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

Hećtor G. Pa´lmer,1 Marta Sańchez-Carbayo,2 Paloma Ordoń˜ez-Morań,1 Marıá Jesu´s Larriba,1 Carlos Cordoń-Cardo´,2 and Alberto Munõz1 1Instituto de Investigaciones Biome´dicas “Alberto Sols,” Consejo Superior de Investigaciones Cientı´ficas-Universidad Autońoma 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: Fundacioń Cientı´fica de la Asociacioń Espanõla contra el Cańcer and isopropanol. Extraction of total RNA was performed using Trizol and purified SAF2001-2291 from Ministerio de Ciencia y Tecnologıá, 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 Munõz, 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. To evaluate the gene expression profiles MAS 5.0. A nonparametric bootstrap technique was used to estimate the ␣ associated with 1 ,25(OH)2D3 treatment in human colon cancer cells robustness of the clusters obtained (27). we chose SW480-ADH cells expressing endogenous VDR (12). Northern Blot Analysis. Northern hybridization was performed using ϩ ␣ SW480-ADH cells grow independently and responded to poly(A) RNA from control and 1 ,25(OH)2D3-treated colon cancer cell lines ϩ 1␣,25(OH) D exposure with an inhibition in their proliferation rate used in the analysis and probes generated of the genes of interest. Poly (A) 2 3 RNA was purified as reported elsewhere (28). Northern blots were performed and a phenotypic change to a more adherent, differentiated epithelial ␣ on nylon membranes (Nytran; Schleicher and Schuell, Keene, NH) following phenotype (Fig. 1A). 1 ,25(OH)2D3 also induced E-cadherin expres- standard protocols (29). All probes were labeled by the random priming sion and the export of ␤-catenin from the nucleus to the plasma ␣ method. Hybridizations were carried out overnight at 65°C in 7% SDS, 500 membrane (Fig. 1). Short exposure (4 h) to 1 ,25(OH)2D3 induced mM sodium phosphate buffer (pH 7.2) and 1 mM EDTA, as described by substitution of the ␤-catenin-TCF-4 dimers by ␤-catenin-VDR Church and Gilbert (30). Filters were washed twice for 30 min each in 1% SDS dimers, leading to partial inhibition of the expression of ␤-catenin and 40 mM sodium phosphate buffer (pH 7.2) at 65°C. The sizes of respective target genes (12). E-Cadherin protein was not induced after 4 h mRNAs were calculated using RNA ladder markers (Invitrogen, Carlsbad, exposure and cells remained elongated and nonadherent. Once E- CA). Membranes were exposed to Hyperfilm MP films (Amersham Pharmacia cadherin protein accumulates, after 16 h of treatment, ␤-catenin Biotech, Piscataway, NJ). Complete human cDNA probes were used for protease M and NES-1 [Georgia Sotiropoulou, University of Patras, (Patras,

Greece], G0S2 [Scott Heximer, Washington University School of Medicine (St. Louis, MO)], Keratin 13 [Jos´e Luis Jorcano, Centro de Investigaciones Ener- ge´ticas, Medioambientales y Tecnolo´gicas (Madrid, Spain)], FREAC-1/FoxF1 [Javier Rey, Centro de Investigaciones Biolo´gicas (Madrid, Spain)], MRG-1 [Toshi Shioda, Massachusetts General Hospital (Charlestown, MA)], and GAPDH; a fragment (nucleotides 2209–2649) of human cDNA for E-cadherin; full-length mouse cDNAs for c-JUN, JUNB, and JUND [Rodrigo Bravo, Pharmacia (Milan, Italy)]; human clones IMAGE 2286742, 34370, and 898281 for ZNF44/KOX7, plectin, and filamin A, respectively [Orlando Domı´nguez, Centro Nacional de Investigaciones Oncolo´gicas Madrid, Spain)]. Quantifica- tions were carried out using a La Cie scanner connected to a Power Macintosh G4 computer and Adobe PhotoShop 4.0 and NIH Image programs. Western Blot Analysis. Whole-cell extracts were prepared by washing cell monolayers twice in PBS, and the cells were lysed by incubation in radioimmunoprecipitation assay buffer as previously described (12), fol- lowed by centrifugation at 13,000 rpm for 10 min at 4°C and analysis in 10% SDS-PAGE gels. Immunoblotting of cell lysates was performed by protein transfer to Immobilon P membranes (Millipore Corp., Bedford, MA) and incubation with mouse monoclonal anticytokeratin 13 antibody (ab1384, 1:50; abcam Ltd., Cambridge, United Kingdom) and goat antifilamin antiserum (F2762, 1:1000; Sigma, St. Louis, MO). Blots were devel- oped using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). Histochemical Studies. Protein patterns of expression of identified targets were assessed for filamin, c-Jun, and ␤-catenin, using fixed cells. Cells were grown and treated on slides. For filamin and ␤-catenin, cells were rinsed four times in PBS, fixed in cold methanol for 30 s at Ϫ20°C, and rinsed in PBS. The nonspecific sites were blocked by incubation with PBS containing 1% BSA for1hatroom temperature. Cells were incubated with mouse monoclonal antifilamin (RDI-CBL229, 1:50; Research Diagnostics, Fig. 1. Experimental design. A, top, phase-contrast micrographs of control SW480- ␤ Ϫ7 ␣ Inc., Flanders, NJ) or mouse monoclonal anti- -catenin antibody (C19220, ADH cells and those treated with 10 M 1 ,25(OH)2D3 for the indicated times. Bar: 50 ␮ ␣ 1:100; Transduction Laboratories, Lexington, KY) diluted in PBS contain- m. Summary of changes induced by 1 ,25(OH)2D3 in SW480-ADH cells (12) and immunofluorescence and confocal microscopy analysis of ␤-catenin localization during ing 1% BSA overnight at 4°C. After four washes in PBS, cells were ␣ the differentiation induced by 1 ,25(OH)2D3. B, phase-contrast micrographs of control incubated with secondary antibodies for 45 min at room temperature. For Ϫ7 ␣ LS-174T cells and those treated with 10 M 1 ,25(OH)2D3 for the indicated times. Bar: c-Jun, cells were washed twice in PBS, fixed with 3.7% formaldehyde 25 ␮m. 7800

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. VITAMIN D3 TARGET GENES IN COLON CANCER CELLS redistributes progressively from the nucleus to the plasma membrane regulated. Some target genes encode proteins involved in chroma- (Fig. 1A). ␤-Catenin target genes are then further inhibited and cells tin remodeling such as the metastasis-associated gene (MTA-1) and acquire a differentiated epithelial phenotype (Ref. 12; Fig. 1A). HIRA (34, 35) or transcription activator or repressors (TIF1␤, We used oligonucleotide arrays to analyze gene expression ATF3, C/EBP␥, GATA-2, TGIF, and GCF-2). These results indi- ␣ ␣ profiles induced by 1 ,25(OH)2D3 before and after the phenotypic cate a major regulatory role of 1 ,25(OH)2D3 on gene trans- change. Total RNA was obtained from control SW480-ADH cells cription. ␣ or from cells treated with 1 ,25(OH)2D3 for 4 or 48 h. These time A large subset of regulated genes may be involved in the ␣ points allowed us to identify early response genes putatively reg- epithelial differentiation induced by 1 ,25(OH)2D3 treatment. ulated transcriptionally (4 h) and those responding with slow These include cytoskeletal and small GTP proteins and related, kinetics that be indirectly and/or posttranscriptionally regulated several cell-cell or cell-matrix adhesion proteins (plectin, zyxin, (48 h). Additionally, in view of the kinetics of E-cadherin protein filamin, keratins 13 and 15, laminin B3, and ␤-myosin) and E- ␣ induction, the time points chosen might also discriminate between cadherin. 1 ,25(OH)2D3 also regulated a group of genes that ␣ 1 ,25(OH)2D3 target genes that may be E-cadherin-dependent participate in signaling pathways such as receptor and nonreceptor (regulated only after 48 h of exposure) or -independent (regulated kinases, phosphatases, and small GTP proteins and their regulators. already after 4 h). Furthermore, we treated cells with the transcrip- Several genes with antiproliferative or proapoptotic effect were tion inhibitor actinomycin D alone or in combination with induced upon treatment, observations that support the antitumoral ␣ ␣ 1 ,25(OH)2D3 for4htoidentify early genes transcriptionally role of 1 ,25(OH)2D3. Among them, the TP53 tumor suppressor, regulated by the hormone. myc-associated zinc-finger protein, protease M, SEL1, several in- We also studied the gene expression profiles induced by sulin-like growth factor binding proteins, and the proapoptotic ␣ 1 ,25(OH)2D3 in LS-174T colon cancer cells. This cell line contains BAX, DAP-1, and PAR-4 genes. Certain genes thought to play a functional VDR as shown by Western blotting and the activation of role in tumorigenesis such as L-MYC oncogene were repressed by ␣ ␣ transfected vitamin D response elements after 1 ,25(OH)2D3 treat- 1 ,25(OH)2D3, as were several putative tumor suppressor genes: ment (12). However, LS-174T cells do not express detectable E- SYK, known to be down-regulated in breast cancer (36); NF-2 and cadherin protein in either basal or treated conditions (12), and they do NM23H1; and also TOB-1, a negative cell cycle regulator. The ␣ not differentiate in response to 1 ,25(OH)2D3 (Fig. 1B). The expres- expression of several proteases thought to play a role in tumori- ␣ sion profiles of this cell line served as a control not only to evaluate genesis was induced by 1 ,25(OH)2D3. Protease M and NES-1 the correlation between gene expression and phenotype changes (normal epithelial cell-specific 1 gene or kallikrein 10) are down- ␣ caused by 1 ,25(OH)2D3 but also to estimate the contribution of regulated in tumor cells and so thought to contribute to the main- ␣ E-cadherin to the gene expression profile induced by 1 ,25(OH)2D3 tenance of the cell phenotype (37–39). ␣ in SW480-ADH cells. Control cells grown in parallel and collected at In concordance with its growth inhibitory action, 1 ,25(OH)2D3 the same time points were included for both SW480-ADH and LS- down-regulated several genes involved in DNA replication (thymi- 174T experiments. dine kinase, thymidylate synthase) and cell cycle (cyclin F, CDC21) Gene Expression Analyses of SW480-ADH Cells Treated with and some histone genes. Supporting the proposed cross-talk between ␣ ␣ ␤ 1 ,25(OH)2D3. We compared the expression profiles of SW480- 1 ,25(OH)2D3 and the TGF- (40), the expression of at least three ␣ ␤ ADH cells treated with 1 ,25(OH)2D3 for4or48hversus control genes of this signaling pathway (TGF- type I receptor, SARA, ␣ cells. As in previous studies (12), the response to 1 ,25(OH)2D3 was SMAD6) increased, although another (TGIF corepressor gene) de- ␣ verified by studying E-cadherin gene expression by Northern blot creased upon 1 ,25(OH)2D3 addition. Moreover, the level of expres- analysis and ␤-catenin localization assessed by immunofluorescence sion of MDMX gene encoding a RING finger ubiquitin ligase that (data not shown). inhibits small mothers against decapentaplegic homologue-(SMAD)- Tables 1 (n ϭ 2) and 2 (n ϭ 1) summarize the expression changes induced transactivation (41) and controls the ubiquitination of p53 ␣ ␤ at4and48hof1 ,25(OH)2D3 exposure taking a 3.5-fold difference protein was repressed. Given the critical roles played by the TGF- in cutoff. These preliminary data show that most target genes were controlling cell proliferation, matrix production and interaction and up-regulated, and only a few were down-regulated. Little overlap was the acquisition of the transformed phenotype, the regulation of this ␣ found between the lists of genes regulated at 4 and 48 h of pathway might contribute to the beneficial effect of 1 ,25(OH)2D3. ␣ 1 ,25(OH)2D3 treatment. Those whose expression changed at both Besides MDMX, the genes coding the ubiquitin-conjugating enzyme time points appear in italics. Remarkably, some genes that were UbcH2 and the ubiquitin ligase Cbl-b were also induced by ␣ up-regulated at 4 h became down-regulated at 48 h such as insulin-like 1 ,25(OH)2D3, whereas those for ubiquitin carrier protein E2-EPF growth factor binding protein 2, Ha-RAS, calgizzarin, and ubiquitin- and ubiquitin-activating enzyme E1 were up-regulated at4hbut activating enzyme E1. These findings indicate that the wide regulatory down-regulated at 48 h. Cbl-b mediates down-regulation of receptor ␣ effects of 1 ,25(OH)2D3 are exerted through the control of a primary tyrosine kinases and, therefore, could be involved in the control of the set of genes, which in turn modulate others in a cascade. proliferative response to growth factors (42). These results suggest a ␣ ␣ Several genes regulated by 1 ,25(OH)2D3 or analogues in other broad effect of 1 ,25(OH)2D3 on proteasome-mediated protein turn- cell types have been identified (indicated with asterisks in Tables 1 over. ␣ ␤ and 2), such as 1 ,25(OH)2D3 24-hydroxylase, E-cadherin, insulin- Tables 1 and 2 do not include TCF-4- -catenin target genes, ␣ like growth factor-binding protein-3, TGF type I receptor, GADD45, although 1 ,25(OH)2D3 has been shown to regulate them in an and protease M (30–33). opposite way than ␤-catenin (12). The explanation is that the regula- ␣ Many genes involved in transcription were found overexpressed tion by 1 ,25(OH)2D3 of c-MYC, CD44, vascular endothelial growth after 1,25(OH)2D3 exposure. HIRA and ZNF44/KOX7 were the factor, engrailed-2, and Brachyury, which appeared in the microarray only such genes up-regulated at both time points, whereas the analysis, did not reach the 3.5-fold cutoff used in the present study. TIF1␤ corepressor gene was up-regulated at 4 h but down- This result agrees with the regulation found previously (12). regulated at 48 h. Three members of the AP-1 family (c-JUN, Besides its effects on genes involved in processes that are related to ␣ JUNB, and JUND) and several zinc-finger and homeobox proteins cell transformation, survival, or oncogenesis, 1 ,25(OH)2D3 regulates (ASCL2, FREAC-1/FoxF1, LD5–1, and ZNF44/KOX7) were up- genes involved in basic cellular functions such as the control of the 7801

Table 1 Gene fold changes of SW480-ADH cells after exposure to Table 1 Continued 1␣,25(OH) D for4h 2 3 Fold Genes are grouped by functionality. The GenBank accession no. for each gene is change Descriptions shown in the first column. The fold change is included. Previously reported ␣ 1 ,25(OH)2D3 target genes are indicated by asterisks. E-Cadherin used as control gene of Metabolism/redox ␣ 1 ,25(OH)2D3 action is in bold. Genes that are regulated also at 48 h of treatment appear L13286 18,38 Mitochondrial 1,25-dihydroxyvitamin D3 24- in italics. hydroxylase* L25879 12,13 p53/HEH epoxide hydrolase Fold D00632 8,00 Glutathione peroxidase* change Descriptions AF059202 6,06 ACAT related gene product 1 Transcription and related D10523 6,06 2-oxoglutarate dehydrogenase U77629 22,63 Achaete-Scute homologue 2 D26535 4,92 Dihydrolipoamide succinyltransferase AF000561 21,11 TTF-I interacting peptide 21 Z49835 4,59 Disulfide isomerase S77763 9,19 Nuclear factor erythroid 2 isoform f AB029821 4,29 Phosphatidylethanolamine N-methyltransferase X16281 8,57 ZNF44/KOX7 L16842 4,00 Ubiquinol cytochrome-c reductase core I Ϫ X89887 8,57 HIRA X5983 3,73 Glutamine synthase Ϫ AF069735 7,46 PCAF associated factor 65 alpha U02882 6,50 Rolipram-sensitive 3,5-cyclic AMP U35113 6,50 MTA1 phosphodiesterase M73077 6,50 Glucocorticoid receptor repression factor 1 DNA/cell cycle D85131 6,50 Myc-associated zinc-finger protein of human islet D64142 18,38 Histone H1x X56681 6,06 JUND* M69199 10,56 G0S2* AB014591 6,06 CCR4-NOT transcription complex, subunit 3 AB017430 10,56 Kinesin-like DNA binding protein (NOT3) M80397 9,85 DNA polymerase delta catalytic subunit X79201 4,29 SYT U14658 5,66 PMS2 M29039 4,29 JUNB AA255502 5,66 Histone H4 Ϫ M77810 4,00 GATA-2 D38305 4,29 Tob X97548 3,73 TIF1␤ RNA/translation D50495 3,73 Transcription elongation factor S-II, hS-II-T1 X79865 5,66 Mitochondrial ribosomal protein L7L12 (Mrp17) M68891 3,73 GATA-binding protein (GATA2) X15331 4,92 Phosphoribosylpyrophosphate synthetase subunit one Ј X64318 Ϫ3,73 E4BP4 L36055 4,29 Regulator of 5 -cap function (4E-binding protein 1) ␥ X89750 Ϫ3,73 TGIF L19161 3,73 Translation initiation factor eIF-2 subunit X68560 Ϫ4,29 SPR-2 AL022318 3,73 Apolipoprotein B mRNA editing protein and Cytoskeleton/adhesion extracellular matrix Phorbolin (APOBEC1) AJ131186 13,00 Nuclear matrix protein NMP200 Chanels/transporters AI683743 8,00 Katanin U62531 4,59 AE2 anion exchanger (SLC4A2) X98507 6,50 Myosin-I beta AF043250 4,59 Mitochondrial outer membrane protein (TOM40) Ϫ Z54367 6,50 Plectin X89066 4,92 TRPC1 Ϫ X81420 5,66 Type II keratin (hHKb1) AF027153 9,19 Sodium/myo-inositol cotransporter (SLC5A3) X53416 4,29 Filamin Others X95735 4,29 Zyxin AA829286 10,56 Serum amyloid lipoprotein precursor (SAP) D38583 4,00 Calgizzarin AB002559 5,66 Syntaxin binding protein 2 (hunc18b2) D29963 3,73 CD151 U63825 4,92 Hepatitis delta antigen interacting protein A (dipA) Z35402 3,73 E-cadherin* U87947 4,29 Hematopoietic neural membrane protein (HNMP-1) X00351 Ϫ4,92 ␤-actin M95627 4,00 Angio-associated migratory cell protein (AAMP) GTPases and related Z23090 3,73 28 kDa heat shock protein J00277 9,19 Rho GDP-dissociation inhibitor 1 X02469 3,73 p53 cellular tumor antigen X69550 4,59 c-Ha-RAS1 L42379 3,73 Bone-derived growth factor (BPGF-1) Ϫ X66436 3,73 Hsr1 G protein AJ012755 3,73 TL132 Ϫ U17032 Ϫ3,73 p190-B AF369661 3,73 Neurofibromatosis 2 tumor suppressor Ϫ U43083 Ϫ3,73 G ␣-q (Gaq) D90070 4,00 ATL-derived PMA-responsive (APR) peptide Ϫ Kinases and related AF006010 4,00 Progestin induced protein (DD5) Ϫ U33635 9,19 Colon carcinoma kinase-4 AF039843 5,66 Sprouty 2 (SPRY2) U33053 7,46 Lipid-activated protein kinase PRK1 X66363 5,28 PCTAIRE-1 X54637 5,28 TYK2 AF097738 4,59 Nonreceptor tyosine kinase (TNK1) cellular redox status and intermediary metabolism, RNA splicing and U37352 Ϫ3,73 Phosphatase 2AB ␣1 regulatory subunit translation, or protein turnover and folding. Some of these genes Ϫ ␣ M33336 4,29 cAMP-dependent protein kinase type I- subunit might potentially be attributed to posttranscriptional regulatory ac- (PRKAR1A) ␣ U48251 Ϫ4,59 Protein kinase C-binding protein (RACK7) tions of 1 ,25(OH)2D3. Receptors and related ␣ Effect of Actinomycin D on 1 ,25(OH)2D3 Treatment. We com- L41147 4,29 5-HT6 serotonin receptor Y00097 3,73 p68 Annexin VI pared the expression profiles of SW480 cells treated with Ϫ ␣ S59184 4,00 Related to receptor tyrosine kinase (RYK) 1 ,25(OH)2D3 alone or in the presence of actinomycin D for 4 h. A M11507 Ϫ4,92 Transferrin receptor few genes showed substantial level of expression under actinomycin Proteases and related ␣ U62801 18,38 Protease M* D exposure. Certain genes that were up-regulated by 1 ,25(OH)2D3 J03870 8,57 Cystatin SA-I alone (Table 1), including 1␣,25(OH) D 24-hydroxylase, E-cad- ␣ 2 3 X01683 4,59 1-antitrypsin herin, cystatin D, protease M, JUNB, and GATA-2, were inhibited AF055481 4,00 Epithelial cell-specific 1 (NES1) ␣ X64364 4,00 M6 antigen/EMMPRIN under combined treatment with 1 ,25(OH)2D3 and actinomycin D. M58028 4,00 Ubiquitin-activating enzyme E1 (UBE1) ␣ These genes are candidates to be regulated by 1 ,25(OH)2D3 at the X70377 3,73 Cystatin D M91670 3,73 Ubiquitin carrier protein (E2-EPF) transcription level. In contrast, those genes unaffected by actinomycin Apoptosis D are probably regulated posttranscriptionally or indirectly. L22475 25,99 BAX ␥ Expression Profiles Associated with 1␣,25(OH) D in LS-174T M35878 13,00 Insulin-like growth factor-binding protein-3 2 3 (IGFBP3)* Cells. We also evaluated the expression profiles of LS-174T cells X16302 7,46 Insulin-like growth factor binding protein-2 that express comparable VDR levels to SW480-ADH cells (IGFBP2)* ␣ but do not undergo differentiation after 48 h of exposure to L22473 5,66 BAX ␣ U19599 5,28 BAX ␦ 1 ,25(OH)2D3. The number of expression changes regulated by M62402 4,00 Insulin-like growth factor binding protein 6 ␣ 1 ,25(OH)2D3 in LS-174T cells was lower than in SW480-ADH (IGFBP6)* cells (Table 3; n ϭ 1). The comparison between the two cell lines revealed that only four of the genes regulated at least 3.5-fold by 7802

␣ Table 2 Gene fold changes of SW480-ADH cells after exposure to 1 ,25(OH)2D3 Table 2 Continued for 48 h Fold Genes are grouped by functionality. The GenBank accession no. for each gene is change Descriptions shown in the first column. The fold change are included. Previously reported ␣ 1 ,25(OH)2D3 target genes are indicated by asterisks. E-Cadherin used as control gene of M60974 6,96 gadd45* ␣ 1 ,25(OH)2D3 action is in bold. Genes that are regulated also at4hoftreatment appear D50840 6,06 Ceramide glucosyltransferase in italics. U63809 5,66 Prostate apoptosis response protein par-4 U82938 Ϫ4,59 CD27BP (Siva) Fold X16302 Ϫ4,59 Insulin-like growth factor binding protein (IGFBP-2)* change Descriptions M62403 Ϫ9,19 Insulin-like growth factor binding protein 4 (IGFBP4)* Transcription and related Metabolism/redox ␤ X83877 18,38 ZFAB (ABP/ZF) L40802 73,52 17- -hydroxysteroid dehydrogenase (17-HSD)* Y09538 13,00 ZNF 185 L13286 55,72 Mitochondrial 1,25-dihydroxyvitamin D3 24- M63896 12,13 Transcriptional enhancer factor (TEF1) hydroxylase* X59244 9,85 ZNF43 J04813 19,70 Cytochrome P450 IIIA L19871 7,46 Activating transcription factor 3 (ATF3) M68840 13,00 Monoamine oxidase A (MAOA) X16281 6,96 ZNF44/KOX7 Z49835 8,00 Disulfide isomerase J04111 6,96 C-JUN U67963 5,28 Lysophospholipase homologue (HU-K5) AF035528 6,50 SMAD6 M57951 4,29 Bilirubin UDP-glucuronosyltransferase isozyme 2 X89887 5,66 HIRA D26535 3,73 Dihydrolipoamide succinyltransferase U20240 5,66 C/EBP ␥ S79639 3,73 Tumour suppressor/hereditary multiple exostoses D16815 4,92 EAR-1r candidate D63391 Ϫ4,00 Platelet activating factor acetylhydrolase IB ␥-subunit U65093 4,92 Msg1-related gene 1 (MRG1) Ϫ U13219 4,59 FREAC-1/Fox F1 X71973 4,29 Phospholipid hydroperoxide glutathione peroxidase L16842 Ϫ4,29 Ubiquinol cytochrome-c reductase core I X52560 4,00 Nuclear factor NF-IL6 AF038406 Ϫ4,59 NADH dehydrogenase-ubiquinone Fe-S protein 8 U69609 4,00 Transcriptional repressor (GCF2) (NDUFS8) X97548 Ϫ4,29 TIF1 ␤ BC000192 Ϫ4,59 Dihydrofolate reductase Cytoskeleton/adhesion/extracellular matrix DNA/cell cycle X81420 39,40 Type II keratin (hHKb1) M69199 24,25 G S2* U81607 13,93 Gravin 0 D00596 Ϫ4,00 Thymidylate synthase Z35402 12,13 E-cadherin* Z36714 Ϫ4,00 Cyclin F* X07696 6,96 Keratin 15 D38073 Ϫ4,00 DNA replication licensing factor beta subunit (hRlf, U17760 6,50 Laminin S B3 chain (LAMB3) p102 protein) X14640 4,92 Keratin 13 AL009179 Ϫ4,29 Histone H2B D38583 Ϫ4,00 Calgizzarin X74794 Ϫ4,59 P1-Cdc21 M95178 Ϫ4,00 Nonmuscle ␣-actinin X14850 Ϫ4,92 Histone H2A.X* X00734 Ϫ4,00 ␤-tubulin (␤-5) M15205 Ϫ7,46 Thymidine kinase X02344 Ϫ4,29 ␤-tubulin (␤-2) RNA/translation M94362 Ϫ4,29 Lamin B2 (LAMB2) U97670 19,70 Translation initiation factor eIF3, p35 subunit AJ131186 Ϫ4,59 Nuclear matrix protein NMP200 X90858 12,13 Uridine phosphorylase M13452 Ϫ4,59 Lamin A Ϫ ␣ ␣ U63289 5,66 RNA-binding protein CUG-BP/hNab50 (NAB50) K00558 5,28 -tubulin ( -1, isoform 44) ␥ Ϫ L19161 4,29 Translation initiation factor eIF-2 subunit X97074 7,46 Clathrin-associated protein AL031681 Ϫ3,73 Splicing factor, arginine/serine-rich 6 (SRP55-2) GTPases and related M69040 Ϫ4,00 ASF/SF2 (SF2p33) AF070629 42,22 RAB2 Z37166 Ϫ4,59 BAT1 mRNA for nuclear RNA helicase (DEAD family) AF010233 21,11 RalBP1-interacting protein (POB1) AF006751 Ϫ4,92 ES/130 U10550 6,06 Gem GTPase X79865 Ϫ5,66 Mitochondrial ribosomal protein L7L12 (Mrp17) U42390 5,28 Trio Chanels and transporters U90920 5,28 PTPL1-associated RhoGAP U59185 29,86 Putative monocarboxylate transporter (MCT) U72206 4,29 ADP-ribosylation factor (hARF6) L77567 Ϫ4,59 Mitochondrial citrate transport protein (CTP) M57763 4,92 Guanine nucleotide regulatory factor (LFP40) U53347 Ϫ9,19 Neutral amino acid transporter B U92715 4,00 Breast cancer antiestrogen resistance 3 protein (BCAR3) Others L22075 3,73 Guanine nucleotide regulatory protein (G13) Ϫ ␣ AF059293 51,98 Cytokine-like factor-1 precursor (CLF-1) M63167 4,00 Rac protein kinase U57029 27,86 T-cell leukemia virus enhancer factor (HTLF)* J00277 Ϫ4,92 c-Ha-RAS1 Ϫ U11037 21,11 Sel-1-like (SEL1L) X69550 6,06 Rho GDP-dissociation inhibitor 1 AB013924 19,70 TSC403 Kinases and related AF104304 13,93 Smad anchor for receptor activation (SARA) L33881 4,00 Protein kinase C iota isoform AF007111 13,93 MDM2-like p53-binding protein (MDMX) Ϫ AF011468 3,73 Serine/threonine kinase (BTAK) U52969 11,31 PEP19 (PCP4) Ϫ S80267 8,57 p72SYK AF049611 8,00 Huntingtin interacting protein (HYPE) Phosphatases AF042273 6,96 Signal transducing adaptor molecule 2A (STAM2) AB026436 10,56 Dual specificity phosphatase MKP-5 D12763 6,96 ST2 U27193 8,57 Protein-tyrosine phosphatase hVH-5 M31516 6,50 Decay-accelerating factor (DAF; CD55) Ϫ J02902 4,00 Protein phosphatase 2A subunit alpha (alpha-PR65) U43916 5,66 Tumor-associated membrane protein homolog (TMP) Receptors and related U04735 4,29 Microsomal stress 70 protein ATPase core (stch) ␤ AF054598 5,66 TGF- type I receptor (TGFBR1)* Y11307 4,29 CYR61 X00588 4,92 Precursor of epidermal growth factor receptor X02469 4,29 p53 cellular tumor antigen J02958 4,59 Hepatocyte growth factor receptor AF045583 4,00 Tubby like protein 3 (TULP3) M76125 4,59 Tyrosine kinase, receptor Axl, Alt. Splice 2 L20941 4,00 Ferritin heavy chain X64116 4,00 Poliovirus receptor (PVR) U33821 4,00 Tax1-binding protein TXBP151 AF095448 4,00 Putative G protein-coupled receptor (RAIG1) X87949 4,00 BiP chaperone M57230 3,73 Glycoprotein gp130 U73682 4,00 Meningioma-expressed antigen 11 (MEA11) Proteases and related AL021977 3,73 v-maf musculoaponeurotic fibrosarcoma F LIKE protein U62801 14,93 Protease M* (MAFF) AF055481 6,50 Normal epithelial cell-specific 1 (NES1) AJ131244 3,73 Sec24 protein (Sec24A isoform) U26710 4,59 Cbl-b D88153 Ϫ3,73 HYA22 Z29331 4,92 Ubiquitin-conjugating enzyme (UbcH2)* U79528 Ϫ4,00 SR31747 binding protein 1 D29012 Ϫ4,00 Proteasome subunit Y X04412 Ϫ4,29 Plasma gelsolin X71345 Ϫ4,59 Trypsinogen IV b-form X73066 Ϫ4,29 NM23-H1 M91670 Ϫ4,59 Ubiquitin carrier protein (E2-EPF) Y11681 Ϫ4,59 Mitochondrial ribosomal protein S12 X64364 Ϫ5,28 M6 antigen/EMMPRIN M25915 Ϫ6,06 Cytolysis inhibitor (CLI) M58028 Ϫ5,66 Ubiquitin-activating enzyme E1 (UBE1) X57351 Ϫ6,50 1-8D gene from interferon-inducible gene family* Apoptosis M26252 Ϫ6,96 Cytosolic thyroid hormone-binding protein M35878 24,25 Insulin-like growth factor-binding protein-3 (IGFBP-3)* J04164 Ϫ7,46 Interferon-inducible protein 9-27* AB000277 11,31 DAP-1 ␣ M97815 Ϫ7,46 28 kDa heat shock protein U69611 10,56 TNF-␣ converting enzyme Z23090 Ϫ9,19 Retinoic acid-binding protein II (CRABP-II) 7803

␣ Table 3 Gene fold changes of LS-174T cells after exposure to 1 ,25(OH)2D3 for 48 h 1/FoxF1,c-JUN, MRG1, and keratin-13 (Fig. 3B) and at both time Genes are grouped as induced or repressed. The GenBank accession no. for each gene points of E-cadherin, G0S2, NES-1, protease M, and ZNF-44/KOX7 is shown in the first column. The fold change values are included. Previously reported ␣ (Fig. 3C). In addition, the negative regulation of SPROUTY-2 was 1 ,25(OH)2D3 target genes are indicated by asterisks. Genes that are regulated also in SW480-ADH cells appear in italics. confirmed but at a different time (48 h) from that suggested by the Fold microarray screening (4 h; data not shown). Three other genes, change Descriptions TP53, NMP200, and PMS2, did not show differential expression. ␣ Induced genes As a negative control, we confirmed that 1 ,25(OH)2D3 did not L13286 73,52 Mitochondrial 1,25-dihydroxyvitamin D3 24- induce FREAC-1/FoxF1 and NES-1 expression in VDR-negative hydroxylase* X83877 59,71 ZFAB (ABP/ZF) SW480-R cells or in LS-174T cells in which the microarray J04813 32,00 Cytochrome P450 IIIA analysis showed no regulation (Table 3). In addition, protease M, ␤ M38180 14,93 3- -hydroxysteroid dehydrogenase/delta-5-delta-4- regulated in LS-174T cells according to the microarray analysis isomerase (3-beta-HSD) J03037 9,19 Carbonic anhydrase II (Table 3), was validated by Northern blotting and remained unin- U62801 6,50 Protease M* duced in SW480-R cells (Fig. 3D). In agreement with the results of Y09788 5,28 MUC5B X56667 4,92 Calretinin the microarray analysis indicated above, the inhibition of ␣ W28589 4,59 48h12 Homo sapiens cDNA 1 ,25(OH)2D3 action by actinomycin D confirmed the transcrip- M29874 4,59 Cytochrome P450-IIB (hIIB1) tional regulation of several genes such as JUNB, FREAC-1/FoxF1, X90579 4,29 DNA for cyp related pseudogene M57951 3,73 Bilirubin UDP-glucuronosyltransferase isozyme 2 and protease M (Fig. 3E). The translation inhibitor cycloheximide ␣ U64197 3,73 Chemokine exodus-1 failed to block 1 ,25(OH)2D3 action on these three genes, sug- BT007051 3,73 Caveolin-2 gesting that this does not require protein synthesis de novo. Repressed genes AF004230 Ϫ3,73 Monocyte/macrophage Ig-related receptor MIR-7 (MIR cl-7) M57417 Ϫ3,73 Mucin 6, Gastric X83618 Ϫ5,66 3-Hydroxy-3-methylglutaryl coenzyme A synthase

␣ 1 ,25(OH)2D3 in SW480-ADH cells listed in Table 2 were also ␣ regulated in LS-174T cells: 1 ,25(OH)2D3 24-hydroxylase, protease M, Bilirubin UDP-glycorosyltransferase isoenzyme 2 and ZFAB. This finding shows the correlation between genotype and phenotype and the differences between the two cell lines and suggests that putative cell-specific genetic and/or epigenetic alter- ␣ ations define the response to 1 ,25(OH)2D3 of colon carcinoma cells. Hierarchical Clustering. The application of a bootstrapping technique provided robustness to the clusters identified based on ␣ time exposure to 1 ,25(OH)2D3 (Fig. 2A). Two main clusters were ␣ observed. The first was associated with4hof1,25(OH)2D3 exposure and the second with longer exposure (48 h). This finding supports the differential phenotype associated with time of expo- ␣ sure of SW480-ADH cells to 1 ,25(OH)2D3 (12), which prompted us to perform this study. It was also possible to segregate the effect ␣ of actinomycin D from cells exposed only to 1 ,25(OH)2D3. Interestingly, the expression profiles of each control cell type at 4 ␣ or 48 h was more similar to that of the paired 1 ,25(OH)2D3- treated cells than those belonging to the other control cell type. The gene expression patterns of cells after4hofexposure were significantly different from those obtained after 48 h of treatment. It was possible to segregate the two different cell types under study within the 48 h exposure cluster, and the differences between treated and control cells were smaller in LS-174T cells. Overall, the hierarchical clustering confirmed the hypothesis that the phenotypic differences between SW480-ADH and LS-174T cells upon ␣ 1 ,25(OH)2D3 exposure are genetically regulated. Growth did not affect cell phenotype: the increase in cell confluency during the 48-h incubation period did not induce differentiation of control ␣ cells, as compared with 1 ,25(OH)2D3-treated ones (Fig. 2B). Validation of Results. We screened 17 available cDNAs corre- Fig. 2. Hierarchical clustering of expression profiles of the colon cancer cell lines sponding to genes regulated by 1␣,25(OH) D in the microarray ␣ 2 3 treated with 1 ,25(OH)2D3 for 4 and 48 h. A, to assess the robustness of the clustering study. Filters containing RNA from control SW480-ADH cells or analysis, a bootstrap resampling technique was applied. First, a large number (1000 in this cells treated with 1␣,25(OH) D for different periods of time were analysis) of copies of the data are generated using a Monte Carlo resampling technique. 2 3 Each of these generated data sets is then clustered using the standard hierarchical method. probed. Thirteen of the 17 genes were validated (Fig. 3). There was The count at each node of the tree represents how many of the 1000 trees had a specific overall concordance between data from the microarrays screening bipartition. Nodes with values close to 1000 are more significant than others displaying lower values. The higher the number at each node of the tree, the more similar the and Northern blotting. This analysis confirmed the induction at 4 h expression patterns of the cells within clusters. B, phase-contrast micrographs of control ␣ ␮ of JUNB, JUND, filamin, and plectin (Fig. 3A)at48hofFREAC- and 1 ,25(OH)2D3-treated SW480-ADH cells during the incubation period. Bar: 50 m. 7804

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. VITAMIN D3 TARGET GENES IN COLON CANCER CELLS ␣ We also examined whether the induction by 1 ,25(OH)2D3 was restricted to the RNA level or extended to differential protein ␣ expression. 1 ,25(OH)2D3 led to increases in filamin A and keratin-13 protein levels as assessed by Western blotting (Fig. 4A, top panel). As control, neither of these two proteins nor E-cadherin increased in LS-174T or SW480-R cells upon treatment (Fig. 4A, bottom panel). Immunofluorescence analysis confirmed the in- ␣ crease in filamin A expression after 1 ,25(OH)2D3 treatment and also a change in its subcellular distribution from a homogeneous cytosolic localization to a highly preferential presence in the ␣ periphery of the epithelioid islands formed upon 1 ,25(OH)2D3 ␣ treatment (Fig. 4B). Likewise, 1 ,25(OH)2D3 increased the nuclear content of c-Jun protein (Fig. 4B). Taken together, these results confirmed those obtained in the gene array screening. The gene expression patterns regulated by ␣ 1 ,25(OH)2D3 in colon SW480-ADH cells have similarities with ␣ those found in 1 ,25(OH)2D3- or EB1089-treated head and neck cancer cells (32, 33). The differences could be attributed to the sets of genes (coregulators and others) expressed and the signaling pathways activated and alterations present in both cell types. In conclusion, this study has revealed novel molecular targets ␣ associated with 1 ,25(OH)2D3 exposure in human colon cancer cells. ␣ Results suggest a pleiotropic regulatory role for 1 ,25(OH)2D3. ␣ 1 ,25(OH)2D3 regulates a series of genetics events involved in inhib- iting cell proliferation, inducing cell adhesion, and modulating apoptosis. As a whole, it induces a phenotypic change toward a normal epithelial phenotype. Although additional research is warranted to

␣ Fig. 4. Validation of gene regulation by 1 ,25(OH)2D3 at the protein level. A, Western blot analysis (50 ␮g/lane) of the increase in filamin, keratin 13, and E-cadherin proteins ␣ at the indicated times of treatment with 1 ,25(OH)2D3 in SW480-ADH, SW480-R, and LS-174T cells. B, immunofluorescence and confocal microscopy analysis of filamin (top panels) and c-Jun (bottom panels) expression in SW480-ADH cells. Representative Ϫ7 ␣ immunostaining patterns of in cells treated or not with 10 M 1 ,25(OH)2D3 for 48 h. Bar,10␮m.

␣ elucidate the role of 1 ,25(OH)2D3 target genes in colon tumorigenesis and treatment, data shown here support the ongoing clinical

studies using nonhypercalcemic vitamin D3 derivatives for the prevention and treatment of colon cancer.

ACKNOWLEDGMENTS

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Héctor G. Pálmer, Marta Sánchez-Carbayo, Paloma Ordóñez-Morán, et al.

Cancer Res 2003;63:7799-7806.

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