Studies On The Effect And Mechanism Of Orphan Receptor TR3/Nur77 Targeted Angiogenesis To Tumor Growth

Background Pathological angiogenesis is a hallmark of many diseases and vascular endothelial growth factor(VEGF)have been demonstrated as a strong pro-angiogenesis factor,VEGF and its receptor VEGFR kinase inhibitors such as bevacizumab,aflibercept and ramucirumab have been widely used in the clinic.However,in addition to the side effects of those drugs,VEGF-targeted therapies are suffering a series of problems such as intrinsic refractoriness,resistance and insufficient efficacy.Therefore,it is desirable to identify additional angiogenesis targets for anti-tumor therapy.TR3/Nur77 is a member of the nuclear receptor IV subfamily of the transcriptional factors,and has been demonstrated play a critical role in VEGF-mediated angiogenesis.Our previous study found that VEGF-mediated angiogenesis and tumor growth could be inhibited by down-regulating expression of TR3/Nur77,demonstrating that TR3/Nur77 is apotential target for pro-angiogenic and anti-angiogenic therapies.TR3/Nur77 has three dominant domains: ligand binding domain LBD,DNA binding domain DBD,transactivation domain TAD.Our previous study showed that TAD and DBD play more important role in VEGF-mediated angiogenesis.However,none of the TR3/Nur77 targeted molecules has been in clinical trial so far.Here we extend our previous findings by designing and generating four TR3/Nur77-derived biomolecules that had therapeutic potential for cancer treatment,aiming to further investigate the structure-function relationships and mechanism of TR3/Nur77 in VEGF-mediated angiogenesis and set foundation for the subsequent angiogenesis-targeted cancer therapy.Objectives Our study is aimed to demonstrate the effect of TR3/Nur77 in VEGF-mediated angiogenesis and tumor growth and further investigate the structure-function relationships and mechanism,identify the potential of TR3/Nur77 to be anti-angiogenic target for cancer therapy.Methods 1.Analyze TR3/Nur77 expression in human melanoma and colorectal cancer tissues with normal para-tumor tissues by immune-staining with TR3/Nur77 antibody.2.Mouse lung carcinoma cell line LLC was transplanted to TR3/Nur77 knockout mice and wild type mice to observe the tumor growth.3.Mouse colorectal carcinoma cell line CMT93 was transplanted to TR3/Nur77 knockout mice and wild type mice to observe tumor metastasis to lung.4.Mouse lung carcinoma cell line LAP0297 was transplanted to EC-Nur77-S,EC-Nur77-DN transgenic mice and wild type mice to observe tumor growth.5.Design and generate four TR3/Nur77-derived biomolecules with adenovirus vector: F-TR3-M1 expressing TAD ? F-TR3-M3 p expressing DBD and two sh RNAs(sh TR3-176 for human TR3/Nur77,sh TR3-297 for mouse TR3/Nur77).6.Prepare,purify and collect the recombinant adenovirus.7.Western-blot to testify the correct expression of protein in cells infected with different adenovirus.8.Monolayer cell scratching assay was employed to determine the migrating ability of cells infected with different adenovirus.9.CCK-8 proliferation assay was employed to determine the proliferating ability of cells infected with different adenovirus.10.In vivo VEGF-induced angiogenesis models were employed on mice ears to study the effect of four TR3/Nur77-derived molecules on VEGF-induced angiogenesis.11.Transplanted tumor assay was employed on nude mice to study the effect of four TR3/Nur77-derived molecules on tumor growth and angiogenesis.12.Analyze angiogenic molecules expression in tumor,skinand liver tissues of sacrificed mice by IHC and q PCR.Results 1.TR3/Nur77 was detected in both of vasculature and tumor cells in human cancer tissues,but not in normal para-tumor tissues.2.LLC grew to large size in wild type mice but growth in Nur77-/-mice was greatly inhibited.3.Tumor metastasis to lungs was detected four months after injection of CMT93 cells in wild type mice but not in Nur77-/-mice.4.Compared to wild type littermates,tumor growth were accelerated in EC-Nur77-S mice and decreased in EC-Nur77-DN mice.5.TR3/Nur77-derived biomolecules inhibited proliferation and migration of endothelial and tumor cells.6.TR3/Nur77-derived biomolecules inhibited angiogenesis induced by VEGF in vivo.7.F-TR3-M1?F-TR3-M3p?sh TR3-176?sh TR3-297 inhibited melanoma growth in mice.8.Tumor angiogenesis was inhibited by adenoviruses expressing F-TR3-M1 ?F-TR3-M3p?sh TR3-176?sh TR3-297.9.F-TR3-M1?F-TR3-M3p?sh TR3-176?sh TR3-297 down regulated expression of angiogenic molecules.Conclusions Based on the main findings of our study,we can conclude as below: TR3/Nur77 is highly expressed in tumor tissues but not in normal para-tumor tissues.TR3/Nur77 regulates tumor growth and metastasis,targeting both tumor cells and tumor microenvironment.TR3/Nur77-derived biomolecules F-TR3-M1 ? F-TR3-M3 p ?sh TR3-176?sh TR3-297 inhibits proliferation and migration of endothelial cells,inhibits VEGF-induced angiogenesis and tumor growth in mice.