Inactivation of the vitamin D receptor in APC(min/+) mice reveals a critical role for the vitamin D receptor in intestinal tumor growth

Abstract

Emerging evidence supports an inhibitory role for vitamin D in colorectal carcinogenesis; however, the mechanism remains unclear. The adenomatous polyposis coli (APC)/β-catenin pathway plays a critical role in colorectal carcinogenesis. The purpose of our study is to explore the interactions of vitamin D and APC/β-catenin pathways in intestinal tumor development. APC(min/+) mice with genetic inactivation of the vitamin D receptor (VDR) were generated through breeding. Intestinal tumorigenesis was compared between APC(min/+) and APC(min/+) VDR(-/-) mice at different ages. No differences were seen in the number of small intestinal and colonic tumors between APC(min/+) and APC(min/+) VDR(-/-) mice aged 3, 4, 6 and 7 months. The size of the tumors, however, was significantly increased in APC(min/+) VDR(-/-) mice in all age groups. Immunostaining showed significant increases in β-catenin, cyclin D1, phosphorylated Stat-3 and MSH-2 levels and decreases in Stat-1 in APC(min/+) VDR(-/-) tumors compared to APC(min/+) tumors. These observations suggest that VDR signaling inhibits tumor growth rather than tumor initiation in the intestine. Thus, the increased tumor burden in APC(min/+) VDR(-/-) mice is likely due to the loss of the growth-inhibiting effect of VDR. This study provides strong evidence for the in vivo relevance of the interaction demonstrated in vitro between the vitamin D and β-catenin signaling pathways in intestinal tumorigenesis.

Figure 1

Interaction of liganded VDR with β-catenin. (A) Co-IP assay in SW480 cells. Cells were treated with ethanol (EtOH) or 2×10⁻⁸ M 1,25(OH)₂D₃ (VD) for 4 hours and cell lysates were immunoprecipitated (IP) with (+) or without (−) antibody against β-catenin. The precipitated proteins were analyzed by Western blot (WB) with anti-VDR antibody. (B) Co-IP assays in CCD-18Co cells. Cells were treated with EtOH or 2×10⁻⁸ M 1,25(OH)₂D₃. Total cell lysates were precipitated with anti-VDR antibody, following by WB analysis with anti-β-catenin antibody. (C) Co-IP of cytoplasmic and nuclear extracts from CCD-18Co cells. Cytoplasmic and nuclear extracts were prepared from EtOH- or VD-treated CCD-18Co cells, and analyzed by Co-IP assays as in (B). (D) Effect of 1,25(OH)₂D₃ treatment on β-catenin nuclear translocation. CCD-18Co cells were treated with EtOH or 1,25(OH)₂D₃ for 24 hours, and the cytoplasmic and nuclear extracts were prepared for Western blot analyses with antibodies against β-catenin, VDR, β-tubulin or histone-3 as indicated. Upper panel: Cytoplasmic fractions; Lower panel: Nuclear fractions. Each lane represents an independent sample.

Figure 2

H&E staining of representative intestinal tumors. (A) Small intestinal adenoma from 3-month old APC^min/+ mouse (Magnification: 50x); (B) Colonic aberrant crypt focus (ACF) from 3-month old APC^min/+VDR^−/− mouse (50x); (C) Colonic adenoma from 4-month old APC^min/+VDR^−/− mouse (100x). (D) Small intestinal adenoma from 4-month old APC^min/+VDR^−/− mouse (50x).

Figure 3

Levels of VDR, β-catenin, cyclin D1 and phospho-Stat-3 in the tumors. (A) VDR, β-catenin and BrdU immunostaining. Tumors from 4-month old APC^min/+ (a, c and e) and APC^min/+VDR^−/− (b, d and f) mice were immunostained with antibodies against VDR (a and b), β-catenin (c and d) and BrdU (e and f). Magnification 200x. The insets in panels c and d are in higher magnification (400x). (B) Western blot analysis for β-catenin. Cell lysates from tumors and neighboring normal intestinal tissues from APC^min/+ and APC^min/+VDR^−/− mice were subject to Western blotting analysis with anti-β-catenin antibody. Fold-increase of β-catenin for each group is presented below the blot. Each lane represents one mouse. (C) Cyclin D1 and phospho-Stat-3 immunostaining analysis. Tumors from 4-month old APC^min/+ (a and c) or APC^min/+VDR^−/− (b and d) mice were immunostained with antibodies against cyclin D1 (a and b) or p-Stat-3 (c and d). Magnification, 200x. (D) Quantitative data from immunostained slides shown in (C) for relative levels of cyclin D1 and p-Stat-3, using computer assisted imaging system. * P<0.05, ** P<0.01 vs. APC^min/+.

Figure 4

Levels of BCL-2, Stat-1, MSH-2 and vimentin in tumors. Tumors from 4-month old APC^min/+ (A, C, E and G) and APC^min/+VDR^−/− (B, D, F and H) mice were immunostained with antibodies against BCL-2 (A and B), Stat-1 (C and D), MSH-2 (E and F), and vimentin-1 (G and H). Magnification 200x. (I) Quantitative results of Stat-1, vimentin-1 and MSH-2 immunostaining using the Image J software. * P<0.05; ** P<0.01 vs. APC^min/+. Note the decreased Stat-1 and increased MSH-2 and vimentin-1 staining in APC^min/+VDR^−/− tumors compared to APC^min/+ tumors.

Figure 5

Quantification of p21, p27 and snail-1 mRNA levels. Total RNAs were extracted from intestinal tumors from APC^min/+ and APC^min/+VDR^−/− mice at 4-month of age, and levels of p21^waf1/cip1 (A), p27^kip1 (B) and snail-1 (C) were quantified by real time RT-PCR. *, P ≤0.05 vs. APC^min/+.