Role of c-Myc in Intestinal Tumorigenesis of the ApcMin/+ Mouse
Natalia A. Ignatenko1,*Hana Holubec1
David G. Besselsen4
Karen A. Blohm-Mangone2
Jose L. Padilla-Torres2
Raymond B. Nagle5 Ignacio Moreno de Alboránç6
Jose M. Guillen-R2 Eugene W. Gerner1,3
1Department of Cell Biology and Anatomy, 2Cancer Biology Division, and 3Department of Biochemistry and Molecular Biophysics of the Arizona Cancer Center; The University of Arizona, Tucson, Arizona USA
4University Animal Care and 5Department of Pathology; The University of Arizona, Tucson, Arizona USA
6Department of Immunology and Oncology; Centro Nacional de Biotecnologia Consejo Superior de Investigaciones Cientificas; Universidad Autonoma de Madrid; Madrid Spain
*Correspondence to: Natalia A. Ignatenko; The University of Arizona; Arizona Cancer Center; 1515 N. Campbell Avenue; Tucson, Arizona 85724; Tel.: 520.626.8044; Fax: 520.626.4480; Email: [email protected]
Original manuscript submitted: 09/07/06Manuscript accepted: 09/07/06
Previously published online as a Cancer Biology & Therapy E-publication:http://www.landesbioscience.com/journals/cc/abstract.php?id=3376
FAP familial adenomatous polyposis; Apc rodent Adenomatous Polyposis Coli gene Fabpl liver fatty acid-binding protein gene c-Myc rodent oncogene c-MYC human oncogenec-MYC rodent or human protein
AcKNoWLEDGMENTs
See page 1663.
ABsTRAcTThe c‑MYC oncogene plays an important role in tumorigenesis and is commonly
highly expressed in gastrointestinal cancers. In colon cells, c‑MYC is regulated by the adenomatous polyposis coli (Apc) tumor suppressor gene. Multiple intestinal neoplasia (ApcMin/+ or Min) mice are heterozygous for a truncating Apc mutation and serve as a model of familial adenomatous polyposis (FAP) disease. To study the role of c‑Myc in the mutant Apc‑mediated colon tumorigenesis, we have developed a transgenic mouse with the conditional deletion of the floxed c‑Myc alleles in the intestinal crypts of ApcMin/+ mice (ApcMin/+; c‑Mycfl/fl). The floxed c‑Myc deletion was initiated via a Cre recombinase controlled by the intestine‑specific transcriptional regulatory elements of the liver fatty acid‑binding protein gene (Fabpl4×at‑132). Fabpl4×at‑132‑mediated Cre expression and recombination resulted in a two‑fold decrease in c‑MYC protein expression with no effect on intestinal tract morphology. Small intestinal tumorigenesis was significantly suppressed throughout the small intestinal tract of ApcMin/+; c‑Mycfl/fl mice compared to c‑Myc wild type littermates. In ApcMin/+; c‑Mycfl/fl mice, the intestinal apoptosis was higher in the areas of the small intestine with the decreased c‑Myc protein expression (p = 0.0016, compared to their littermates with the wild type c‑Myc). Thus, conditional inactivation of c‑Myc, mediated by Fabpl4×at‑132‑driven Cre‑recombinase, suppresses Apc‑dependent intestinal tumorigenesis in adult ApcMin/+ mice, without apparent effect on normal intestinal mucosa.
INTRoDucTIoNThe proto-oncogene c-MYC regulates several distinct and even counter-acting
cellular processes, including growth, differentiation, proliferation, and apoptosis.1 The c-MYC gene belongs to the Myc family, which includes also L-MYC, N-MYC, B-MYC, and S-MYC. The c-MYC gene encodes a transcription factor that is widely expressed in proliferating embryonic and mature cells and possesses a potent transforming and transactivation ability.2 Under normal physiological conditions, c-MYC participates with the DNA binding proteins MAX and MAD to regulate specific gene transcriptional activation or repression.3 MYC-MAX dimers bind to the sequence CACGTG (E-boxes) in the regulatory region of many genes, particularly cyclin D2 and ornithine decarboxylase, and induce their transcription.4,5 Heretodimerization of MAX with MAD and its binding to E-boxes block most functions of the MYC-MAX dimers, including cell transformation, via repression of gene transcription.6
In malignant tissues, c-MYC expression is elevated or deregulated and is often associated with aggressive, poorly differentiated tumors. The c-MYC oncogene is overexpressed in many types of human cancers[Liao, 2000 #187; Nesbit, 1999 #186], including colorectal cancer [Brabletz, 2000 #188]. In gastric and esophageal tumors and in hepatocellular carcinomas, elevated c-MYC is associated with the presence of metastatic or recurrent tumors.10-12 c-MYC is expressed in pancreatic cancer cells and tumors and has been linked to advanced tumor grade in pancreatic cancer patients.13,14 c-MYC is overexpressed in adenomas of FAP patients and in sporadic colorectal tumors.15,16 Activation of the c-MYC gene during tumorigenesis occurs through different mechanisms, including translocation, amplification, enhanced translation or protein stability.17-21 In colorectal cancer, c-MYC transcription is activated by the b-catenin/TCF complex and is inhibited by APC.22
A series of genetically engineered mice has been generated to study the role of c-Myc in human cancers. Transgenic mice, which overexpress c-Myc in liver, pancreatic beta cells, skin, or prostate, show increased proliferation with progression from precancerous lesions to invasive cancers.23-26 In skin, sustained activation of c-Myc is sufficient to induce
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papillomatosis together with angiogenesis, which are commonly observed in humans during the development of precancerous epithelial lesion hyperplastic actinic keratosis. All of these premalig-nant changes spontaneously regress upon deactivation of c-Myc.24 Transgenic mice expressing human c-MYC in the mouse prostate developed murine prostatic intraepithelial neoplasia followed by invasive adenocarcinoma.26 The c-Myc RNA transcript along with cyclin D1 transcript was found to be elevated in colon polyps compared with the normal epithelium in a new mouse model of FAP with a germline Apc inactivation via exon 14 deletion (Apc D14/+).27
Direct assessment of c-Myc function in cellular proliferation, differentiation, and embryogenesis by using homologous recom-bination in mouse embryonic stem cells indicates that c-Myc inactivation is lethal in homozygotes between 9.5 and 10.5 days of gestation.28 The conditional knockout of c-Myc in mouse epidermis and liver provided genetic evidence of the role of c-Myc in tissue homeostatis.29,30 In mammalian epidermis, c-Myc controls cell size, growth, endoreplication and stem cell amplification.29 In the liver, postnatal hepatoproliferation does not require c-Myc, whereas it was necessary for liver regeneration in adult mice.30 The role of c-Myc during the intestinal tract development and homeostasis has been evaluated using the mouse model with the conditional knockout of the one c-Myc allele.31 This intestine-specific c-Myc knockout via tamoxifen-induced activation of Cre-ERT2 recombinase, controlled by the villin promoter, showed that mice with deleted c-Myc were able to form and maintain normal epithelium in the absence of c-MYC activity. However, the role of c-Myc during the intestinal tumorigenesis has not been directly evaluated. In order to study the role of c-Myc in the development of intestinal tumors, we condition-ally knocked out c-Myc gene in the ApcMin/+ mouse using a fatty acid binding promoter (Fabp)-mediated Cre recombination. We report the suppression of Apc-mediated tumorigenesis in the intestinal epithelium of ApcMin/+ mice with the mosaic loss of c-Myc protein. We also show that the Fabpl4×at -132-mediated mosaic c-Myc deletion did not alter either intestinal development or intestinal crypt size and appearance in ApcMin/+ mice, although it was associated with the elevated apoptosis in the areas where c-Myc activity was lost.
MATERIALs AND METHoDsGeneration of conditional intestinal c‑Myc knockout mice.
C57BL/6J-Apc Min/+ mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and bred in the University of Arizona’s Animal Care Facility in accordance with The University of Arizona Institutional Animal care Utilization Committee guidelines. These mice have a mutation at codon 850 of the murine Apc gene that converts a leucine into a stop codon and causes the truncation of an APC protein. Affected ApcMin/+ animals were obtained by crossing Min males with C57BL/6J females. The presence of the mutant Apc allele was detected in tail-snip DNA using an allele-specific PCR assay.32 Mice carrying the floxed c-Myc alleles (c-Mycfl/fl)33 were bred to transgenic mice which express Cre recombinase under the control of transcriptional regulatory elements derived from a liver fatty acid-binding protein gene (Fabpl 4x at -132).34 The genetic background was B6.129 for c-Mycfl/fl mice and FVB/N for Fabpl4x at-132Cre mice. Heterozygous progeny were then interbred to initiate the deletion of loxP flanked c-Myc sequences in rapidly self-renewing mouse small intestinal epithelium. The Fabpl4×at -132Cre; c-Mycfl/fl mice were subsequently bred to ApcMin/+ mice and then to their progeny to create ApcMin/+ mice with an intestine-specific knockout of the c-Myc
oncogene (ApcMin/+; c-Mycfl/fl). Genotyping of ApcMin/+; c-Mycfl/fl mice for the c-Myc floxed alleles was performed by PCR analysis and has been previously reported.33 Mice were of mixed background and only littermates ApcMin/+; c-Myc+/+ and ApcMin/+; c-Mycfl/fl were used for the analysis. Animals were housed in accordance with institutional guidelines. Mice were fed the defined diet AIN-93G. Irradiated distilled water was available ad libitum for the duration of experiment. Mice used in the experiments were 110–115 day old.
Immunofluorescent staining for Cre recombinase. The proximal, middle and distal areas of the small intestine were opened and their luminal contents were removed by washing the tissues in phos-phate-buffered saline (PBS). One-centimeter portions of each area were embedded in a Tissue-Tek OCT compound and snap frozen in isopropyl alcohol. Tissue blocks were cut into 3 mm sections and placed on SuperFrost Plus slides. Staining for Cre recombinase was performed according to El Marjou et al,35 with modifications. Briefly, after fixation in 2% paraformaldehyde for 7 min, tissue sections were treated with PBS containing 50 mM NH4Cl for 5 min, followed by permeabilization with 0.2% Triton X-100 in PBS for 4 min and blocking with PBS containing 2% BSA for 5 min. A rabbit anti-Cre recombinase polyclonal antiserum (Covance, Berkeley, CA) was diluted in PBS with 2% BSA (final dilution 1:1,000). The secondary antibody was goat anti-rabbit IgG coupled to Alexa Fluor 488 (Invitrogen-Molecular Probes, Carlsbad, CA) diluted in PBS (final dilution 1:400). A ProLong Gold antifade mounting reagent with DAPI (Invitrogen-Molecular Probes, Carlsbad, CA) was used to visualize nuclei. Representative images were captured digitally using an Olimpus BX50 camera with 60x objective and imaging software ImagePro (Media Cybernetics, Silver Spring, MD).
Histology and crypt scoring. Whole small intestinal rolls were collected from 110 day old ApcMin/+; c-Myc+/+and ApcMin/+; c-Mycfl/fl mice, flushed with PBS, and fixed in 10% formalin at 4˚C over-night. Fixed tissue was processed for paraffin embedding, cut in 3 mm sections and stained with H&E. Morphological evaluation was performed using a bright field Nikon microscope. Representative images were captured digitally using a Sony 3CCD camera and imaging software ImagePro (Media Cybernetics, Silver Spring, MD). Morphometric analysis of crypt length was performed on digitally captured images (Sony 3CCD color video camera) of H&E stained sections of the distal small intestine using ImagePro software. Three mice per genotype were used and fifteen measurements (in mm) of full-length crypts were obtained from each sample.
Tissue preparation for immunohistochemistry and apoptosis. The distal portions of small intestinal segments from ApcMin/+ mice with different c-Myc genotypes were fixed in 10% buffered formalin, paraffin-embedded, and sectioned at 3 mm. Serial sections were used for c-Myc and cleaved caspase 3 immunochistochemistry as well as for determination of apoptosis.
Immunohistochemistry. These serial sections were deparaffinized in xylene, followed by a rehydration in a graded series of ethanol, ending with water immersion. Antigen retrieval was performed by microwave exposure in Na citrate buffer, pH 6.1. Serial tissue sections were processed for c-Myc and cleaved caspase 3 staining according to protocols described below. Immunohistochemical staining for c-Myc was done using the Discovery XT staining module (Ventana Medical Systems, Inc., Tucson, AZ). Briefly, sections were incubated with a rabbit polyclonal antibody for c-MYC, 1:20 dilu-tion (BD Transduction Labs, Upstate), followed by a secondary goat anti-rabbit-HRP conjugated IgG antibody, 1:200 dilution (BD Transduction Labs, Upstate), and detected using the diaminobenzi-
Conditional Intestinal c-Myc Knockout in the Apc Min/+ Mouse
dine (DAB) detection system. Representative images were captured digitally using a Sony 3CCD camera and imaging software ImagePro (Media Cybernetics, Silver Spring, MD). For the cleaved caspase-3 staining, endogenous peroxidase blocking was performed using 3.0% H2O2 in methanol, and sections were blocked with 1.5% normal goat serum (Vector Laboratories, Burlingame, CA). Then sections were incubated with a rabbit polyclonal antibody at a concentration of 1.2 mg/ml (Cat.# 9661, Cell Signaling Technology), followed by a biotinylated secondary antibody (Vector Laboratories). Sections were then treated with a Vectastain Elite ABC reagent used according to manufacturer instructions (Vector Laboratories), diaminobenzidine (DAB) activated with H2O2, and counterstained by hematoxylin. Cleaved caspase-3 expression was measured in the crypts directly adjacent to the muscularis (250 crypts per mouse in average). C-Myc and cleaved caspase staining were analyzed in three or four mice from each genotype.
Analysis of c‑MYC positive cells. To evaluate the degree of reduction in c-MYC expression level, the stained tissue was evalu-ated at random locations under 100x magnification using imaging software Image-Pro-Plus, Version 4.5 (Media Cybernetics, Silver Spring, MD). After counting each individual tissue sample, the area which stained positively for c-MYC in the distal portion of the small intestine was expressed as a percent ± SD of the mean of areas count. Three mice per genotype were scored, and twelve areas per mouse were evaluated.
Determination of apoptosis. The serial sections, described earlier, were stained with hematoxylin and eosin (H&E). Morphologic criteria were used to identify the cells undergoing apoptosis featuring the late apoptotic stage, especially for the presence of fragmented nuclei and apoptotic bodies. The apoptotic index in the crypt region was determined by counting the crypts directly adjacent to the muscularis (200 crypts per mouse in average). Apoptosis was quanti-fied under a 100x light objective and was expressed as an apoptotic index (AI), the percent of crypts with at least one apoptotic cell. Apoptosis was confirmed by immunochistochemistry for cleaved caspase 3.
Western blot analysis. Individual adenomas and apparently normal intestinal tissues were collected from 110 day old ApcMin/+; c-Myc+/+ mice. Adenomas were located in the middle portion of the small intestine. Samples were sonicated for 30 sec and kept on ice for 30 min followed by centrifugation at 13,000 x g for 10 min. Supernatants were collected and protein concentrations were deter-mined using a Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA). 50 mg of cell lysate was loaded per lane and run on a 12.5% gel. The proteins were transferred electrophoretically to Hybon-C nitrocellulose membrane (Amersham, Arlington Heights, IL) overnight. The membrane was blocked in BlottoA (5% w/v nonfat dry milk, 0.1% Tween 20, and Tris-buffered saline (TBS) consisting of 10 mM Tris-HCl, pH 8.0, 150 mM NaCl) for 2 hr at room temperature. A c-MYC mouse monoclonal antibody (Cat.#sc-40, 1:500 dilution, Santa Cruz Biotechnology Inc., Santa Cruz, CA) was incubated with the blot overnight. The blot was washed in TBS/0.1% Tween-20. The primary antibody was detected with an anti-mouse immunoglobulin G antibody and visualized with an enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech Inc., Piscataway, NJ). To control for variations in loading, the blot was probed with an anti b-actin antibody (1:1000 dilution, Sigma Life Sciences, Saint Louis, MS). Protein bands were quantified using Scion Image Analysis software (Scion Corporation, Frederick, Maryland). The expression of c-MYC was normalized
against expression of b-actin in the same samples. Western blots were performed from three mice, and a representative blot is shown.
Tumor scoring. Mice were sacrificed at the age of 110 days by CO2 inhalation, and whole small intestine and colon segments were removed, flushed with buffered saline, opened longitudinally and laid flat, mucosal surface up. Tissues were fixed in 10% neutral buffered formalin for 24 h and then placed in 70% ethanol. Tumor number per mouse was assessed using a dissecting microscope at 20x magni-fication. Tumors were counted starting at the size of 0.5 mm.
Statistics. All of the statistical analyses were carried out with Statistical Analysis System software (SAS Institute, Cary, NC) and p values <0.05 were considered significant. For multiple comparison, the Tukey Student range test was used. Poisson regression was used for analyzing the difference in the number of tumors in Apc Min/+ mice with different c-Myc genotypes. ANOVA test was used for the analysis of the c-Myc expression and apoptosis rate.
REsuLTsConditional inactivation of c‑Myc in the intestinal epithelium.
Mice expressing the wild type c-Myc gene flanked by loxP sequences (c-Mycfl/fl)33 were crossed with mice expressing Cre recombinase under the control of a derivative of the promoter of a liver fatty acid binding protein that contained four copies of nucleotides -177 to -132 inserted at position -132 (Fabpl 4x at -132).36 As has been previously shown by crossing the transgenic Fabpl 4x at -132 mice with mice expressing the floxed LacZ reporter, the Fabpl 4x at -132 - directed Cre-recombination had a mosaic pattern37 of expression. However, the majority of intestinal crypts displayed a homogenous population of Cre-recombinase expressing cells in the lower quarter of crypts.37
We bred the Fabpl4×at -132 Cre; c-Mycfl/fl mice to ApcMin/+ mice, and the compound ApcMin/+; Fabpl4×at -132 Cre; c-Mycfl/fl (ApcMin/+; c-Mycfl/fl) mice were used for further experiments. It should be noted that all genotype comparisons have been made in mice with this compound background in order to account for possible tumor modifying alleles not present in C57BL/6J mice. In the ApcMin/+ mouse, tumors are mostly detected in the small intestine and only a few are present in the colon. Therefore, we conducted the analysis of the conditional intestine-specific c-Myc knockout in the small intestine but not in colon of ApcMin/+; c-Mycfl/fl mice. Cre recom-binase was expressed in the different portions of the small intestine of ApcMin/+; c-Mycfl/fl mice (Fig. 1A) and was colocalized with nuclei in the undifferentiated progenitor cell compartment of crypts and epithelial cells throughout the crypt-villus axis (Fig. 1B).
To determine the extent of c-Myc protein inactivation, we performed immunohistochemical staining for c-Myc in the distal 2 cm segment of the small intestine. We scored the positively stained areas using imaging software Image-Pro-Plus, Version 4.5 (Media Cybernetics, Silver Spring, MD) (Fig. 2A). C-Myc expression was decreased in a statistically significant manner in ApcMin/+ mice homozygous for the floxed c-Myc allele compared to the expression in mice with the wild type c-Myc (p > 0.00001). Western blot analysis of N-Myc, L-Myc and B-Myc was also performed to investigate whether other members of the Myc family can compensate for the loss of c-Myc expression in the intestinal tract of ApcMin/+; c-Mycfl/fl mice. These proteins were not detected in the intestinal tissue of 110 day old ApcMin/+; c-Mycfl/fl mice (data not shown).
We performed histological evaluation of the intestinal tract morphology in ApcMin/+; c-Mycfl/fl mice using a whole intestinal roll preparation stained by hematoxylin and eosin (H&E). Intestinal
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morphology and crypt appearance in ApcMin/+; c-Mycfl/fl mice were within normal limits and similar to the intestinal tract morphology of littermates with wild type c-Myc (Fig. 2B). We also measured the crypt size in ApcMin/+ mice with the floxed c-Myc genotype using H&E-stained sections of the distal 2 cm of the small intestine. We found no statistically significant difference in the crypt size between ApcMin/+; c-Myc+l+ and ApcMin/+; c-Mycfl/fl mice in the randomly scored crypts (60.31 ± 6.69 mm and 66.5 ± 16.7 mm, respectively).
Loss of c‑Myc in the small intestine is associated with increased apoptosis. To evaluate the apoptotic pathway in ApcMin/+; c-Mycfl/fl mice, we performed immunohistochemistry analysis for the cleaved form of caspase 3, as a marker of cellular apoptosis38 in the same regions of the distal small intestine where c-MYC expression was
evaluated. Activated caspase-3 positive cells were present at the bottom and middle section of the intestinal crypt in the ApcMin/+; c-Mycfl/fl mouse, compared to the middle section in the control c-Myc wild type mouse (Fig. 3A and B). The localization of apoptotic cells was similar in c-Myc wild type anf knockout mice. The serial sections of distal portions of the small intestines were also subjected to apop-totic cell counts by morphological criteria using H&E staining. In ApcMin/+; c-Myc+/+ mice, typically, 2% of intestinal crypts contained apoptotic cells in the crypt bases. In ApcMin/+; c-Mycfl/fl mice, the percent of intestinal crypts with apoptotic cells in the proliferating zone was higher (4%) (Fig. 3C, p = 0.0016).
Intestinal specific c‑Myc gene knockout suppresses intestinal tumorigenesis in the Min mouse. The control ApcMin/+ transgenic mice with wild type c-Myc on the mix C57BL6.129 FVB back-ground exhibited the FAP phenotype similar to that observed in the C57BL/6J-Apc Min/+ mice, such that they develop tumors predomi-nantly in the small intestine. We measured the expression of c-Myc protein in the intestinal tissue and intestinal adenomas of ApcMin/+; c-Myc+/+ mice. Mice were harvested at 110 days of age, and the apparently normal intestine and individually collected adenomas of size ≥3.0 mm were processed for the c-Myc analysis. In ApcMin/+; c-Myc+/+ mice, the c-Myc protein level in the intestinal adenomas was elevated by 5 fold compared with the apparently normal intestinal
Figure 1. (A) Immunofluorescent detection of Cre recombinase in the proximal, middle and distal segments of the small intestine of ApcMin/+; c-Mycfl/fl mouse. Small intestinal tissue segments were collected from 110 day old ApcMin/+ c-Mycfl/fl mice, and Cre recombinase staining on cryosections was performed as described in Materials and Methods. DAPI was used to visualize nuclei. 2oAb+DAPI indicates secondary goat anti-rabbit IgG coupled to Alexa Fluor 488. Cre recombinase staining of the small intestinal tissue of ApcMin/+; c-Mycfl/fl mice, which did not have Cre recombinase gene, was used as a negative control (Cre + DAPI). Data shown is representative of three independent experiments performed from tissue isolated from three mice. (B) Fluorescent images of distal segments of the small intestine from ApcMin/+ animals with the wild type c-Myc and animals homozygous for a c-Myc allele showing the nuclear localization of Cre recombinase. Distal 2 cm segments of the small intestine were collected from110 day old mice and stained with a Cre recombinase antibody as described in Materials and Methods. Secondary goat anti-rabbit IgG coupled to Alexa Fluor 488 was used as a negative control. Nuclei were visualized by staining with DAPI and were pseudocolored as red. Colocalization of Cre recombinase (green fluorescence) and nuclei appears as yellow fluorescence. Images shown were acquired at 60x objective. Data shown is representative of three independent experiments performed from tissue isolated from three mice of each genotype.
Figure 2. Immunohistochemical and histological analysis in the small intestines of ApcMin/+ animals with wild type and deleted c-Myc. (A) Immunohistochemical analysis of the c-Myc protein expression in ApcMin/+ animals with the wild type c-Myc and animals homozygous for a floxed c-Myc allele. The distal segments (about 2 cm proximally to the cecum) of the small intestine from 110 days old ApcMin/+; c-Myc+/+ and ApcMin/+; c-Mycfl/fl mice were collected and stained for c-MYC as described in Materials and Methods. A secondary goat anti-rabbit polyclonal antibody was used as a negative control. Images shown were acquired at 100x objective. Data shown is representative of three independent experiments performed from tissue isolated from three mice of different c-Myc genotypes. Scoring of c-Myc positive cells was performed as described in Materials and Methods. *p < 0.00001. (B) Intestinal tract morphology of the ApcMin/+ animals with the wild type c-Myc and animals homozygous for a floxed c-Myc allele. Swiss small intestinal rolls were prepared from 110 day old ApcMin/+; c-Myc+/+ and ApcMin/+; c-Mycfl/fl mice and processed for H&E staining as described in Materials and Methods. Images presented were obtained at 40x magnification from the region located 2 cm proximally from the cecum. Data shown is representative of three independent experiments performed from tissue isolated from three mice of different c-Myc genotypes.
Conditional Intestinal c-Myc Knockout in the Apc Min/+ Mouse
tissue (Fig. 4A). ApcMin/+; c-Myc+/+ mice developed on average 7.33 + 2.1 adenomas per mouse in the small intestine when fed AIN-93G diet (Mean ± SD, n = 12 mice/group) which is 4 times less then Min mice on a pure C57BL/6 inbred genetic background (C57BL/6J-Apc Min/+ mice). ApcMin/+c-Mycfl/fl mice developed on average 3.59 + 0.72 (n = 18 mice/group) adenomas per mouse that is significantly lower than the c-Myc wild type littermates (p = 0.0003). The average tumor number per mouse was found to be significantly lower in the proximal, middle and distal areas of the small intestine of ApcMin/+ mice after c-Myc deletion compared with their wild-type c-Myc litter-mates (Fig. 4B).
DIscussIoNOverexpression of the c-MYC proto-oncogene occurs in a variety
of human cancers and is frequently observed in colon and other gastrointestinal cancers.10-13,15 Truncating mutations in APC lead to the formation of constitutive nuclear b-catenin/TCF complexes.42,43 In the intestinal epithelium, TCF-4 is the most prominently expressed TCF family member, and c-MYC is a major downstream target of Tcf-4.42 It is known that the c-MYC transcriptional factor regulates expression of many genes related to cell growth, division and apoptosis.39,40 Among them are ornithine decarboxylase, cyclin D2
and CDK genes, which are involved in cell proliferation, cell cycle progression, and tumorigenesis.4,5,41 Knockout of c-Myc in mice is embryonic lethal.28 To understand the contribution of c-MYC in colorectal transformation we evaluated intestinal tumorigenesis in the ApcMin/+ mouse with the conditional c-Myc knockout. We generated a Min mouse model with Fabpl 4x at -132Cre driven Cre-recombinase expression which mediated intestine-specific knockout of the c-Myc gene. Wong et al37 provided a detailed characterization of the FVB-Fabpl 4x at -132Cre transgenic mouse indicating that Cre is expressed in multipotent crypt stem cells and has a mosaic pattern.37 We observed a similar pattern of Cre recombinase expression in Fabpl4×at -132Cre; c-Mycfl/fl mice as well as in their crosses to ApcMin/+ mice. Immunohistochemical analysis of histological tissue sections of the ApcMin/+; c-Mycfl/fl mice showed a more than two-fold decrease in the number of regions with c-Myc positive crypts. Although c-Myc protein expression was reduced, histological analysis of the whole intestinal tract of ApcMin/+; c-Mycfl/fl mice did not show any changes in the intestinal tract morphology or the crypt size. This implies that the conditional loss of c-Myc in the intestinal epithelium mediated by the transcriptional regulatory elements of a liver fatty acid-binding protein gene does not alter intestinal development and homeostasis in adult mice. Our conclusions are similar to the finding reported recently by Bettes and coworkers, who observed a Myc-independent
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Figure 3. Effect of the intestine-specific c-Myc deletion on the small intestinal apoptosis in the ApcMin/+mouse. (A) Cleaved caspase-3 expression in the small intestine of the ApcMin/+mouse with wild type and deleted c-Myc. Cleaved caspase-3 was found at the base and in the middle of the crypt in the ApcMin/+; c-Mycfl/fl mouse. Apoptotic cells positively stained are marked with arrows. A biotinylated secondary anti-rabbit antibody was used as a negative control. (B) Images of cleaved caspase 3 staining captured at 40x magnification. (C) Analysis of apoptosis in distal segments of the small intestine of ApcMin/+ animals with wild type c-Myc and animals homozygous for a floxed c-Myc allele. Analysis was performed on 110 day old ApcMin/+ c-Myc+/+ and ApcMin/+ c-Mycfl/fl mice. The apoptotic index was evaluated in the serial section of distal small intestinal region 2 cm proximally to cecum by morphologic criteria as described in Materials and Methods and was expressed as an apoptotic index. The statistical significance (p = 0.0016) was calculated using ANOVA.
Figure 4. Effect of the intestine-specific c-Myc deletion on small intestinal tumorigenesis in the ApcMin/+mouse. 4A. Expression of c-MYC in the normal intestinal tissue and in adenomas of transgenic ApcMin/+ animals with wild type c-Myc. Normal intestinal tissue (N) and adenomas (Tumors) were iso‑lated from 110 day old ApcMin/+ c-Myc+/+ mice and processed for Western blot analysis of c-Myc as described in Materials and Methods. b-actin level was used as loading control. Densitometric analysis was performed with Scion Image Analysis software. 4B. Intestinal tumorigenesis in ApcMin/+ ani‑mals with wild type c-Myc and deleted floxed c-Myc alleles. Tumor counts were performed on 110 day old ApcMin/+ c-Myc+/+ (n=12 mice/group) and ApcMin/+ c-Mycfl/fl (n=17 mice/group) mice fed AIN-93G diet. The statistical significance was calculated for the average tumor number per mouse in the proximal, middle and distal portions of the small intestine between c-Myc+/+ and c-Mycfl/fl groups using the Poisson regression.
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proliferation and expansion of intestinal epithelial cells in the mouse model with a knockout of one c-Myc allele via tamoxifen-induced activation of the Cre-ERT2 recombinase, driven by the villin promoter.31 Tumor analysis showed that intestinal tumorigenesis was suppressed in ApcMin/+; c-Mycfl/fl mice compared with the wild type c-Myc littermates. Immunohistochemical analysis for c-Myc and evaluation of apoptosis was performed in serial sections of ApcMin/+ animals with floxed c-Myc and showed a 2-fold increase in the number of apoptotic cells in the regions of the small intestine with decreased c-MYC protein expression level.
The other members of Myc family, N-, L-, and B-Myc, which all encode for basic region/helix-loop-helix/leucine zipper transcrip-tion factors, share similar biological activity and have oncogenic potential. N- and L-Myc genes were found to be amplified in human neuroblastomas and in a subset of human small-cell lung carcinomas, respectively.47,48 N-Myc has been shown to functionally replace c-Myc in vivo.49 In the present study we did not observe a compensatory increase in N-, L- or B-Myc protein expression, suggesting a unique role of c-Myc in colon tumorigenesis.
We present here a mouse model of intestinal tumorigenesis with an intestine-specific knockout of the c-Myc gene. In this mouse model, the c-Myc knockout in the intestinal epithelium is not essential for normal epithelial development, but is important for the development of intestinal tumors. Our data suggests that c-MYC plays an important role in the development of intestinal tumors with activating mutations in the APC gene, and this role is associated with decreased apoptosis in the normal intestinal epithelium.
C-MYC presents an attractive target for pharmacological inhibi-tion. Transient c-Myc inactivation can be an effective therapy for certain cancers as has been shown in the transgenic mouse model of osteogenic sarcoma.50 Repression of the c-MYC promoter is regulated in part via formation of a G-quadruplex structure.51 Therapeutically selective transcriptional silencing of the c-MYC promoter has been achieved using a porphyrin analogue that selectively binds to the c-MYC G-quadruplex.52 Our observations support the rationale for targeting c-MYC as a possible therapeutic strategy for suppression of APC-dependent intestinal cancers.
AcknowledgementsThe authors gratefully acknowledge Dr. Elliott Epner for his help
in obtaining the c-Mycfl/fl mice. The authors thank Carl Boswell, Ph.D. and the Confocal Imaging Shared Service at AZCC for the technical help in obtaining Cre recombinase images and William Meek, Cancer Biology Division, Arizona Cancer Center, for the technical help with scoring of c-Myc immunohistochemistry slides. We are also greatiful to Kim Nicolini for editorial assistance with manuscript preparation. This work was supported by the NIH grant CA-72008 and a contract from the Arizona Disease Control Research Commission (ADCRC) #8004.
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