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Cancer Genes and Genomics

Inhibition of Intestinal Polyposis with Reduced Angiogenesis in Apc^Min/+ Mice Due to Decreases in c-Myc Expression

Department of Cell and Developmental Biology and Anatomy, University of South Carolina School of Medicine, Columbia, South Carolina

Requests for reprints: Troy A. Baudino, Department of Cell and Developmental Biology and Anatomy, University of South Carolina School of Medicine, 6439 Garners Ferry Road, Building #1, C-57, Columbia, SC 29209. Phone: 803-733-1562; Fax: 803-733-1533. E-mail: tbaudino{at}med.sc.edu

	Abstract

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
The c-myc oncogene plays an important role in tumorigenesis and is frequently deregulated in many human cancers, including gastrointestinal cancers. In humans, mutations of the adenomatous polyposis coli (Apc) tumor suppressor gene occur in most colorectal cancers. Mutation of Apc leads to stabilization of β-catenin and increases in β-catenin target gene expression (c-myc and cyclin D1), whose precise functional significance has not been examined using genetic approaches. Apc^Min/+ mice are a model of familial adenomatous polyposis and are heterozygous for an Apc truncation mutation. We have developed a model for examining the role of c-Myc in Apc-mediated tumorigenesis. We crossed c-myc^+/– mice to Apc^Min/+ to generate Apc^Min/+ c-myc^+/– animals. The compound Apc^Min/+ c-myc^+/– mice were used to evaluate the effect of c-myc haploinsufficiency on the Apc^Min/+ phenotype. We observed a significant reduction in tumor numbers in the small intestine of Apc^Min/+ c-myc^+/– mice compared with control Apc^Min/+ c-myc^+/+ mice. In addition, we observed one to three polyps per colon in Apc^Min/+ c-myc^+/+ mice, whereas only two lesions were observed in the colons of Apc^Min/+ mice that were haploinsufficient for c-myc. Moreover, reduction in c-myc levels resulted in a significant increase in the survival of these animals. Finally, we observed marked decreases in vascular endothelial growth factor, EphA2, and ephrin-B2 expression as well as marked decreases in angiogenesis in intestinal polyps in Apc^Min/+ c-myc^+/– mice. This study shows that c-Myc is critical for Apc-dependent intestinal tumorigenesis in mice and provides a potential therapeutic target in the treatment of colorectal cancer. (Mol Cancer Res 2007;5(12):1296–303)

	Introduction

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Mutation and inactivation of the adenomatous polyposis coli (Apc) tumor suppressor gene is an early event in the development of colorectal cancer (1). Inactivation of Apc occurs in 80% of adenomatous polyps and it is also responsible for the genetic disorder familial adenomatous polyposis, which leads to the formation of adenomatous polyps in the small and large intestine that results in colon cancer (2-4). It has been shown that the major function of Apc is to degrade cytosolic levels of β-catenin, thus preventing formation of the β-catenin/T-cell factor-4 complex and effectively blocking β-catenin/T-cell factor-4–meditated transcription (5). Interestingly, mice lacking T-cell factor-4 display no proliferating cells in their intestinal crypts, suggesting that this signaling pathway is essential for intestinal proliferation (6). Multiple downstream targets for β-catenin have been identified, including cyclin D1 and c-myc, two factors that are critical for cell cycle traverse and other biological processes (7, 8). Indeed, previous studies have shown that c-myc expression is repressed by Apc and is increased by overexpression of β-catenin in human colon cancer cells (7, 9). Although these two β-catenin target genes are likely to be important for colorectal tumorigenesis, the exact functional significance of these targets has yet to be clearly defined. The c-myc gene encodes a transcription factor with a basic helix-loop-helix leucine zipper domain that mediates heterodimerization with its obligate DNA-binding partner Max. This basic helix-loop-helix leucine zipper domain is also essential for direct DNA binding. Myc activates or represses the transcription of a wide array of target genes that elicit a variety of biological responses, including cell growth, proliferation, differentiation, metabolism, and apoptosis (10-12). In addition, the role of Myc during development has been examined in several tissues, including the gastrointestinal tract (13). Previous data from the Trumpp laboratory showed that c-Myc is essential for intestinal crypt formation but is dispensable for maintenance and homeostasis of the adult intestinal epithelium (13). These data were surprising because c-Myc is generally essential for cell cycle traverse. Thus, the role of c-Myc during development and homeostasis of the intestine remains to be discovered. Additionally, c-Myc has also been shown to modulate the expression of various angiogenic factors, including vascular endothelial growth factor (VEGF; refs. 14-16). Moreover, c-Myc has been shown to down-regulate antiangiogenic factors, such as thrombospondin-1 (14, 17, 18). The importance of Myc in cancer is evident by its increased expression in many human cancers, including colon, breast, prostate, and lung (19). Furthermore, Myc overexpression in various cell types in mice induces tumors that display increased proliferation, delayed differentiation, and increased angiogenesis (16, 20). Indeed, c-Myc levels are increased in the tumors of Apc^Min/+ mice and deletion of c-myc results in reduced tumorigenesis due to increases in apoptosis (21). However, these previous studies from the Gerner and Trumpp laboratories did not examine other biological processes in which Myc is involved, specifically angiogenesis. Therefore, because c-Myc expression is increased in the Apc^Min/+ mouse model of familial adenomatous polyposis, it is possible that this leads to increases in c-Myc target gene expression and promotes progression through the adenoma-carcinoma sequence and subsequent metastasis. Thus, the aim of the present study was to evaluate the role of c-Myc on tumorigenesis in the Apc^Min/+ murine model.

	Results

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
c-myc Haploinsufficiency Increases the Survival of Apc^Min/+ Mice and Greatly Decreases Tumor Incidence and Size
It has been previously shown that c-myc is a β-catenin target gene, and in Apc^Min/+ mice, the levels of c-myc are significantly increased when compared with wild-type (WT) animals (7, 9). Moreover, it has been previously shown that complete deletion of c-myc in the intestinal tract does not perturb normal intestinal homeostasis but does disrupt the formation of intestinal crypts (13). In addition, recent studies from Sansom et al. (22) show that c-myc deletion blocks tumorigenesis in Apc-deficient mice in the small intestine, suggesting that Myc is a critical mediator of neoplasia following Apc loss. Furthermore, recent studies from the Gerner laboratory showed that c-myc loss (2-fold reduction in protein) resulted in reduced tumorigenesis in Apc^Min/+ mice due to increased levels of apoptosis (21). Thus, to further investigate the role of c-Myc in Apc-mediated tumorigenesis, we crossed Apc^Min/+ mice with c-myc^+/– animals to generate Apc^Min/+ c-myc^+/– mice. We then followed the life span of these animals, with mice being humanely sacrificed when moribund. Mice that were WT for c-myc and Apc^Min/+ had a median life span of 169 days, whereas animals that were heterozygous for c-myc and Apc^Min/+ lived an average of 291 days (Fig. 1 ). Amazingly, many of the animals that were Apc^Min/+ c-myc^+/– were still alive at 1 year of age (Fig. 1).

View larger version (9K): [in this window] [in a new window] [Download PPT slide]   FIGURE 1. Survival curve of ApcMin/+ mice that are WT or haploinsufficient for c-myc. Loss of a single allele of c-myc results in a dramatic increase in the life span of ApcMin/+ animals. ApcMin/+ mice that are WT for c-myc (gray) or heterozygous for c-myc (black) are shown. Mice that were WT for c-myc and ApcMin/+ had a median life span of 169 d (n = 65), whereas animals that were heterozygous for c-myc had a median life span of 291 d (n = 37).

View larger version (57K): [in this window] [in a new window] [Download PPT slide]   FIGURE 2. Intestinal and colonic polyp numbers are reduced in ApcMin/+ c-myc+/– animals. A. Intestinal polyps were counted according to size in 12-wk-old age-matched animals that were either WT (black columns) or heterozygous (gray columns) for c-myc (n = 10 for each group). We observed a 67% reduction in polyps <0.5 mm in size, a 75% reduction in polyps 0.5 to 2.0 mm in size, and a 90% reduction in polyps >2.0 mm in size in c-myc+/– animals. B. Intestinal polyps were counted according to size in moribund animals that were either WT (black columns; n = 29) or heterozygous (gray columns; n = 20) for c-myc. Again, notice the marked reduction in the number of polyps present in c-myc+/– animals. C. Total numbers of colonic polyps were counted in moribund animals. There was a >90% reduction in the number of colonic polyps per mouse in animals that were heterozygous for c-myc. In fact, only two c-myc+/– animals (n = 20) displayed any colonic polyps. D. Representative images of intestines isolated from age-matched (12 wk) ApcMin/+ mice that were either WT (left) or heterozygous (right) for c-myc. Notice the marked decrease in the number and size of polyps present in c-myc+/– animals. E. Representative H&E-stained sections for ApcMin/+ mice that were WT (left) or heterozygous (right) for c-myc. Notice the decrease in polyp size (white arrows) in c-myc+/– animals.

View larger version (58K): [in this window] [in a new window] [Download PPT slide]   FIGURE 3. Cell proliferation is reduced in intestinal polyps of ApcMin/+ c-myc+/– animals. A. Immunofluorescent images showing phospho-histone H3 (P-Histone H3)-positive cells (red) in intestinal polyps from WT (left) or c-myc+/– animals (right) that are ApcMin/+. Sections were counterstained with 4',6-diamidino-2-phenylindole (blue) to visualize nuclei. B. Cell proliferation is reduced in intestinal polyps present in ApcMin/+ c-myc+/– mice. Graph showing the percentage of cells undergoing proliferation in ApcMin/+ c-myc+/+ (black columns) or ApcMin/+ c-myc+/– (gray columns) mice. Data were obtained from z-series confocal sections (n = 10) of multiple intestinal tumors (n = 5) from multiple mice (n = 5 per group) and quantified using Image-Pro Plus software.

View larger version (51K): [in this window] [in a new window] [Download PPT slide]   FIGURE 4. Effect of c-myc haploinsufficiency on apoptosis in the normal intestinal crypts and polyps of ApcMin/+ mice. A, B, D, and E. TUNEL-positive nuclei were quantified in the normal intestinal crypts or polyps of WT (black columns) or c-myc+/– (gray columns) mice and expressed as percentage of total number of nuclei counted using Image-Pro Plus software in the representative x40 confocal images. There is no significant difference in the percentage of TUNEL-positive nuclei in the normal intestinal crypts between ApcMin/+ c-myc+/+ and ApcMin/+ c-myc+/– (C), but the percentage of TUNEL-positive nuclei is dramatically increased with loss of one allele of c-myc in polyps isolated from ApcMin/+ intestines (F). n = 5 mice for each group; n = 5 crypts or polyps from each mouse. Bar, 50 µm.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   FIGURE 5. c-myc and cyclin E, but not cyclin D1, expression is increased in the intestinal polyps of ApcMin/+ mice. A. Normal intestinal tissue or polyps of equal sizes (0.5 mm, 0.5-2.0 mm, or >2.0 mm) were examined for c-myc expression by real-time PCR. All samples are normalized to ARPP P0 and then expression is plotted as fold of normal WT or c-myc+/– tissue. c-myc expression is reduced in ApcMin/+ c-myc+/– (gray columns) when compared with ApcMin/+ mice that are WT for c-myc (black columns). However, in the larger polyps, c-myc expression is amplified. B. Cyclin D1 expression is unchanged regardless of c-myc expression. C. Cyclin E expression is reduced in intestinal polyps that are heterozygous for c-myc. Notice that the reduction in cyclin E expression correlates with levels of c-myc expression. Statistical significance between samples as determined by Student's t test (n = 4).

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   FIGURE 6. Reduced levels of c-myc result in decreased expression of angiogenic factors in tumors in the small intestine. A. Levels of VEGF present in the serum of age-matched (8 wk) WT (solid black columns), c-myc+/– (solid gray columns), or ApcMin/+ mice that were either WT for c-myc (black hatched columns) or heterozygous for c-myc (gray hatched columns) are shown. Notice the increase in VEGF levels in ApcMin/+ mice that are WT for c-myc (n = 5 per group). B. Normal intestinal tissue or intestinal polyps were examined for VEGF expression by real-time PCR. Total RNA was isolated from normal intestinal tissue from ApcMin/+ c-myc+/+ (solid black columns) or pooled intestinal polyps (black hatched columns) or normal tissue from ApcMin/+ mice that were heterozygous for c-myc (solid gray columns) or pooled intestinal polyps (gray hatched columns). All samples were normalized to ARPP P0 and then expression was plotted as fold of normal WT tissue. Significance between samples as determined by Student's t test (n = 5). C. Normal intestinal tissue or polyps of equal sizes (0.5 mm, 0.5-2.0 mm, or >2.0 mm) were examined for VEGF expression by real-time PCR. Notice that VEGF expression is reduced in ApcMin/+ c-myc+/– (gray columns) when compared with ApcMin/+ mice that are WT for c-myc (black columns). However, in the larger polyps, VEGF expression is increased, similar to that pattern observed for c-myc expression. Statistical significance between samples as determined by Student's t test (n = 4). D. Expression of several Ephs and ephrins in normal intestinal tissue and polyps. Total RNA was isolated and examined for EphA2, EphB4, ephrin-A1, and ephrin-B2 expression. As in B, all samples were normalized to ARPP P0. c-myc expression only alters the expression of EphA2 and ephrin-B2. Asterisks, significance between the sample and all other samples as determined by Student's t test (n = 5).

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   FIGURE 7. Reduced c-myc expression in ApcMin/+ mice restores spleen size and a normal hematocrit. A. Blood was isolated from age-matched animals (12 wk) that were either Apc+/+ c-myc+/+, ApcMin/+ c-myc+/+, Apc+/+ c-myc+/–, or ApcMin/+ c-myc+/– and hematocrit was determined. B. WBC counts were restored in ApcMin/+ mice on c-myc loss. C. Loss of c-myc eliminated splenomegaly in ApcMin/+ mice. Following eye bleeds, hematocrit and WBC counts were done using a VetScan HM2 Hematology System. Following blood isolation, animals were humanely sacrificed and spleens were isolated and weighed. Asterisks, significance between the sample and all other samples as determined by Student's t test (n = 5).

	Discussion

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

Myc activates or represses the transcription of a wide array of genes, including proangiogenic and antiangiogenic factors (14-16). Indeed, Myc overexpression in various cell types in mice induces tumors that display increased proliferation, delayed differentiation, and increased angiogenesis (16, 20). To better understand the role that c-Myc plays in Apc-mediated tumorigenesis, we examined tumor formation and survival in Apc^Min/+ animals that were either WT or heterozygous for c-myc. Our results clearly showed that c-Myc is critical for Apc-mediated tumorigenesis, as loss of a single allele of c-myc resulted in a dramatic increase in survival (Fig. 1). Additionally, c-myc^+/– animals displayed markedly fewer overall polyps than their WT littermates, as well as showing dramatic decreases in the number of larger polyps (>2.0 mm; 90% decrease; Fig. 2A and B). Surprisingly, we only observed a 2-fold change in the level of proliferation within the intestinal polyps following the loss of a single c-myc allele (Fig. 3A and B). We also examined the normal intestinal crypts as well as polyps for apoptosis. We observed no differences in the apoptotic indices in the normal intestinal crypts between WT and c-myc heterozygotes in Apc^Min/+ mice (Fig. 4A-C). However, the number of TUNEL-positive cells was significantly increased in adenomatous polyps in animals that were heterozygous for c-myc (Fig. 4D-F). These results are interesting, as previous studies from Alan Clarke's laboratory showed that there is a decrease in levels of apoptosis in the small intestine following loss of both Apc and c-myc in the adult animal (22). Furthermore, previous studies from our laboratory and others showed that c-myc loss has no effect on levels of apoptosis either in E9.5 embryos (14) or in the normal intestinal epithelium (13).

These effects on proliferation and apoptosis are due to changes in c-myc expression in the polyps of Apc^Min/+ mice. Indeed, when we examined c-myc expression in intestinal polyps from Apc^Min/+ c-myc^+/– mice, we observed a significant decrease in c-myc expression in the smaller polyps when compared with those isolated from Apc^Min/+ mice that are WT for c-myc (Fig. 5A). However, in the larger polyps, the difference in c-myc expression is not as great (Fig. 5A). This increase in c-myc expression that is observed in the larger polyps of the Apc^Min/+ c-myc^+/– mice could be due to loss of the WT Apc allele or could also be due to increases in c-Myc expression due to the hypoxic environment of the larger polyps. Indeed, several recent articles have shown that hypoxia can increase c-Myc expression and/or transcriptional activity (26, 27).

In addition to the observed proliferative and apoptotic changes in the Apc^Min/+ c-myc^+/– mice, the polyps that did form in these animals (Apc^Min/+ c-myc^+/–) showed marked decreases in vascularization when compared with Apc^Min/+ c-myc^+/+ mice and the blood vessels present in the polyps from Apc^Min/+ c-myc^+/– animals seem to be nonfunctional (data not shown). Finally, these decreases in vascularization seem to be due to significant reductions in levels of VEGF, EphA2, and ephrin-B2 expression (Fig. 6B-D). Taken together, these studies show that c-Myc is an important downstream target of β-catenin and that c-Myc functions as an important mediator of polyp growth and colorectal cancer initiation by regulating factors that are essential for angiogenesis to allow for adequate vascularization, supply of nutrients, and tumorigenesis. These studies further strengthen the importance of c-Myc as a target for novel therapeutic modalities in the treatment of colorectal cancers as well as other cancers with deregulated Myc expression.

	Materials and Methods

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Animal Studies
Apc^Min/+ mice on a pure C57BL/6 background were mated to c-myc^+/– mice on a mixed C57BL/6 x SV129 background, and the resulting pups were screened for the Min mutation (28) and for the c-myc–null gene (14) by PCR as previously described. Animals that were homozygous for the WT c-myc gene and heterozygous for the Min mutation served as controls. Animals were housed and bred within the Animal Resources Facility at the University of South Carolina School of Medicine. Primer sequences for genotyping are available on request.

Polyp Number Determination
Intestinal adenomas and colonic adenomas were scored for numbers and size (diameter) at postnatal day 84 (age matched) or when the animal was moribund. The mice were weighed and sacrificed by cervical dislocation and blood and serum were obtained. In addition, the small and large intestines and colon were isolated. The intestinal tract was rinsed with cold PBS using a blunt-end syringe and opened along the longitudinal axis. The opened intestine was spread flat between sheets of filter paper and fixed in fresh 4% paraformaldehyde. Paraformaldehyde-fixed intestinal sections were rinsed in deionized water and stained with 0.2% methylene blue and polyps were counted at x20 magnification under a dissecting microscope using tweezers to pick through the intestinal villi and identify polyps. Polyps were categorized as >0.5 mm, 0.5 to 2.0 mm, or >2.0 mm in size. After polyps were counted, intestinal sections were placed in 70% ethanol for further analysis. For each experimental group, the incidence of tumors, defined as the number of mice with tumors/number of mice in the group, the mean number of tumors/mouse ± SD, and the mean tumor diameter (mm) in the group ± SD were calculated for the intestine and colon separately. Student's t test was used to compare means of each group; differences were considered to be significant if P values were <0.05. For histology, segments of intestine and colon were paraffin embedded; 5-µm sections were cut and stained with H&E. In addition, whole mount images were taken at x20 to show differences in polyp size and number.

Measurement of Hematocrit and Spleen Weight
For measurement of hematocrit and WBC counts, retro-orbital eye bleeds (200 µL) were done on WT, Apc^Min/+ c-myc^+/+, Apc^+/+ c-myc^+/–, and Apc^Min/+ c-myc^+/– age-matched animals (12 weeks) and samples were analyzed using the VetScan HM2 Hematology System (Abaxis). Animals were then sacrificed and their spleens were isolated and weighed (n = 5). In addition, their small and large intestines and colon were isolated as described above for further analyses.

ELISA Measurement of VEGF
Retro-orbital bleeds were done as described above on age-matched animals (8 weeks) and blood samples were allowed to coagulate and samples were centrifuged at 14,000 x g for 10 min to isolate blood serum. Serum was then assayed for VEGF protein levels using an ELISA assay specific for murine VEGF as described by the manufacturer (R&D Systems).

Real-time PCR Analyses
Total RNA was isolated from normal intestinal tissue or intestinal tumor tissue using Trizol reagent (Invitrogen) as described by the manufacturer. Real-time PCR analyses were done using a Bio-Rad reverse transcription-PCR kit for probes and the Bio-Rad iQ5 Real-time Thermocycler (Bio-Rad) as described by the manufacturer. We used primer/probe sets that were specific for murine c-myc, cyclin D1, cyclin E, VEGF, EphA2, EphB4, ephrin-A1, ephrin-B2, and ARPP P0. ARPP P0 served as our internal control for normalization. Primer and probe sequences are available on request.

Tissue Isolation and Cell Proliferation Analyses
Immunofluorescent analyses were done on intestines isolated from 12-week-old Apc^Min/+ c-myc^+/+ and Apc^Min/+ c-myc^+/– animals. Briefly, intestines were fixed in fresh 4% paraformaldehyde overnight at 4°C and 120-µm-thick sections were cut using a vibratome (Oxford Instruments). Sections were permeabilized using PBS containing 0.01 mol/L glycine and 0.1% Triton X-100 for 1 h at room temperature. The sections were then blocked using PBS containing 5% bovine serum albumin for 1 h at room temperature. Antibody against phospho-histone H3 (Santa Cruz Biotechnology) was used at a 1:200 concentration. Sections were incubated with primary antibody in 1% blocking buffer (1% bovine serum albumin/PBS) overnight at 4°C. Sections were washed and incubated with donkey anti-rabbit FITC secondary antibody (Zymed) at a 1:100 concentration in 1% blocking buffer for 2 h at room temperature. In addition, sections were stained with Texas red phalloidin for actin visualization and nuclei were visualized through staining with 4',6-diamidino-2-phenylindole (Molecular Probes). Intestinal sections were imaged using a Leica LSM 510 laser scanning microscope. Image-Pro Plus software was used to quantify total number of nuclei and nuclei that were phospho-histone H3 positive per representative x40 high magnification field. Five polyps were analyzed from five individual mice for each genotype (Apc^Min/+ c-myc^+/+ and Apc^Min/+ c-myc^+/–).

Cell Death Detection In situ Using TUNEL
Apoptotic cells in the intestinal polyps were visualized using the TUNEL method (Roche Molecular Biochemicals) as described by the manufacturer. Briefly, deparaffinized tissue sections were permeabilized using Triton X-100 (Sigma). The sections were then incubated with TUNEL reaction mixture containing terminal deoxynucleotidyl transferase and fluorescein-dUTP, at 37°C for 30 min, rinsed in PBS for 10 min, mounted, and analyzed using a Leica LSM 510 laser scanning microscope. Image-Pro Plus software was used to quantify the number of TUNEL-positive nuclei. DNase I–treated tissue sections were used as positive controls. TUNEL-positive nuclei were expressed as percent of total number of nuclei counted per representative x40 higher magnification image. Scale bars are set at 50 µm.

	Acknowledgements

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
We thank Drs. Frank Berger and Tom Borg for their mentorship and valuable comments and Arti Intwala for her work in breeding, maintaining, and genotyping our mouse colonies.

	Notes

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Grant support: NIH grant RR017698.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5/24/07; revised 7/31/07; accepted 8/ 7/07.

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Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 

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