases evaluated, 347 received postoperative adjuvant chemotherapy. All 546 gastric adenocarcinoma cases were then evaluated by tissue microarray and immunohistochemistry (IHC) for FBXW7 expression. Clinicopathological features and diagnoses were confirmed by histopathologic evaluation and review of clinical data. Overall survival (OS) was then evaluated in the 546 gastric cancer patients.ResultsBy immunohistologic evaluation, low expression of FBXW7 in primary gastric cancer significantly correlated with poor differentiation of tumor cells. Moreover, low FBXW7 expression was associated with worse survival as well as worse adjuvant chemotherapy response.ConclusionOur findings suggest that FBXW7 may serve as an important predictor in chemotherapeutic responses.