Autocrine Wnts Contribute To Renal Cell Carcinoma Cell Proliferation Via Stimulating Fzd7 Endocytosis

Wnt proteins are secreted glycoproteins that bind to the N-terminal extra-cellular cysteine-rich domain of the Frizzled(Fzd) receptor family. The Fzd receptors can respond to Wnt proteins in the presence of Wnt co-receptors to activate the canonical and non-canonical Wnt pathways. Overexpression of Wnts and Fzds or the expression of a constitutively active Fzd have been linked to Wnt/β-catenin pathway activation in various tumors. There are several researches suggest the involvement of Wnt signaling pathway in RCC. As a member of Fzd receptor family, Fzd7 plays an important role in cellular proliferation, differentiation, translocation, polarity and cancer development and progression. But the direct research on the Fzd7 protein expression in cc RCC has not been investigated previously. It is known that Wnts is capable to activate canonical and non-canonical signaling pathway in an autocine mechanism in various tumor cells. Wnts are capable to transduce signaling pathways through binding with surface Fzd receptors and being internalized. However some researches find that internalized Wnt-receptor complex strengthens the Wnt signal pathway. Both canonical and non-canonical Wnt ligands are able to induce endocytosis of corresponding Fzd receptors and the alterations in Fzd endocytosis or intracellular trafficking seem to have effect on the signaling pathway. The Wnt-induced endocytosis of Fzd receptors appears to be an integral component of both canonical and noncanonical signaling pathways. However, little is known about the regulation and mechanism of Wnt on Fzd7 in RCC cell lines.This study aimed to detect the Fzd7 protein expression in clinical cc RCC tissues, peritumor tissues and RCC cell lines. Furthermore we explore the effect of Wnts on Fzd7 by controlling the activity of Wnts in RCC cell lines.The paper includes two parts as following:PartⅠ Detection the expression of Fzd7 in clinical cc RCC tissues, peritumor tissues and RCC cell linesObjective: To assay the Fzd7 protein expression in clinical cc RCC tissues, peritumor tissues and RCC cell lines.Metholds: Immunohistochemistry was performed on 53 cc RCC tissues and peritumor tissues to assay the Fzd7 expression. And Fzd7 protein expression in 786-O、OS-RC-2、Caki-1 RCC cell lines were detected using Western blot. To investigate whether the protein bands detected in the three RCC cell lines was glycosylated forms, the cell lysates were pretreated by endoglycosidase H and N-glycosidase F.Results: Immunohistochemical staining of Fzd7 demonstrated that about 36% of cc RCC tissues expressed Fzd7. Notably, analysis of Fzd7 expression level in tumor tissues in comparison to peritumor tissues revealed the overexpression of Fzd7 in tumor tissues, which meant that Fzd7 played a role in tumor progression. Results of Western blot showed that in the three renal cell carcinoma cells the main bands of Fzd7 was about 64 k D, 60 k D and 35 k D. Both of the endoglycosidase H and N-glycosidase F did not affect the distribution of Fzd7 bands in the three RCC cell lines, which suggested that none of the three bands were the glycosylated immature forms of Fzd7. According the results obtained above, the 60 k D and 35 k D might be the uncompleted synthesis form of Fzd7 or the degraded form of Fzd7 through endocytosis.Conclusion: The Fzd7 expression in cc RCC tissues and RCC cell lines was confirmed and supplied the data for the further study of the mechanism that Endogenous Wnts stimulated Fzd7 to endocytosis in RCC cells.PartⅡ Mechanism of endogenous Wnts on endocytosis of Fzd7 in RCC cellsObjective: To investigate the mechanism of Fzd7 endocytosis induced by Wnts in RCC cells.Methods: The expression of representative Wnts were analyzed by RT-PCR and real-time PCR using primers specific for each. The three RCC cell lines were pretreated with the specific Wnt processing and secretion inhibitor IWP-2, the clathrin inhibitor chlopromazine and the caveolae inhibitor nystatin, the Fzd7 expression was detected by Western blot. The expression of total phospho-β-catenin, total β-catenin, nuclear β-catenin and cytoplasmic β-catenin were detected after IWP-2 treating RCC cell lines. The expression of Fzd7 was analyzed by real-time PCR. When Fzd7 sh RNA/ GFPsh RNA was transduced into cells, the growth rate of 786-O cells was showed by the cell proliferation assay. Then Fzd7 sh RNA/786-O cells and GFPsh RNA/786-O cells were treated with Wnt3 a and cells growth rate were detected in the same way.Results: The results of Western blot demonstrated that IWP-2 significantly increased the 64 k D bands in the three RCC cell lines which represented the total monomer of Fzd7. when 786-O cells were treated with chlopromazine, the monomer of Fzd7 increased in a dose dependent manner. However, variouse concentrations of nystatin did not affect the monomer level of Fzd7 in 786-O cells. Both of the results suggested that the endocytosis of Fzd7 induced by endogenous Wnts was primarily mediated by clathrin. RCC cells were treated with variouse concentrations of IWP-2 and 10 μM IWP-2 treatment significantly increased phospho-β-catenin level and decreased the total β-catenin in 786-O, OS-RC-2 and Caki-1 cells. Furthermore, IWP-2 inhibited the nuclear translocation of β-catenin in OS-RC-2 cells. These results suggested that IWP-2 treatment inhibited the pathway activation. Real-time PCR analysis showed that the Fzd7 m RNA expression level in 786-O cell line was higher than in OS-RC-2 and Caki-1 cell lines. The cell proliferation assay showed that growth rate of 786-O cells was significantly reduced when Fzd7 sh RNA was transduced into cells when compared with GFPsh RNA control plasmid. Then Fzd7 sh RNA/786-O cells and GFPsh RNA/786-O cells were treated with Wnt3 a and cells were induced to proliferate. As the control medium, Fzd7 sh RNA/786-O cells proliferation was more quickly than GFPsh RNA/786-O cells when induced by Wnt3 a. These results suggested that Fzd7 played a role in RCC cell proliferation.Conclusion: Endogenous Wnts of RCC cells activated the canonical pathway and the endocytosis of Fzd7 stimulated by endogenous Wnts was mediated by clathrin。Besides, Fzd7 played a role in RCC cell proliferation.