P-gp/RACK1/Src Complex Regulates The Proliferation And Migration Of Drug-resistant Breast Cancer Cell

ObjectiveMultidrug resistance is a huge obstacle to cancer treatment.Among studies about multidrug resistance,P-glycoprotein(P-gp)was proved that it played a very important role in multidurg resistance.In our precious study,we found that P-gp not only play a role in drug resistance,but also has a relationship with cell invasion and proliferation.We have found that P-gp via binding Src to activate downstream Anxa2 to promote the invasion of breast cancer resistant cells.But the detailed molecular mechanism is unclear.Therefore,we speculate P-gp may interact with other proteins to involve in other aspects of cancer cells.The purpose of this study is to search P-gp interaction proteome by co-immunoprecipitation combined with mass spectrometry and use bioinformatic methods to analyse proteins which were identified.We also identified the interaction of RACK1 and P-gp.In addition,we observed the effect caused by RACK1 reduction in breast cancer drug resistance cells.Method1.RT-PCR technology was used to detect mRNA levels of MDR1 and Anxa2 in normal breast tissue and breast cancer tissue.2.We successfully constructed the vector named MDR1-Flag.Western blottingting and Immunofluresence staining were used to investigate the expression of MDR1-Flag in cells.3.Using co-immunoprecipitation to obtain P-gp interaction protein complex.Using protein database to analyse protein molecular composition,functional classification and signaling pathways.Using software Osprey to form the network of P-gp interaction proteome.4.Co-Immunoprecipitation method was used to investigate the interaction between P-gp and other proteins.Immunofluresence method was used to investigate the co-location of P-gp and RACK1.5.siRNA was used to knockdown the expression of RACK1 in drug resistance breast cancer cells.Western blottingting method was used to detect the expression of RACK1 and P-gp,then MTT,colony assay,wound healing and Transwell assay were used to examine the effect of RACK deletion on cell proliferation and migration.Using MTT and flow cytometry methods to detect the sensibility of cancer cells to doxorubicin and P-gp activity.7.After RACK 1 knockdown,Co-Immunoprecipitation and Western blottingting methods were used to detect the interaction of P-gp,Anxa2 and Src.Results1.Using Co-IP combine with mass spectrometry to identify 465 proteins interacting with P-gp.System analysis result was completed through a database search.Using professional software to get P-gp protein interaction network.2.Co-IP experiment show that there is a interaction between the endogenous P-gp and RACK1,and immunofluorescence experiment show colocalization of P-gp and RACK1.3.RACK1 knockdown did not change expression levels of P-gp,but increases cells sensitivity to doxorubicin,and also found that P-gp activity decreased.4.RACK1 knockdown suppressed migration and proliferation of MCF-7/ADR cells and interaction of P-gp,Src and Anxa2,and phosphorylation of Anxa2 and ERK1/2 induced by doxorubicin was delayed compared to control.Conclusion1.The P-gp interaction network was build using co-immunoprecipitation,mass spectrometry and proteomics analysis.2.In breast cancer drug resistance cells,P-gp interact and co-localize with RACK1.RACK1 decrease did not affect P-gp expression levels,but suppresse the activity of P-gp,making the cells sensitive to doxorubicin.RACKl plays an important role in maintaining the multidrug resistance process.3.P-gp and RACK1,Src form a protein complexes.RACK1,as a scaffold protein,mediate the interaction between P-gp and Src/Anxa2 to affect cell proliferation and migration via the activation of Erk1/2 signaling pathway.