Oncogenes and Tumor Suppressors

7 Abstract 8 miRNAs (miR) play a critical role in human cancers, including hepatocellular carcinoma. Although miR-302b 9 has been suggested to function as a tumor repressor in other cancers, its role in hepatocellular carcinoma is 10 unknown. This study investigated the expression and functional role of miR-302b in human hepatocellular 11 carcinoma. The expression level of miR-302b is dramatically decreased in clinical hepatocellular carcinoma 12 specimens, as comparedwith their respective nonneoplastic counterparts, and in hepatocellular carcinoma cell lines. 13 Overexpression of miR-302b suppressed hepatocellular carcinoma cell proliferation and G1–S transition in vitro, 14 whereas inhibition ofmiR-302b promoted hepatocellular carcinoma cell proliferation andG1–S transition. Using a 15 luciferase reporter assay, AKT2was determined to be a direct target ofmiR-302b. Subsequent investigation revealed 16 that miR-302b expression was inversely correlated with AKT2 expression in hepatocellular carcinoma tissue 17 samples. Importantly, silencing AKT2 recapitulated the cellular and molecular effects seen upon miR-302b 18 overexpression, which included inhibiting hepatocellular carcinoma cell proliferation, suppressing G1 regulators 19 (Cyclin A, Cyclin D1, CDK2) and Q4 increasing p27Kip1 phosphorylation at Ser10. Restoration of AKT2 20 counteracted the effects of miR-302b expression. Moreover, miR-302b was able to repress tumor growth of 21 hepatocellular carcinoma cells in vivo. Mol Cancer Res; 1–13. 2013 AACR. 22 23 24 25 Introduction

[1]  Bill Bynum,et al.  Lancet , 2015, The Lancet.

[2]  J. Bruix,et al.  Hepatocellular carcinoma , 2012, The Lancet.

[3]  Yuanzhi Lao,et al.  Akt: A Double-Edged Sword in Cell Proliferation and Genome Stability , 2012, Journal of oncology.

[4]  Hui Zhang,et al.  microRNA-320a inhibits tumor invasion by targeting neuropilin 1 and is associated with liver metastasis in colorectal cancer. , 2012, Oncology reports.

[5]  C. Croce,et al.  MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review , 2012, EMBO molecular medicine.

[6]  Ajit Kumar,et al.  MicroRNA in HCV infection and liver cancer. , 2011, Biochimica et biophysica acta.

[7]  C. Verslype,et al.  Molecular classification of hepatocellular carcinoma anno 2011. , 2011, European journal of cancer.

[8]  Robert L. Judson,et al.  Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells , 2011, Nature Biotechnology.

[9]  I. Ng,et al.  miR-130b Promotes CD133(+) liver tumor-initiating cell growth and self-renewal via tumor protein 53-induced nuclear protein 1. , 2010, Cell stem cell.

[10]  Donald C. Chang,et al.  MicroRNA miR-302 inhibits the tumorigenecity of human pluripotent stem cells by coordinate suppression of the CDK2 and CDK4/6 cell cycle pathways. , 2010, Cancer research.

[11]  M. Salto‐Tellez,et al.  MicroRNA-130b regulates the tumour suppressor RUNX3 in gastric cancer. , 2010, European journal of cancer.

[12]  S. Lowe,et al.  579 MIR-221 OVEREXPRESSION CONTRIBUTES TO LIVER TUMORIGENESIS , 2010 .

[13]  K. Choy,et al.  MiR-222 Overexpression Confers Cell Migratory Advantages in Hepatocellular Carcinoma through Enhancing AKT Signaling , 2010, Clinical Cancer Research.

[14]  S. Lowe,et al.  miR-221 overexpression contributes to liver tumorigenesis , 2009, Proceedings of the National Academy of Sciences.

[15]  S. Thorgeirsson,et al.  Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties , 2009, Oncogene.

[16]  G. Mills,et al.  Akt1 and akt2 play distinct roles in the initiation and metastatic phases of mammary tumor progression. , 2009, Cancer research.

[17]  Kathryn A. O’Donnell,et al.  Therapeutic microRNA Delivery Suppresses Tumorigenesis in a Murine Liver Cancer Model , 2009, Cell.

[18]  Ping Jin,et al.  MicroRNA and gene expression patterns in the differentiation of human embryonic stem cells , 2009, Journal of Translational Medicine.

[19]  G. Melen,et al.  Embryonic Stem Cell-Specific miR302-367 Cluster: Human Gene Structure and Functional Characterization of Its Core Promoter , 2008, Molecular and Cellular Biology.

[20]  Leping Li,et al.  Oct4/Sox2-Regulated miR-302 Targets Cyclin D1 in Human Embryonic Stem Cells , 2008, Molecular and Cellular Biology.

[21]  S. Hsieh,et al.  Profiling the proteome dynamics during the cell cycle of human hepatoma cells , 2008, Proteomics.

[22]  Nathalie Wong,et al.  MicroRNA-223 is commonly repressed in hepatocellular carcinoma and potentiates expression of Stathmin1. , 2008, Gastroenterology.

[23]  Mihee M. Kim,et al.  Phosphorylation of MDMX Mediated by Akt Leads to Stabilization and Induces 14-3-3 Binding* , 2008, Journal of Biological Chemistry.

[24]  R. Weissleder,et al.  Imaging in the era of molecular oncology , 2008, Nature.

[25]  L. Hansen,et al.  Akt-mediated phosphorylation of CDK2 regulates its dual role in cell cycle progression and apoptosis , 2008, Journal of Cell Science.

[26]  Krista A. Zanetti,et al.  Identification of metastasis‐related microRNAs in hepatocellular carcinoma , 2008, Hepatology.

[27]  Mihee M. Kim,et al.  RING domain-mediated interaction is a requirement for MDM2's E3 ligase activity. , 2007, Cancer research.

[28]  Y. Atlasi,et al.  OCT‐4, an embryonic stem cell marker, is highly expressed in bladder cancer , 2007, International journal of cancer.

[29]  A. Clark,et al.  Human embryonic stem cell genes OCT4, NANOG, STELLAR, and GDF3 are expressed in both seminoma and breast carcinoma , 2005, Cancer.

[30]  L. Olson,et al.  Errarum: Oct4 expression in adult stem cells: Evidence in support of the stem cell theory of carcinogenesis (Carcinogenesis (2005) Vol. 26 (495-502)) , 2005 .

[31]  J. Wengel,et al.  LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. , 2004, Biochemistry.

[32]  S. Moon,et al.  Human embryonic stem cells express a unique set of microRNAs. , 2004, Developmental biology.

[33]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[34]  A. Gartel,et al.  Mechanisms of c-myc-mediated transcriptional repression of growth arrest genes. , 2003, Experimental cell research.

[35]  P. Pandolfi,et al.  Activation of Akt/Protein Kinase B Overcomes a G2/M Cell Cycle Checkpoint Induced by DNA Damage , 2002, Molecular and Cellular Biology.

[36]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[37]  M. Hung,et al.  Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells , 2001, Nature Cell Biology.

[38]  P. Tsichlis,et al.  Transduction of interleukin-2 antiapoptotic and proliferative signals via Akt protein kinase. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[39]  G. Luker,et al.  Luciferase protein complementation assays for bioluminescence imaging of cells and mice. , 2011, Methods in molecular biology.

[40]  N. Coleman,et al.  Identification of microRNAs From the miR-371~373 and miR-302 clusters as potential serum biomarkers of malignant germ cell tumors. , 2011, American journal of clinical pathology.

[41]  M. Eder,et al.  Lentivirus-mediated antagomir expression. , 2010, Methods in molecular biology.

[42]  R. Cetin-Atalay,et al.  Inhibition of Akt signaling in hepatoma cells induces apoptotic cell death independent of Akt activation status , 2010, Investigational New Drugs.

[43]  Jinu Kim,et al.  MicroRNA Expression, Survival, and Response to Interferon in Liver Cancer , 2010 .

[44]  Donald C. Chang,et al.  Pluripotent Stem Cells by Coordinate Suppression of the MicroRNA miR-302 Inhibits the Tumorigenecity of Human , 2010 .

[45]  Robert L. Judson,et al.  Embryonic stem cell–specific microRNAs promote induced pluripotency , 2009, Nature Biotechnology.

[46]  H. Willenbring,et al.  A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice , 2008, Nature Protocols.

[47]  J. Testa,et al.  Activation of AKT kinases in cancer: implications for therapeutic targeting. , 2005, Advances in cancer research.