Skip Navigation


Carcinogenesis Advance Access originally published online on January 19, 2008
Carcinogenesis 2008 29(3):666-672; doi:10.1093/carcin/bgn001
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
29/3/666    most recent
bgn001v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Oyama, T.
Right arrow Articles by Mori, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oyama, T.
Right arrow Articles by Mori, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Further upregulation of β-catenin/Tcf transcription is involved in the development of macroscopic tumors in the colon of ApcMin/+ mice

Takeru Oyama, Yasuhiro Yamada*, Kazuya Hata, Hiroyuki Tomita, Akihiro Hirata1, HongQiang Sheng, Akira Hara, Hitomi Aoki2, Takahiro Kunisada2, Satoshi Yamashita3 and Hideki Mori

Department of Tumor Pathology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan
1 Division of Animal Experiment, Life Science Research Center, Gifu University, Yanagido 1-1, Gifu, 501-1193, Japan
2 Department of Tissue and Organ Development, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
3 Carcinogenesis Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, Japan

* To whom correspondence should be addressed. Email: y-yamada{at}gifu-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
ApcMin/+ mouse, a mouse model for human familial adenomatosis polyposis, contains a truncating mutation in the Apc gene and spontaneously develops intestinal tumors. Our previous study revealed two distinct stages of tumorigenesis in the colon of ApcMin/+ mouse: microadenomas and macroscopic tumors. Microadenomas already have lost their remaining allele of the Apc and all microadenomas show accumulation of β-catenin, indicating that activation of the canonical Wnt pathway is an initiating event in the tumorigenesis. This study shows that expression of nuclear β-catenin in macroscopic tumors is further upregulated in comparison with that in microadenomas. Furthermore, transcriptional activity of β-catenin/T-cell factor (Tcf) signaling, assessed using β-catenin/Tcf reporter transgenic mice, is higher in the macroscopic tumors than that in microadenomas. In addition, the expression level of Dickkopf-1, which is known to be a negative modifier of the canonical Wnt pathway, was reduced only in colon tumors. These results suggest that activation of β-catenin/Tcf transcription plays a role not only in the initiation stage but also in the promotion stage of colon carcinogenesis in ApcMin/+ mice.

Abbreviations: Dkk1, Dickkopf-1; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RT–PCR, reverse transcription—polymerase chain reaction; Tcf, T-cell factor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Colorectal carcinogenesis is a multistep process (1). In humans, APC, KRAS oncogene and p53 genes are thought to play important roles at different stages of colorectal carcinogenesis (2,3). Of these, mutations in the APC gene found in the earliest stages of the adenoma–carcinoma sequence are recognized to play a gate-keeping role in tumor formation and progression (4). Moreover, germ line mutations in the APC gene are known to be responsible for familial adenomatous polyposis, a dominantly inherited autosomal condition characterized by the formation of multiple colonic adenomatous polyps with a high likelihood to develop colon carcinomas (5,6).

A mutant mouse lineage predisposed to Min is regarded as one of the models for colorectal tumorigenesis (7). Originally, this lineage was established from an ethylnitrosourea-treated C57BL/6J mouse. The dominant mutation is known to be located in Apc, the mouse homologue of the human APC gene, resulting in truncation of the gene product at amino acid 850 (8). ApcMin/+ mice develop multiple intestinal neoplasia in the intestinal tracts. Therefore, APC is now regarded as a tumor suppressor gene, and inactivation of both alleles is considered to be necessary for tumor formation (9). In mice heterozygous for a mutant allele of Apc, the loss of Apc function occurs almost exclusively by LOH (10,11).

ApcMin/+ mice have been reported to develop intestinal tumors primarily in their small intestine and only a few tumors arise in the colon (12). Our previous study revealed the presence of a number of intramucosal microadenomas (~100 microadenomas) in the colon of ApcMin/+ mice (12). In that study, microadenomas in the colon of the ApcMin/+ mice consisted of a few dysplastic crypts (12). Based on the multistep carcinogenesis theory in the colon (2–4), such findings suggested that there are, at least, two distinct stages for colon tumorigenesis in ApcMin/+ mice. Importantly, such microadenomas in the colon were found to have lost the remaining allele of Apc, thus indicating that a loss of the Apc function has already occurred in such crypts (12–14). In agreement with the presence of Apc LOH, the accumulation of β-catenin is observed in all microadenomas in the colon of the ApcMin/+ mice (12). These findings suggest that activation of the Wnt signaling pathway by Apc LOH is the initiating event in the carcinogenesis but is not sufficient for the development of macroscopic tumors.

It is not clear at this time which event is required for the development of macroscopic tumors in the colon of ApcMin/+ mice. Although it has been reported that K-ras, p53 and B-raf genes are sometimes mutated in colorectal cancers and such alterations are expected to cause progression of colon carcinogenesis (2), no genetic alterations of these genes are detected in colon tumors of the ApcMin/+ mice model (unpublished data).

The purpose of this study was to identify alterations that are responsible for the development of macroscopic tumors in the colon of ApcMin/+ mice. It is possible that the increased expression of the canonical Wnt pathway, accompanied by an increased nuclear β-catenin level and a decreased Dickkopf-1 (Dkk1) expression, is associated with the development of macroscopic tumors.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Animal maintenance and treatments
ApcMin/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). They were bred and maintained in a pathogen-free animal facility under standard 12:12 h light:dark cycle and fed on a basal diet, CE-2 (CLEA Japan, Tokyo, Japan), and water ad libitum until termination of the study (12,15).

Dextran sodium sulfate treatment
Dextran sodium sulfate (DSS) with a molecular weight of 36 000–50 000 (ICN Biochemicals) was dissolved in distilled water at a concentration of 2% (wt/vol). Eight male ApcMin/+ mice were divided into experimental and control groups. The animals of experimental groups were administered 2% (wt/vol) DSS in drinking water for 1 week from 5 weeks of age. The dose was determined based on the results of previous studies (16).

β-Catenin/T-cell factor reporter transgenic mice, ApcMin/+: β-catenin/T-cell factor reporter mice
For the generation of β-catenin/T-cell factor (Tcf) reporter mice, the Tcf-binding site and intron were polymerase chain reaction (PCR) amplified and cloned using a pTOPFLASH plasmid (Upstate Biotechnology, Lake Placid, NY) and CDM8 into a pEGFP-N3 plasmid (Clontech Japan, Tokyo, Japan) (3,17). The gene construct was excised from the vector and the fragment was microinjected into the pronuclei of fertilized C57BL/6 mouse eggs. Male β-catenin/Tcf reporter transgenic mice (strain C57BL/6) were bred with C57BL/6 females to produce transgenic mice on a C57BL/6 background. Genotyping was done using DNA extracted from tail biopsies of 3- to 4-week-old pups, and new breeding harems of 5- to 6-week-old mice were established to expand the β-catenin/Tcf reporter population before initiating separate crosses of the mice with the ApcMin/+ mice. The offspring from these crosses yielded β-catenin/Tcf reporter +/–:ApcMin/+ mice.

Flow cytometry
Splenocytes of β-catenin/Tcf reporter mice were dissected and dissociated with phosphate-buffered saline (PBS). They were plated at a density of 106 cells/ml and maintained for 24 h in Dulbecco’s modified Eagle’s medium with 50 mmol/l LiCl, 50 mmol/l NaCl or H2O only for controls (18,19). After 24 h, the cells were washed and resuspended in PBS containing 3% fetal calf serum (staining medium). Cells were filtered through nylon mesh to remove large clumps, washed and resuspended in staining medium containing 0.5 µl/ml propidium iodide (Calbiochem-Novabiochem Corp., San Diego, CA) to eliminate dead cells. These cells were analyzed by FACS using a Vantage SE flow cytometer (Becton Dickinson, San Jose, CA) (20), and then the data were analyzed using CELLQuest software (Becton Dickinson). Gating was implemented based on wild-type litter mice as a negative control (data not shown).

Tumor analysis and tissue processing
Animals were killed and their colons were removed and cut open along their longitudinal axis. For paraffin sections, they were fixed flat in 10% buffered formalin for 24 h at room temperature, and for frozen sections, they were fixed in 4% buffered paraformaldehyde for 1 h at room temperature (12,14). Colon tumors that were macroscopically identified were divided into three groups. The first was fixed in 10% buffered formalin with the surrounding normal mucosa; the second was fixed in 4% buffered paraformaldehyde; and the third was snap frozen in liquid nitrogen, stored at –80°C and used for either DNA or RNA extraction (14,21).

Immunohistochemical analysis of tissue sections
Paraffin sections (4 µm) were treated with 0.01 M sodium citrate buffer (pH 6.0) four times in a microwave oven at high power for 6 min. They were treated with a methanol solution containing 5% H2O2 for 10 min, to block endogenous peroxidases. After blocking with 2% bovine serum albumin in PBS for 40 min at room temperature, they were treated with primary antibodies, anti-β-catenin (Transduction Laboratories, Lexington, KY; 1:1000), anti-enhanced green fluorescent protein antibody (Molecular Probes, Eugene, OR; 1:1000), anti-Myc N262 antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1:100) and anti-cyclin D1 antibody (Santa Cruz Biotechnology; 1:100) overnight at 4°C. For β-catenin staining, they were incubated with a tetramethyl rhodamine-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA; 1:250) for 30 min, or for enhanced green fluorescent protein staining, they were incubated with fluorescein isothiocyanate-conjugated secondary antibody (Dakocytomation Japan, Kyoto, Japan; 1:250) for 30 min, before detection was done using a fluorescence microscope (Olympus Optical Co., Ltd., Tokyo, Japan) after 4',6-diamidino-2-phenylindole (Nacalai Tesque, Kyoto, Japan) staining for 5 min (22). For cyclin D1 and Myc, the sections were incubated with a biotin-conjugated anti-rabbit secondary antibody (1:250) for 30 min and avidin conjugate of horseradish peroxidase for 30 min at room temperature (Vector Laboratories, Burlingame, CA; 1:250). Detection was done with 3,3-diaminobenzidine tetrahydrochloride (Dakocytomation Japan) for 0.5–10 min at room temperature and counterstained with hematoxylin (21). After blocking with 2% bovine serum albumin in PBS for 40 min at room temperature, they were incubated with the primary antibody, anti-mDkk1 antibody (R&D Systems, Minneapolis, MN; 1:100) overnight at 4°C. They were incubated with tetramethyl rhodamine-conjugated anti-rat secondary antibody for 30 min at room temperature. Detection was done using a fluorescence microscope after 4',6-diamidino-2-phenylindole staining for 5 min. For β-catenin and GFP staining, digital images of microadenomas (n = 7), macroscopic tumors (n = 17) and the surrounding normal crypts were acquired using a fluorescence microscope (DP-70, Olympus Optical Co.). In the measurement of fluorescent intensities, the mean intensity of the whole nuclear area of a single cell in microadenomas, macroscopic tumors and their adjacent normal crypts was measured by an image-processing software program (NIH image). The average values of the nuclear fluorescent intensity in each crypts of two distinct lesions and adjacent control mucosa were calculated and the ratio of the value in each lesion to that in adjacent control crypts were compared by a statistical analysis using Mann–Whitney’s U-test.

Real-time reverse transcription–polymerase chain reaction
Total RNA was isolated from snap-frozen tissues using the RNA queous-4PCR (Ambion, Austin, TX) according to the manufacturer’s protocol. Up to 200 ng of total RNA was subjected to reverse transcription using Superscript III Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA). Real-time reverse transcription–polymerase chain reaction (RT–PCR) amplification was carried out in a final volume of 20 µl containing 10 µl of 2x SYBR green master mix (Takara, Kyoto, Japan), 1 µl of primers (10 µmol/l) and 5 µl of cDNA using a LightCycler 1.0. (Roche Diagnostics, Indianapolis, IN) according to the protocols described previously (21). The primers used in the present study are shown in supplementary Table 1, available at Carcinogenesis Online. Reaction conditions were activation at 95°C for 10 min, denaturation at 95°C for 10 s, annealing 60°C for 10 s and extension 72°C for 6 s. All PCR amplifications were done for 40 cycles and a melt curve analysis was used to examine the specificity of an amplified product. Standard curves were generated to quantify the expression levels of each target gene in comparison with the 18S rRNA or β-actin reference genes in each sample. The relative expression levels of each gene were calculated, dividing the value of these genes by those of the internal control genes (18S rRNA or β-actin).

Sodium bisulfite treatment and sequencing analysis
Genomic DNA from tumors and control mucosa were subjected to sodium bisulfite modification (EZ DNA Methylation-Gold Kit, Zymo Research, Orange, CA) as described previously (23). After PCR amplification using primers listed in supplementary Table 1, available at Carcinogenesis Online, the products were cloned into the TOPO vector. The inserted PCR fragments of the individual clones obtained from each sample were sequenced with both M13 reverse and M13 forward primers using the ABI Prism Dye Terminator Cycle Sequencing Kit and an ABI Prism 3100 DNA Sequencer.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Two distinct stages in the colon of ApcMin/+ mice
In order to verify the two-stage model of tumorigenesis in the colon of ApcMin/+ mice, colonic mucosa of ApcMin/+ mice at three different ages (5, 20 and 35 weeks) were examined in en face histological sections. The longest diameter of the intramucosal lesions that were hardly detectable in whole-mount preparations were measured on the histological sections of the mice at each of the investigated ages. The sizes of most intramucosal lesions remained within 300 µm and majority of microadenomas did not grow into a macroscopic tumor even at 35 weeks of age, thus suggesting that microadenomas are self-limiting lesions (Figure 1A). We further treated ApcMin/+ mice with a potent tumor promoter, DSS in order to determine whether such microadenomas have a potential to progress into larger lesions and microadenomas are precursors of macroscopic lesions. Importantly, the size of microadenomas significantly increased by the DSS treatment, indicating that microadenomas started to grow by a well-known strong promoter of the tumorigenesis (Figure 1B). These findings represent two distinct stages of tumorigenesis in the colon of ApcMin/+ mice.


Figure 1
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Two distinct stages of tumorigenesis in the colon of ApcMin/+ mice. (A) Size distribution of intramucosal lesions in the colon of ApcMin/+ mice at 5, 20 and 35 weeks. The sizes of almost all intramucosal lesions remain <300 µm in their diameter and do not progress to a macroscopic tumor. Bars indicate average sizes of microadenomas. (B) DSS initiates the growth of microadenomas in the colon of ApcMin/+ mice. The size distribution of the ‘self-limiting’ microadenomas increases by the treatment of DSS.

 
Increased expression of β-catenin and its target genes in macroscopic tumors in the colon of ApcMin/+ mice
Immunohistochemical analysis of β-catenin protein demonstrated that although microadenomas already revealed increased levels of nuclear β-catenin (n = 7), macroscopic tumors had further increased levels of nuclear β-catenin (n = 17) on the same slide of en face sections (Figure 2C and D). This was followed by a comparison of fluorescent signals of nuclear β-catenin. The results showed that the signal intensity of β-catenin in macroscopic tumors was higher than that of microadenomas (Figure 2E) (P < 0.005 by Mann–Whitney U-test). Immunostaining revealed that Myc and cyclin D1, well-known targets of β-catenin/Tcf transcription, were detectable in the nucleus of both microadenomas (n = 16) and macroscopic tumors (n = 15). However, the strong nuclear staining was observed only in macroscopic tumors (Figure 2F–I).


Figure 2
View larger version (115K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Increased nuclear β-catenin and β-catenin/Tcf targets in colon tumor cells of ApcMin/+ mice. (A and B) Histological appearance of microadenoma (A) and macroscopic tumor (B). Hematoxylin and eosin staining. Bars = 30 µm. (C and D) β-Catenin immunostaining of both lesions. Fluorescent intensities of nuclear β-catenin are higher in macroscopic tumor cells than those in microadenoma cells. Bars = 30 µm. (E) Measurement of fluorescent intensity of both lesions. The relative intensity of nuclear β-catenin staining is shown. *P < 0.005 by Mann–Whitney U-test. (F–I) Myc and cyclin D1 immunostaining of both lesions. Nuclear staining of both Myc and cyclin D1 protein further increases in macroscopic tumor cells in comparison with microadenoma cells. Bars = 30 µm.

 
Reporter mice for β-catenin/Tcf transcriptional activity
In the present study, transgenic mice were generated with a reporter for β-catenin/Tcf transcriptional activity (Figure 3A). To confirm that the reporter mice actually represent transcriptional activity of β-catenin/Tcf signals in vivo, primary splenocytes of the transgenic mice were treated with LiCl, an inhibitor of GSK-3β and an activator of β-catenin/Tcf transcription. Flow cytometry revealed an increased GFP signal in the splenocytes with the LiCl treatment, suggesting that GFP reporter works in vivo (Figure 3B).


Figure 3
View larger version (76K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. β-Catenin/Tcf reporter mice. (A) Structure of the transgene. (B) β-Catenin/Tcf reporter works in the transgenic mice. LiCl, a Wnt signal activator, induces GFP expression in splenocytes of β-catenin/Tcf reporter transgenic mice. (C) Fluorescent signal of GFP stainings. GFP signal is higher in microadenomas in comparison with the surrounding normal crypts. Note that the reporter expression is significantly upregulated in macroscopic tumors when compared with microadenomas. *P < 0.005, **P < 0.005 and ***P < 0.005, by Mann–Whitney U-test. (D–I) GFP signals are detectable in both microadenomas and macroscopic tumors. Note that the strong GFP expression is observed only in macroscopic tumors. Bars = 30 µm.

 
β-Catenin/Tcf transcriptional activity in two distinct stages of colon carcinogenesis
The mRNA expression for GFP in colon tumors and normal colonic mucosa in ApcMin/+ mice with GFP reporter allele was analyzed by semiquantitative RT–PCR. The GFP expression in colon tumors (n = 12) was significantly higher than that in the normal colonic mucosa (n = 8, supplementary Figure 1, available at Carcinogenesis Online) (P < 0.01 by Mann–Whitney U-test). We next measured the fluorescence intensity of immunofluorescent staining of GFP at normal crypts, microadenomas and macroscopic tumors. The fluorescent signals in microadenomas have already increased when compared with those in adjacent normal crypts (Figure 3C and E) (P < 0.005 by Mann–Whitney U-test). However, GFP signals in macroscopic tumor were further upregulated in comparison with those in microadenomas (Figure 3C and H) (P < 0.005 by Mann–Whitney U-test). In this study, three different lines of transgenic mice were generated. GFP expression in all lines showed the same pattern, excluding the possibility that the observation depends on the locus in which transgenes were integrated.

Altered expressions of Wnt antagonist genes in colon tumors of ApcMin/+ mice
Previous studies have shown that loss of Apc function and consequent accumulation of β-catenin occurs in the microadenomas, suggesting that the β-catenin/Tcf signaling pathway is already activated in the initiation stage (12). Wnt antagonist genes are epigenetically silenced in most human colorectal cancers (24–28), and such silencing has been shown to further increase the transcriptional activity of the β-catenin/Tcf signaling pathway, regardless of Apc inactivation (28–30). To investigate the involvement of Wnt antagonists in further upregulation of β-catenin/Tcf signaling in the macroscopic tumors of this model, mRNA levels for Wnt antagonist genes in macroscopic tumors and control mucosa were compared by semiquantitative real-time RT–PCR. The Wnt antagonist genes examined in the present study were Sfrp1, Sfrp2, Sfrp4, Sfrp5, Dkk1, Dkk2, Dkk3 and Dkk4. Sfrp4, Dkk1 and Dkk4 expression was significantly decreased in macroscopic tumors in comparison with control mucosa (Figure 4) (P < 0.05, P < 0.001 and P < 0.001, respectively, by Mann–Whitney U-test). In contrast, Dkk2 expression was significantly increased in macroscopic tumors (Figure 4) (P < 0.05 by Mann–Whitney U-test).


Figure 4
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. Expression of Wnt antagonist genes in colon tumors of ApcMin/+ mice was assessed by semiquantitative real-time RT–PCR. The expression of Sfrp4, Dkk1 and Dkk4 was upregulated, whereas the expression of Dkk2 was downregulated. The expression was normalized to S18 or β-actin mRNA expression. Samples were analyzed in triplicate. (Columns, mean of three independent experiments; bars, SEM; n = 12 for tumor and n = 8 for control. *P < 0.05, **P < 0.001, ***P < 0.05 and ****P < 0.001; by Mann–Whitney U-test.)

 
Dkk1 expression in two distinct stages of colon tumorigenesis
Among three Wnt antagonist genes (Sfrp4, Dkk1 and Dkk4) that are downregulated in macroscopic tumors, only Dkk1 is shown to have the ability to functionally suppress β-catenin/Tcf transcription in the intestine of mice (29,30). Additionally, it is interesting to note that decreased Dkk1 expression is frequently observed in human colon cancers (26,28). Therefore, the Dkk1 expression was examined in two distinct lesions by immunostaining to determine when the Dkk1 expression decreased in the course of multistage carcinogenesis in the colon of ApcMin/+ mice. Consistent with the results in the RT–PCR analysis, decreased fluorescent intensities of Dkk1 were observed in macroscopic tumors in comparison with those in the surrounding control mucosa (5/8) (Figure 5). This was consistent with the previous RT–PCR results. In contrast, the Dkk1 expression in microadenoma is higher than that of the surrounded control mucosa (6/6) (Figure 5). These results suggest that Dkk1 expression is decreased in the course of the transition from microadenomas to macroscopic tumors.


Figure 5
View larger version (43K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. Immunohistochemistry of Dkk1 in microadenomas and macroscopic tumors in the colon of ApcMin/+ mice. (A and B) Fluorescent signal levels decrease in the macroscopic tumor. Note that Dkk1 is upregulated in microadenomas. Bars = 30 µm. (C) Summary of Dkk1 staining in two lesions.

 
Methylation status of Dkk1 and Sfrp4 promoter regions in colon tumors of ApcMin/+ mice
Epigenetic silencing of Wnt antagonist genes in human colon cancers is often accompanied by DNA hypermethylation (24,30). Since mouse Dkk4 has no apparent clustering of CpG sites in the promoter region, the methylation status of Dkk1 and Sfrp4 promoters was examined by bisulfite sequencing. The promoter region of Dkk1 (27 clones from three colons) and Sfrp4 (nine clones from two colons) was unmethylated in the control mucosa. However, no evidence of DNA hypermethylation was detected in colon tumors in the Dkk1 (28 clones from three colon tumors) and Sfrp4 (6 clones from two colon tumors) promoters, regardless of the mRNA expression status (Figure 6).


Figure 6
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6. Results of bisulfite DNA sequencing of the promoter CpG island region of Dkk1 and Sfrp4. Numbers represent the positions relative to the transcription start site. Square, CpG dinucleotide (potential target of methylation); closed square, methylated CpG dinucleotide and open square, unmethylated CpG dinucleotide.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
Colorectal tumors progress through a series of clinical and histopathological stages, ranging from single crypt lesions to invasive cancers (2). Accumulating evidence suggests that such progression results from a series of genetic and epigenetic changes (2,31). However, how such alterations are linked to the histopathological progression is not fully understood. A previous study has shown that two lesions, microadenomas and macroscopic tumors, are distinguishable in the colon of ApcMin/+ mice (12). In this study, we have shown that microadenomas themselves are self-limiting lesions, but such microadenomas can potentially grow into larger lesions by inflammatory stimuli. Together with previous lines of experiments, these findings indicate the existence of two stages of tumorigenesis in the colon of ApcMin/+ mice and suggest that additional stochastic alterations are required for microadenomas to progress into macroscopic tumors. Although the loss of the Apc function through LOH is associated with the initial microadenoma formation (12), the underlying mechanism of the development of macroscopic tumors remained elusive. A previous study using a rat model for colon cancer demonstrated that intramucosal lesions possess a wide spectrum of mutations in the β-catenin gene, but only lesions with specific mutations that directly regulate β-catenin levels developed macroscopic tumors (32). Based on those findings, it is assumed that sufficient β-catenin accumulation may be required for the development of macroscopic tumors. The present study showed that the nuclear accumulation of β-catenin and the expression of Myc and cyclin D1, well-known downstream targets of the β-catenin/Tcf signaling pathway, were significantly upregulated in macroscopic tumors when compared with microadenomas. Using β-catenin/Tcf reporter mice, the β-catenin/Tcf transcriptional activity was measured directly as the amount of GFP in vivo. This demonstrated an increased GFP expression in macroscopic tumors in comparison with that in microadenomas. These results suggest that further activation of the β-catenin/Tcf signaling activity might be responsible for the development of macroscopic tumors from microadenomas. Although significant evidence suggests that the activation of β-catenin signaling is an initiating event in the colon tumorigenesis, the present findings may shed light on the role of β-catenin/Tcf transcription on the later stage of the tumorigenesis. There may be a threshold of β-catenin/Tcf transcriptional activity when small lesions progress into macroscopic tumors, and only lesions that exceed that threshold could grow into a macroscopic lesion.

A recent study indicated that β-catenin/Tcf signaling in colon cancer cells could be further activated by upstream signals regardless of the constitutive activation of the pathway by downstream mutations (30). The present study showed altered expression of a number of Wnt antagonist genes in the colon tumors of ApcMin/+ mice. Importantly, although the level of Dkk1 protein was decreased in most macroscopic tumors, Dkk1 expression in microadenomas was increased in comparison with the adjacent normal mucosa. This study suggests that the decreased levels of Dkk1 play a role in further activation of β-catenin/Tcf transcription. Detailed analyses are necessary to elucidate the functional significance of the decreased Dkk1 on the β-catenin/Tcf transcription in colon tumors of ApcMin/+ mice.

The present findings also raised additional questions of the underlying mechanism that induces the silencing of Dkk1 in macroscopic tumors. Recently, epigenetic inactivation of Wnt antagonist genes has been reported in most human colorectal cancers (26). In addition, mice deficient DNA methyltransferases have been shown to block the development of macroscopic tumors in the colon of ApcMin/+mice (14,33), suggesting that DNA methylation plays a role in the development of macroscopic tumors. In order to determine whether the inactivation of Wnt antagonist genes is associated with DNA hypermethylation, the methylation status of the promoter region of Dkk1 and Sfrp4, which were silenced in colon tumors of ApcMin/+ mouse model, was investigated. However, no significant difference in the DNA methylation status between macroscopic tumors and control mucosa was detectable, thus suggesting that Dkk1 downregulation is independent of DNA hypermethylation. Accordingly, it is possible that other epigenetic silencing, such as histone modifications may be associated with the inactivation and this might provide a useful model to study the epigenetic silencing that is independent of DNA hypermethylation in these tumors.

Although the fact that most microadenomas have the ability to progress into macroscopic tumors supports the idea that macroscopic tumors arise from a subset of microadenomas, we cannot exclude a possibility that there are alternative pathways in which normal colon epithelium is converted into macroscopic tumor cells. Indeed, recent evidences strongly suggest that histological changes are preceded by epigenetic alterations that are associated with the activation of β-catenin/Tcf transcription in the colon (30,34). Meanwhile, it is also possible that microadenomas and macroadenomas may originate from different cell types in terms of the differentiation state. Therefore, small adenomatous lesions may not be uniform lesions and such differences might affect the later progression; thus, some microadenomas may more easily progress into macroscopic tumors than others. Further investigations are required to elucidate the multiple genetic and/or epigenetic pathways of tumor development in the colon.

In summary, these findings suggest that activation of β-catenin/Tcf transcription, which is accompanied by an increased nuclear β-catenin level and decreased Dkk1 expression, plays a role in the development of macroscopic tumors in the colon of ApcMin/+ mice. In addition, the β-catenin/Tcf reporter transgenic mice described here would be useful to elucidate the biological role of β-catenin/Tcf transcription in vivo.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Figure 1 and Table 1 can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
Ministry of Health, Labour and Welfare of Japan; The Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Acknowledgments
 
We thank Dr Toshikazu Ushijima for helpful comments and critical reading of the manuscript. We also thank Kyoko Takahashi, Ayako Suga and Yoshitaka Kinjyo for the technical assistance and the animal care.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

  1. Vogelstein B, et al. Genetic alterations during colorectal-tumor development. N. Engl. J. Med. (1988) 319:525–532.[Abstract]
  2. Fearon ER, et al. A genetic model for colorectal tumorigenesis. Cell (1990) 61:759–767.[CrossRef][Web of Science][Medline]
  3. Morin PJ, et al. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science (1997) 275:1787–1790.[Abstract/Free Full Text]
  4. Powell SM, et al. APC mutations occur early during colorectal tumorigenesis. Nature (1992) 359:235–237.[CrossRef][Medline]
  5. Groden J, et al. Identification and characterization of the familial adenomatous polyposis coli gene. Cell (1991) 66:589–600.[CrossRef][Web of Science][Medline]
  6. Nishisho I, et al. Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients. Science (1991) 253:665–669.[Abstract/Free Full Text]
  7. Moser AR, et al. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science (1990) 247:322–324.[Abstract/Free Full Text]
  8. Su LK, et al. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science (1992) 256:668–670.[Abstract/Free Full Text]
  9. Levy DB, et al. Inactivation of both APC alleles in human and mouse tumors. Cancer Res. (1994) 54:5953–5958.[Abstract/Free Full Text]
  10. Luongo C, et al. Loss of Apc+ in intestinal adenomas from Min mice. Cancer Res. (1994) 54:5947–5952.[Abstract/Free Full Text]
  11. Oshima M, et al. Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene. Proc. Natl Acad. Sci. USA (1995) 92:4482–4486.[Abstract/Free Full Text]
  12. Yamada Y, et al. Microadenomatous lesions involving loss of Apc heterozygosity in the colon of adult Apc(Min/+) mice. Cancer Res. (2002) 62:6367–6370.[Abstract/Free Full Text]
  13. Yamada Y, et al. Multistep carcinogenesis of the colon in Apc(Min/+) mouse. Cancer Sci. (2007) 98:6–10.[CrossRef][Medline]
  14. Yamada Y, et al. Opposing effects of DNA hypomethylation on intestinal and liver carcinogenesis. Proc. Natl Acad. Sci. USA (2005) 102:13580–13585.[Abstract/Free Full Text]
  15. Boivin GP, et al. Pathology of mouse models of intestinal cancer: consensus report and recommendations. Gastroenterology (2003) 124:762–777.[CrossRef][Web of Science][Medline]
  16. Tanaka T, et al. Dextran sodium sulfate strongly promotes colorectal carcinogenesis in Apc(Min/+) mice: inflammatory stimuli by dextran sodium sulfate results in development of multiple colonic neoplasms. Int. J. Cancer (2006) 118:25–34.[CrossRef][Web of Science][Medline]
  17. Korinek V, et al. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma. Science (1997) 275:1784–1787.[Abstract/Free Full Text]
  18. Stambolic V, et al. Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr. Biol. (1996) 6:1664–1668.[CrossRef][Web of Science][Medline]
  19. Klein PS, et al. A molecular mechanism for the effect of lithium on development. Proc. Natl Acad. Sci. USA (1996) 93:8455–8459.[Abstract/Free Full Text]
  20. Walker PR, et al. Topoisomerase II-reactive chemotherapeutic drugs induce apoptosis in thymocytes. Cancer Res. (1991) 51:1078–1085.[Abstract/Free Full Text]
  21. Tomita H, et al. Development of gastric tumors in Apc(Min/+) mice by the activation of the beta-catenin/Tcf signaling pathway. Cancer Res. (2007) 67:4079–4087.[Abstract/Free Full Text]
  22. Yamada Y, et al. Pre-cancerous lesions for colorectal cancers in rodents: a new concept. Carcinogenesis (2003) 24:1015–1019.[Abstract/Free Full Text]
  23. Yamashita S, et al. Chemical genomic screening for methylation-silenced genes in gastric cancer cell lines using 5-aza-2'-deoxycytidine treatment and oligonucleotide microarray. Cancer Sci. (2006) 97:64–71.[CrossRef][Medline]
  24. Caldwell GM, et al. The Wnt antagonist sFRP1 in colorectal tumorigenesis. Cancer Res. (2004) 64:883–888.[Abstract/Free Full Text]
  25. He B, et al. Blockade of Wnt-1 signaling induces apoptosis in human colorectal cancer cells containing downstream mutations. Oncogene (2005) 24:3054–3058.[CrossRef][Web of Science][Medline]
  26. Aguilera O, et al. Epigenetic inactivation of the Wnt antagonist DICKKOPF-1 (DKK-1) gene in human colorectal cancer. Oncogene (2006) 25:4116–4121.[CrossRef][Web of Science][Medline]
  27. Nojima M, et al. Frequent epigenetic inactivation of SFRP genes and constitutive activation of Wnt signaling in gastric cancer. Oncogene (2007).
  28. Gonzalez-Sancho JM, et al. The Wnt antagonist DICKKOPF-1 gene is a downstream target of beta-catenin/TCF and is downregulated in human colon cancer. Oncogene (2005) 24:1098–1103.[CrossRef][Web of Science][Medline]
  29. Kuhnert F, et al. Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1. Proc. Natl Acad. Sci. USA (2004) 101:266–271.[Abstract/Free Full Text]
  30. Suzuki H, et al. Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer. Nat. Genet. (2004) 36:417–422.[CrossRef][Web of Science][Medline]
  31. Jones PA, et al. The fundamental role of epigenetic events in cancer. Nat. Rev. Genet. (2002) 3:415–428.[Web of Science][Medline]
  32. Yamada Y, et al. Beta-catenin mutation is selected during malignant transformation in colon carcinogenesis. Carcinogenesis (2003) 24:91–97.[Abstract/Free Full Text]
  33. Lin H, et al. Suppression of intestinal neoplasia by deletion of Dnmt3b. Mol. Cell. Biol. (2006) 26:2976–2983.[Abstract/Free Full Text]
  34. Linhart HG, et al. Dnmt3b promotes tumorigenesis in vivo by gene-specific de novo methylation and transcriptional silencing. Genes. Dev. (2007) 21:3110–3122.[Abstract/Free Full Text]
Received July 11, 2007; revised December 16, 2007; accepted December 28, 2007.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 CarcinogenesisHome page
X. Liu, L. Wang, S. Zhang, J. Lin, S. Zhang, M. A. Feitelson, H. Gao, and M. Zhu
Mutations in the C-terminus of the X protein of hepatitis B virus regulate Wnt-5a expression in hepatoma Huh7 cells: cDNA microarray and proteomic analyses
Carcinogenesis, June 1, 2008; 29(6): 1207 - 1214.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
29/3/666    most recent
bgn001v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Oyama, T.
Right arrow Articles by Mori, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oyama, T.
Right arrow Articles by Mori, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?