title: Aspartate β-hydroxylase modulates cellular senescence through glycogen synthase kinase 3β in hepatocellular carcinoma.
abstract: Aspartate β-hydroxylase (ASPH) is an enzyme overexpressed in human hepatocellular carcinoma (HCC) tumors that participates in the malignant transformation process. We determined if ASPH was a therapeutic target by exerting effects on cellular senescence to retard HCC progression. ASPH knockdown or knockout was achieved by short hairpin RNAs or the CRISPR/Cas9 system, respectively, whereas enzymatic inhibition was rendered by a potent second-generation small molecule inhibitor of ASPH. Alterations of cell proliferation, colony formation, and cellular senescence were evaluated in human HCC cell lines. The potential mechanisms for activating cellular senescence were explored using murine subcutaneous and orthotopic xenograft models. Inhibition of ASPH expression and enzymatic activity significantly reduced cell proliferation and colony formation but induced tumor cell senescence. Following inhibition of ASPH activity, phosphorylation of glycogen synthase kinase 3β and p16 expression were increased to promote senescence, whereas cyclin D1 and proliferating cell nuclear antigen were decreased to reduce cell proliferation. The mechanisms involved demonstrate that ASPH binds to glycogen synthase kinase 3β and inhibits its subsequent interactions with protein kinase B and p38 upstream kinases as shown by coimmunoprecipitation. In vivo experiments demonstrated that small molecule inhibitor treatment of HCC bearing mice resulted in significant dose-dependent reduced tumor growth, induced phosphorylation of glycogen synthase kinase 3β, enhanced p16 expression in tumor cells, and promoted cellular senescence.
  
  
  CONCLUSIONS
  We have identified a new mechanism that promotes HCC growth and progression by modulating senescence of tumor cells; these findings suggest that ASPH enzymatic activity is a novel therapeutic target for HCC.
authors.affiliation: Division of Gastroenterology & Liver Research Center, Warren Alpert Medical School of Brown University and Rhode Island Hospital, Providence, RI.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Iwagami
authors.affiliation: Division of Gastroenterology & Liver Research Center, Warren Alpert Medical School of Brown University and Rhode Island Hospital, Providence, RI.
authors.forename: Chiung-Kuei
authors.initials: CK
authors.lastname: Huang
authors.affiliation: Department of Medical Chemistry, College of Pharmacy Glendale, Midwestern University, Glendale, AZ.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Olsen
authors.affiliation: Department of Medical Chemistry, College of Pharmacy Glendale, Midwestern University, Glendale, AZ.
authors.forename: John-Michael
authors.initials: JM
authors.lastname: Thomas
authors.affiliation: Department of Medical Chemistry, College of Pharmacy Glendale, Midwestern University, Glendale, AZ.
authors.forename: Grace
authors.initials: G
authors.lastname: Jang
authors.affiliation: Division of Gastroenterology & Liver Research Center, Warren Alpert Medical School of Brown University and Rhode Island Hospital, Providence, RI.
authors.forename: Miran
authors.initials: M
authors.lastname: Kim
authors.affiliation: Department of Internal Medicine, College of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, OK.
authors.forename: Qiushi
authors.initials: Q
authors.lastname: Lin
authors.affiliation: Division of Gastroenterology & Liver Research Center, Warren Alpert Medical School of Brown University and Rhode Island Hospital, Providence, RI.
authors.forename: Rolf I
authors.initials: RI
authors.lastname: Carlson
authors.affiliation: Arizona State University, Glendale, AZ.
authors.forename: Carl E
authors.initials: CE
authors.lastname: Wagner
authors.affiliation: Department of Internal Medicine, College of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, OK.
authors.forename: Xiaoqun
authors.initials: X
authors.lastname: Dong
authors.affiliation: Division of Gastroenterology & Liver Research Center, Warren Alpert Medical School of Brown University and Rhode Island Hospital, Providence, RI.
authors.forename: Jack R
authors.initials: JR
authors.lastname: Wands
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000201:enzymology* / Q000473:pathology; D049109:Cell Proliferation; D016922:Cellular Senescence / Q000502:physiology*; D004195:Disease Models, Animal; D005260:Female; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D064593:Heterografts; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology* / Q000473:pathology; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011897:Random Allocation; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
chemical_list: D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D009124:Muscle Proteins; D006899:Mixed Function Oxygenases; C419389:ASPH protein, human; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3