HINT1 inhibits β‐catenin/TCF4, USF2 and NFκB activity in human hepatoma cells

In this study we explored the relevance of Hint, a novel tumor suppressor gene, to human hepatoma. The human hepatoma cell lines Hep3B and HepG2 express very low levels of the HINT1 protein but the Huh7 cells express a relatively high level. In Hep3B and HepG2 cells, but not in Huh7 cells, the promoter region of Hint1 is partially methylated and treatment with 5‐azadcdeoxycytidine increased expression of the HINT1 protein and Hint1 mRNA in Hep3B and HepG2 cells. Increased expression of HINT1 in HepG2 cells markedly inhibited their growth. It also inhibited the transcriptional activities of β‐catenin/TCF4, and USF2, and inhibited the expression of endogenous cyclin D1 and TGFβ2. Furthermore, HINT1 co‐immunoprecipitated with USF2 in extracts of Hep2 cells. HINT1 also inhibited NFκB transcription factor reporter activity and inhibited translocation of the endogenous p65 protein to the nucleus of HepG2 cells. Therefore, decreased expression of the Hint1 gene through epigenetic silencing may play a role in enhancing the growth of a subset of human hepatoma by increasing the expression of genes controlled by the transcription factors β‐catenin, USF2, and NFκB. © 2008 Wiley‐Liss, Inc.

[1]  Y. Dragan,et al.  Mutational analysis of three tumor suppressor genes in two models of rat hepatocarcinogenesis , 1999, Molecular carcinogenesis.

[2]  M. Buendia,et al.  Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[3]  S. Jacob,et al.  The dual role of helix – loop – helix-zipper protein USF in ribosomal RNA gene transcription in vivo , 1997, Oncogene.

[4]  B. Viollet,et al.  Regulation of cdc2 gene expression by the upstream stimulatory factors (USFs) , 1999, Oncogene.

[5]  K. Bhoola,et al.  Genealogy, expression, and cellular function of transforming growth factor-beta. , 2003, Pharmacology & therapeutics.

[6]  O. Huber,et al.  The tumor suppressor Fhit acts as a repressor of β-catenin transcriptional activity , 2007, Proceedings of the National Academy of Sciences.

[7]  Bonnie F. Sloane,et al.  Isolation of a novel USF2 isoform: repressor of cathepsin B expression. , 2004, Gene.

[8]  S. Fan,et al.  β‐catenin mutation and overexpression in hepatocellular carcinoma , 2001 .

[9]  A. Tsamandas,et al.  TGFbeta1, TGFbeta2, and TGFbeta3 protein expression in gastric carcinomas: correlation with prognostics factors and patient survival. , 2007, The Journal of surgical research.

[10]  A. Rizzino,et al.  Transcription of the Transforming Growth Factor-β2 Gene Is Dependent on an E-box Located between an Essential cAMP Response Element/Activating Transcription Factor Motif and the TATA Box of the Gene* , 1996, The Journal of Biological Chemistry.

[11]  R. Beauchamp,et al.  Role of Smad proteins in the regulation of NF-κB by TGF-β in colon cancer cells , 2006 .

[12]  L. Jeng,et al.  Absence of APC gene mutation in the mutation cluster region in hepatocellular carcinoma. , 1998, Cancer letters.

[13]  A. Gudkov,et al.  Secreted transforming growth factor β2 activates NF-κB, blocks apoptosis, and is essential for the survival of some tumor cells , 2004 .

[14]  J. Rivera,et al.  Suppression of Microphthalmia Transcriptional Activity by Its Association with Protein Kinase C-interacting Protein 1 in Mast Cells* , 1999, The Journal of Biological Chemistry.

[15]  Y. Konishi,et al.  Different frequencies and patterns of beta-catenin mutations in hepatocellular carcinomas induced by N-nitrosodiethylamine and a choline-deficient L-amino acid-defined diet in rats. , 1999, Cancer research.

[16]  J. Taub,et al.  Physical and Functional Interactions between USF and Sp1 Proteins Regulate Human Deoxycytidine Kinase Promoter Activity* , 2003, Journal of Biological Chemistry.

[17]  L. Matherly,et al.  Transcriptional regulation of the human reduced folate carrier A1/A2 promoter: Identification of critical roles for the USF and GATA families of transcription factors. , 2005, Biochimica et biophysica acta.

[18]  O. Huber,et al.  The histidine triad protein Hint1 interacts with Pontin and Reptin and inhibits TCF–β-catenin-mediated transcription , 2005, Journal of Cell Science.

[19]  S. Straus,et al.  The cellular transcription factor USF cooperates with varicella-zoster virus immediate-early protein 62 to symmetrically activate a bidirectional viral promoter , 1994, Molecular and cellular biology.

[20]  M. Masuda,et al.  Growth inhibition of human hepatoma cells by acyclic retinoid is associated with induction of p21(CIP1) and inhibition of expression of cyclin D1. , 2002, Cancer research.

[21]  L. Babiss,et al.  Upstream stimulatory factor regulates expression of the cell cycle-dependent cyclin B1 gene promoter , 1995, Molecular and cellular biology.

[22]  G. Stark,et al.  Secreted transforming growth factor beta2 activates NF-kappaB, blocks apoptosis, and is essential for the survival of some tumor cells. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[23]  J. Kuiper,et al.  KLF2 Suppresses TGF-&bgr; Signaling in Endothelium Through Induction of Smad7 and Inhibition of AP-1 , 2007, Arteriosclerosis, thrombosis, and vascular biology.

[24]  Chih-Hsin Tang,et al.  Osteoblasts‐derived TGF‐β1 enhance motility and integrin upregulation through Akt, ERK, and NF‐κB‐dependent pathway in human breast cancer cells , 2008 .

[25]  T. Mäkelä,et al.  Interactions of Cdk7 and Kin28 with Hint/PKCI-1 and Hnt1 Histidine Triad Proteins* , 2000, The Journal of Biological Chemistry.

[26]  Randall T Moon,et al.  Mechanism and function of signal transduction by the Wnt/β-catenin and Wnt/Ca2+ pathways , 1999, Oncogene.

[27]  Yu-Nee Lee,et al.  Nonconventional Involvement of LysRS in the Molecular Mechanism of USF2 Transcriptional Activity in FcεRI-Activated Mast Cells , 2005, Molecular and Cellular Biology.

[28]  Jörg Stappert,et al.  β‐catenin is a target for the ubiquitin–proteasome pathway , 1997 .

[29]  T. Su,et al.  Deletion of histidine triad nucleotide-binding protein 1/PKC-interacting protein in mice enhances cell growth and carcinogenesis , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[30]  P. Futreal,et al.  Novel consensus DNA‐binding sequence for BRCA1 protein complexes , 2003, Molecular carcinogenesis.

[31]  B. Viollet,et al.  Immunochemical Characterization and Transacting Properties of Upstream Stimulatory Factor Isoforms (*) , 1996, The Journal of Biological Chemistry.

[32]  J. Deng,et al.  Overlapping roles and asymmetrical cross-regulation of the USF proteins in mice. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[33]  H. Hermeking,et al.  Evidence for a cancer-specific switch at the CDK4 promoter with loss of control by both USF and c-Myc , 2004, Oncogene.

[34]  C. Knabbe,et al.  TGF‐Beta Signaling in Breast Cancer , 2006, Annals of the New York Academy of Sciences.

[35]  M. Colombo,et al.  Epidemiology of hepatocellular carcinoma. , 1995, The Italian journal of gastroenterology.

[36]  S. Reynolds,et al.  Aberrant gene expression in human non small cell lung carcinoma cells exposed to demethylating agent 5-aza-2'-deoxycytidine. , 2004, Neoplasia.

[37]  J. Foley,et al.  Beta-catenin mutations and protein accumulation in all hepatoblastomas examined from B6C3F1 mice treated with anthraquinone or oxazepam. , 2000, Cancer research.

[38]  J. M. Scholtz,et al.  Energetic Implications for Protein Phosphorylation , 1996, The Journal of Biological Chemistry.

[39]  S. Fan,et al.  beta-Catenin mutation and overexpression in hepatocellular carcinoma: clinicopathologic and prognostic significance. , 2001, Cancer.

[40]  Michael Karin,et al.  NF-κB in cancer: from innocent bystander to major culprit , 2002, Nature Reviews Cancer.

[41]  Michael Karin,et al.  NF-kappaB in cancer: from innocent bystander to major culprit. , 2002, Nature reviews. Cancer.

[42]  A. Rizzino,et al.  Transcriptional regulation of the transforming growth factor-β2 gene in glioblastoma cells , 2001, In Vitro Cellular & Developmental Biology - Animal.

[43]  M. Karin,et al.  IKKα is a critical coregulator of a Smad4-independent TGFβ-Smad2/3 signaling pathway that controls keratinocyte differentiation , 2008, Proceedings of the National Academy of Sciences.

[44]  J. Shimazaki,et al.  Expression and prognostic roles of beta-catenin in hepatocellular carcinoma: correlation with tumor progression and postoperative survival. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[45]  K. Jungermann,et al.  The upstream stimulatory factor-2a inhibits plasminogen activator inhibitor-1 gene expression by binding to a promoter element adjacent to the hypoxia-inducible factor-1 binding site. , 2001, Blood.

[46]  I. Weinstein,et al.  Hint1 inhibits growth and activator protein-1 activity in human colon cancer cells. , 2007, Cancer research.

[47]  S. Vaulont,et al.  Differential Roles of Upstream Stimulatory Factors 1 and 2 in the Transcriptional Response of Liver Genes to Glucose* , 1998, The Journal of Biological Chemistry.

[48]  Haiyang Li,et al.  Hint1 is a haplo-insufficient tumor suppressor in mice , 2006, Oncogene.

[49]  R Kemler,et al.  beta-catenin is a target for the ubiquitin-proteasome pathway. , 1997, The EMBO journal.

[50]  K. Jungermann,et al.  Modulation of glucokinase expression by hypoxia-inducible factor 1 and upstream stimulatory factor 2 in primary rat hepatocytes , 2004, Biological chemistry.

[51]  G. Stark,et al.  Secretion of cytokines and growth factors as a general cause of constitutive NFκB activation in cancer , 2004, Oncogene.

[52]  A. Tsamandas,et al.  TGFβ1, TGFβ2, and TGFβ3 Protein Expression in Gastric Carcinomas: Correlation with Prognostics Factors and Patient Survival , 2007 .

[53]  P. Higgins,et al.  Upstream stimulatory factor regulates E box‐dependent PAI‐1 transcription in human epidermal keratinocytes , 2005, Journal of cellular physiology.