Pathogenic Roles Of CUL4B In Bladder Cancer

Bladder cancer(BC)has the highest incidence of urinary system malignancy in China and the incidence shows an increased tendency annually in recent years.However,the tumorigenic mechanisms of BC have not been fully explicated.Therefore,investigating the molecular mechanisms underlying BC is crucial for the development of novel diagnostic and treatment strategies for patients with BC.CUL4B acts as scaffolding protein for modular cullin ring ligase 4B(CRL4B)complexes which plays a significant role in cell proliferation and tumorigenesis,and targets several tumor suppressor proteins.It could be classified as oncogene in certain contexts.However,the roles and mechanisms of CUL4B in BC has not been fully elucidated.In this study,we did the following observations:1.We analyzed the expression level of CUL4B in BC using tissue microarray.The results of immunohistochemistry showed that the expression level of CUL4B was correlated with the pathological grade,T grade and clinical stage of BC.Moreover,the expression level of CUL4B was found to be positively correlated with the malignancy of BC cells,suggesting that CUL4B plays an important role in development of BC.2.The results of Cck-8 and EdU assays showed that CUL4B promotes cell proliferation in 5637,but not other four BC cell lines,indicating that promotion of BC by CUL4B is not mainly attributed to proliferation regulation.3.Tumor metastases are the most common causes of death in cancer patients and represent the utmost challenge for cancer treatment.Wound healing,transwell and matrigel invasion assays showed that downregulation of CUL4B significantly attenuated migration and invasion ability of BC cells.Besides,the role of CUL4B in chemo-drug sensitivity of BC cells was studied.Cck-8 results showed that the viability of BC cells was much lower in CUL4B knockdown cells than in control after the treatment of chemotherapeutic drugs.TUNEL assay revealed that downregulation of CUL4B significantly increased apoptosis caused by cisplatin.These results suggest that CUL4B is involved in regulating the chemo-drug sensitivity of BC cells.Moreover,downregulation of CUL4B significantly inhibited the formation of BC tumorspheres and decreased the level of the sternness marker CD44 in BC cells,indicating the important roles of CUL4B in maintaining sternness of BC cells.4.RNA-Seq analysis showed that the number of differentially expressed genes in the PI3K-Akt pathway was riched in CUL4B knockdown BC cells,suggesting that CUL4B may regulated PI3K-Akt pathway.Western Blot confirmed that CUL4B could positively regulate the level of p-Akt,and the p-Akt inhibitor,MK2206,effectively reversed the enhancement of migration and invasion ability and decrease of chemo-drug sensitivity of BC cells induced by CUL4B upregulation.Furthermore,CUL4B also could positively regulate the expression level of the PIK3CA,which regulated the phosphorylation of Akt.These results indicate that CUL4B promotes migration,invasion,and decreases chemo-drug sensitivity of bladder cancer cells through activation of p-Akt.Collectively,these experiments indicated that CUL4B has a positive correlation with the malignancy of BC and is able to promote the migration,invasion,sternness and inhibit chemo-drug sensitivity of BC cells.More importantly,our results also showed that CUL4B may promote BC progression through activating p-Akt pathway.All these results suggest that CUL4B function as an oncogene in BC,which can be considered as a promising target for therapeutic intervention in BC patients.