UBE2S activates NF-κB signaling by binding with IκBα and promotes metastasis of lung adenocarcinoma cells.

PURPOSE: Nuclear factor (NF)-κB signaling in cancer cells has been reported to be involved in tumorigenesis. Phosphorylation and degradation of inhibitor of NF-κBα (IκBα) is a canonical pathway of NF-κB signaling. Here, we aimed to identify and characterize noncanonical activation of NF-κB signaling by ubiquitin-conjugating enzyme E2S (UBE2S) in lung adenocarcinoma cells. METHODS: TCGA and the Human Atlas Protein Database were used to analyze the survival rate of lung adenocarcinoma patients in conjunction with UBE2S expression. In addition, PC9, H460, H441 and A549 lung adenocarcinoma cells were used in this study. PC9 and H460 cells were selected for further analysis because they expressed different UBE2S protein levels. Specific IKK inhibitors, PS1145 and SC514, were used to assess IκBα phosphorylation. Western blot analysis was used to assess protein levels in PC9 and H460 cells. A scratch wound-healing assay was used to analyze the migrative abilities of PC9 and H460 cells. Overexpression and knockdown of UBE2S in H460 and PC9 cells were used to analyze their effects on downstream protein levels. Immunoprecipitation, immunofluorescent staining, glutathione S transferase (GST) pull-down and in vitro binding assays were used to analyze the interaction between UBE2S and IκBα. A luciferase assay was used to analyze activation of NF-κB signaling regulated by UBE2S. An in vivo zebrafish xenograft model was used to assess metastasis of PC9 cells regulated by UBE2S. RESULTS: We found that UBE2S expression in lung adenocarcinoma patients was negatively related to survival rate. The protein level of UBE2S was higher in PC9 cells than in H460 cells, which was opposite to that observed for IκBα. PC9 cells showed a higher UBE2S expression and migrative ability than H460 cells. Phosphorylation of IκBα was not changed by treatment with the IKK-specific inhibitors PS1145 and SC514 in PC9 and H460 cells. Overexpression and knockdown of UBE2S in H460 and PC9 cells revealed that the protein levels of IκBα were inversely regulated. Immunoprecipitation, immunofluorescent staining, GST pull-down and in vitro binding assays revealed direct binding of UBE2S with IκBα. Nuclear P65 protein levels and luciferase assays showed that NF-κB signaling was regulated by UBE2S. The expression of epithelial-to-mesenchymal (EMT) markers and the migrative ability of lung adenocarcinoma cells were also regulated by UBE2S. A zebrafish xenograft tumor model showed a reduction in the metastasis of PC9 cells that was induced by UBE2S knockdown. CONCLUSIONS: Higher UBE2S expression in lung adenocarcinomas may lead to increased binding with IκBα to activate NF-κB signaling and promote adenocarcinoma cell metastasis. UBE2S may serve as a potential therapeutic target for lung adenocarcinomas.

Flavonoids Luteolin and Quercetin Inhibit RPS19 and contributes to metastasis of cancer cells through c-Myc reduction.

Flavonoids luteolin and quercetin can inhibit growth and metastasis of cancer cells. In our previous report, luteolin and quercetin was shown to block Akt/mTOR/c-Myc signaling. Here, we found luteolin and quercetin reduced protein level and transactivation activity of RPS19 in A431-III cells, which is isolated from parental A431 (A431-P) cell line. Further investigation the inhibitory mechanism of luteolin and quercetin on RPS19, we found c-Myc binding sites on RPS19 promoter. The Akt inhibitor LY294002, mTOR inhibitor rapamycin and c-Myc inhibitor 10058-F4 significantly suppressed RPS19 expression and transactivation activities. Overexpression and knockdown of c-Myc in cancer cells show RPS19 expression was regulated by c-Myc. Furthermore, Knockdown and overexpression of RPS19 was used to analyze of the function of RPS19 in cancer cells. The epithelial-mesenchymal transition (EMT) markers and metastasis abilities of cancer cells were also regulated by RPS19. These data suggest that luteolin and quercetin might inhibit metastasis of cancer cells by blocking Akt/mTOR/c-Myc signaling pathway to suppress RPS19-activated EMT signaling.

IRF9-Stat2 Fusion Protein as an Innate Immune Inducer to Activate Mx and Interferon-Stimulated Gene Expression in Zebrafish Larvae.

Virus infection often causes large amounts of mortality during teleost larvae stage. Strong induction of innate immunity to increase survival rates of teleost larvae has been less reported. In this study, we present a zebrafish IRF9-Stat2 fusion protein (zIRF9-S2C) as a strong innate immunity inducer and characterized induction of interferon-stimulated genes (ISGs) in zebrafish larvae. zIRF9-S2C could mimic IFN-stimulated gene factor 3 (ISGF3) complex to constitutively activate transcription of Mx promoter through IFN-stimulatory element (ISRE) sites. Mutation of two ISRE sites on Mx promoter reduced transactivation activities of Mx promoter induced by zIRF9-S2C. An electrophoretic mobility shift assay experiment shows that zIRF9-S2C could directly bind to two ISRE sites of Mx promoter. Induction of transactivation of Mx promoter by zIRF9-S2C shows significantly higher activity than by zebrafish IFN1 (zIFN1), IFNγ (zIFNγ), and Tetraodon IRF9-S2C (TnIRF9-S2C). zIRF9-S2C raises transcription of Mxa, Mxb, Mxc, Ifnφ1, Ifnφ2, and Ifnφ3 in zebrafish liver ((ZFL) cell line) cells and zebrafish larvae. Collectively, we suggest that IRF9-S2C could activate transcription of ISGs with species-specific recognition and could be an innate immunity inducer in teleost larvae.