The Mechanisms Study Of Rack1 In Regulating The Activity Of P-glycoprotein To Confer Drug-Resistance Of Breast Cancer Cells

Objectives:Breast cancer is the first killer of women’s health in the worldwide.Chemotherapy plays an important role in the treatment of breast cancer.However,the failure of chemotherapy and the emergence of multidrug resistance（MDR）are the major obstacles for effective therapy in locally advanced and metastatic breast cancer.Overexpression of the drug transporter in cancer cells is one of the main causes of MDR due to its ability to efflux anticancer drugs out of cells.Although the signaling node that regulates the expression of P-gp has been intensively investigated,the regulatory mechanism underlying P-gp transport activity in drug-resistant cells remains obscure.Receptor for Activated C Kinase 1（Rack1）is a multifunctional scaffold protein in mediating different proteins interaction.In addition,Rack1 plays an important role in the resistance of tumor cells to chemotherapy drugs,however,whether Rack1 regulates the transport activity of P-gp in drug-resistant cells remains unknown.In this study,we performed in vitro cellular experiments and in vivo animal xenograft tumor derived from MDR breast cancer cells to clarify the function of Rack1in regulating the transfer activity of P-gp.Furthermore,we explored the molecular mechanism underlying P-gp transfer activity,which may provide guidance for treatment of advanced and drug-resistant breast cancer patient in clinical practice.Methods:1.Specific small interfering RNA was used to knockdown the expression of Rack1 in drug-resistant cell lines,and western blotting was used to analyze the effect of Rack1 silencing on the expression level of P-gp.2.CCK-8 based assay was used to analyze the sensitivity of MDR breast cancer cell line to epirubicin after silencing of Rack1 expression,and the corresponding IC₅₀ value was calculated by using the Prism 6 software.3.Rhodamine123（Rh123）,a fluorescence substrate of P-gp,was added to the cells and incubated for a period of time.Flow cytometry was used to detect the intensity changes of Rh123 intracellular before and after down-regulation of Rack1,and then the transport activity of P-gp was analyzed accordingly.4.We took advantage of the innate fluorescence characteristics of epirubicin,a chemotherapeutic drug,and added it to the cells for incubation for a period of time.The fluorescence intensity of epirubicin was photographed by fluorescence microscopy before and after Rack1 down regulation,and then the accumulation of epirubicin in MDR breast cancer cells was analyzed.5.Small interfering RNA was used to silence the expression of Src in MDR breast cancer cell lines,and the protein levels of Src and P-gp in cells were analyzed by immunoblotting.6.After the down-regulated expression of Src in MDR breast cancer cell lines,CCK-8 based assay was used to detect the vitality of cells to epirubicin;flow cytometry was used to detect the transport activity of P-gp;and immunofluorescence was used to analyze the intracellular accumulation of epirubicin.7.Saracatinib and Dasatinib,two Src kinase-specific inhibitors,were used to block the kinase activity of Src,and the protein levels of total-Src,phosphate-Src and P-gp were analyzed by Western Blotting.8.After inhibiting Src kinase activity in MDR breast cancer cell lines,CCK-8 based assay was used to analyze the effect on sensitivity of cells to chemotherapy drugs;flow cytometry was used to analyze the changes in intracellular fluorescence intensity of Rh123 and the transport activity of P-gp;immunofluorescence technology was used to analyze the accumulation of chemotherapy drugs within cells.9.The expression of intracellular Rack1 was silenced by transfection specific small interference RNA,and the interaction between P-gp/Src/Cav1（caveolin-1）was analyzed by co-immunoprecipitation.Further,knocked down the expression of Src in MDR cells and then the interaction between P-gp/Rack1/Cav1 was analyzed by immunoprecipitation.10.Short hairpin RNA（shRNA）was designed for targeting the non-coding region sequences of Rack1,and the MDR breast cancer cell lines with stably knockout of Rack1 were constructed.Expression plasmids of flag-labeled Rack1^WT and Rack1^Y246F（which interfered with Rack1 binding to Src after mutation）were constructed.Rack1^WT and Rack1^Y246F were successfully re-expressed in Rack1 stably knockout cells,after which,the interaction between P-gp/Src/Cav1 was analyzed by co-immunoprecipitation.11.Silencing the expressions of Rack1 or Src,or inhibiting the kinase activity of Src,then analyzed the expressions of Rack1,total-Src,phosphate-Src,total-cav1 and phosphate-cav1 by Western Blotting.12.Rack1^WT and Rack1^Y246F were rescued respectively in stably knockout Rack1 cells,western blotting was used to analyze the expressions of Rack1,total-Cav1,and phosphor-Cav1.13.We constructed wild-type Cav1(Cav1^WT)and phosphorylation mimicking mutant Cav1^Y14E expression plasmids and introduced those plasmids into Cav1 silenced cells,and then the binding ability of Cav1 to P-gp was analyzed in the rescued cells by immunoprecipitation.In addition,Src kinase inhibitors were added into the two rescued cells,and the transport activity of P-gp was analyzed by flow cytometry.14.After the rescue the expression of Rack1^WT and Rack1^Y246F,CCK-8 based assay used to detect the changes in cells sensitivity to epirubicin in each treatment group;flow cytometry was used to detect the intensity of fluorescence molecules in cells,and then analyze the changes in P-gp transport activity.15.Silencing Rack1expression group and its control group cells,Rack1^WT-and Rack1^Y246F-rescued groups and their control groups derived from drug-resistant cells were transplanted subcutaneously in nude mice to construct xenograft model.Tumor volume was analyzed after the treatment with epirubicin in different groups mice.Results:1.After 72 h transfection of siRack1 into MDR breast cancer cell lines,the expression level of Rack1 was decreased significantly,while the protein level of P-gp remained unchanged.2.Knockdown of Rack1 expression in MDR breast cancer cell lines increased the sensitivity of cells to epirubicin and decreased the IC₅₀0 value.3.Down-regulation of Rack1 expression in MDR breast cancer cell lines,increased the proportion of for Rh123 fluorescence molecule positive cells,and the ability of P-gp to transport Rh123 out of cells was weakened.4.Knocked down of Rack1 expression in MDR breast cancer cell lines,increased the accumulation of epirubicin intracellular observed by immunofluorescence.5.Silencing the expression of Src in MDR breast cancer cell lines had no effect on the protein level of P-gp.6.Down-regulation of Src expression in MDR breast cancer cell lines increased the sensitivity of cells to epirubicin and decreased the IC₅₀ value.The proportion of Rh123 fluorescence molecules positive cells was increased detected by flow cytometry,and the ability of P-gp to transport Rh123 out of cells was decreased;Immunofluorescence showed that the fluorescence intensity of epirubicin in cells was increased.7.Both Saracatinib and Dasatinib inhibitors can inhibit the kinase activity of Src in a dose-dependent manner,without affecting the basic expression of Src or the expression level of P-gp.8.After blocking the kinase activity of Src in MDR breast cancer cells increased cell sensitivity to epirubicin and decreased the IC₅₀ value;the intracellular interception of Rh123 fluorescence molecule was increased;and the transport activity of P-gp was decreased;the accumulation of epirubicin observed by immunofluorescence was increased.9.Silencing Rack1 expression in MDR breast cancer cell lines had no effect on the protein levels of P-gp and Src,but the binding ability of the two molecules was decreased,and the interaction between P-gp and Cav1 was increased.After down- regulated the expression of Src,the interaction between P-gp and Rack1 was not affected,and the ability of P-gp to combine with Cav1 was increased.10.MDR breast cancer cell line with stable Rack1 knockout was successfully established.The expression levels of exogenous Rack1^WT and Rack1^Y246F expression plasmid in this cell line were close to the endogenous Rack1 expression levels of wild type cells.The interaction between P-gp and Src was restored in Rack1^WT rescued cells and inhibiting the combination of P-gp and Cav1,while the interaction between P-gp and Src could not be restored in Rack1^Y246F246F rescued cells,and there was still a strong interaction between P-gp and Cav1.11.Silencing of Rack1 or Src expression,or inhibiting of Src kinase activity,inhibited phosphorylation of Cav1 and without effected on its basic expression.12.The phosphorylation of Cav1 was inhibited in the stably knockout of Rack1 expressed MDR cells,and the re-expression of Rack1^WTT protein restored the phosphorylation level of Cav1,while the phosphorylation of Cav1 was still inhibited in cells rescued with the mutant Rack1^Y246F form.13.We silenced the endogenous expression of Cav1 and successfully re-expressed the exogenous Cav1^WT and Cav1^Y14E in the silenced cells;The binding ability between Cav1 and P-gp in Cav1^Y14E mutant rescued cells was weakened compared with Cav1^WT rescued cells.Flow cytometry results showed that the transport ability of P-gp was enhanced when knockdown of Cav1 expression.Compared with Cav1^WT,the re-expression of the Cav1^Y14E mutant promoted the expulsion of Rh123 and increased the transport capacity of P-gp.In addition,P-gp efflux activity in Cav1^WT-expressing cells can be quenched by Src inhibitor,while Src inhibition has little effect on the efflux function of P-gp in Cav1^Y14E-expressing cells14.Compared with Rack1^Y246F mutant,cells rescued with Rack1^WT were able to recover resistance to epirubicin,with an increase in IC₅₀ value;the ability of P-gp to transport Rh123 outside was increased,as well as increased in the proportion of Rh123-positive cells.15.We successfully constructed xenograft tumor models derived from MDR breast cancer cells pretreatment differently within nude mice.Mice were treated with epirubicin for a period and the tumor volume was significantly reduced in stably silencing Rack1 expression group as well as the Rack1^Y246F-rescue group,however,the xenograft tumors derived from Rack1^WT-rescued cells were resistant to epirubicin therapy.Conclusions:This study demonstrated that the expression of Rack1 and Src positively regulate P-gp transport activity and promote drug resistance in drug-resistant breast cancer cells.Rack1 acts as a scaffold protein and mediates the interaction between Src and P-gp.In the Rack1/Src/P-gp interaction complex,Src phosphorylates Cav1,which changed from non-phosphorylated state to phosphorylated state,resulting in a decrease in its interaction with P-gp,and the transport activity of P-gp changed from inhibited state to activated state.Silencing Rack1 expression or destroying the interaction between Rack1/Src/P-gp can attenuate the transport activity of P-gp,and the ability of P-gp to pump chemotherapy drugs out of cells was decreased,which increases accumulation of chemotherapy drugs within cells and weakens drug resistance.Therefore,Rack1 can be used as a new potential therapeutic target to develop anti-tumor drugs and reverse multi-drug resistance of tumor cells.