Mesencephalic Astrocyte-Derived Neurotrophic Factor Inhibits Liver Cancer Through Small Ubiquitin-Related Modifier (SUMO)ylation-Related Suppression of NF-κB/Snail Signaling Pathway and Epithelial-Mesenchymal Transition.

BACKGROUND AND AIMS: Endoplasmic reticulum (ER) stress is associated with liver inflammation and hepatocellular carcinoma (HCC). However, how ER stress links inflammation and HCC remains obscure. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER stress-inducible secretion protein that inhibits inflammation by interacting with the key subunit of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) p65. We hypothesized that MANF may play a key role in linking ER stress and inflammation in HCC. APPROACH AND RESULTS: Here, we found that MANF mRNA and protein levels were lower in HCC tissues versus adjacent noncancer tissues. Patients with high levels of MANF had better relapse-free survival and overall survival rates than those with low levels. MANF levels were also associated with the status of liver cirrhosis, advanced tumor-node-metastasis (TNM) stage, and tumor size. In vitro experiments revealed that MANF suppressed the migration and invasion of hepatoma cells. Hepatocyte-specific deletion of MANF accelerated N-nitrosodiethylamine (DEN)-induced HCC by up-regulating Snail1+2 levels and promoting epithelial-mesenchymal transition (EMT). MANF appeared in the nuclei and was colocalized with p65 in HCC tissues and in tumor necrosis factor alpha (TNF-α)-treated hepatoma cells. The interaction of p65 and MANF was also confirmed by coimmunoprecipitation experiments. Consistently, knockdown of MANF up-regulated NF-κB downstream target genes TNF-α, interleukin (IL)-6 and IL-1α expression in vitro and in vivo. Finally, small ubiquitin-related modifier 1 (SUMO1) promoted MANF nuclear translocation and enhanced the interaction of MANF and p65. Mutation of p65 motifs for SUMOylation abolished the interaction of p65 and MANF. CONCLUSIONS: MANF plays an important role in linking ER stress and liver inflammation by inhibiting the NF-κB/Snail signal pathway in EMT and HCC progression. Therefore, MANF may be a cancer suppressor and a potential therapeutic target for HCC.

Positive and neutral updating reconsolidate aversive episodic memories via different routes.

Aversive memories are long-lasting and prone to burden our emotional wellbeing and mental health. Yet, how to remedy the maladaptive effects of aversive memories remains elusive. Using memory reactivation and emotional updating manipulations, we investigated how positive and neutral emotion may update aversive memories for reconsolidation in humans. We found that positive updating after reactivation was equivalent to neutral updating in impairing true memories of a previous aversive event after a 12-hour wakeful delay, but induced more false memory. Moreover, additional 12-hour delay with overnight sleep did not further enlarge true memory differences, but attenuated the effect of reactivation and updating on false memory. Interestingly, the neutral rather than the positive updating reduced the emotional arousal of the aversive memory 24 h later. Our findings could serve as a reference for real-world therapeutic applications regarding how positive and neutral updating may reshape aversive memories, especially when taking wake- and sleep-filled reconsolidation into account.

Small ubiquitin-related modifier 2/3 interacts with p65 and stabilizes it in the cytoplasm in HBV-associated hepatocellular carcinoma.

BACKGROUND: SUMOylation, an important post-translational modification, associates with the development of hepatocellular carcinoma (HCC). p65, one of the most important subunits of NF-κB, is a key regulator in the development of HCC and has been reported to be SUMOylated by exogenous small ubiquitin-related modifier 3 (SUMO3) in HEK 293T cells. However, the relationship between p65 and SUMO2/3 in HCC remains unknown. This study was to investigate the interaction between p65 and SUMO2/3 and explore the potential roles involved in HCC. METHODS: The expressions of p65 and SUMO2/3 in the liver tissues were detected by using immunohistochemistry. We performed double-labeled immunofluorescence and co-immunoprecipitation assay to verify the interaction between p65 and SUMO2/3. The extraction of nuclear and cytoplasmic proteins was performed, and the subcellular localization of p65 was detected. The proliferation and migration of hepatoma cells were observed using MTT, colony formation, and transwell assays. RESULTS: We found a strong SUMO2/3-positive immunoreactivity in the cytoplasm in the non-tumor tissues of HCC. However, SUMO2/3 level was down regulated in the tumor tissues as compared with the adjacent non-tumor tissues. In accordance with this finding, p65 was up regulated in the adjacent non-tumor tissues and almost localized in the cytoplasm. There was a close correlation between SUMO2/3 and p65 expressions in the liver tissues (R = 0.800, p = 0.006). The interaction between p65 and SUMO2/3 was verified by co-immunoprecipitation and double-labeled immunofluorescent assays. TNF-α (10 ng/ml) treatment for 30 min not only up regulated the cytoplasmic conjugated SUMO2/3, but also enhanced SUMO2/3-p65 interaction. Furthermore, we found that SUMO2/3 up regulated the cytoplasmic p65 protein level in a dose-dependent manner, but not affected its mRNA level. The increase of p65 protein by SUMO2/3 was abolished by MG132 treatment, a reversible inhibitor of proteasome. Meanwhile, TNF-α-induced increase of SUMO2/3-conjugated p65 was along with the reduction of the ubiquitin-conjugated p65. The further study showed that SUMO2/3 over-expression decreased the proliferative ability of hepatoma cells, but did not affect the migration. CONCLUSION: SUMO2/3-p65 interaction may be a novel mechanism involved in the transformation from chronic hepatitis B to HCC via stabilizing cytoplasmic p65, which might shed light on understanding the tumorigenesis and development.

Small ubiquitin-related modifier 1 is involved in hepatocellular carcinoma progression via mediating p65 nuclear translocation.

Small ubiquitin-related modifier (SUMO) proteins participate in a post-translational modification called SUMOylation and regulate a variety of intracellular processes, such as targeting proteins for nuclear import. The nuclear transport of p65 results in the activation of NF-κB, and p65 contains several SUMO interacting motifs (SIMs). However, the relationship between p65 and SUMO1 in hepatocellular carcinoma (HCC) remains unclear. In this study, we demonstrated the potential roles of SUMO1 in HCC via the regulation of p65 subcellular localization. We found that either SUMO1- or p65-positive immunoreactivity was remarkably increased in the nuclei of tumor tissues in HCC patients compared with non-tumor tissues, and further analysis suggested a correlation between SUMO1- and nuclear p65-positive immunoreactivities (R = 0.851, P = 0.002). We also verified the interaction between p65 and SUMO1 in HCC by co-immunoprecipitation. TNF-α and hypoxia increased SUMO1 protein levels and enhanced SUMO1-modified p65 SUMOylation. Moreover, the knockdown of SUMO1 decreased p65 nuclear translocation and inhibited NF-κB transcriptional activity. Further the results of this study revealed that the knockdown of SUMO1 suppressed the proliferation and migration of hepatoma cells. These results suggest that SUMO1 contributes to HCC progression by promoting p65 nuclear translocation and regulating NF-κB activity.