Associations among β-TrCP, an E3 ubiquitin ligase receptor, β-catenin, and NF-κB in colorectal cancer

BACKGROUND The ubiquitin-proteasome pathway is important in regulating protein signaling pathways that are involved in tumorigenesis. beta-transducin repeat-containing proteins (beta-TrCP) are components of the ubiquitin ligase complex targeting beta-catenin and IkappaBalpha for proteasomal degradation and are thus a negative regulator of Wnt/beta-catenin signaling and a positive regulator of NF-kappaB signaling. We analyzed expression of beta-TrCP in colorectal cancers and its association with types of beta-catenin subcellular localization, an indirect measure of activation. METHODS Levels of beta-TrCP1 mRNA and protein were measured by quantitative reverse transcription-polymerase chain reaction and immunoblotting, respectively, in samples of tumor and normal tissues from 45 patients with colorectal cancer. Types of beta-catenin activation (diffuse or invasion edge) and NF-kappaB activation were examined by immunohistochemistry. Apoptosis was determined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) assay. All statistical tests were two-sided. RESULTS Compared with the beta-TrCP1 levels in normal tissues, 25 (56%) of 45 tumors had increased beta-TrCP1 mRNA and protein levels. Of the 22 (49%) tumors with beta-catenin activation, 12 had the diffuse type (i.e., nuclear accumulation throughout the tumor) and 10 had the invasion edge type (i.e., nuclear accumulation predominantly in the tumor cells that formed the invasion edge). Increased beta-TrCP1 levels were statistically significantly associated with beta-catenin activation (P =.023) and decreased apoptosis (P =.035). beta-TrCP accumulated in the nuclei of tumor cells that contained increased levels of beta-TrCP1 mRNA and the active form of NF-kappaB. Higher levels of beta-TrCP1 mRNA were detected in primary tumors of patients who had metastases (0.960 arbitrary units, 95% confidence interval = 0.878 to 1.042) than in the tumors of patients who did not (0.722 arbitrary units, 95% confidence interval = 0.600 to 0.844; P =.016). CONCLUSION In colorectal cancer, increased expression of beta-TrCP1 is associated with activation of both beta-catenin and NF-kappaB, suggesting that the integration of these signaling pathways by increased beta-TrCP expression may contribute to an inhibition of apoptosis and tumor metastasis.

[1]  S. Fuchs,et al.  The many faces of β-TrCP E3 ubiquitin ligases: reflections in the magic mirror of cancer , 2004, Oncogene.

[2]  T. Minamoto,et al.  β-catenin and ras oncogenes detect most human colorectal cancer , 2003 .

[3]  K. Nakayama,et al.  Impaired degradation of inhibitory subunit of NF-κB (IκB) and β-catenin as a result of targeted disruption of the β-TrCP1 gene , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[4]  J. Peters.  Emi1 proteolysis: how SCF(beta-Trcp1) helps to activate the anaphase-promoting complex. , 2003, Molecular cell.

[5]  K. Shigemasa,et al.  Skp2 overexpression is a prognostic factor in patients with ovarian adenocarcinoma. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[6]  K. Nakayama,et al.  A novel route for connexin 43 to inhibit cell proliferation: negative regulation of S-phase kinase-associated protein (Skp 2). , 2003, Cancer research.

[7]  Hong Sun,et al.  Expression of the F-box protein SKP2 induces hyperplasia, dysplasia, and low-grade carcinoma in the mouse prostate. , 2003, Cancer research.

[8]  Akira Kikuchi,et al.  Tumor formation by genetic mutations in the components of the Wnt signaling pathway , 2003, Cancer science.

[9]  R. Hay,et al.  βTrCP-Mediated Proteolysis of NF-κB1 p105 Requires Phosphorylation of p105 Serines 927 and 932 , 2003, Molecular and Cellular Biology.

[10]  T. Minamoto,et al.  Detection of oncogenes in the diagnosis of cancers with active oncogenic signaling , 2002, Expert review of molecular diagnostics.

[11]  B. Leggett,et al.  Emerging concepts in colorectal neoplasia. , 2002, Gastroenterology.

[12]  M. Wolter,et al.  Molecular genetic analysis of malignant melanomas for aberrations of the WNT signaling pathway genes CTNNB1, APC, ICAT and BTRC , 2002, International journal of cancer.

[13]  S. Fuchs.  The role of ubiquitin-proteasome pathway in oncogenic signaling. , 2002, Cancer biology & therapy.

[14]  A. Fong,et al.  Genetic Evidence for the Essential Role of β-Transducin Repeat-containing Protein in the Inducible Processing of NF-κB2/p100* , 2002, The Journal of Biological Chemistry.

[15]  S. Fuchs.  The Role of the Ubiquitin-Proteasome Pathway in Oncogenic , 2002 .

[16]  Kenneth Pienta,et al.  APC/CTNNB1 (β‐catenin) pathway alterations in human prostate cancers , 2002 .

[17]  Z. Ronai,et al.  p53-Mdm2--the affair that never ends. , 2002, Carcinogenesis.

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

[19]  Michael Karin,et al.  NF-κB at the crossroads of life and death , 2002, Nature Immunology.

[20]  M. Mann,et al.  Pseudosubstrate regulation of the SCF(beta-TrCP) ubiquitin ligase by hnRNP-U. , 2002, Genes & development.

[21]  M. Pignatelli,et al.  β-catenin - A linchpin in colorectal carcinogenesis? , 2002 .

[22]  T. Slaga,et al.  Inhibition of HOS expression and activities by Wnt pathway , 2002, Oncogene.

[23]  A. Ciechanover,et al.  Ubiquitin‐mediated degradation of cellular proteins in health and disease , 2002, Hepatology.

[24]  R. Knuechel,et al.  Variable β-catenin expression in colorectal cancers indicates tumor progression driven by the tumor environment , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[25]  A. Duval,et al.  Extensive characterization of genetic alterations in a series of human colorectal cancer cell lines , 2001, Oncogene.

[26]  J. Piette,et al.  MDM2: life without p53. , 2001, Trends in genetics : TIG.

[27]  T. Slaga,et al.  Induction of β-Transducin Repeat-containing Protein by JNK Signaling and Its Role in the Activation of NF-κB* , 2001, The Journal of Biological Chemistry.

[28]  A. Adjei,et al.  Blocking oncogenic Ras signaling for cancer therapy. , 2001, Journal of the National Cancer Institute.

[29]  J. Taipale,et al.  The Hedgehog and Wnt signalling pathways in cancer , 2001, Nature.

[30]  M. Katoh,et al.  Expression profiles of betaTRCP1 and betaTRCP2, and mutation analysis of betaTRCP2 in gastric cancer. , 2001, International journal of oncology.

[31]  Michele Pagano,et al.  Inverse relation between levels of p27Kip1 and of its ubiquitin ligase subunit Skp2 in colorectal carcinomas , 2001, Cancer.

[32]  Tsonwin Hai,et al.  ATF4 Degradation Relies on a Phosphorylation-Dependent Interaction with the SCFβTrCPUbiquitin Ligase , 2001, Molecular and Cellular Biology.

[33]  Alexander Varshavsky,et al.  The ubiquitin system. , 1998, Annual review of biochemistry.

[34]  D. Chung,et al.  The genetic basis of colorectal cancer: insights into critical pathways of tumorigenesis. , 2000, Gastroenterology.

[35]  Shinsei Minoshima,et al.  Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase , 2000, Nature Genetics.

[36]  R. Schmid,et al.  NF-κB/Rel/IκB: Implications in gastrointestinal diseases , 2000 .

[37]  M. Pagano,et al.  Five human genes encoding F-box proteins: chromosome mapping and analysis in human tumors , 2000, Cytogenetic and Genome Research.

[38]  Michele Pagano,et al.  Wnt/β-Catenin Signaling Induces the Expression and Activity of βTrCP Ubiquitin Ligase Receptor , 2000 .

[39]  A. Ciechanover,et al.  Ubiquitin‐mediated proteolysis: biological regulation via destruction , 2000, BioEssays : news and reviews in molecular, cellular and developmental biology.

[40]  H. Pahl.  Activators and target genes of Rel/NF-κB transcription factors , 1999, Oncogene.

[41]  V. Soldatenkov,et al.  Inhibition of homologue of Slimb (HOS) function sensitizes human melanoma cells for apoptosis. , 1999, Cancer research.

[42]  T. Iwama,et al.  Frequent Mutation of β-Catenin and APC Genes in Primary Colorectal Tumors from Patients with Hereditary Nonpolyposis Colorectal Cancer , 1999 .

[43]  Graeme J. Poston,et al.  β‐catenin expression in primary and metastatic colorectal carcinoma , 1999 .

[44]  J. Palazzo,et al.  Frequent loss of expression of the cyclin-dependent kinase inhibitor p27 in epithelial ovarian cancer. , 1999, Cancer research.

[45]  Wilhelm Krek,et al.  p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells , 1999, Nature Cell Biology.

[46]  Michele Pagano,et al.  SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27 , 1999, Nature Cell Biology.

[47]  A. Millar,et al.  Beta-catenin mutations are specific for colorectal carcinomas with microsatellite instability but occur in endometrial carcinomas irrespective of mutator pathway. , 1999, Cancer research.

[48]  Hong Sun,et al.  p27Kip1 ubiquitination and degradation is regulated by the SCFSkp2 complex through phosphorylated Thr187 in p27 , 1999, Current Biology.

[49]  Bruce A. Yankner,et al.  β-Trcp couples β-catenin phosphorylation-degradation and regulates Xenopus axis formation , 1999 .

[50]  K. Milde-Langosch,et al.  Strongly reduced expression of the cell cycle inhibitor p27 in endometrial neoplasia , 1999, Virchows Archiv.

[51]  R. Benarous,et al.  Inducible Degradation of IκBα by the Proteasome Requires Interaction with the F-box Protein h-βTrCP* , 1999, The Journal of Biological Chemistry.

[52]  T. Maniatis,et al.  A ubiquitin ligase complex essential for the NF-kappaB, Wnt/Wingless, and Hedgehog signaling pathways. , 1999, Genes & development.

[53]  R. Benarous,et al.  The F-box protein β-TrCP associates with phosphorylated β-catenin and regulates its activity in the cell , 1999, Current Biology.

[54]  Paul Polakis,et al.  The oncogenic activation of β-catenin , 1999 .

[55]  Stephen J. Elledge,et al.  The SCFβ-TRCP–ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IκBα and β-catenin and stimulates IκBα ubiquitination in vitro , 1999 .

[56]  Gregor Eichele,et al.  Mutation of the Angelman Ubiquitin Ligase in Mice Causes Increased Cytoplasmic p53 and Deficits of Contextual Learning and Long-Term Potentiation , 1998, Neuron.

[57]  Yanping,et al.  Loss of the Cyclin-dependent Kinase Inhibitor 27Kq Protein in Human Prostate Cancer Correlates with Tumor Grade’ , 2005 .

[58]  A. Ciechanover,et al.  Inhibition of NF‐κB cellular function via specific targeting of the IκB‐ubiquitin ligase , 1997 .

[59]  Inder M. Verma,et al.  IκB kinase: Beginning, not the end , 1997 .

[60]  K. Mimori,et al.  p27 expression and gastric carcinoma , 1997, Nature Medicine.

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

[62]  M. Loda,et al.  Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinomas , 1997, Nature Medicine.

[63]  Herman Yeger,et al.  Decreased levels of the cell-cycle inhibitor p27Kip1 protein: Prognostic implications in primary breast cancer , 1997, Nature Medicine.

[64]  James M. Roberts,et al.  Expression of cell-cycle regulators p27Kip1 and cyclin E, alone and in combination, correlate with survival in young breast cancer patients , 1997, Nature Medicine.

[65]  A. Iavarone,et al.  Kip/Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-beta. , 1995, Genes & development.

[66]  C. Thompson,et al.  Apoptosis in the pathogenesis and treatment of disease , 1995, Science.

[67]  L. Houdebine,et al.  An improvement of the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1990, BioTechniques.

[68]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[69]  T. Minamoto,et al.  Oncogenic beta-catenin and MMP-7 (matrilysin) cosegregate in late-stage clinical colon cancer. , 2002, Gastroenterology.

[70]  Ahmedin Jemal,et al.  Cancer Statistics, 2002 , 2002, CA: a cancer journal for clinicians.

[71]  Z. Ronai,et al.  K-ras mutation: early detection in molecular diagnosis and risk assessment of colorectal, pancreas, and lung cancers--a review. , 2000, Cancer detection and prevention.

[72]  T. Minamoto,et al.  Altered expression of p53 and p27 proteins, alone or combined, as a predictor of metastatic potential in early invasive carcinoma of colon and rectum--a comparative clinicopathologic and molecular analysis. , 2000, Cancer Detection and Prevention.

[73]  E. Harlow,et al.  Using Antibodies: A Laboratory Manual , 1999 .

[74]  R. Schmidt,et al.  Simultaneous detection of TDT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive cells and multiple immunohistochemical markers in single tissue sections. , 1995, BioTechniques.