Phosphorylation of Nucleophosmin at Threonine 234/237 is associated with HCC metastasis
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Rachel Hiu Ha Ching1,2, Eunice Yuen Ting Lau1,2, Patrick Ming Tat Ling3, Joyce Man Fong Lee1,2, Mark Kin Fai Ma1,2, Bowie Yik Ling Cheng1,2, Regina Cheuk Lam Lo1,2, Irene Oi Lin Ng1,2, Terence Kin Wah Lee1,2
1State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China
2Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
3Australian Prostate Cancer Research Centre-Queensland & Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia
Terence Kin Wah Lee, e-mail: tkwlee@hku. hk
Irene Oi Lin Ng, e-mail: iolng@hku. hk
Keywords: NPM, HCC, CDK1, metastasis, hepatocellular carcinoma, immunohistochemistry
Abbreviations: cyclin dependent kinase 1, CDK1; hepatocellular carcinoma, HCC; nucleophosmin, NPM; LIM kinase, LIMK; immunohistochemistry, IHC; Rho kinase II, ROCK2
Received: May 01, 2015 Accepted: October 19, 2015 Published: October 30, 2015
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.
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