ReviewThe effect of 1,25 dihydroxyvitamin D3 treatment on the mRNA levels of β catenin target genes in mice with colonic inactivation of both APC alleles
Introduction
Studies show that lifestyle factors such as diet can decrease the development of colorectal cancer [1], [2]. One factor proposed to suppress colorectal cancer development is vitamin D. Epidemiological studies show an inverse association between high serum 25 hydroxyvitamin D (25(OH)D), the marker of vitamin D status, and colon cancer risk [3], [4], [5], [6], [7], [8]. The anti-cancer effects of vitamin D are mediated through the vitamin D receptor (VDR) which binds to the active form of vitamin D, 1,25 dihydroxyvitamin D (1,25(OH)2D), and regulates the transcription of genes involved in anti-cancer events (e.g., growth arrest, metastasis, angiogenesis) in colon cancer cells [9]. However, an alternate hypothesis to explain vitamin D-mediated growth arrest is that VDR disrupts β-catenin signaling and blocks the Wnt-signaling pathway.
In normal colonic cells, β-catenin is retained in the cytoplasm by interacting with adenomatous polyposis coli (APC) or at the plasma membrane by binding to E-cadherin at adherens junctions. A destruction complex of Axin, APC, and glycogen synthase kinase-3 keeps β-catenin levels low in the nucleus by promoting its proteasomal degradation thereby reducing transcriptional regulation of genes by β-catenin [10], [11]. Activation of Wnt signaling releases β-catenin from the destruction complex, permits β-catenin nuclear accumulation, and increases binding of β-catenin to the TCF/LEF-1 transcriptional complex to regulate genes controlling cell proliferation (e.g., c-Myc) [12]. APC inactivating gene mutations that inhibit β-catenin-APC interactions are found in 85 percent of sporadic colorectal cancers [13].
Several in vitro studies have demonstrated that 1,25(OH)2D inhibits β-catenin transcriptional activity in colon cancer cells [14], [15]. In addition, siRNA-mediated knockdown of VDR in SW480-ADH cells increased β-catenin-mediated reporter gene activity and inhibited 1,25(OH)2D-mediated suppression of transcript levels from β-catenin target genes [15]. Consistent with this observation, downregulation of VDR gene expression by overexpression of Snails 1 or 2 blunted 1,25(OH)2D-mediated suppression of β-catenin transcriptional activity [16], [17]. In SW480-ADH colon cancer cells, 1,25(OH)2D treatment induced an interaction between VDR and β-catenin [14], [18] which reduced the interaction between β-catenin and TCF-4 necessary for gene transcription [14]. In addition, two-hybrid assays show that the VDR AF-2 domain directly interacts with the C terminus of β-catenin [19]. The interaction of VDR with β-catenin and the suppression of β-catenin-mediated gene transcription in the presence of 1,25(OH)2D has also been shown in other colon cancer cell lines with mutant APC genes, such as Caco-2 and HT-29 [20] and in human embryonic kidney cells (HEK-293) [19]. However, direct testing of the ability of 1,25(OH)2D to reduce β catenin-regulated transcript levels in vivo has not been reported.
Here we examined whether activating VDR with 1,25(OH)2D can inhibit β-catenin transcriptional activity in the absence of APC function in vivo. To do this, we used the CAC; APCΔ580/Δ580 mouse model we developed in which both APC alleles are inactivated specifically in the epithelial cells of the colon [21]. These mice develop dysplastic lesions in the distal colon that are characterized by high levels of nuclear and cytoplasmic β-catenin and increased cellular proliferation. Although β-catenin regulated transcripts are elevated in the distal colonic lesions from these mice, we found that a short course of three 1,25(OH)2D injections did not decrease expression of known β-catenin targets in either wild type mice or in CAC; APCΔ580/Δ580 mice in vivo.
Section snippets
Reagents
Cell culture reagents were obtained from Cambrex (Rockland, ME) and Invitrogen (Carlsbad, CA). Cell culture plastic ware was obtained from BD Biosciences (Sparks, MD). 1,25(OH)2D was purchased from Enzo Life Science (Farmingdale, NY), dissolved in ethanol, and kept in light-protected vials at −80 °C until use. 10% neutral buffered formalin was purchased from VWR (Radnor, PA).
Cell culture
Caco-2 cells were obtained from American Type Culture Collection (Rockville, MD) and cultured as previously described [22]
1,25(OH)2 treatment reduces mRNA levels of β-catenin target genes in colon cancer cells
We examined whether 1,25(OH)2D treatment reduces mRNA level of β-catenin target genes in SW480-ADH cells and in Caco-2 cells. Both of these cell lines express the VDR (data not shown) and contain an APC inactivating mutation [26]. 1,25(OH)2D treatment (100 nM, 24 h) significantly up-regulated 25 hydroxyvitamin D-24 hydroxylase (CYP24A1) mRNA expression in both Caco-2 and SW480-ADH cells (Fig. 1(A)) and significantly reduced the mRNA level of four β-catenin target genes (Axin2, TCF-1, Lef-1, and
Discussion
APC mutations are a common initiating event in colon cancer [28] and loss of APC allele heterozygosity is common in human colon tumors [29]. Epidemiological and cell culture studies suggest that vitamin D and vitamin D metabolites may inhibit colon cancer development [9 and that vitamin D analogs may be useful therapeutic agents [30]. Although the mechanism for this protection is not known with certainty, several in vitro studies show that 1,25(OH)2D-induced growth arrest in colon cancer cells
Conflict of interest
Drs. DeWitt, Snyder, and Fleet have no conflicts to report.
Funding
The work was supported by grants from the American Institute for Cancer Research (Award # 09A098 to JCF), and the National Cancer Institute (NCI) (CA156240 to JCF) and a fellowship from the Purdue Interdisciplinary Cancer Prevention Internship Program from National Institutes of Health (R25CA128770). Histological analysis of the samples was conducted by The Purdue Histology and Phenotyping Laboratory, a core facility supported by the Purdue University Center for Cancer Research, and NCI
Acknowledgement
The authors would like to thank Mrs. Rebecca Replogle for her technical assistance.
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Present address: Department of Dermatology, Indiana University School of Medicine, 550 N. University Blvd., Suite 3240, Indianapolis, IN 46202, USA.
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Present address: Eli Lilly and Company, Lilly Corporate Center, 355 East Merrill St., Drop Code 0438, Indianapolis, IN 46225, USA.