Molecular Mechanisms Of TNFRSF 10B Regulation By YIPF2 In Human Cancer Cells

PurposeTRAIL receptors, also called death receptors(DRs), are important in extrinsic pathway of cell apoptosis. TRAIL-R2, also called TNFRSF10B or DR5, transduces apoptotic signal to downstream CASP cascades upon activated. Translocation of TNFRSF10B to plasma membrane is required for its function.YIPF2 (YIP1 Protein Family member 2)belongs to YEP1 Protein Family which, mainly is locolizedin ER and Golgi Apparatus and may function in vesicular transport.In the previous study, we found the YIPF2 mRNA level increased due to chemotherapeutical agent treatment in NSCLC cells. Meanwhile these treatments induce TNFRSF10B up regulation and trigger the apoptosis.The purpose of this project is to investigate the role of YIPF2 in the transport of TNFRSF10B between intracellular compartments and the regulation of chemotherapeurical agent-induced apoptosis.Methods1) Detected the expression level of YIPF2 and TNFRSF10B in NSCLC cells after chemotherapeutical agent treatment by Western Blot.2) Knockdown YIPF2 with siRNA or overexpressed YIPF2 in NSCLC cells followed by the treatment of chemotherapeutical agent pemetrexed (PEM). Then detected the expression level of TNFRSF10B and activation of CASP cascades by Western Blot and analyse the apoptosis by flowcytometry. The plasma membrane localization of TNFRSF10B were analyzed with confocal microscopy and flowcytometry.3) The interaction of YIPF2 and TNFRSF10B were detected by Co-Immunoprecipitation.4) To figure out which RABs are involved in YIPF2-regulated TNFRSF10 vesicular transportation, knock down YIPF2 with siRNA or overexpressed YIPF2 in NSCLC cells, then the same treatment and experiments as 2) were conducted.5) To verify which RABs are in the YIPF2-TNFRSF10B complex, Co-Immunoprecipitation assays were performed.Results1) The expression of YIPF2 increase in chemotherapeutical agent treated NSCLC cells in dose- and time-dependent manner, sooner than the up-regulation of TNFRSF10B.2) Inhibiton or enhancement of YIPF2 expression in non-Small Cell Lung Cancer Cells alleviates or enhances chemotherapeutical agent induced TNFRSF10B up-regulation and the resulting apoptosis, respectively.3) YIPF2 interacts with TNFRSF10B and they co-localize in cytoplasm in NSCLC cells.4) RAB8 binds to YIPF2-TNFRSF10B complex and is negatively regulated by YIPF2.5) RAB8 negatively regulates YDPF2 and TNFRSF10B levels, TNFRSF10B localization on plasma membrane, and chemotherapeutical agent induced apoptosis.6) YIPF2 positively regulates RAB18 and RAB40c, which in turn positively regulats TNFRSF10 translocation to plasma membrane.Conclusion1) YIPF2 regulates the expression and vesicular transport of TNFRSF10B in NSCLC cells.2) YIPF2 regulates cancer cell apoptosis through regulation of TNFRSF10B.3) RAB8, RAB18 and RAB40c are involved in TNFRSF1 OB regulation by YIPF2.