The Molecular Mechanisms Of G3BP1-Mediated EMT In Breast Cancer

Breast cancer has become the most common cancer and the principal cause of death among women. The predominant death cause of this disease is metastasis. The epithelial-mesenchymal transition (EMT) can induce carcinoma in situ develop into invasive and metastatic tumors, involved in the regulation of metastasis and drug resistance. G3BP1, an essential Ras mediator, has been implicated in cancer development, including cell proliferation, motility, invasion and apoptosis. Our previous study found that G3BP1 knockdown inhibited the migration and invasion of H1299 cells which indicated that G3BP1 might be associated with EMT. Here, we investigated the relationship between G3BP1 and EMT, and the molecular mechanism. Furthermore, we also found that G3BP1 could regulate the cell proliferation. Given that P-Catenin could participate in cell proliferation, we preliminarily studied G3BP1-mediated regulation of β-Catenin signaling pathway and its mechanism.Purpose:1. To clarify the molecular mechanism of G3BP1-induced EMT.2. To investigate the regulation of Smad signaling pathway mediated by G3BP1 and its mechanism.3. Identify the function of G3BP1 in the metastasis of breast cancer and evaluate the potential of G3BP1 exhibited as a metastasis biomarker.4. To investigate the regulation of β-Catenin signaling pathway mediated by G3BP1 and its mechanism.Methods:1. Investigate whether G3BP1 participated in the regulation of EMT. First of all, we constructed the human G3BP1 expression plasmid and stable G3BP1-overexpressing MDCK cell lines by transfecting with pCDNA3.1-G3BP1. In the model cells, we observed the morphology changes, investigated the expression changes of EMT markers via Western blot assay, detected the changes of cytoskeleton protein via Immunofluorescence assay and detected cell migration and invasion capabilities via Transwell migration and Matrigel transwell invasion assay. Next, we used the similar method to investigate the regulation of G3BP1 in EMT in the G3BP1 transient/stable overexpressing MCF-7 cells and G3BP1 transient/stable silencing MDA-MB-231 cells respectively.2. Study EMT-related Smad signaling pathway. Investigate the phosphorylation level changes of Smads induced by transient/stable overexpression or knockdown via Western blot assay in MCF-7 and MDA-MB-231 cells. Using Real-time PCR assay, we tested the transcriptional level of the signaling-related genes. Detect the interaction between G3BP1 and Smads via CO-IP assay. Find the key transcription factors and Smad proteins responsible for G3BP1-induced-EMT via RNA interference method.3. Evaluate the effect of G3BP1 on the metastasis model of breast cancer. We constructed stable G3BP1 shRNA-expressing clones and mouse metastasis model of breast cancer in vivo. Evaluate the effect of G3BP1 knockdown on the tumor in situ and metastatic tumor via optical imaging system. Evaluate the pathological changes in tumor in situ and metastatic tumor via HE assay. Detect the expression of EMT markers in tumor in situ via Western blot assay.4. Investigate the regulation of β-Catenin signaling pathway mediated by G3BP1. Evaluate the effect of G3BP1 on proliferation in MCF-7 cells via SRB assay. Detect the expression changes of β-Catenin signaling-related proteins and target genes via Western blot assay. Investigate the activation of signaling pathways via Dual-luciferase reporter assay and β-Catenin nuclear accumulation changes. Test the effect of G3BP1 on phosphorylation level of GSK-3β via Western blot assay and the effect of GSK-3β kinase inhibitors on the expression of G3BP1. Investigate the interaction between G3BP1 and GSK-3β via CO-IP assay.Results:1. The results indicated that overexpression of G3BP1 induced EMT in MDCK cells. The transient/stable overexpression of G3BP1 activated EMT in MCF-7 cells, while G3BP1 knockdown suppressed the mesenchymal phenotype in MDA-MB-231 cells.2. The overexpression of G3BP1 activated Smad signaling pathway in MCF-7 cells. G3BP1 knockdown inhibited Smad signaling pathway in MDA-MB-231 cells. G3BP1 could regulate the activation of Smads independently of exogenous TGF-β1. CO-IP results indicated that G3BP1 could interact with Smads, especially Smad2/4. Silencing Slug almost inversed G3BP1-induced EMT. Silencing Smad3 almost completely blocked G3BP1-induced EMT.3. G3BP1 knockdown suppressed the mesenchymal phenotype in 4T1 cells. The stable knockdown of G3BP1 suppressed tumor growth and lung metastasis of 4T1 cells in vivo. Cells could keep their properties of G3BP1 stable knockdown in tumor in vivo.4. The overexpression of G3BP1 had a tendency to promote the proliferation, while G3BP1 knockdown significantly inhibited the proliferation of MCF-7 cells. The overexpression of G3BP1 could promote the activation of β-Catenin signaling and G3BP1 knockdown could inhibit its activation. G3BP1 could regulate the phosphorylation levels of GSK-3β and interact with GSK-3β. GSK-3β specific inhibitor SB216763 induced the degradation of G3BP1 in concentration- and time-dependent manners.Conclusion:1. In vitro experiment indicated that G3BP1 could regulate the migration and invasion of breast cancer cells; G3BP1 could regulate the metastasis of breast cancer cells in mouse metastasis model of breast cancer in vivo. These findings identified a novel function of G3BP1 in the metastasis of breast cancer, indicating that G3BP1 might represent a potential therapeutic target for human metastatic breast cancer. In the further study, we found that G3BP1 could perform the regulation of metastasis via EMT process, which was regulated mainly via the activation of Smads and the upregulation of Slug.Our study revealed the mechanisms of G3BP1-induced EMT in the molecular and cellular levels. G3BP1 could recruit Smad2/4, which suggested that G3BP1 might act as a novel co-factor of Smads.2. G3BP1 might affect the activity of P-Catenin signaling via the regulation of GSK-3P kinase activity. G3BP1 might regulate the proliferation via β-Catenin signaling. Our findings might identify a novel regulation of G3BP1 in the proliferation. The degradation of G3BP1 mediated by GSK-3β specific inhibitor SB216763 indicated that SB216763 might exhibit as a novel G3BP1 regulator.