Sunitinib Promotes VM Formation Via Triggering The PABPN1/TRSM/AR/HIF-2α Signaling Axis In Clear Cell Renal Cell Carcinoma

Background and purposeClear cell renal cell carcinoma is one of the most common malignant tumors of the urinary system Its incidence rate and rnortality rate are continually increasing in China.Clinically,surgical resection is the most effective treatment for localized renal clear cell carcinoma.Metastatic clear cell renal cell carinoma,the targeted therapy,including the classic sunitinib,is still the main first-line treatment Howevr,the therapeutic efficacy is not satisfactory,and the 5-year survival rate is less than 10%.Except for tumor blood vessels,vascular mimicry is confirmed to be one of the altemative ways to provide nutrition for tumor cells,which promotes tumor invasion,metastasis and drug sensitivity.However,it is still unclear whether sunitinib could affect the formation ofv ascular mimicry to reduce the clinical efficacy Therefore,for the first time,we will explore whether sunitinib could promote vascular mimicry,and clarify the potential molecular mechanism,to provide a new insight for the clinical treatment of clear cell renal cell carcinoma.Methods1.The effects of different concentrations of sunitinib on the formation of vascular mimicry in renal clear cell carcinoma cell lins SW839 and OSRC-2 were observed by matrix gel 2D culture experiment.2.Based on the previous observed results of our lab,we further confirmed whether sunitinib could affect the formation of tumor vascular mimicry through androgen receptor(AR),by constructing a stable knock-downAR cell line and AR inhibitor enzalutamide.3.According to a genome-wide landscape on the expression of long non coding RNA(lncRNA)in renal clear cell carcinoma cells after sunitinib treatment,the RT-qPCR was performed on both SW839 and OSRC-2 cell lines to find out the target dysregulated lncRNAs that could be affected by sunitinib in expression and interacted with AR mRNA,which were firstly selected through the bioinformatics analysis of RNA-RNA interaction.Then,the interaction between lncRNAs and AR was further observedby RIP experiment and double luciferase reporter gene system experiment.4.The expression of HIF-2a and miRNA-145-5p was detected by constructing a stable HIF-2a knock-down cell line and using HIF-2a small molecule inhibitor,to explore their correlations with the sunitinib/PABPN1/TRSM/AR signaling pathway.Results1.0.625-1 25 μM of sunitinib could both increase formation of vascular mimicry in clear cell renal cell carinoma cell lines SW839 and OSRC-2.2.Sunitinib could promote the formation of vascular mimicry by increasing the expression of AR in clear cell renal cell carcinoma cells.3.Sunitinib could increase AR expression via increasing TRSM stability through the RNA binding protein PABPN1 to increase the expression of TRSM that binds to 5’UTR of AR mRNA to neutralize the negative regulation of 5’uORF onAR translation.4.The increased androgen receptor AR could suppress the miRNA-145-5p expression to increase the HIF-2a expression,which increase the formation of vascular mimicry in clear cell renal cell carcinoma cells.ConclusionsThe TRSM may play a key role in mediating the sunitinib-increased clear cell renal cell carcinoma vascular mimicry formation.Targeting this newly identified sunitinib/PABPN1/TRSM/AR/miRNA145-5p/HIF-2a signaling may help us to develop new therapies to better suppress the clear cell renal cell carcinoma vascular mimicry formation and to increase the sunitinib therapectic efficacy.