The homeodomain transcription factor Cdx1 does not behave as an oncogene in normal mouse intestine

Abstract

The Caudal-related homeobox genes Cdx1 and Cdx2 are intestine-specific transcription factors that regulate differentiation of intestinal cell types. Previously, we have shown Cdx1 to be antiproliferative and to promote cell differentiation. However, other studies have suggested that Cdx1 may be an oncogene. To test for oncogenic behavior, we used the murine villin promoter to ectopically express Cdx1 in the small intestinal villi and colonic surface epithelium. No changes in intestinal architecture, cell differentiation, or lineage selection were observed with expression of the transgene. Classic oncogenes enhance proliferation and induce tumors when ectopically expressed. However, the Cdx1 transgene neither altered intestinal proliferation nor induced spontaneous intestinal tumors. In a murine model for colitis-associated cancer, the Cdx1 transgene decreased, rather than increased, the number of adenomas that developed. In the polyps, the expression of the endogenous and the transgenic Cdx1 proteins was largely absent, whereas endogenous Villin expression was retained. This suggests that transgene silencing was specific and not due to a general Villin inactivation. In conclusion, neither the ectopic expression of Cdx1 was associated with changes in intestinal cell proliferation or differentiation nor was there increased intestinal cancer susceptibility. Our results therefore suggest that Cdx1 is not an oncogene in normal intestinal epithelium.

Figure 1

Development of a transgenic mouse line ectopically expressing Cdx1 throughout the mouse intestine. (A) Mouse Cdx1 cDNA was cloned into pCMV-Tag3C to add an N-terminal human cMyc tag and SV40 PolyA, then placed behind 12.4 kb of mouse villin gene regulatory sequences, which included the promoter, untranslated exon 1, intron 1, and the start of exon 2. Genotyping primers, indicated by black arrows, amplified the junction of villin and Cdx1. RT-PCR primers, represented by gray arrows, amplified the junction of Cdx1 and SV40 PolyA. An N-terminal cMyc tag was added to differentiate transgenic from endogenous Cdx1. (B) The villin-mycCdx1 transgene expresses a functioning Cdx1 protein. mycCdx1 protein is detected in nuclear extracts from cells after transfection and is detected in the nucleus by immunofluorescence using anti-Cdx1 or anti-myc antibodies. Equal protein loading was confirmed by YY1 immunoblot analysis. VE, Villin-empty; VMC, Villin-mycCDX1. (C) Transient transfection of mycCdx1 into 293T induces luciferase activity from a canonical Cdx reporter, SI. Gray bar, empty vector control; white bar, mycCdx1. Cotransfection of the canonical β-catenin/TCF reporter TOPFLASH with mycCdx1 constructs demonstrates that the chimera can inhibit β-catenin/TCF transcriptional activity. Black bar, empty vector alone; gray bar, empty vector with β-catenin expression vector; white bar, mycCdx1 with β-catenin expression vector. (D) Transgene expression is shown by RT-PCR. RNA was isolated from the distal jejunum and colon of 1-month-old offspring from two different founders. RT-PCR with Cdx1/SV40 primers demonstrated expression of the transgene in both the small intestine and colon. (E) Overexpression of Cdx1 in the intestine in the Villin-Cdx1 transgenic mice. Jejunum tissue from offspring of two different founders. Nuclear extracts were isolated and loaded on sodium dodecyl sulfate-polyacrylamide gels. Western blot with Cdx1 antibody demonstrates overexpression of Cdx1 in transgenic animals. The same blot was stripped and reprobed with an antibody to nuclear protein YY1 to demonstrate even loading. F3138 and F3139 are the designations of the founder line serving as the source of the jejunal tissue.

Figure 2

Cdx1 ectopic expression in the small intestine villi and colon surface epithelium of Villin-Cdx1 transgenic mice. The small intestine and colon from transgenic or littermate control mice were excised at 1 and 12 months, fixed overnight in 4% paraformaldehyde, then processed for immunohistochemical studies. (A) Anti-Cdx1 antibody (CPSP) demonstrating that Cdx1 protein is limited to the crypt epithelial cells in 1-month-old wild-type mice (arrow). In contrast, in villin-Cdx1 transgenic mice, we see darkly stained nuclei in the upper crypt and in the villus epithelial cells (arrowhead). A mouse monoclonal antibody against the human cMyc-tag does not recognize endogenous mouse cMyc in the crypt cells of 1-month-old wild-type offspring (gray arrow) but detects nuclear-localized transgene expression in the small intestinal villi of transgenic mice (arrowhead). (B) These patterns of Cdx1 expression persist in 1-year-old wild-type and transgenic animals. Cdx1 protein is limited to the colon crypts of 1-year-old wild-type mice but is highly expressed in the colon surface epithelium of transgenic mice.

Figure 3

No change in markers for enterocyte differentiation or cell lineages due to ectopic Cdx1 expression. (A) Immunohistochemistry with anti-sucrase isomaltase antibody shows no change in pattern of sucrase isomaltase gene expression in the small intestine brush border between 7-month-old wild-type and villin-Cdx1 transgenic mice. No difference in activity of brush border enzyme alkaline phosphatase noted in 7-month-old wild-type and transgenic mice. (C) Seven-month-old wild-type and villin-Cdx1 transgenic mice show no difference in the distribution of immunohistochemical staining for an enteroendocrine cell marker (chromagranin-A) or a paneth cell marker (lysozyme). (B) Similarly, staining with Alcian blue, a marker for goblet cells in the ileum of the small intestine and colon, was unchanged by ectopic Cdx1 expression in wild-type and transgenic small intestine.

Figure 4

Ectopic expression of Cdx1 reduces Cdx2 expression in the small intestine villi and colon surface epithelium. Immunohistochemistry with either Cdx1 or Cdx2 antibodies on adjacent sections from 9-month-old nontransgenic and Villin-Cdx1 littermates. (A) Small intestine of nontransgenic mice with normal Cdx1 and Cdx2 expressions. Arrow points to reduced Cdx2 staining. (B) Cdx1 and Cdx2 expression pattern in the colon of transgenic and nontransgenic mice. Arrowhead points to reduced Cdx2 staining. (C) Real-time RT-PCR quantification of Cdx2 mRNA levels in the small intestine of transgenic and nontransgenic littermates. (D) Endogenous Cdx1 and total mRNA levels in the small intestine determined by quantitative RT-PCR. WT, wild-type; Tg, transgenic; NS, nonsignificant differences.

Figure 5

No change in crypt proliferation pattern due to ectopic expression of Cdx1. (A) Immunohistochemistry with anti-BrdU antibody shows that proliferating cells remain restricted to the crypts in both 6-month-old wild-type and transgenic mice small intestine and colon. (B) Quantification of epithelial proliferation based on BrdU incorporation in the ileum and colon. BrdU-positive cells per crypt were counted in 15 well-shaped crypts from three 6-month-old mice for each founder. Matched wild-type littermates were used as controls. Data for the combined analysis from all three transgenic lines, from the ileum and colon (n = 9) are presented. Dark gray bar, wild-type mice; white bar, Villin-Cdx1 transgenic mice.

Figure 6

Ectopic Cdx1 expression directed by the villin-Cdx1 transgene reduced the number of adenomas elicited by AOM/DSS treatment. (A) Significant transgene expression demonstrated in rectum to squamo-columnar junction. Immunohistochemical staining for Cdx1 (CPSP) and the myc-tagged transgene protein (anti-myc). (B) Combined analysis of both treatment groups. Black squares, wild-type littermates receiving two rounds of 3% DSS; white squares, wild-type littermates receiving three rounds of 3% DSS; black circles, villin-Cdx1 transgenic mice receiving two rounds of 3% DSS; white circles, villin-Cdx1 transgenic mice receiving three rounds of 3% DSS. Statistical significance was determined by Student's t test using a two-tailed distribution. (C) Subgroup analysis of treatment groups using Student's t test using a single-tailed distribution. TG, transgenic; WT, wild-type.

Figure 7

Expression of the transgene villin-Cdx1 transgene was lost in the adenomas elicited by AOM/DSS treatment. (A) Sections were obtained from the AOM/DSS polyps and were immunohistochemically stained for Cdx1, the Myc-tagged Cdx1, or Cdx2 proteins. A region of normal epithelium was included for comparison. (B) Staining for β-catenin and villin expression in the polyps from transgenic mice. Insets: higher magnification photo of neoplastic cells.