Decreased Expression Of CHIP Leads To Increased Angiogenesis Via VEGF-VEGFR2 Pathway And Poor Prognosis In Human Renal Cell Carcinoma

Context:New vascular formation is needed to force solid tumor grow each 1-2mm,and this process is called "angiogenesis".There is increasing amount of data show that in the process of cancer development,many molecules are activated and interact with each other,which consist a complex signal network.Determining the signal network and key mechanism in the cancer angiogenesis pathways may aid in inhibiting development and curing cancer.RCC is a highly vascular tumor,and the elucidation of the VEGF-VEGFR2 pathway has led to the development of targeted therapy in RCC.Ubiquitin need the function of E1 activating enzyme,E2 binding enzyme and E3 ligase,processing specific modification of the target protein.The present studies demonstrates that the ubiquitination of proteins is a common mechanism of down-regulation of its activation,and also plays an important role in the development of cancer.CHIP is a member of E3 ubiquitin ligase,functioning as a link between the chaperone(heat shock protein70/90)and proteasome systems.CHIP targets a set of tumor-related proteins,including p53,Erb B2,ER?,Met and HIF-1?,for ubiquitylation and degradation.Through regulation of several important proteins,CHIP controls diverse cellular processes,including cell-cycle progression,cell proliferation,differentiation,DNA damage repair,angiogenesis,and apoptosis.However,little is known about the role of CHIP and the relationship among CHIP,angiogenesis and VEGF-VEGFR2 pathway in RCC.Objective: To test the hypothesis that CHIP regulates cell migration,invasion and angiogenesis by inhibiting VEGF-VEGFR2 signaling pathway in RCC.Methods:The study material consists of 304 consecutive cases of RCC and 34 cases of NRT.IHC was performed on the TMA slides to investigate whether CHIP expression is changed in RCC,then we studied if CHIP expression correlates with clinicopathologic features and patient survival.To determine the effect of CHIP overexpression or knockdown on RCC cells migration and invasion,we transiently transfected 786-O and OS-RC-2 cells with Myc-control and Myc-CHIP plasmids or control si RNA and CHIP si RNA,respectively.Cell migration or invasion was determined by using a modified two chamber migration or invasion assay with a pore size of 8 ?m.Furthermore,we used HUVECs growth and endothelial cell tube formation assay to test the effect of CHIP expression in RCC angiogenesis.Moreover,we investigated whether the role of CHIP on angiogenesis was mediated by regulating VEGF secretion using ELISA test.And the expression of VEGFR2 was detected by WB and the mechanisms of CHIP on angiogenesis was further investigated.Results:CHIP staining was mainly localized in the cytoplasm and the expression of CHIP was significant lower in the carcinoma tissues than the NRT.Positive CHIP staining was recorded in 94.1%(32/34)and 51.0%(155/304)of the biopsies in NRT and RCC tissue,respectively.Furthermore,CHIP staining was significantly correlated with p T status(P = 0.022)and TNM stage(P = 0.022).Moreover,decreased CHIP staining correlated with both 5-year OS and DSS in RCC(P = 0.029 and P = 0.030,respectively)and low CHIP expression was an independent prognostic marker for RCC patients(hazard ratio,0.738;95% confidence interval,0.559-0.974;P = 0.032).In vitro,the ability of cell migration was drastically decreased after CHIP overexpression in both 786-O and OS-RC-2 RCC cell line.In contrast,knockdown CHIP promoted cell migration.Meanwhile,the results of the cell invasion assay corresponded with the cell migration assay.In the HUVECs growth and tube formation assay,we found that conditioned medium from 786-O and OS-RC-2 cells overexpressing or knockdown CHIP significantly inhibited or promoted proliferation of endothelial cells and the average number of complete tubular structures formed by HUVECs.In addition,restoration of CHIP inhibited,while knockdown promoted,the secreted level of VEGF.Furthermore,the VEGFR2 protein level was dramatically reduced in 786-O and OS-RC-2 cells after being transfected with Myc-CHIP.Conclusion:Decreased expression of CHIP in RCC speciments constitute a strong unfavorable prognostic factor for RCC patients.Upregulation of CHIP in RCC cells inhibits migration,invasion,proliferation and tube formation of cocultured endothelial cells by decreasing VEGFR2 expression and VEGF secretion.Our observations support the hypothesis that CHIP may interact with VEGF-VEGFR2 signaling pathway and regulate angiogenesis in RCC.Meanwhile,our results provided the first in vitro evidences that CHIP may be used as a promising prognostic marker for RCC patients and targeting CHIP might represent a new therapy to suppress RCC progression.