Trb3 And Its Mechanism In Tumor Progression

As a member of Tribbles homologous protein family, TRB3can interact with a variety of signaling molecules to regulate cell functions. The transcription factor Smad3is the key protein in the downstream of TGF-β signal pathway, and participates in many physiological and pathological processes. In the previous study, we searched proteins interacting with Smad3by the Yeast Double-Hybrid System, and found that TRB3could interact with Smad3, which has not been reported before. We further verified the interaction in mammalian cells, and results revealed that TRB3could interact with Smad3specifically through the kinase-like domain (KD). By the Dual-Luciferase Reporter assay and Western blot, we found that TRB3positively regulated Smad3-mediated transcriptional activity and expression of TGF-β1target genes. And also, silencing TRB3could reduce Smad3-mediated transcriptional activity and expression level of TGF-P1target genes significantly. By detecting the activity of pTRB3-Luc luciferase, we found that the transactivation of TRB3was enhanced by Smad3overexpression in a concentration-dependent manner and TGF-β1could further promote this positive effect. There existed a positive feedback loop between TRB3and TGF-β-Smad3signaling.We carried out GST-Pull down, cell immunonfluorescence and nucleolus-cytoplasm separation to further explore the regulation mechanisms of Smad3-mediated transcriptional activity by TRB3. Results indicated that TRB3could bind with the MH2domain of Smad3, suppress Smad3export from the nucleolus, promote the aggregation of Smad3, and stabilize its level in the nucleolus. At the same time, the immunoprecipitation (IP) displayed that TRB3enhanced pSmad3stability through promoting the degradation of Smurf2, an E3ubiquitin ligase, thus exerting its positive effect on Smad3-mediated transcriptional activity.Through BrdU cell proliferation detection, the Transwell cell migration and invasion experiment and Matrigel three-dimension cell culture in vitro, we found that TRB3promoted the proliferation vitality and the migration and invasion ability of tumor cells. We established the subcutaneous transplanted tumor model and the experimental pulmonary metastasis model of tumor cells in nude mice in vivo, and found that TRB3could promote tumor growth and metastasis. When silencing TRB3, the growth and metastasis ability of tumor was inhibited obviously.EMT process helps tumor cells to penetrate adjacent tissues, obtain invasion features and form metastasis finally. TGF-β could maintain tumor invasion through inducing EMT in the late stage of tumor progression. We investigated the regulation of EMT process by TRB3through cell immunofluorescence, the Dual-Luciferase Reporter assay, RT-PCR, Western blot, and so on. Results displayed that TRB3inhibited expression of the epithelium markers, E-cadherin and a-catenin, enhanced expression of mesenchymal phenotype markers, Fibronectin, Vimentin and N-cadherin, and promoted transcription of E-cadherin suppressors, Snail and Twist-1, thus indicating that TRB3played an important role in sustaining the mesenchymal phenotype of tumor cells. Wnt/β-catenin signaling pathway can enhance the migration and invasion ability of cells through activating the transcription of target genes. By cell immunofluorescence, IP and the separation of subcellular components, we found that TRB3stabilized the expression of β-catenin on cell membrane and in the cytoplasm at the post-transcription level and reduced the expression level of p-β-catenin. The mechanisms might be associated with TRB3-GSK-3β and TRB3-β-catenin interaction, as well as inhibition of β-catenin degradation.Autophagy promoted protein degradation, inhibited anabolism and growth of tumor cells, and also, the dysfunction of autophagy may lead to tumorigenesis. TRB3suppressed the autophagy signals of cells. TRB3silencing could enhance expression of LC3-II, Beclin1and PI3KC3, and inhibit expression of Akt, mTOR and P62, and there were a large number of autophagic vacuoles in the cytoplasm. Associated with Annexin V-FITC and TUNEL apoptosis detection and Caspase-3expression, we presumed that TRB3silencing promoted apoptosis of tumor cells, and this kind of programmed cell death showed autophagic features. TRB3inhibited the autophagy-associated cell death. P62is a key molecule to reflect the dynamic procedure of autophagy. TRB3could interact with P62and the interaction was mainly mediated by the UBA and LIR domains of P62.In a word, TRB3is one of the interacting proteins of Smad3and the interaction between them has not been reported before. Through the interaction, TRB3could enhance the transcriptional activity mediated by Smad3, promote the ability of proliferation, invasion and metastasis of tumors, exert crucial effects on EMT, and suppress the autophagy-associated death of tumor cells. TRB3might become a potential target for preventing tumor metastasis.