The Role Of Angiopoietin-like Protein 3 In Sorafenib Resistance Of Renal Cell Carcinoma

PurposesRenal Cell Carcinoma(RCC)is a common human malignant tumor,and its incidence has been increasing year by year.Approximately 20% of patients were found to have metastasis at initial diagnosis,and nearly 30% of localized RCC still undergo recurrence or metastasis after surgical removal of the tumor.Sorafenib was the first approved first-line therapy drug of RCC by the China Food and Drug Administration(CFDA)in 2006.However,about 20% of patients showed intrinsic sorafenib resistance on first course of treatment and the remaining majority of patients showed acquired resistance to sorafenib after about 6-15 months of treatment.Therefore,for the patients treated with sorafenib,the extension of the total survival is limited.At present,the difficulty of targeted therapy for RCC is that the mechanism of drug resistance is unknown,and there is a lack of effective biomarkers to predict the efficacy of sorafenib.There is no good way to reverse the drug resistance after emergence of resistance.The role and mechanism of Angiopoietin-like proteins(ANGPTLs)in targeting resistance to RCC have not been reported yet.This study was to investigate the role of Angiopoietin-like proteins in sorafenib-resistant RCC and to search of related molecular markers and the target of reversing drug-resistant combination therapy.So that we can provide the basis for improving the effect of targeted therapy and further prolonging the overall survival of patients.Methods1.Seven common cell lines of RCC were selected and the IC50 values of sorafenib was measured.The mRNA expression levels of ANGPTL1-8 in these cells were also determined.The IC50 values and mRNA expression levels were combined to screen for members that may be associated with sorafenib in RCC.2.Twenty tumor samples of RCC patients receiving sorafenib were selected,of which ten were resistant and the other ten were sensitive to sorafenib.The mRNA level of ANGPTL1-8 was determined,and the members of ANGPTLs related to sorafenib resistance in RCC were further screened.Combined with the result 1,we found that ANGPTL3 and RCC sorafenib resistance were most related,so ANGPTL3 was chosed for further validation.3.Further the relationship between ANGPTL3 mRNA expression level and sorafenib resistance was verified in a cohort including 68 RCC samples.4.The relationship between ANGPTL3 protein levels and sorafenib resistance was verified in RCC.32 RCC tissue samples(16 drug-resistant and 16 sensitive)were selected for Western blot detection.RCC data of 136 patients with renal cell carcinoma were collected.The ANGPTL3 levels were detected by immunohistochemistry and the survival analysis was performed in another cohort including 136 RCC patients(70 patients treated with sorafenib and 66 patients without adjuvant therapy).5.We used two independent short hairpin RNAs(shRNAs)to knockdown ANGPTL3 expression in RCC cell lines.CCK-8 proliferation assay,Western blot assay of apoptosis protein and flow cytometry assay of apoptosis were used to detect the change of RCC cells sensitivity to sorafenib.6.RCC cell lines stably over-expressing ANGPTL3 were constructed by lentivirus.CCK-8 proliferation assay,Western blot detection of apoptosis proteins and flow cytometry were used to detect the change of RCC cells sensitivity to sorafenib.7.OS-RC-2 cells stably over-expressing ANGPTL3 and control EGFP were subcutaneously injected into nude mice to construct a subcutaneous tumor-bearing model.Nude mice were given sorafenib intragastric(80mg / kg / day)to simulate clinical administration.Tumor size was plotted weekly to plot tumor growth and tumor size was measured by live animal imaging.8.Human proteomic microarray was used to screen ANGPTL3-binding proteins and siRNA screening was used to narrow the screening range.Finally,protein binding to ANGPTL3 was verified by protein co-immunoprecipitation and confocal laser scanning microscopy.And the domain of ANGPTL3 to combine the target protein was explored by constructing the truncation of ANGPTL3.9.Rescue method was used to demonstrate that the above-identified protein plays a role in the process of ANGPTL3 promoting sorafenib sensitivity in RCC,and further clarify its specific mechanism of regulating the sensitivity of sorafenib in RCC.Result1.In RCC cell lines ANGPTL3 and ANGPTL7 mRNA expression levels were correlated with IC50 values of sorafenib.2.A small sample of RCC tissue screening showed ANGPTL3 mRNA expression was related with RCC sorafenib resistance.Combined with the results 1 we found ANGPTL3 may be related to sorafenib sensitivity of RCC.So ANGPTL3 was chosen to further study.3.Further the relationship between ANGPTL3 mRNA expression levels and sorafenib resistance in a cohort including 68 RCC samples was verified: ANGPTL3 is low expressed in sorafenib-resistant tissues and cell lines,whereas ANGPTL3 is high expressed in sorafenibsensitive tissues and cell lines.4.Protein levels validation also confirmed that ANGPTL3 expression correlated with sorafenib sensitivity in RCC.ANGPTL3 expression levels were related to prognosis of RCC patients received sorafenib treatment: in ANGPTL3 high expression group,the prognosis of patients received sorafenib treatment was better than those who did not receive sorafenib treatment in renal cell carcinoma;but in ANGPTL3 low expression group,the prognosis showed limited differences between patients received sorafenib treatment and not received treatment.5.The knockdown of ANGPTL3 led to resistance to sorafenib in RCC cells: IC50 increased and apoptosis decreased.6.Overexpression of ANGPTL3 enhances the sensitivity of RCC cells to sorafenib: IC50 decreased and apoptosis increased.7.In vitro experiments confirmed that overexpression of ANGPTL3 increased the sensitivity of RCC to sorafenib.RCC xenografts of ANGPTL3 overexpression showed slower growth rate than the control xenografts upon sorafenib treatment.8.The FBG-like domain of ANGPTL3 binds to Focal adhesion kinase(FAK).9.Overexpression of FAK can promote RCC cell resistance to sorafenib,and overexpression of ANGPTL3 can reverse this effect.Knockdown of ANGPTL3 increased resistance to sorafenib in RCC cells and knockdown of FAK counteracted this effect,whereas PF-562271,a FAK kinase inhibitor,did not reverse ANGPTL3-knockdown induced sorafenib resistance.Further study showed that sorafenib can inhibit FAK phosphorylation,and promote the FAK nuclear localization,which led to the degradation of p53.Overexpression of ANGPTL3 can inhibit FAK into the nucleus,thereby inhibiting the degradation of p53 ubiquitination and increasing p53 expression.ConclusionANGPTL3 is highly expressed in sorafenib-sensitive tissues of RCC and low expressed in sorafenib-resistant tissues.Sorafenib has better efficacy in the treatment of RCC with high expression of ANGPTL3.Sorafenib induced FAK into the nucleus to promote the degradation of p53 via ubiquitination,leading to sorafenib resistance.ANGPTL3 combined to FAK and inhibited FAK nucleus translocation.So ANGPTL3 inhibited the sorafenib induced ubiquitination of p53,which led to p53 maintaining in a higher levels,promoting RCC cell apoptosis processes and improving the therapeutic effect of sorafenib.Therefore,ANGPTL3 may be used as a biomarker to predict the sensitivity of sorafenib,and its downstream FAK nucleus translocation and p53 ubiquitination degradation are also expected to become new targets for targeted therapy of RCC.