Activation of AXIN2 Expression by β-catenin/TCF: A Feedback ...

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Activation of AXIN2 Expression by β-catenin/TCF: A Feedback Repressor Pathway Regulating Wnt Signaling Janet Y. Leung 2 , Frank T. Kolligs 1 , Rong Wu 3 , Yali Zhai 3 , Rork Kuick 4 , Samir Hanash 4,5 , Kathleen R. Cho 1,3,5 , Eric R. Fearon 1,2,3,5 Departments of Internal Medicine 1 , Human Genetics 2 , Pathology 3 , and Pediatrics 4 and The Cancer Center 5 , University of Michigan Medical School, Ann Arbor, MI 48109-0638 Running Title: AXIN2 is a β-catenin/TCF-regulated target gene Keywords: AXIN2, Conductin, Axil, Wnt signaling, β-catenin, TCF, cancer Abbreviations used: APC, adenomatous polyposis coli; CCND1, cyclin D1 gene; CHX, cycloheximide; ER, estrogen receptor; GAPDH, glyceraldehyde-3- phosphate dehydrogenase; GSK3β, glycogen synthase kinase 3β; MMR, mismatch repair; N-terminal, amino terminal; OEA, ovarian endometriod adenocarcinoma; TCF, T cell factor; 4-OH-T, 4-hydroxy-tamoxifen Correspondence: Eric R. Fearon Division of Medical Genetics University of Michigan Medical Center 4301 MSRB III, Box 0638 1150 W. Medical Center Drive Ann Arbor, MI 48109-0638 Tel. 734-764-1549 FAX 734-647-7979 [email protected] Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc. JBC Papers in Press. Published on April 8, 2002 as Manuscript M200139200 by guest on February 19, 2018 http://www.jbc.org/ Downloaded from

Transcript of Activation of AXIN2 Expression by β-catenin/TCF: A Feedback ...

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Activation of AXIN2 Expression by β-catenin/TCF: A Feedback Repressor Pathway Regulating

Wnt Signaling

Janet Y. Leung2, Frank T. Kolligs1, Rong Wu3, Yali Zhai3, Rork Kuick4, Samir Hanash4,5,

Kathleen R. Cho1,3,5, Eric R. Fearon1,2,3,5

Departments of Internal Medicine1, Human Genetics2, Pathology3, and Pediatrics4 and The

Cancer Center5, University of Michigan Medical School, Ann Arbor, MI 48109-0638

Running Title: AXIN2 is a β-catenin/TCF-regulated target gene

Keywords: AXIN2, Conductin, Axil, Wnt signaling, β-catenin, TCF, cancer

Abbreviations used: APC, adenomatous polyposis coli; CCND1, cyclin D1 gene; CHX,

cycloheximide; ER, estrogen receptor; GAPDH, glyceraldehyde-3-

phosphate dehydrogenase; GSK3β, glycogen synthase kinase 3β; MMR,

mismatch repair; N-terminal, amino terminal; OEA, ovarian endometriod

adenocarcinoma; TCF, T cell factor; 4-OH-T, 4-hydroxy-tamoxifen

Correspondence:Eric R. FearonDivision of Medical GeneticsUniversity of Michigan Medical Center4301 MSRB III, Box 06381150 W. Medical Center DriveAnn Arbor, MI 48109-0638Tel. 734-764-1549FAX [email protected]

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 8, 2002 as Manuscript M200139200 by guest on February 19, 2018

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ABSTRACT

The Wnt pathway regulates cell fate, proliferation and apoptosis, and defects in the pathway play

a key role in many cancers. While Wnts act to stabilize β-catenin levels in the cytosol and

nucleus, a multi-protein complex containing adenomatous polyposis coli (APC), GSK3β, and

Axin1 or its homolog Axin2/Axil/Conductin promotes β-catenins phophorylation and

subsequent proteasomal degradation. We found the rat Axil gene was strongly induced upon

neoplastic transformation of RK3E cells by mutant β-catenin or γ-catenin or following ligand-

induced activation of a β-catenin-estrogen receptor fusion protein. Expression of Wnt1 in

murine breast epithelial cells activated the Conductin gene, and human cancers with defective β-

catenin regulation had elevated AXIN2 gene and protein expression. Expression of AXIN2/Axil

was strongly repressed in cancer cells by restoration of wild type APC function or expression of

a dominant negative form of TCF-4. TCF-binding sites in the AXIN2 promoter played a key

role in β-catenins ability to activate AXIN2 transcription. In contrast to AXIN2/Axil,

expression of human or rat Axin1 homologs was nominally affected by β-catenin/TCF. Because

Axin2 can inhibit β-catenin abundance and function, the data implicate AXIN2 in a negative

feedback pathway regulating Wnt signaling. Additionally, though Axin1 and Axin2 have been

thought to have comparable functions, the observation that Wnt pathway activation elevates

AXIN2 but not AXIN1 expression suggests there may be potentially significant functional

differences between the two proteins.

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INTRODUCTION

The Wnt signaling pathway plays an important role in cellular proliferation,

differentiation and morphogenesis, and control of β-catenin stability is central to Wnt

signaling1-6. In brief, Wnts activate transmembrane frizzled receptors and the disheveled protein, leading

to inhibition of glycogen synthase kinase 3β (GSK3β) activity. Typically, GSK3β, when active

and present in a multi-protein complex containing the APC (adenomatous polyposis coli) tumor

suppressor and Axin1 and/or Axin2 (also known as axil or conductin), can phosphorylate

specific serine and/or threonine residues near the β-catenin amino (N)-terminus6-10. The

phosphorylated forms of β-catenin bind to the F-box protein β-TrCP, a subunit of the SCF-

type E3 ubiquitin ligase complex, resulting in ubiquitination of β-catenin and its ultimate

degradation by the proteasome5,6,11-14. Wnt pathway activation inhibits GSK3β activity,

causing β-catenin to accumulate in the cytoplasm and nucleus, where it can bind to members of

the TCF (T cell factor)/LEF (lymphoid enhancer family) transcription factor family (referred to

hereafter collectively as TCFs)1-5. In the nucleus, TCFs mediate sequence-specific DNA

binding and β-catenin, via its interaction with TCFs, affects transcription of genes with TCF-

binding sites in their regulatory regions. Thus far, it appears that β-catenin generally activates

TCF-regulated genes and suggested β-catenin/TCF target genes include c-MYC, cyclin D1

(CCND1), matrilysin/MMP-7, Tcf-1, PPARδ, PEA3, ENC1, c-ETS2, c-MYB, and c-KIT15-

23.

Defects that interfere with β-catenin regulation have been reported in various human

cancers. In a subset of many different cancer types, mutations at or nearby the serine and

threonine residues in β-catenins N-terminal domain alter its ability to be phosphorylated by

GSK3β4,5. In other cancers, particularly colorectal cancers, inactivation of the APC tumor

suppressor gene appears to be the predominant mechanism leading to β-catenin

deregulation4,5,24. In yet other cancers, mutations in the genes encoding one of the two Axin proteins have bee

reported, including the AXIN1 gene in hepatocellular carcinomas and medulloblastomas25,26

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and the AXIN2 gene in a small fraction of colorectal cancers lacking APC or β-catenin

mutations27. A prime consequence of the mutational defects in β-catenin regulation is

constitutive activation of downstream β-catenin/TCF-regulated target genes, particularly genes

with major effects on cell growth regulation and tumorigenesis, such as c-MYC, CCND1, and

MMP-74,5.

In an effort to better understand the effects of Wnt/β-catenin/TCF pathway activation in

cancer cells, we undertook studies to identify novel β-catenin/TCF-regulated target genes. We

used oligonucleotide microarrays to identify transcripts with elevated expression following

neoplastic transformation of the rat E1A-immortalized RK3E cell line by mutant β-catenin or

γ-catenin or following ligand-induced activation of a β-catenin-estrogen receptor (ER) fusion

protein. We found expression of the rat Axil gene was strongly induced in the RK3E cell line in

all three of these settings. Further studies established the mouse and human homologs of Axil,

known as Conductin and AXIN2, respectively, were consistently induced by Wnt pathway

activation. TCF proteins played a key role in AXIN2 induction. Unlike AXIN2, AXIN1 was not

found to be a β-catenin/TCF-regulated gene. Prior studies have shown the Axin1 and Axin2

proteins have roughly 45% amino acid identity and essentially identical functions in regulating

β-catenin levels7-10,28. In addition to showing that AXIN2 functions in a feedback repressor

pathway regulating Wnt signaling, our findings on the differential effects of Wnt pathway

activation on AXIN2 versus AXIN1 expression suggest potentially significant functional

differences may exist between their protein products.

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EXPERIMENTAL PROCEDURES

Plasmids

Expression vectors for wild type and mutant (codon 33 substitution of tyrosine for serine S33Y)

forms of β-catenin and dominant negative Tcf-4 (Tcf-4∆N31) have previously been

described29. The pBabe-S33Y-ER-puro expression vector encoding a chimeric β-catein/ER protein, in

which full-length S33Y β-catenin sequences are fused in-frame to a mutated ER ligand binding

domain, was generated by cloning the S33Y β-catenin cDNA into the BamHI and EcoRI sites of

the retroviral plasmid pBABE-puro30. The reporter constructs pTOPFLASH, which contains

three copies of an optimal TCF-binding motif (CCTTTGATC), and pFOPFLASH, which

contains three copies of a mutant motif (CCTTTGGCC), have been previously described31.

Plasmid pCH110 (Pharmacia, Piwscataway, N.J.) contains a functional lacZ gene cloned

downstream of a cytomegalovirus early-region promoter-enhancer element. The

Axin2pcDNA3.1mycHis(-)B expression vector was a kind gift from Wanguo Liu (Mayo Clinic,

Rochester, MN)27. DNA fragments containing human AXIN2 promoter sequences cloned

upstream of a luciferase reporter gene were obtained by PCR amplication of genomic DNA,

using primers generated from AXIN2 sequences in GenBank (accession AC00485). AXIN2

genomic DNA fragments were subcloned upstream of the luciferase reporter gene in the

pGL3Basic reporter vector (Promega, Madison, WI), using the KpnI and NheI sites. The

reporter gene vector AX2(1078WT)/Luc contains AXIN2 sequences from -1078 to +5 relative

to the presumed transcription start site, and the vector AX2(181WT)/Luc contains AXIN2

sequences from -181 to +5. The forward primer for generating the AX(1078WT)/Luc vector

was 5’-CCCGTTCAGCCCCTACCCTTCTTAG-3’ and the forward primer for the

AX(181WT)/Luc vector was 5’-CAGCGCCTGATACTTAGATG-AGC-3’; the reverse primer

for generating both vectors was 5’-CAAGTCAGCAGGGGCTCAT-CTG-3’. Mutations in a

presumptive TCF DNA-binding site at basepairs (bp) –108 to -102 were obtained in vitro via a

standard PCR-based mutagenesis strategy, generating the reporter gene vectors

AX2(1078Mut)/Luc and AX2/(181Mut)/Luc. All plasmid sequences were confirmed by

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automated sequencing of double-stranded DNA templates.

Cell Culture

All cell lines were obtained from American Type Culture Collection (Rockville, MD), with the

exception of the following: the amphotropic Phoenix packaging cell line, which was obtained

from G. Nolan (Stanford University School of Medicine); the RAC311, RAC311/Wnt-1,

RAC311/Wnt-1 #9, C57/Vect and C57/Wnt-1 lines32, all of which were obtained from L.

Howe (Weill Medical College of Cornell University); Gli-transformed RK3E cells33, which

were obtained from J.M. Ruppert (University of Alabama at Birmingham); and the HT-29/β-gal

and HT29/APC lines34, which were obtained from B. Vogelstein (Johns Hopkins University

School of Medicine). All cells were grown in 5% C02 with medium containing 10% fetal bovine

serum and penicillin/streptomycin, unless otherwise stated. HEK293, Phoenix, parental RK3E

cells, RK3E cells neoplastically transformed by K-ras, Gli, β-catenin, and γ-catenin29,35,

RAC311 lines, C57 lines, and all human colon cancer lines, except for HT-29, LS174T, RKO

and SW48 cells, were grown in DMEM (Life Technologies, Gaithersburg, MD). LS174T cells

were grown in MEMα (Life Technologies), and SW48 cells were grown in L15 medium (Life

Technologies) in the absence of CO2. RKO, HT29, HT29/β-Gal and HT29/APC cells were

cultured in McCoy’s medium (Life Technologies). Hygromycin B (Sigma, St. Louis, MO) was

included at a concentration of 0.6 mg/ml for the HT29/β-Gal and HT29/ APC cells. Insulin (10

µg/ml; Sigma) was added to the media for the C57 lines. A clonal RK3E cell line expressing the

β-catenin S33Y/ER fusion protein was obtained following retroviral transduction of RK3E cells

with supernatants from amphotrophic Phoenix cells transfected with pBabe-S33Y-ER-puro.

Drug selection on the pBabe-S33Y-ER-puro-transduced RK3E cells was carried out in

puromycin (Sigma, St. Louis, MO) at a concentration of 1.0 µg/ml. A single resistant colony

was isolated by ring cloning and expanded into a stable cell line, termed RK3E/S33Y-ER. The

RK3E/S33Y-ER line was subsequently maintained in 0.5 µg/ml puromycin. To activate the

S33Y-ER fusion protein, the RK3E/S33Y-ER cells were treated with media supplemented with

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0.5 µM 4-OH-tamoxifen (4-OH-T) (Sigma), made from a stock concentration of 100 µM 4-

OH-T in 100% ethanol. To inhibit new protein synthesis in RK3E/S33Y-ER cells, media was

supplemented with cycloheximide (Sigma) at a concentration of 1 µg/ml. To assess effects of

dominant negative TCF-4 on AXIN1 and AXIN2 gene expression, a retroviral TCF-4∆N31

expression construct29 was used to transduce two RK3E lines that had been neoplastically

transformed by mutant β-catenin (RK3E/∆N47-B and RK3E/ ∆N132-A)29 as well as the

SW480 and DLD1 colon cancer lines. Empty vector (pPGS-Neo) control transductions of the

two RK3E and two colon lines were carried out in parallel. The TCF4∆N31- and empty vector-

transduced cells were subsequently selected for 7-10 days in 1.0-1.5 mg/ml G418 (Sigma). To

assess effects of wild type APC gene function on AXIN1 and AXIN2 gene expression, HT29/β-

Gal and HT29/APC cells were treated with 150 µM of ZnCl2 for induction of the control lacZ

and wild type APC genes, respectively.

DNA Array Expression Analysis

Trizol (Life Technologies) extraction and purification with the RNeasy Cleanup Kit (Qiagen,

Chatsworth, CA) was used to prepare total RNA from 5 samples: parental RK3E cells;

RK3E/S33Y-ER cells either mock (ethanol) treated or 4-OH-T-treated for 24 hr; a pool of

equal masses of RNA from 7 clonal RK3E lines neoplastically transformed by mutant β-

catenin29; and a pool of equal masses of RNA from 5 clonal RK3E lines neoplastically transformed by

γ-catenin35. Gene expression analyses on the 5 samples were carried out with commercial high-

density oligonucleotide arrays (Affymetrix, Santa Clara, CA), using protocols and methods

developed by the supplier. Arrays were scanned using the GeneArray scanner (Affymetrix), and

image analysis was performed with GeneChip 4.0 software (Affymetrix), which stores the results

for each feature in .CEL files. Each RG_U34A chip consists of 534 x 534 probes (24 x 24 um

each) that are 25 base long single-stranded DNA sequences. There are typically 16 pairs of

features (probe-pairs) for each of the transcripts (probe-sets), and a total of 8799 probe-sets.

Half of the features are complementary to a specific sequence (perfect match = PM features), the

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other half have a identical except a central base has been altered (mismatch = MM features). We

have developed software to read .CEL files and perform some processing of the data, available

at: http://dot.ped.med.umich.edu:2000/ourimage/pub/shared/Affymethods.html. The chip for the

parental RK3E sample was selected as a standard. Probe-pairs for which PM-MM < -100 on

the standard were excluded from the analysis. One-sided signed-rank tests of the PM-MM

values for each probe-set on each chip were obtained to help judge if transcripts were detectable.

The average intensity for each probe-set was computed as the mean of the PM-MM

differences, after trimming away the 25% highest and lowest differences. A set of 3692

reference probe-sets were selected for use in normalization, these being the probe-sets that gave

p<.05 for all 5 chips for the test of detectability. A normalization factor for each chip was

obtained using the reference probe-sets by computing the anti-logarithm of the mean log ratios

of the average intensities for the selected chip divided by the standard. The average intensities

were divided by this factor to obtain the normalized intensities for the probe-sets. When

computing fold-change indices, we replaced intensities less than 10 by 10 before forming ratios,

in order to avoid negative or spuriously large fold change figures.

Northern Blot Analysis

Total RNA was extracted from cells with Trizol and Northern blot analysis was performed.

Approximately 15-20 ug of total RNA was separated on a 1.2% formaldehyde-agarose gel and

transferred to Zeta-Probe GT membrane (Bio-Rad, Hercules, CA) by capillary action. cDNA

probes to detect rat Axil, mouse Conductin, rat Axin1, human AXIN1 expression were generated

by RT-PCR, using primers derived from sequences in GenBank. The probe to detect AXIN2

was generated by PCR using the Axin2-pcDNA3myc3.1 plasmid (provided by W. Liu; Mayo

Clinic). The sequence of all PCR products probes were confirmed by automated sequencing.

All probes were random-labeled with α32P-dCTP using Rediprime (Amersham Biosciences,

Piscataway, NJ) and hybridized to the membrane with RapidHyb Buffer (Amersham

Biosciences) according to the manufacturer’s protocol. All Northern blots were stripped and

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hybridized to species-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA

probes to control for RNA loading and transfer efficiency.

Western Blot Analysis

Whole cell lysates were prepared in radioimmunoprecipitation assay buffer [Tris-buffered saline

(TBS), 0.5% deoxycholic acid, 0.1% SDS, and 1% NP-40 with Complete protease inhibitors

(Roche Molecular Biochemicals, Indianapolis, IN)]. Protein concentration was determined by

the bicinchoninic acid assay (Pierce Biochemicals, Rockford, IL) and 50 ug of total protein from

each sample was separated on 10% SDS/polyacrylamide gels. Proteins were transferred to

Immobilon P membranes (Millipore, Bedford, MA) by semidry electroblotting. Immunoblot

analyses were carried out with the anti-Conductin (S-19) or anti-Conductin (M-20) goat

polyclonal antibodies (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) at a 1:500 dilution in 1X

TBS with 5% dry milk and 0.5% Tween followed by incubation with a horseradish peroxidase-

conjugated donkey anti-goat antibody (Pierce Biochemicals) at a 1:10,000 dilution. To verify

equal loading of the samples, membranes were incubated with a rabbit polyclonal antibody

against β-actin (Sigma), followed by a horseradish peroxidase-conjugated donkey anti-rabbit

IgG antibody (Pierce Biochemicals). Antibody complexes were detected with the ECL Western

blot kit (Amersham Biosciences) and exposure to X-OMAT-AR film (Kodak, Rochester, NY).

Real-time RT-PCR Analysis of AXIN2 Expression

Total RNA was isolated with Trizol from 42 snap-frozen, primary ovarian endometrioid type

adenocarcinomas (OEAs) that had previously been analyzed in detail for β-catenin nuclear

localization and mutational defects in the β-catenin, APC, AXIN1, and AXIN2 genes36. The

RNA was used for real-time RT-PCR studies of AXIN2 and HPRT gene expression. In brief,

first strand cDNA was synthesized from DNase I treated mRNA samples using random hexamer

primers (Pharmacia Biosciences) and Superscript II (Life Technologies, Inc.). For PCR with a

Prism 7700 Sequence Detector (Applied Biosystems, Foster City, CA), 5 ng of cDNA from each

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tumor sample was used in each reaction. For AXIN2, PCR was performed in 96-well plates in a

25 µl reaction volume containing 1X TaqMan Universal PCR Master Mix (Applied Biosystems),

0.2 µM AXIN2 forward primer (5-CAAGGGCCAGGTCACCAA-3), 0.2 µM reverse AXIN2

primer (5CCCCCAACCCATCTTCGT-3), and 0.2 µM of the dye-labeled AXIN2 probe (5-

CCCATGTCTGTCTCTTCCAACACCAGG-3) (Synthetic Genetics, San Diego, CA). For

HPRT, the 25 µl reaction contained 1X TaqMan Universal PCR Master Mix, 0.2 µM forward HPRT

primer (5-TTCCTCGAGATGTGATGAAGGA -3), 0.2 µM reverse HPRT primer (5-

CCAGCAGGTCAGCAAAGAATT-3), and 0.2 µM of the dye-labeled HPRT probe (5-

CCATCACATTGTAGCCCTCTGTGTGCTC-3) (Applied Biosystems). The AXIN2 probe had

a carboxyfluorescein label at its 5 end and the HPRT probe had a VICTM label at its 5 end. Both

probes had carboxytetramethyl rhodamine labels at their 3’ ends. The AXIN2 and HPRT PCRs

was performed in duplicate for each tumor sample, and AXIN2 and HPRT reactions were

performed in adjacent wells. The following PCR conditions were used: 2 min at 50oC, 10 min at

95oC, followed by 40 cycles of 15 sec at 95oC and 1 min at 60oC. Using the software

accompanying the Prism 7700 detector, the HPRT signals were used for normalization. The

Student’s t test was used to determine the significance of differences in AXIN2 expression

between the 12 OEAs with strong nuclear staining for β-catenin and mutations in the β-catenin,

APC, AXIN1, or AXIN2 genes and the 30 OEAs lacking strong nuclear β-catenin staining and

pathway mutations.

Immunohistochemical Analysis

Immunohistochemcial analysis of AXIN2 expression in OEAs was performed as described

previously36. In brief, 5 µm sections of formalin-fixed, paraffin-embedded tissues were

mounted on Probe-On slides (Fisher Scientific, Hanover Park, IL), deparaffinized in xylene, and

then rehydrated into distilled water through graded alcohols. Antigen retrieval was enhanced by

microwaving the slides in citrate buffer (pH 6.0; Biogenex, San Ramon, CA) for 15 min.

Endogenous peroxidase activity was quenched with 6% hydrogen peroxide in methanol and the

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slides were blocked with 1.5% normal horse serum for 1 hour. Sections were then incubated

with the anti-Conductin (M-20) goat polyclonal antibody (Santa Cruz Biotechnology, Inc.) at a

1:500 dilution overnight at 4 oC, followed by a biotinlylated horse anti-goat secondary antibody

at a 1:200 dilution for 30 min at room temperature. Antigen-antibody complexes were detected

with the avidin-biotin peroxidase method using 3,3’-diaminobenzidine as a chromogenic

substrate (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). Immunostained sections

were lightly counterstained with hematoxylin and then examined by light microscopy.

Luciferase Reporter Gene Assays

For all luciferase reporter assays, cells were plated in 35-mm 6-well plates 12-24 hr prior to

transfection. Transfections were performed with FuGENE6 (Boehringer Mannheim,

Indianapolis, IN) for 24-36 hr according to the manufacturer’s protocol. Lysates were collected

in 1X Reporter Lysis Buffer (Promega, Madison, WI). TCF transcriptional activity was

measured as the ratio of luciferase activity from the pTOPFLASH vector to the pFOPFLASH

vector. All luciferase activities were normalized for transfection efficiency by co-transfection

with pCH110 and measurement of β-galactosidase activity. To assess effects of AXIN2 on wild

type and mutant β-catenin-induced TCF activity, 293 cells were co-transfected with 0.25 µg of

pTOP- or pFOPFLASH, 0.5 µg of a pcDNA3 vector encoding wild type or S33Y mutant β-

catenin29, 1 µg of Axin2pcDNA3.1mycHis(-)B, and 0.25 µg of pCH110. To confirm stable

expression of TCF-4∆N31in β-catenin transformed RK3E cells as well as SW480 and DLD1

cells, cells were co-transfected with 1 µg of pTOPFLASH or pFOPFLASH and 1 µg of

PCH110. For reporter gene assays with AXIN2 promoter constructs, DLD1 cells were co-

transfected with 1 µg of AX2(1078WT)/Luc or AX2(1078Mut)/Luc and 1 µg of pCH110, while

SW480/Neo or SW480/Tcf-4∆N31 cells were co-transfected with 1 µg of AX2(181WT)/Luc or

AX2(181Mut)/Luc and 1 µg of pCH110. The total mass of transfected DNA in each well was

kept constant by adding empty vector plasmid DNA, when necessary. All experiments were

done in triplicate and mean and standard deviation value were determined.

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RESULTS

Induction of Axil Expression by β- or γ-catenin Deregulation or Ligand-induced Activation of a β-

catenin-ER fusion protein

Our prior studies have shown that N-terminal mutant forms of β-catenin akin to those

found in cancers, but not wild type β-catenin, will promote neoplastic transformation of RK3E

cells, a rat E1A-immortalized epithelial cell line29. Unlike β-catenin, its close functional

relative γ-catenin (also known as plakoglobin), will promote neoplastic transformation of RK3E

cells when overexpressed, without a need for N-terminal mutations in the presumptive GSK3β

phosphorylation consensus sites to activate γ-catenins transforming potential35. In an effort to

define novel downstream target genes in the Wnt pathway, we used commercial oligonucleotide

microarrays to identify genes with elevated expression in β- and γ-catenin transformed RK3E

cells compared to parental RK3E cells. Because some observed changes in gene expression in

individual β- and/or γ-catenin transformed RK3E lines might simply reflect the clonal origin of

the transformed line under study, for the array analysis, we pooled equal masses of RNA from 7

independent β-catenin-transformed lines and 5 independent γ-catenin-transformed lines. In

addition, as some changes in gene expression following transformation of RK3E cells by β- or

γ-catenin might be due to alterations in signaling pathways unrelated to catenin/TCF deregulation,

we also assessed the consequences of transient activation of β-catenin. Transient activation of

β-catenin in RK3E cells was achieved by treatment of an RK3E cell line expressing a chimeric β-

catenin-estrogen receptor (ER) fusion protein (RK3E/S33Y-ER) with the ligand 4-hydroxy-

tamoxifen (4-OH-T) for 24 hr. For our studies, we used the Affymetrix U34A rat GeneChip

array, which contains roughly 8,000 known genes and expressed sequence tags. By comparing

gene expression in parental RK3E cells and mock-treated RK3E/S33Y-ER cells (i.e., the two

control cell populations) to gene expression in β- and γ-catenin transformed RK3E and 4-OH-

T-treated RK3E/S33Y-ER cells, we identified only 14 genes predicted to have greater than 2-

fold increases in expression over control levels following catenin/TCF activation.

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Northern blotting was used to assess the 14 candidate genes identified by the array

analysis, and the most promising data were obtained for the rat Axil gene. When compared to

parental RK3E cell lines or RK3E lines transformed by mutated K-ras or Gli1, marked increases

in Axil expression were seen in 8 of 10 independent β-catenin transformed RK3E lines (Fig. 1A)

and all 8 γ-catenin-transformed RK3E lines studied (Fig. 1C). In contrast to the strong

induction of Axil, expression of the rat Axin1 gene was not altered in RK3E lines transformed by

β-catenin (Fig. 1B). Confirmation that induction of Axil gene expression resulted in changes in

expression of the Axil protein was documented in selected β-catenin transformed RK3E lines

(Fig. 1D). Rapid induction of Axil expression was also seen in the RK3E/S33Y-ER cell line

following treatment with the 4-OH-T ligand (Fig. 2A). The observation that the protein

synthesis inhibitor cycloheximide did not block 4-OH-T-mediated induction of Axil

expression in RK3E/S33Y-ER cells (Fig. 2A) indicates Axil is very likely to be a gene directly

activated by β-catenins accumulation in the nucleus. Consistent with the view that Axil is

induced via β-catenins interaction with TCF transcription factors, expression of a dominant

negative form of TCF-4 in RK3E cell lines stably transformed by mutant β-catenin inhibited

TCF transcriptional activity (Fig. 2B) inhibited Axil expression (Fig. 2C).

Axil Homologues in Mouse and Man are Downstream Targets of the Wnt Pathway

In an effort to establish the Wnt pathway regulates expression of Axil or its homologues

in other systems and settings, we analyzed expression of Conductin, the mouse homologue of

Axil, in breast epithelial cells expressing high levels of the Wnt-1 protein. In both RAC311 and

C57 cells, Wnt-1 expression was associated with high levels of Conductin expression (Fig. 3).

As noted above, inactivating mutations in the APC gene are common in human colon

cancers and a subset of the 20-25% of colon cancers that lack APC mutations have gain-of-

function mutations in β-catenins N-terminus4,5. Both the inactivating mutations in APC and

the activating mutations in β-catenin lead to β-catenin deregulation and constitutive activation

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of β-catenin/TCF transcripton. Consistent with the notion that the human AXIN2 gene might

also be a target of the Wnt pathway, we found variable but readily detectable expression of

AXIN2 in all 12 colon cancer cell lines studied (Fig. 4A and data not shown). To further explore

the relationship between β-catenin deregulation and AXIN2 expression in colon cancers, we

took advantage of a colon cancer cell line with regulated expression of the wild type APC gene.

The HT29 colon cancer line has truncating mutations in both of its APC alleles. Morin et al.

generated a variant HT29 line (HT29/APC) in which, following zinc treatment, expression of an

exogenous wild type APC protein is rapidly induced to roughly the same level as that of the

endogenous truncated APC proteins34. Using HT29/APC cells and a matched control line (i.e.,

HT29/β-Gal), we found AXIN2 expression was strongly downregulated following APC

induction. Zinc treatment of the control HT29/β-gal cell line had no detectable effect on AXIN2

expression. In contrast to the AXIN2 results, restoration of APC function in HT29 cells had only

modest effects on AXIN1 expression. Furthermore, expression of a dominant negative form of

TCF-4 in DLD1 and SW480 colon cancer cells strongly inhibited TCF transcriptional activity

and AXIN2 expression, but had at best minimal effects on AXIN1 expression (Fig. 5).

Nearly all candidate β-catenin/TCF-regulated genes described in the literature have been

proposed based on data from in vitro and/or animal model studies. Thus far, few studies have

evaluated expression of presumptive β-catenin/TCF target genes in primary human tumors that

have been thoroughly characterized for mutational defects in β-catenin regulation. We choose

to assess AXIN2 expression in primary ovarian endometrioid adenocarcinomas (OEAs), because

while OEAs share similar histological features, only about 30-40% of the lesions have

mutational defects affecting β-catenin regulation36-38. This contrasts with the picture in

primary colorectal carcinomas which almost uniformly carry mutational defects in β-catenin

regulation4,5. Hence, comparision of gene expression in OEAs with intact β-catenin regulation

versus OEAs with defective β-catenin regulation should permit a more definitive assessment to

be made about the relationship between β-catenin regulatory defects and expression of candidate

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β-catenin/TCF target genes. Using real time RT-PCR assays to assess AXIN2 expression in a

panel of 42 OEAs previously characterized for β-catenin nuclear localization and mutations in

the β-catenin, APC, AXIN1, and AXIN2 genes36, we found AXIN2 expression was on average

roughly 20-fold higher in the OEAs with β-catenin regulatory defects than in OEAs with

apparently intact β-catenin regulation (Fig. 6). To confirm induction of AXIN2 gene expression

resulted in demonstrable changes in AXIN2 protein expression in primary tumors with β-catenin

defects, we performed immunohistochemistry studies on a subset of the OEAs analyzed in the

real time RT-PCR studies. AXIN2 expression was found to be increased in the majority of

OEAs with β-catenin regulatory defects compared to those OEAs with intact β-catenin

regulation (examples in Figure 7).

Critical Role of TCF Binding Sites in AXIN2 Proximal Promoter in β-catenin-mediated

Induction

To further establish the role of TCFs in regulating AXIN2 expression, we examined

AXIN2 genomic sequences for candidate TCF-binding sites. The only consensus TCF binding site

identified in a search of sequences located from –1500 bp to +500 bp (relative to the presumed

transcriptional start site) was found at –108 to –102 (i.e., CTTTGAT; Fig. 8A). Luciferase

reporter gene constructs containing this element as well as reporter gene constructs in which the

element was mutated to CTTTGGC were generated. In DLD1 and SW480 colon cancer cells,

we found that mutation of the consensus TCF site in the AXIN2 promoter markedly decreased

the activity of a reporter construct containing roughly 1.0 kb of AXIN2 promoter sequence (Fig.

8B and data not shown). Similar results were obtained in DLD1 and SW480 colon cancer cells

with wild type and mutant reporter gene constructs containing only 181 bp of AXIN2 promoter

sequences (Fig. 8B and data not shown). Moreover, while expression of a dominant negative

TCF-4 mutant protein (dnTCF-4) inhibited the activity of the wild type AXIN2 reporter

construct, the dnTCF-4 protein had no major effect on the activity of the reporter gene construct

harboring mutations in the consensus TCF binding site (Fig. 8B). Interestingly, co-transfection

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experiments in HEK293, COS, and Hela cells, in which an expression vector encoding the S33Y

mutant β-catenin protein was co-transfected with the AXIN2 reporter gene constructs revealed

that β-catenin was not sufficient on its own for activation of AXIN2 transcription via the

proximal TCF element1. The differing results in colon cancer versus other cell lines suggest that

cellular context, perhaps including the expression of other cellular proteins, may play a role in β-

catenins ability to activate AXIN2 transcription (see Discussion).

The Axin2 Protein Regulates Wild Type But Not Mutant β-catenin

Prior work has shown the rat Axil and mouse Conductin proteins can negatively regulate

Wnt signaling perhaps in large part as a result of the ability of Axil/Conductin to serve as a

scaffold for efficient coordination of the interactions of GSK3β, APC, and β-catenin, resulting

in β-catenins phosphorylation at critical N-terminal sites10,28. To confirm that the human

Axin2 protein had analogous function, we assessed its ability to antagonize β-catenins effects on

TCF transcription. As shown in Figure 9, while the ability of wild type β-catenin to activate

TCF transcription was strongly inhibited by Axin2, the S33Y mutant form of β-catenin was not

significantly inhibited by Axin2. There findings indicate the Axin2 protein is, as expected, a

negative regulator of wild type β-catenin and Wnt signaling.

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DISCUSSION

The critical role of the Wnt pathway in development has long been appreciated.

Nevertheless, only in the recent past has it become abundantly clear that mutations in Wnt

pathway components play a prominent role in the pathogenesis of a rather broad array of human

cancers3-5. A principal effect of the loss-of-function mutations in APC or the gain-of-

function mutations in β-catenin is to elevate β-catenin levels in the cytoplasm and nucleus. As

a result of its deregulation, β-catenins ability to complex with TCFs is enhanced and altered

transcription of TCF-regulated genes ensues. Thus far, it appears that activation of β-

catenin/TCF-regulated target genes is a major consequence following Wnt pathway deregulation

in cancer. Candidate β-catenin/TCF target genes described in the literature include c-MYC,

CCND1, MMP-7, Tcf-1, PPARδ, PEA3, ENC1, c-ETS2, c-MYB, and c-KIT15-23.

In this manuscript, we have presented a substantial body of data implicating the human

AXIN2 gene (and the rat Axil and mouse Conductin genes) as a downstream target of the Wnt/β-

catenin/TCF pathway. Findings consistent with those we report here on AXIN2/Conductin/Axil

expression and its regulation by the Wnt pathway were recently published by others39-41. We

initially found the rat Axil gene was strongly induced upon neoplastic transformation of RK3E

cells by mutant β-catenin or γ-catenin or following 4-OH-T-induced activation of a β-

catenin-estrogen receptor fusion protein. In murine breast epithelial cells, we found

overexpression of Wnt1 strongly activated the Conductin gene. Human colon cancer cell lines

had elevated AXIN2 expression and restoration of APC function or expression of a dominant

negative form of TCF-4 in the cells strongly inhibited AXIN2 expression. Primary ovarian

carcinomas with defective β-catenin regulation were found to have elevated AXIN2 gene and

protein expression when compared to a similar cohort of ovarian carcinomas with intact β-

catenin regulation.

Consistent with the notion that the AXIN2/Axil/Conduction genes are directly activated

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as a result of binding of the β-catenin/TCF protein complex to regulatory elements within or

nearby the genes, we found Axil was robustly activated by β-catenin in the absence of new

protein synthesis. Use of reporter gene constructs containing proximal promoter sequences from

the AXIN2 gene established the ability of β-catenin to activate AXIN2 transcription as well as

the key role of TCFs in AXIN2 activation. While our findings indicate β-catenin and TCFs play

a vital role in the activation of AXIN2 expression in colon and ovarian cancer cells, our

observation that the activity of the AXIN2 proximal promoter was not demonstrably affected by

β-catenin in several other epithelial cell types, namely HEK293, COS, and HeLa cells1, suggests

the regulation of AXIN2 transcription by β-catenin/TCF may be complex. For example, it is

possible that only certain TCF isoforms may bind to and regulate the AXIN2 promoter and these

TCF isoforms display tissue- or cell type-restricted patterns of expression. Alternatively, other

transcription factors that bind to specific sites in the AXIN2 promoter may play a key role in

cooperating with β-catenin/TCF to activate TCF transcription. Prior studies have suggested that

cooperation between β-catenin/TCF and other transcription factors may be important for

activation of certain genes, such the cooperation between β-catenin/TCF and PEA3 in the

activation of MMP-742.

In light of prior data in the literature and the data presented here showing that Axin2 can

negatively regulate β-catenin function, our findings imply AXIN2 is a negative feedback

regulator of the Wnt pathway. Interestingly, despite the fact that the Axin1 and Axin2 proteins

appear to have similar functions in negatively regulating β-catenin levels, via the ability of the

Axins to complex GSK3β, APC, and β-catenin, we obtained no clear-cut evidence the human

AXIN1 gene or its rat homolog rAxin1 were induced by Wnt pathway activation. The differential

effects of the Wnt pathway on AXIN1 and AXIN2 suggests there may be potentially important

functional differences between the proteins. For instance, while the two proteins share roughly

45% amino acid identity, they may differ in their ability to interact with other cellular proteins.

Thus far, the Axin1 protein has been shown to bind to multiple other proteins besides Wnt

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pathway factors (i.e., APC, GSK3β, β-catenin, disheveled). These other Axin1-interacting

proteins include the following: the mitogen activated protein kinase (MAPK) kinase kinase

(MEKK1) protein43; the GSK3β binding protein (GBP)44; the PR61β and PR61γ regulatory

subunits of protein phosphatase 2A45,46; the low-density lipoprotein receptor-related protein-5

(LRP-5)47, which function as a Wnt co-receptor; the transforming growth factor-β pathway

transcription factor Smad348; and a novel protein termed Axam49. Given the apparently large

number of Axin1-interacting proteins, if Axin1 expression was strongly induced by Wnt

pathway activation, there might be significant effects on many other signaling pathways besides

the Wnt pathway. Thus far, it is not clear whether the Axin2 protein binds any or all of these

other Axin1-interacting proteins. However, some of the interactions between Axin1 and the

non-Wnt pathway interacting proteins are mediated via regions that are not highly conserved

between Axin1 and Axin2. As such, perhaps the differential interactions of Axin1 and Axin2

with certain of the non-Wnt pathway proteins, accounts for why Axin2 function as a major

negative feedback regulator of Wnt signaling and Axin1 does not.

While bi-allelic inactivation of AXIN1 has been seen in some hepatocellular carcinomas

and medulloblastomas25,26, indicating that AXIN1 functions as a tumor suppressor gene, bi-

allelic inactivation of AXIN2 in cancers has not yet been noted. To date, mutations in the

AXIN2 gene appear to be restricted to colon and perhaps other cancers with mismatch repair

(MMR) pathway defects27,36. The truncated AXIN2 alleles seen in cancers with MMR defects

have been proposed to encode proteins that function in a dominant negative fashion to interfere

with β-catenin regulation27. Because the ability of Axin2 to regulate β-catenin appears to

depend upon intact APC function and wild type β-catenin N-terminal sequences, in those

cancers with inactivating mutations in APC or oncogenic mutations in β-catenin, elevated Axin2

expression is quite unlikely to have any major inhibitory effect on β-catenin levels and function.

Even in cancers with AXIN1 or AXIN2 mutations, because the Axin proteins have been

suggested to dimerize28,50, it is possible that β-catenin cannot be downregulated by induction

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of AXIN2, because wild type function of both the Axin1 and Axin2 proteins is required. In light

of the observations indicating that the elevated expression of AXIN2 in cancers with Wnt

pathway defects is not sufficient to downregulate β-catenin levels and function, it is possible that

Axin2s induction might have other important effects in cancer cells, potentially even growth

promoting effects. Further studies of the interactions between Axin2 and other cellular proteins

should offer insights into Axin2’s function, as well as the consequences of its induction by Wnt

pathway activation in normal and cancer cells.

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ACKNOWLEDGEMENTS

This work was supported by NIH grants CA85463, CA84953, and DK58771. The following

investigators generously provided plasmid and cell line reagents using in the studies described

here: G. Nolan, B. Vogelstein, W. Liu, L. Howe, and J.M. Ruppert.

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FOOTNOTES

1 Leung, J. Y., and Fearon, E. R. Unpublished observations.

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FIGURE LEGENDS

Figure 1. Expression of the rat Axil and Axin1 (rAxin1) genes and Axil protein in β- and γ-

catenin transformed RK3E cell lines. Northern blot analysis of Axil (panel A) and rAxin1 (panel

B) were carried out on total RNA isolated from parental rat E1A-immortalized RK3E epithelial

cells (RK3E), RK3E cells transformed by codon 12 mutant K-ras (RK3E/Kras) and Gli1

(RK3E/Gli), as well as 10 independent clonal RK3E lines transformed by mutant β-catenin

proteins29. In panel C, Northern blot analysis of Axil was carried out on parental RK3E cells

and 8 independent clonal γ-catenin transformed RK3E cell lines35. Northern blots in panels A-

C were stripped and rehybridized to a rat Gapdh probe to control for loading. In panel D,

Western blot analyses of Axil protein expression were carried out on parental RK3E cells and 2

independent clonal β-catenin-transformed RK3E lines, using the S-19 (left) and M-20 (right)

goat polyclonal antibodies raised against amino-terminal mouse Conductin sequences. A single

band of roughly 97 kDa was detected with the S-19 antibody (left) and the larger of the two

bands detected with the M-20 antibody (right) in the β-catenin-transformed RK3E lines (right)

migrated at approximately 97 kDa. Blots were stripped and incubated with an anti-actin

antibody to confirm equal loading of protein samples.

Figure 2. Role of β-catenin and TCFs in activation of Axil expression. (A) Northern blot

analysis of Axil expression in a clonal RK3E cell line that stably expresses a β-catenin-estrogen

receptor (ER) fusion protein (RK3E/S33Y-ER), following 4-OH-tamoxifen (4-OH-T)-

induced activation. Total RNA was collected from the RK3E/S33Y-ER cells at various time

points following mock treatment, treatment with 4-OH-tamoxifen (4-OH-T), or treatment with

4-OH-T and cycloheximide (CHX). Expression of Axil in a clonal RK3E line stably

transformed by mutant β-catenin (RK3E/S33Y-A) is shown in the lane at the far right. The blot

was stripped and rehybridized to a control rat Gapdh probe to control for loading. Inhibition of

TCF transcriptional activity (panel B) and Axil expression (panel C) in β-catenin-transformed

RK3E cell lines that express a dominant negative mutant form of TCF-4 (dnTCF-4; i.e., TCF-

-catenin/TCF-regulated target geneβ is a AXIN2Leung et al.

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4∆N31). The two β-catenin transformed RK3E lines, RK3E/∆N47-B and RK3E/∆N132-A, were

stably transduced with the empty pPGS-Neo retroviral vector or the pPGS-Neo vector

expressing dnTCF-4. In panel B, following drug selection, TCF transcriptional assay was

assessed by transient transfection of the cells with the reporter gene vectors TOPFLASH and

FOPFLASH. Luciferase activities were measured in triplicate and the ratio of luciferase

activities in TOPFLASH-transfected versus FOPFLASH-transfected cells was determined and

reported as the relative TCF activity. The control β-galactosidase-expressing vector pCH110

was used to correct for differences in transfection efficiency. In panel C, Axil expression was

analyzed by Northern blot analysis of total RNA from the cell lines, and a rat Gapdh probe was

used to control for RNA loading and transfer.

Figure 3. Wnt-1 induced activation of Conductin in murine breast epithelial cells. Northern

analysis of Conductin was carried out on total RNA isolated from the following: parental

RAC311 cells; polyclonal populations of RAC311 cells transduced with empty retroviral

expression vector (RAC311/Vect only) or a vector encoding Wnt-1 (RAC311/Wnt-1); a clonal

RAC311 line selected for morphological transformation and high Wnt-1 expression

(RAC311/Wnt-1 #9); and polyclonal populations of C57MG cells transduced with an empty

retroviral expression vector (C57/Vect only) or a vector encoding Wnt-1 (C57/Wnt-1). The blot

was stripped and rehybridized to a control mouse Gapdh probe to control for loading and transfer

efficiency. All cell lines were previously described32.

Figure 4. Expression of AXIN2 in human colon cancer cells and its regulation by APC function.

(A) AXIN2 expression in the indicated human colon cancer cell lines was assessed by Northern

blot analysis. (B) Restoration of wild type APC expression in the HT-29 colon cancer cell line

represses AXIN2 expression, but not AXIN1expression. Northern blot analysis was performed

on RNA isolated from an HT29 cell line that displays ZnCl2-inducible wild type APC

expression (HT-29/APC) and a control HT-29 line with ZnCl2-inducible β-galactosidase

-catenin/TCF-regulated target geneβ is a AXIN2Leung et al.

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expression (HT-29/β-gal). The RNA was isolated prior to ZnCl2 treatment (0 hr) or following

various exposure times (6, 12, 18 hr). Following hybridization to the AXIN2 and AXIN1

probes, the blots were stripped and rehybridized to a human GAPDH probe to control for loading

and transfer.

Figure 5. Expression of dominant negative TCF-4 in human colon cancer cell lines inhibits TCF

transcription activity and AXIN2 expression. (A) The DLD1 and SW480 colon cancer cell lines

were stably transduced with the empty pPGS-Neo retroviral vector or a vector expressing

dnTCF-4. TCF transcriptional assay was assessed by transient transfection of the cells with the

reporter gene vectors TOPFLASH and FOPFLASH. Luciferase activities were measured in

triplicate and the ratio of luciferase activity in TOPFLASH-transfected versus FOPFLASH-

transfected cells was determined and reported as the relative TCF activity. The control β-

galactosidase-expressing vector pCH110 was used to correct for differences in transfection

efficiency. (B) AXIN2 and AXIN1 expression were analyzed by Northern blot analysis of total

RNA from the cell lines, and human GAPDH probe used to control for RNA loading and

transfer.

Figure 6. AXIN2 expression is markedly increased in ovarian endometriod adenocarcinomas

(OEAs) with nuclear β-catenin localization compared to OEAs with non-nuclear β-catenin

localization. cDNA preparations from 42 snap-frozen OEA specimens that had been previously

studied for β-catenin immunohistochemistry and mutations in critical Wnt pathway components

(β-catenin, APC, AXIN1, and AXIN2)36 were subjected to quantitative real-time (TaqMan)

analysis of AXIN2 expression, using primer pairs and flourescent probes for AXIN2 and HPRT

described in Materials and Methods. Using HPRT to normalize, the relative AXIN2

fluorescence of the 12 samples with strong nuclear staining for β-catenin and mutations in the

β-catenin, APC, AXIN1, or AXIN2 genes was compared to the relative fluorescence of the 30

OEAs lacking strong nuclear β-catenin staining and pathway mutations. The Students t test was

-catenin/TCF-regulated target geneβ is a AXIN2Leung et al.

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used to determine the significance of differences in AXIN2 expression between the two groups.

Figure 7. Immunohistochemical staining reveals elevated AXIN2 expression in OEAs with β-

catenin regulatory defects compared to OEAs with intact β-catenin regulation. OEA specimens

that had been previously studied for β-catenin immunohistochemistry and mutations in critical

Wnt pathway components (β-catenin, APC, AXIN1, and AXIN2)36 were used for AXIN2

immunohistochemistry studies. Representative photomicrographs of the staining seen in OEA

specimens with intact β-catenin regulation (panels A-D) and OEAs with defective β-catenin

regulation (panels E-H) are shown.

Figure 8. Critical role of a TCF site in proximal AXIN2 promoter in regulating transcriptional

activity in colon cancer cell lines. (A) Schematic representation of AXIN2 reporter gene

constructs. The AX2(1078WT)/Luc reporter vector contains AXIN2 sequences from -1078 to

+5 relative to the presumptive transcription start site and the vector AX2(181WT)/Luc contains

AXIN2 sequences from -181 to +5. The AX2(1078Mut)/Luc and AX2(181Mut)/Luc vectors

carry mutations in the TCF consensus element. (B) Effects of mutations and dominant negative

TCF-4 (dnTCF-4) on the activity of AXIN2 reporter gene vectors in colon cancer cells. DLD1

(left) and SW480 (right) cells were transfected with the indicated reporter gene constructs and

luciferase activity was measured. In the case of experiments with SW480 cells, the luciferase

constructs were co-transfected with either an empty expression vector or the vector encoding

dnTCF-4. The assays were performed in triplicate, mean and standard deviation values are

shown, and the control β-galactosidase-expressing vector pCH110 was used to correct for

differences in transfection efficiency.

Figure 9. Axin2 can inhibit the activity of wild type β-catenin but not an N-terminal mutant

(S33Y) form of β-catenin. HEK293 cells were co-transfected with the indicated pcDNA3

expression vectors and the TOPFLASH or FOPFLASH flash reporter construct. Equal masses of

-catenin/TCF-regulated target geneβ is a AXIN2Leung et al.

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DNA were used in each transfection, luciferase activities were measured in triplicate, and the

ratio of luciferase activities in TOPFLASH-transfected versus FOPFLASH-transfected cells

was determined and reported as the relative TCF activity. The control β-galactosidase-

expressing vector pCH110 was used to correct for differences in transfection efficiency.

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- RK3E- RK3E/Kras- RK3E/Gli- RK3E/S33Y-A- RK3E/S33Y-B- RK3E/S33Y-C- RK3E/S33Y-D- RK3E/rN47-A- RK3E/rN47-B- RK3E/rN89-A- RK3E/rN89-B- RK3E/rN132-A- RK3E/rN132-B

- RK3E- RK3E/Kras- RK3E/Gli- RK3E/S33Y-A- RK3E/S33Y-B- RK3E/S33Y-C- RK3E/S33Y-D- RK3E/rN47-A- RK3E/rN47-B- RK3E/rN89-A- RK3E/rN89-B- RK3E/rN132-A- RK3E/rN132-B

- RK3E- RK3E/WTγ-A- RK3E/WTγ-B- RK3E/WTγ-C- RK3E/S28Lγ-A- RK3E/S28Lγ-C- RK3E/rN38γ-A- RK3E/rN38γ-B- RK3E/rN38γ-C

Axil

Gapdh

rAxin1

Gapdh

Axil

Gapdh

Figure 1

AB

C

- RK3E

- RK3E/S33Y-A

- RK3E/S33Y-B

- RK3E

- RK3E/S33Y-A

- RK3E/S33Y-B

D

Axil

β−Actin

Axil

β−Actin

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- 0

- 12

- 24

- 48

- 0

- 12

- 24

- 48

- 0

- 12

- 24

- R

K3E

/S33

YA

Time (hr):

Axil

Gapdh

Rel

ativ

e T

CF

Act

ivity

VectOnly

+dnTCF-4

VectOnly

+dnTCF-4

RK3E/rN47-B RK3E/rN132-A

Mock 4-OH-T

4-OH-T + CHX

VectOnly

+dnTCF-4

VectOnly

+dnTCF-4

Figure 2

A

B C

RK3E/rN47-B RK3E/rN132-A

Axil

Gapdh

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- Rac311

- Rac311/Vect only

- Rac311/Wnt-1

- Rac311/Wnt-1 #9

- C57/Vect only

- C57/Wnt-1

Conductin

Gapdh

Figure 3

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- DLD1

- Caco2

- HCT116

- LoVo

- HT-29

- LS174T

- SW48

- SW116

- SW480

- SW1463

- SW837

- WIDR

- HT-29/β-gal- HT-29/APC- HT-29/β-gal- HT-29/APC- HT-29/β-gal- HT-29/APC- HT-29/β-gal- HT-29/APC

Tim

e (hr)follow

ing ZnC

l2:0

612

18

Figure 4

AB

- HT-29/β-gal- HT-29/APC- HT-29/β-gal- HT-29/APC- HT-29/β-gal- HT-29/APC- HT-29/β-gal- HT-29/APC

Tim

e (hr)follow

ing ZnC

l2:

AX

IN2

GA

PD

H

AX

IN2

GA

PD

H

AX

IN1

GA

PD

H

06

1218

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VectOnly

+dnTCF-4

VectOnly

+dnTCF-4

VectOnly

+dnTCF-4

VectOnly

+dnTCF-4

Rel

ativ

e TC

F A

ctiv

ity

DLD1 SW480

A

Figure 5

DLD1 SW480

AXIN2

AXIN1

GAPDH

B

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Nuclear β-catenin localization (N = 12)

Non-nuclear β-catenin localization (N = 30)

p = 0.0000146

Figure 6

Rel

ativ

e A

XIN

2 Fl

oure

scen

ce

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A B

C D

E F

G H

Figure 7

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Rel

ativ

e lig

ht u

nits

Rel

ativ

e lig

ht u

nits

p

GL

3-B

asic

AX

2(10

78W

T)/

Luc

AX

2(10

78M

ut)/

Luc

pG

L3-

Bas

ic

AX

2(18

1WT

)/L

uc

AX

2(18

1Mut

)/L

uc

Luciferase

rCTTTGAT

NheIKpn I

AX2(1078WT)/Luc

_ _-108 -102

-1078

Luciferase

irCTTTGGC

AX2(1078MUT)/Luc

-1078

Luciferase

rCTTTGGC

AX2(1078WT)/Luc

Luciferaser

AX2(1078MUT)/Luc

A

B

Figure 8

ii

iCTTTGGC-1078

-1078

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Relative TCF Activity

Relative TCF Activity

Control

WTβ-CAT

Control

S33Yβ-CAT

WTβ-CAT + AXIN2

S33Yβ-CAT +AXIN2

Figure 9

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Page 42: Activation of AXIN2 Expression by β-catenin/TCF: A Feedback ...

Kathleen R. Cho and Eric R. FearonJanet Y. Leung, Frank T. Kolligs, Rong Wu, Yali Zhai, Rork Kuick, Samir Hanash,

regulating Wnt signalingActivation of AXIN2 expression by beta-catenin/TCF: A feedback repressor pathway

published online April 8, 2002J. Biol. Chem. 

  10.1074/jbc.M200139200Access the most updated version of this article at doi:

 Alerts:

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