title: Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/mTOR signaling pathway in bladder cancer cells.
abstract: Angiogenin (ANG), a member of RNase A superfamily, is the only angiogenic factor that possesses ribonucleolytic activity. Recent studies showed that the expression of ANG was elevated in various types of cancers. Accumulating evidence indicates that ANG plays an essential role in cancer progression by stimulating both cancer cell proliferation and tumor angiogenesis. Human ribonuclease inhibitor (RI), a cytoplasmic protein, is constructed almost entirely of leucine rich repeats (LRRs), which are present in a large family of proteins that are distinguished by their display of vast surface areas to foster protein-protein interactions. RI might be involved in unknown biological effects except inhibiting RNase A activity. The experiment demonstrated that RI also could suppress activity of angiogenin (ANG) through closely combining with it in vitro. PI3K/AKT/mTOR signaling pathway exerts a key role in cell growth, survival, proliferation, apoptosis and angiogenesis. We recently reported that up-regulating RI inhibited the growth and induced apoptosis of murine melanoma cells through repression of angiogenin and PI3K/AKT signaling pathway. However, ANG receptors have not yet been identified to date, its related signal transduction pathways are not fully clear and underlying interacting mechanisms between RI and ANG remain largely unknown. Therefore, we hypothesize that RI might combine with intracellular ANG to block its nuclear translocation and regulate PI3K/AKT/mTOR signaling pathway to inhibit biological functions of ANG. Here, we reported for the first time that ANG could interact with RI endogenously and exogenously by using co-immunoprecipitation (Co-IP) and GST pull-down. Furthermore, we observed the colocalization of ANG and RI in cells with immunofluorescence staining under laser confocal microscope. Moreover, through fluorescence resonance energy transfer (FRET) assay, we further confirmed that these two proteins have a physical interaction in living cells. Subsequently, we demonstrated that up-regulating ANG including ANG His37Ala mutant obviously decreased RI expression and activated phosphorylation of key downstream target molecules of PI3K/AKT/mTOR signaling pathway. Finally, up-regulating ANG led to the promotion of tumor angiogenesis, tumorigenesis and metastasis in vivo. Taken together, our data provided a novel mechanism of ANG in regulating PI3K/AKT/mTOR signaling pathway via RI, which suggested a new therapeutic target for cancer therapy.
authors.affiliation: Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, PR China; The First Clinical College, Chongqing Medical University, Chongqing 400016, PR China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Peng
authors.affiliation: Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, PR China; Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing 400016, PR China.
authors.forename: Lin
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Clinical Medicine, Luzhou Medical College, Luzhou 646000, PR China.
authors.forename: Mengge
authors.initials: M
authors.lastname: Huang
authors.affiliation: Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, PR China; Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing 400016, PR China.
authors.forename: Changzhu
authors.initials: C
authors.lastname: Duan
authors.affiliation: The First Clinical College, Chongqing Medical University, Chongqing 400016, PR China.
authors.forename: Luyu
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, PR China; Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing 400016, PR China. Electronic address: chjunxia@126.com.
authors.forename: Junxia
authors.initials: J
authors.lastname: Chen
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology / Q000556:secondary; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D050505:Mutant Proteins / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010926:Placental Hormones / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation; D001749:Urinary Bladder Neoplasms / Q000201:enzymology* / Q000473:pathology; D023041:Xenograft Model Antitumor Assays
chemical_list: D050505:Mutant Proteins; D019869:Phosphatidylinositol 3-Kinases; D010926:Placental Hormones; D051057:Proto-Oncogene Proteins c-akt; D012259:Ribonuclease, Pancreatic; D058570:TOR Serine-Threonine Kinases; C022052:placental ribonuclease inhibitor; C546842:MTOR protein, human; C046366:angiogenin
keywords: Angiogenin; Bladder cancer cells; Interaction; PI3K/AKT/mTOR; Ribonuclease inhibitor