51592

TRIM33

epithelial-mesenchymal transition (EMT) is a fundamental cellular process that contributes to epithelial tissue morphogenesis during normal development and in tumor invasiveness and metastasis. The transcriptional regulator SnoN robustly influences EMT in response to the cytokine TGFbeta, but the mechanisms that regulate the fundamental role of SnoN in TGFbeta-induced EMT are not completely understood. Here we employ interaction proteomics to uncover the signaling protein TIF1gamma as a specific interactor of SnoN1 but not the closely related isoform SnoN2. A 16-amino acid peptide within a unique region of SnoN1 mediates the interaction of SnoN1 with TIF1gamma. Strikingly, although TIF1gamma is thought to act as a ubiquitin E3 ligase, we find that TIF1gamma operates as a small ubiquitin-like modifier (SUMO) E3 ligase that promotes the sumoylation of SnoN1 at distinct lysine residues. Importantly, TIF1gamma-induced sumoylation is required for the ability of SnoN1 to suppress TGFbeta-induced EMT, as assayed by the disruption of the morphogenesis of acini in a physiologically relevant three-dimensional model of normal murine mammary gland (NMuMG) epithelial cells. Collectively, our findings define a novel TIF1gamma-SnoN1 sumoylation pathway that plays a critical role in EMT and has important implications for our understanding of TGFbeta signaling and diverse biological processes in normal development and cancer biology.CI - (c) 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

TIF1gamma is a novel regulator of transforming growth factor (TGF)-beta/Smad signaling. Our previous studies show that dysregulated expression of transcriptional intermediary factor 1 gamma (TIF1gamma) and abnormal TGF-beta/Smad signaling are implicated in non-small-cell lung cancer (NSCLC) separately. However, how TIF1gamma contributes to NSCLC by controlling TGF-beta/Smad signaling is poorly understood. Here, we investigated the mechanistic role of TIF1gamma in TGF-beta-induced epithelial-mesenchymal transition (EMT), as well as a link between TIF1gamma and SOX2 in NSCLC. We show that TIF1gamma is a downstream target of SOX2 in NSCLC cells. SOX2 overexpression negatively regulated TIF1gamma promoter activity and thereby attenuated TIF1gamma mRNA and protein expression levels; SOX2 knockdown significantly enhanced TIF1gamma promoter activity and augmented TIF1gamma expression. Moreover, TIF1gamma mRNA expression was downregulated in human NSCLC tissues and negatively correlated with SOX2 protein, which was upregulated in NSCLC tissues. Importantly, knockdown of TIF1gamma or SOX2 overexpression augmented SMAD4 (human Mad (mothers against decapentaplegic)-related homologous protein 4)-dependent transcriptional responses, and enhanced TGF-beta-induced EMT and human NSCLC cell invasion; knockdown of SOX2 impaired TGF-beta-induced EMT and NSCLC cell invasion. In an in vivo model of metastasis, knockdown of TIF1gamma promotes NSCLC cell metastasis. In addition, our data suggested that TIF1gamma inhibited TGF-beta-induced EMT through competing with SMAD4 in NSCLC cells. Taken together, our findings reveal a new mechanism by which SOX2-mediated transcription repression of TIF1gamma promotes TGF-beta-induced EMT in NSCLC.

The transcriptional regulator SnoN (also known as SKI-like proto-oncogene, SKIL), a member of the Ski family, has been reported to influence epithelial-mesenchymal transition (EMT) in response to TGF-beta. In the present study, we investigated the role of SnoN in bladder cancer (BC). Differential expression of SnoN was not detected in BC tissues compared with that noted in adjacent non-cancerous tissues. SnoN was upregulated in response to TGF-beta treatment, but had no effect on the TGF-beta pathway, which may be explained by the low level of SnoN SUMOylation. TIF1gamma, which catalyzes the SUMOylation of SnoN, was downregulated in BC tissues. Overexpression of TIF1gamma restored the ability of SnoN to suppress the TGF-beta pathway. Furthermore, TGF-beta-induced EMT and invasion of BC cells were suppressed by TIF1gamma in the presence of SnoN. Collectirely, our data suggest that SnoN suppresses TGF-betainduced EMT and invasion of BC cells in a TIF1gammadependent manner and may serve as a novel therapeutic option for the treatment of BC.

BACKGROUND: TGF-beta promotes tumor invasion and metastasis through inducing epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC). Circular RNAs (circRNAs) are recognized as functional non-coding RNAs involved in human cancers. However, whether and how circRNAs contribute to TGF-beta-induced EMT and metastasis in NSCLC remain vague. Here, we investigated the regulation and function of Circular RNA hsa_circ_0008305 (circPTK2) in TGF-beta-induced EMT and tumor metastasis, as well as a link between circPTK2 and transcriptional intermediary factor 1 gamma (TIF1gamma) in NSCLC. METHODS: Circular RNAs were determined by human circRNA Array analysis, real-time quantitative reverse transcriptase PCR and northern blot. Luciferase reporter, RNA-binding protein immunoprecipitation (RIP), RNA pull-down and fluorescence in situ hybridization (FISH) assays were employed to test the interaction between circPTK2 and miR-429/miR-200b-3p. Ectopic overexpression and siRNA-mediated knockdown of circPTK2, TGF-beta-induced EMT, Transwell migration and invasion in vitro, and in vivo experiment of metastasis were used to evaluate the function of circPTK2. Transcription and prognosis analyses were done in public databases. RESULTS: CircPTK2 and TIF1gamma were significantly down-regulated in NSCLC cells undergoing EMT induced by TGF-beta. CircPTK2 overexpression augmented TIF1gamma expression, inhibited TGF-beta-induced EMT and NSCLC cell invasion, whereas circPTK2 knockdown had the opposite effects. CircPTK2 functions as a sponge of miR-429/miR-200b-3p, and miR-429/miR-200b-3p promote TGF-beta-induced EMT and NSCLC cell invasion by targeting TIF1gamma. CircPTK2 overexpression inhibited the invasion-promoting phenotype of endogenous miR-429/miR-200b-3p in NSCLC cells in response to TGF-beta. CircPTK2 overexpression significantly decreased the expression of Snail, an important downstream transcriptional activator of TGF-beta/Smad signaling. In an in vivo experiment of metastasis, circPTK2 overexpression suppressed NSCLC cell metastasis. Moreover, circPTK2 expression was dramatically down-regulated and positively correlated with TIF1gamma expression in human NSCLC tissues. Especially, circPTK2 was significantly lower in metastatic NSCLC tissues than non-metastatic counterparts. CONCLUSION: Our findings show that circPTK2 (hsa_circ_0008305) inhibits TGF-beta-induced EMT and metastasis by controlling TIF1gamma in NSCLC, revealing a novel mechanism by which circRNA regulates TGF-beta-induced EMT and tumor metastasis, and suggesting that circPTK2 overexpression could provide a therapeutic strategy for advanced NSCLC.