title: Phosphorylation of nucleophosmin at threonine 234/237 is associated with HCC metastasis.
abstract: Hepatocellular carcinoma (HCC) is frequently complicated by the occurrence of intrahepatic and extrahepatic metastases, leading to poor prognosis. To improve the prognosis for HCC patients, there is an urgent need to understand the molecular mechanisms of metastasis in HCC. Since protein Serine/Threonine phosphorylation emerges to be an important posttranslational modification critical in signaling process associated with cell proliferation, survival and metastasis, we employed a pair of primary tumor-derived and corresponding lung-metastatic counterparts (PLC/PRF/5-PT and PLC/PRF/5-LM) and aimed to identify these changes using CelluSpot Serine/Threonine kinase peptide array. Upon analysis, we found phosphorylated level of nucleophosmin (NPM) at Threonine 234/237 (p-NPM-Thr234/237) had remarkably high level in metastatic HCC cells (PLC-LM) than the corresponding primary HCC cell line (PLC-PT). Similar observation was observed in another match primary and their metastatic counterparts (MHCC-97L and MHCC-97H). By immunohistochemical staining, p-NPM-Thr234/237 was consistently found to be preferentially expressed in metastatic HCCs when compared with primary HCC in 28 HCC cases (p < 0.0001). By overexpressing Flag-tagged NPM and its phosphorylation site mutant (Thr234/237A) into low p-NPM-Thr234/237 expressing cells (Hep3B and Huh7) using a lentiviral based approach, we demonstrated that p-NPM-Thr234/237 is critical in invasion and migration of HCC cells, and this effect was mediated by cyclin-dependent kinase 1 (CDK1). Wild-type NPM was found to physically interact with a metastatic gene, ROCK2, and defective in Thr234/237 phosphorylation decreased its binding affinity, resulting in decrease in ROCK2 mediated signaling pathway. Identification of CDK1/p-NPM/ROCK2 signaling pathway provides a novel target for molecular therapy against HCC metastasis.
authors.affiliation: State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China.|Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: Rachel Hiu Ha
authors.initials: RH
authors.lastname: Ching
authors.affiliation: State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China.|Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: Eunice Yuen Ting
authors.initials: EY
authors.lastname: Lau
authors.affiliation: Australian Prostate Cancer Research Centre-Queensland & Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia.
authors.forename: Patrick Ming Tat
authors.initials: PM
authors.lastname: Ling
authors.affiliation: State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China.|Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: Joyce Man Fong
authors.initials: JM
authors.lastname: Lee
authors.affiliation: State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China.|Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: Mark Kin Fai
authors.initials: MK
authors.lastname: Ma
authors.affiliation: State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China.|Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: Bowie Yik Ling
authors.initials: BY
authors.lastname: Cheng
authors.affiliation: State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China.|Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: Regina Cheuk Lam
authors.initials: RC
authors.lastname: Lo
authors.affiliation: State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China.|Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: Irene Oi Lin
authors.initials: IO
authors.lastname: Ng
authors.affiliation: State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China.|Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: Terence Kin Wah
authors.initials: TK
authors.lastname: Lee
mesh_terms: D015153:Blotting, Western; D016203:CDC2 Protein Kinase; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D055785:Gene Knockdown Techniques; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D009361:Neoplasm Invasiveness; D009687:Nuclear Proteins / Q000378:metabolism*; D000090243:Nucleophosmin; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D013912:Threonine / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D000090243:Nucleophosmin; D013912:Threonine; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases
keywords: CDK1; HCC; NPM; hepatocellular carcinoma; immunohistochemistry; metastasis