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Genetic Signatures of Differentiation Induced by 1 ,25-Dihydroxyvitamin D3 in Human Colon Cancer Cells

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Genetic Signatures of Differentiation Induced by 1 ,25-Dihydroxyvitamin D3 in Human Colon Cancer Cells [CANCER RESEARCH 63, 7799–7806, November 15, 2003] ␣ Genetic Signatures of Differentiation Induced by 1 ,25-Dihydroxyvitamin D3 in Human Colon Cancer Cells He´ctor G. Pa´lmer,1 Marta Sa´nchez-Carbayo,2 Paloma Ordo´n˜ez-Mora´n,1 Marı´a Jesu´s Larriba,1 Carlos Cordo´n-Cardo´,2 and Alberto Mun˜oz1 1Instituto de Investigaciones Biome´dicas “Alberto Sols,” Consejo Superior de Investigaciones Cientı´ficas-Universidad Auto´noma de Madrid, Madrid, Spain, and 2Division of Molecular Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York ␣ ABSTRACT and it is accepted that high circulating levels of 1 ,25(OH)2D3 associate with reduced risk of colon cancer (13, 14). Accordingly, Epidemiological and preclinical data indicate that vitamin D and its several clinical trials are under way to assess the activity of various most active metabolite 1␣,25-dihydroxyvitamin D [1␣,25(OH) D ] have 3 2 3 nonhypercalcemic vitamin D derivatives in patients with colorectal anticancer activity. Accordingly, clinical trials are under way using sev- ␣ ␣ carcinoma and other neoplasms (2, 15–18). 1 ,25(OH)2D3 regu- eral nonhypercalcemic 1 ,25(OH)2D3 analogues against various neo- ␣ lates gene expression by binding to specific receptors (VDRs) of plasms including colon cancer. 1 ,25(OH)2D3 induces proliferation arrest and epithelial differentiation of human SW480-ADH colon cancer cells. the nuclear receptor superfamily, which are ligand-modulated tran- ␣ We examined the gene expression profiles associated with 1 ,25(OH)2D3 scription factors (Refs. 19, 20 reviews). Upon ligand activation ␣ exposure using oligonucleotide microarrays. 1 ,25(OH)2D3 changed the VDR binds specific nucleotide sequences (vitamin D response expression levels of numerous previously unreported genes, including elements) in target genes to activate or repress their expression. many involved in transcription, cell adhesion, DNA synthesis, apoptosis, Nongenomic actions and cross-talk between ligand-activated VDR redox status, and intracellular signaling. Most genes were up-regulated, and other transcription factors and signaling pathways have also and only a small fraction were down-regulated. Fourteen of 17 candidate been described previously (11, 20). Moreover, certain polymor- genes studied were validated as 1␣,25(OH) D target genes by Northern 2 3 phisms in the VDR gene have been associated with various neo- and Western blotting or immunocytochemistry. They included c-JUN, JUNB, JUND, FREAC-1/FoxF1, ZNF-44/KOX7, plectin, filamin, keratin- plasms, including colon cancer (21, 22), and expression of VDR 13, G S2, and the putative tumor suppressors NES-1 and protease M. decreases during the late stages of colon carcinogenesis (23) 0 ␣ There was little overlap between genes regulated after short (4 h) or long additionally supporting the relation between 1 ,25(OH)2D3 and ␣ (48 h) exposure. Gene regulatory effects of 1 ,25(OH)2D3 in SW480-ADH cancer. cells differed from those in LS-174T cells, which lack E-cadherin and do We have previously studied the mechanism of action of ␣ ␣ not differentiate in response to 1 ,25(OH)2D3. Data from this study reveal 1 ,25(OH)2D3 and several analogues in human SW480 cells, a widely ␣ that 1 ,25(OH)2D3 causes a profound change in gene expression profiles used model for colon cancer (24, 25). Despite mutations affecting and provide a mechanistic basis to the ongoing clinical studies using TP53, K-RAS, and APC genes, these compounds inhibit the prolifer- nonhypercalcemic vitamin D derivatives for colon cancer prevention and 3 ation and promote the differentiation of a subline of SW480 cells treatment. expressing VDR (SW480-ADH) but not of another VDR-negative subline (SW480-R; Ref. 12). They inhibit the activation of the ␤-cate- INTRODUCTION nin signaling pathway by disrupting the TCF-4/␤-catenin interaction and by decreasing the nuclear content of ␤-catenin through the induc- ␣ 3 1 ,25(OH)2D3 is the most active metabolite of vitamin D3,a tion of E-cadherin (12). More comprehensive understanding of the scarce natural product that is synthesized in the organism mainly in ␣ molecular mechanism of 1 ,25(OH)2D3 action may improve the the skin from 7-dehydrocholesterol by the action of UV sunlight (1, clinical use and selection of patients to be treated with vitamin D 2). In addition to its classical role in the regulation of calcium derivatives. The present study was undertaken to evaluate the gene ␣ homeostasis and bone formation/resorption, 1 ,25(OH)2D3 and sev- expression profiles associated with the protective effects of eral synthetic vitamin D derivatives, which show reduced calcemic ␣ 1 ,25(OH)2D3 on SW480-ADH cells, using oligonucleotide micro- activity, induce cell cycle arrest and differentiation or apoptosis in a arrays. variety of cancer cell lines (3–5). Moreover, they have anti-invasion, antiangiogenesis, and antimetastatic activity in vivo (6–8) and are chemopreventive in animal models of colorectal and breast cancer MATERIALS AND METHODS (9–11). Cell Culture and RNA Extraction. The human colon cancer cell lines Several findings suggest that vitamin D improves colon cancer SW480-ADH, SW480-R and LS-147T were grown in DMEM supplemented ␣ prevention and therapy. In vitro,1 ,25(OH)2D3 induces growth with 10% FCS (12). All cells were grown and harvested at 50–75% confluence Ϫ arrest and differentiation in colon cancer cells (3, 12). Epidemio- no longer than 4–6 passages in culture. Treatment of cells with 10 7 M ␣ logical data indicate an inverse correlation between vitamin D 1 ,25(OH)2D3 [supplied by Dr. Lise Binderup, Leo Pharmaceuticals Products dietary intake or sunlight exposure and human colorectal cancer, (Copenhagen, Denmark)] dissolved in isopropanol was performed in DMEM supplemented with charcoal-treated FCS to remove liposoluble hormones. Control cells were always treated with the corresponding concentration of Received 5/2/03; revised 8/20/03; accepted 8/29/03. Grant support: Fundacio´n Cientı´fica de la Asociacio´n Espan˜ola contra el Ca´ncer and isopropanol. Extraction of total RNA was performed using Trizol and purified SAF2001-2291 from Ministerio de Ciencia y Tecnologı´a, Spain. using RNeasy columns (Qiagen, Valencia, CA). H. G. P., M. S-C., P. O-M., and M. J. L. contributed equally to this work. Oligonucleotide Microarrays Hybridization, Scanning, and Scaling. The costs of publication of this article were defrayed in part by the payment of page cDNA was synthesized from 10 ␮g of total RNA using a T7-promoter tagged charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. oligodeoxythymidylic acid primer. RNA target was synthesized by in vitro Requests for reprints: Alberto Mun˜oz, Instituto de Investigaciones Biome´dicas transcription and labeled with biotinylated nucleotides (Enzo Biochem, Farm- “Alberto Sols,” Arturo Duperier, 4, 28029 Madrid, Spain. Phone: 34-91-585-4451; Fax: ingdale, NY). Labeled target was assessed by hybridization to Test arrays 34-91-585-4401; E-mail: [email protected]. 3 ␣ ␣ (Affymetrix, Santa Clara, CA). Gene expression analysis was carried out using The abbreviations used are: 1 ,25(OH)2D3,1 ,25-dihydroxyvitamin D3; EST, ex- Ͼ pressed sequence tags; TGF, transforming growth factor; VDR, vitamin D receptor; Affymetrix U95A human gene arrays with 12,665 features for individual GAPDH, glyceraldehyde-3-phosphate dehydrogenase. known genes and ESTs. Two main response measures, the average difference 7799 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. VITAMIN D3 TARGET GENES IN COLON CANCER CELLS and absolute call were extracted from each gene on every sample, as deter- (freshly prepared from paraformaldehyde) in PBS for 10 min at room mined by default settings of Affymetrix Microarray Suite 5.0. Average differ- temperature, and subsequently permeabilized with 0.5% Triton X-100 in ence was used as the primary measure of expression, and absolute call was PBS for 20 min at room temperature. Before immunostaining, fixed culture retained as a secondary measure. Expression values of each array were mul- cell samples were sequentially incubated with 0.1 M glycine in PBS for 30 tiplicatively scaled to give an average expression of 500 across the central min, 1% BSA in PBS for 15 min, and 0.01% Tween 20 in PBS for 5 min. 95–99% of all genes on the array. For immunolabeling, cells were rinsed in PBS containing 0.05% Tween 20 Data Analysis. For U95A oligonucleotide arrays, scanned image files were (PBS-Tw), incubated for2hatroom temperature with rabbit polyclonal visually inspected for artifacts and analyzed using Microarray Suite 5.0 (Af- anti-c-Jun antibody (H-79, sc1694; Santa Cruz Biotechnology, Santa Cruz, fymetrix). Differential expression was evaluated using several measures. Final CA; 1:200 diluted in PBS), washed in PBS-Tw, and incubated for 45 min ranking to obtain genes uniform and strongly differentially expressed was with the secondary antibody. Cells were then washed and mounted in determined as follows. The expression dataset was first filtered to include only Vectashield (Vector Laboratories, Peterborough, United Kingdom) and those probe sets detecting genes with mean expression values that differed by sealed with nail polish. Confocal microscopy was performed with a Bio- at least 3.5-fold [corresponding to the increase in E-cadherin RNA levels after Rad MRC-1024 laser scanning microscope, equipped with a Zeiss Axiovert ␣ 4h1 ,25(OH)2D3 exposure] between each pair of samples under comparison. 100 invert microscope (Carl Zeiss, Oberkochen, Germany). Probes were then ranked based on the relative magnitude of the difference (t test) between the means of each comparison set. The relationship between cell RESULTS AND DISCUSSION lines was analyzed by hierarchical clustering using XCluster and Tree View software (26) taking only genes and ESTs displaying present call according to Experimental Design.
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