DEP Domain-containing Protein 1B(DEPDC1B)promotes Migration And Invasion In Pancreatic Cancer Through The Rac1/PAK1-LIMK1-Cofilin1 Signaling Pathway

Objective: With increasing incidence,pancreatic cancer(PC)is one of the most common malignant digestive tract tumors.Because the early symptoms of PC are obscure and because PC is prone to vascular invasion,80%-85% of patients have lost the chance for radical resection at diagnosis.Surgical resection is the only potential cure for 15%-20% of PC patients.However,its high recurrence rate significantly impedes improvements in the postoperative prognosis of PC patients.The specific mechanism of the metastasis and recurrence of PC is still unclear,and it is urgent to find sensitive and effective therapeutic targets for PC.Although previous studies have shown that DEP domain-containing protein 1B(DEPDC1B)plays an important role in a variety of cancers,the function of that in PC remains unknown.This study aimed to explore the role of DEPDC1 B in the migration and invasion of PC cells as well as its mechanism,and looking for new promising molecular targets and potential targeted therapeutic drugs for inhibiting the metastasis and recurrence of PC.Methods: Firstly,western blotting assay was used to analyze the expression of DEPDC1 B in five PC cell lines and normal pancreatic cell line.Then,transfection of PC cells by si RNA or lentivirus down-regulated or up-regulated the expression of DEPDC1 B.Subsequently,wound healing assay,transwell migration and invasion assay were utilized to analyze the migration and invasion ability of PC cells in experimental group and control group.What's more,western blotting assay was used to analyze the expression level of Epithelial-Mesenchymal Transition-related(EMT)protein in PC cells of experimental group and control group.Next,we used the coimmunoprecipitation assay to analyze whether DEPDC1 B and Rac1 could interact directly in PC cells.In addition,western blotting assay was conducted to analyze the expression levels of related proteins in the Rac1/PAK1-LIMK1-Cofilin1 pathway in the experimental and control groups.EHop-016 was selected as an inhibitor of Rac1,and the migration and invasion ability of PC cells in the control group,up-regulation of DEPDC1 B group and up-regulation of DEPDC1 B combined with EHop-016 group were analyzed by wound healing assay and transwell migration and invasion assay.In the above three groups,the expression levels of related proteins in EMT process and Rac1/PAK1-LIMK1-Cofilin1 pathways were analyzed by western blotting assay.Result: 1.DEPDC1 B expression was significantly higher in the five human PC cell lines than that in normal PC line.2.DEPDC1 B expression is correlated with migration and invasion in PC cells.DEPDC1 B knockdown cells migrated more slowly than control cells in the wound healing assay.Similarly,the transwell migration assay(without Matrigel)and invasion assay(with Matrigel)carried out in PC cells indicated that DEPDC1 B knockdown contributed to a decrease in cell migration and invasion.In addition,by the western blotting assay,we discovered that knockdown of DEPDC1 B in PC cells contributed to increased epithelial marker(E-cadherin)protein levels and decreased mesenchymal marker(N-cadherin,Vimentin)and transcription factor(TWIST1)protein levels.However,the results were opposite when DEPDC1 B was overexpressed in PC cells.3.DEPDC1 B mediates the Rac1/PAK1-LIMK1-Cofilin1 signaling pathway via interacting with Rac1 in PC cells in vitro.Coimmunoprecipitation(Co-IP)experiments verified that DEPDC1 B and Rac1 interacted with each other in PC cells.What's more,DEPDC1 B silencing decreased GTP-Rac1,phospho-PAK1(P-PAK1),phospho-LIMK1(P-LIMK1)and phospho-Cofilin1(PCofilin1)protein levels,but the total Rac1,PAK1,LIMK1 and Cofilin1 protein levels were not significantly changed.However,the results were opposite when DEPDC1 B was overexpressed in PC cells.4.EHop-016,a Rac1 inhibitor,suppressed the DEPDC1B-induced Rac1/PAK1-LIMK1-Cofilin1 signaling pathway.The DEPDC1 Binduced high GTP-Rac1,phospho-PAK1,phospho-LIMK1 and phospho-Cofilin1 expression levels were observably inhibited by EHop-016,but the total PAK1,LIMK1 and Cofilin1 protein expression levels did not change in PC cells.Moreover,the cells in the EHop-016-treated LV-DEPDC1 B group migrated more slowly than cells in the LV-DEPDC1 B group in a wound healing assay.The transwell migration assay(without Matrigel)and invasion assay(with Matrigel)also confirmed this finding that EHop-016 treatment resulted in weakened cell migration and invasion abilities.According to western blotting assay,we found that epithelial marker(E-cadherin)protein levels increased,and mesenchymal marker(N-cadherin,Vimentin)and transcription factor(TWIST1)protein levels decreased in the EHop-016-treated LV-DEPDC1 B group compared with those in the LV-DEPDC1 B group.5.EHop-016 suppressed the DEPDC1B-induced liver metastasis of PC in vivo.In PC-derived liver metastasis models,we found that the number of mice that had liver metastatic nodules was higher in the LV-DEPDC1 B group than in the LVCON319 group;however,in the EHop-016-treated LV-DEPDC1 B group,there were no mice that had liver metastatic nodules.Conclusion: 1.DEPDC1 B expression was upregulated in pancreatic cancer.2.DEPDC1 B expression was correlated with migration as well as invasion and regulated he expression levels of EMT-related proteins in PC cells.3.DEPDC1 B mediates the Rac1/PAK1-LIMK1-Cofilin1 signaling pathway via interacting with Rac1 in PC cells in vitro.4.EHop-016,a Rac1 inhibitor,suppressed the DEPDC1B-induced Rac1/PAK1-LIMK1-Cofilin1 signaling pathway.5.EHop-016 suppressed the DEPDC1B-induced liver metastasis of PC in vivo.Therefore,the DEPDC1 B gene can be used as a new and promising molecular target for inhibiting PC metastasis and recurrence.EHop-016 is also a potential targeted therapeutic drug that plays a leading role in the downstream of DEPDC1 B gene.