The most recent reeasearch includes (1)Development of targeted therapy for hepatocellular carcinoma (HCC) remains a major challenge. We have recently identified an elevated expression of the fifth subunit of COP9 signalosome (CSN5) in early HCC as compared with dysplastic stage. In the present study, we explored the possibility of CSN5 being a potential therapeutic target for HCC. Our results show that CSN5 knockdown by small-interfering (si) RNA caused a strong induction of apoptosis and inhibition of cell-cycle progression in HCC cells in vitro. The down-regulation of CSN5 was sufficient to interfere with CSN function as evidenced by the accumulation of neddylated Cullin 1 and changes in the protein levels of CSN-controlled substrates SKP2, p53, p27 and nuclear factor-kappaB, albeit to a different degree depending on the HCC cell line, which could account for the CSN5 knockdown phenotype. The transcriptomic analysis of CSN5 knockdown signature showed that the anti-proliferative effect was driven by a common subset of molecular alterations including down-regulation of cyclin-dependent kinase 6 (CDK6) and integrin beta1 (ITGB1), which were functionally interconnected with key oncogenic regulators MYC and TGFbeta1 involved in the control of proliferation, apoptotic cell death and HCC progression. Consistent with microarray analysis, western blotting revealed that CSN5 depletion increased phosphorylation of Smad 2/3, key mediators of TGFbeta1 signaling, decreased the protein levels of ITGB1, CDK6 and cyclin D1 and caused reduced expression of anti-apoptotic Bcl-2, while elevating the levels of pro-apoptotic Bak. A chemically modified variant of CSN5 siRNA was then selected for in vivo application based on the growth inhibitory effect and minimal induction of unwanted immune response. Systemic delivery of the CSN5 3/8 variant by stable-nucleic-acid-lipid particles significantly suppressed the tumor growth in Huh7-luc+ orthotopic xenograft model. Taken together, these results indicate that CSN5 has a pivotal role in HCC pathogenesis and maybe an attractive molecular target for systemic HCC therapy.Our most recent progress in this project include;and (2) Previously studies have shown that protein level and kinase activity of Wee1 are significantly elevated in HCC compared with surrounding cirrhotic tissues, although the underlying mechanisms are still unknown. Under normal conditions, Wee1 kinase plays an important role in maintaining G2 arrest through the inhibitory phosphorylation of cdc2 on Tyr-15. In the present study, we explored the possibility of Wee1 being a potential therapeutic target for HCC. To inactivate Wee1, three Wee1-specific small interfering (si) RNAs (Wee1-1, Wee1-2 and Wee1-3) were tested for growth inhibition in HCC cell lines as determined by MTT assay, FACS analysis and microscopy. To obtain insights into molecular changes caused by Wee1 silencing, global changes in gene expression were examined by illumina microarray. For in vivo evaluation of Wee1 as a therapeutic target, we employed orthotopic xenograft model using luciferase-expressing HCC reporter cell lines Huh7- and HepG2-luc+ and stable-nucleic-acid-lipid-particle (SNALP) as an optimal carrier of siRNA into liver. Among the tested siRNA molecules, the Wee1-2siRNA was the most effective in inhibiting Huh7 and HepG2 cell growth (80% and 84%, respectively) which was paralleled by a similar decrease in the levels of target mRNA and protein. Wee1 knockdown by siRNA also caused a block in cell cycle progression and induced apoptosis of HCC cells. The comparison of gene expression profiles in HepG2 cells treated with either control siRNA or Wee1-2siRNA identified 506 differentially expressed genes (P <0.05 by bootstrap t-test). Genes functionally involved in cell proliferation, such as cdk2, cyclin B1, and Akt1, were down-regulated while cell cycle inhibitor p21 and tumor suppressor TSC2 were up-regulated. Western blotting showed that Wee1 silencing significantly increased the expression of p53 and p21 and decreased cyclin D1 protein levels in Wee1-deficient HepG2 cells, which could contribute to cell cycle arrest and induction of apoptosis. Wee1 5/6, a modified variant of Wee1-2siRNA, was then selected for in vivo application based on the growth inhibitory effect and minimal induction of unwanted immune response. Systemic delivery of Wee1 5/6 variant by SNALP significantly suppressed both Huh7 and HepG2 tumor growth in orthotopic xenograft model. In addition, administration of Wee1 5/6 SNALP increased the survival of mice bearing HepG2-derived tumors bearing mice in a dose-dependent manner. Taken together, these results indicate that Wee1 maybe an attractive molecular target for systemic HCC therapy.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Andersen, Jesper B; Thorgeirsson, Snorri S (2013) Genomic decoding of intrahepatic cholangiocarcinoma reveals therapeutic opportunities. Gastroenterology 144:687-90
Wang, P; Dong, Q; Zhang, C et al. (2013) Mutations in isocitrate dehydrogenase 1 and 2 occur frequently in intrahepatic cholangiocarcinomas and share hypermethylation targets with glioblastomas. Oncogene 32:3091-100
Andersen, Jesper B; Spee, Bart; Blechacz, Boris R et al. (2012) Genomic and genetic characterization of cholangiocarcinoma identifies therapeutic targets for tyrosine kinase inhibitors. Gastroenterology 142:1021-1031.e15
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Lee, Y-H; Judge, A D; Seo, D et al. (2011) Molecular targeting of CSN5 in human hepatocellular carcinoma: a mechanism of therapeutic response. Oncogene 30:4175-84
Lee, Yun-Han; Andersen, Jesper B; Song, Ho-Taek et al. (2010) Definition of ubiquitination modulator COP1 as a novel therapeutic target in human hepatocellular carcinoma. Cancer Res 70:8264-9
Wang, Xin Wei; Thorgeirsson, Snorri S (2009) Transcriptome analysis of liver cancer: ready for the clinic? J Hepatol 50:1062-4