| Epithelial-mesenchymal transition (EMT) is a process through which cells lose their epithelial phenotypes and gain mesenchymal phenotypes. EMT plays a critical role in tumorigenesis. Tumor cells undergoing EMT become more invasive, resistant to apoptosis induced by anti-tumor drugs, and act as cancer stem-like cells (CSCs).SNAIL is one of the most well established master transcriptional regulators of EMT. It facilitates EMT via transactivation of mesenchymal markers and transinhibition of epithelial markers. Hence it is very important to understand the mechanism that controlling the SNAIL expression. We are interested in the posttranslational regulation of SNAIL, especially the ubiquitination regulation. Like other transcription factors of great importance, SNAIL is a highly unstable protein and has a very short half-life. So far, several E3 ligases have been reported to be responsible for SNAIL ubiquitination, such as FBXW1, FBXL14 and FBXO11. It is noteworthy that previously identified E3 ligases mediating SNAIL degradation are all F-box members. Given the diversity and complexity of protein ubiquitination regulation, we speculate that there may be E3 ligases other than F-box proteins involved in post-translational regulation of SNAIL.In this study, we performed a luciferase-based genome-wide siRNA screening against 195 E3 ligase genes and identified SPSB3 to be a new E3 ligase responsible for the polyubiquitination and degradation of SNAIL. Different from previous identified E3 ligases, SPSB3 belongs to the SOCS box protein family and acts as the substrate recognition subunit of the CRL E3 ligase complex. We demonstrated that SPSB3 directly targets SNAIL for degradation in response to GSK3β through UPS system.Subsequently, we specifically focused on the function of SPSB3-mediated SNAIL degradation in tumorigenesis. As a positive regulator of EMT, SNAIL is considered to act as an oncoprotein by inducing metastasis and maintaining cancer cell sternness. Thus we used premalignant immortalized breast epithelial cell line MCFCAla to investigate the functional compact of SPSB3 on tumorigenesis. The result indicated that SPSB3 plays a tumor suppressor role through mediating SNAIL degradation both in vitro and in vivo. In addition, ability of SPSB3 to down regulate SNAIL led us to investigate whether overexpression of SPSB3 alters metastatic potential of tumor cells. Through transwell assay in vitro and 4T1 lung metastasis mouse model in vivo, we found that SPSB3 blockade SNAIL-induced migration and invasion in vitro and breast cancer lung metastasis in vivo.Mechanistically, we found that SPSB3 mediated SNAIL degradation is dependent on GSK3β phosphorylation of SNAIL. To find out the precise regulation mechanism of SPSB3 dependent SNAIL degradation, we investigate the crucial domain in charge of their interaction. A series of truncation Co-IP assay showed that SRD domain where GSK3β phosphorylation sites locate in is indispensable for SNAIL interaction with SPSB3. Additionally, we found that TNFa could stabilize SNAIL at least partially through inhibiting the expression of SPSB3, although the detailed mechanism still remains to be explored.Finally, we used immunohistochemistry (IHC) staining to test the expression and correlation between SPSB3 and SNAIL in the same esophageal and ovary tumor samples. And we used real time PCR to test the expression of SPSB3 in the paired esophageal cancer.In summary, we identified SPSB3 as a novel ubiquitin ligase that targets SNAIL for ubiquitination and degradation. We further discovered that SNAIL degradation by SPSB3 is dependent on SRD domain phosphorylation of SNAIL by GSK3(3. SPSB3 blocks SNAIL-induced EMT, tumor initiation, and metastasis both in vitro and in vivo. Our study provides new insights into the molecular mechanism of EMT function in tumorigenesis and potential therapeutic target for clinical treatment of cancer. |
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