Comparative Genome Analysis Identifies the Vitamin D Receptor Gene As a Direct Target of P53-Mediated Transcriptional Activation

Research Article

Comparative Genome Analysis Identifies the Vitamin D Receptor Gene as a Direct Target of p53-Mediated Transcriptional Activation

Reo Maruyama,1 Fumio Aoki,3 Minoru Toyota,1,2,5 Yasushi Sasaki,2 Hirofumi Akashi,3 Hiroaki Mita,2 Hiromu Suzuki,1,4 Kimishige Akino,1,2 Mutsumi Ohe-Toyota,2 Yumiko Maruyama,1 Haruyuki Tatsumi,3 Kohzoh Imai,1 Yasuhisa Shinomura,1 and Takashi Tokino2

1First Department of Internal Medicine; 2Department of Molecular Biology, Cancer Research Institute; 3Information Center of Computer Communication; and 4Department of Public Health, Sapporo Medical University, Sapporo, Japan; and 5Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Japan

Abstract Several approaches have been used to successfully identify genes induced by p53 including differential display, representational p53 is the most frequently mutated tumor suppressor gene in human neoplasia and encodes a transcriptional coactivator. difference analysis, cDNA microarrays, and serial analysis of gene Identification of p53 target genes is therefore key to expression (3–6). However, identification of p53-inducible targets understanding the role of p53 in tumorigenesis. To identify by gene expression profiling is highly dependent on the expression novel p53 target genes, we first used a comparative genomics level of the target gene, potentially overlooking genes of which the expression level is relatively low in the tissue analyzed. Alterna- approach to identify p53 binding sequences conserved in the in silico human and mouse genome. We hypothesized that potential tively, p53 target genes should be identifiable using an p53 binding sequences that are conserved are more likely to analysis of p53 response elements (p53RE) as probes (7). However, be functional. Using stringent filtering procedures, 32 genes a complete data set of functional p53REs in the human genome is were newly identified as putative p53 targets, and their not yet available. Comparison of orthologous genomic sequences responsiveness to p53 in human cancer cells was confirmed has become a powerful tool for identifying functional elements by reverse transcription-PCR and real-time PCR. Among them, within the genome (8, 9). Using a computational approach, coding we focused on the vitamin D receptor (VDR) gene because regions, regulatory elements, and noncoding RNA conserved in different vertebrates have been identified (10). We hypothesized vitamin D3 has recently been used for chemoprevention of human tumors. VDR is induced by p53 as well as several other that both known and novel functional p53REs important for p53 p53 family members, and analysis of chromatin immunopre- function are likely to exhibit greater sequence conservation than cipitation showed that p53 protein binds to conserved intronic nonfunctional sequences (11). A comparative genomic analysis of sequences of the VDR gene in vivo. Introduction of VDR into putative p53 binding sequences may thus be a way to identify the cells resulted in induction of several genes known to be downstream mediators of p53 function. p53 targets and suppression of colorectal cancer cell growth. Epidemiologic studies indicate that vitamin D3 and its most active analogue, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], have In addition, p53 induced VDR target genes in a vitamin D3- In vitro dependent manner. Our in silico approach is a powerful antitumor activity (12, 13). , vitamin D3 induces cell cycle method for identification of functional p53 binding sites arrest, differentiation, and apoptosis of cancer cells (14–17). Clinical trials are under way to assess the activity of various vitamin D and p53 target genes that are conserved among humans and other organisms and for further understanding the function analogues against tumors. Vitamin D3 exerts its activity through of p53 in tumorigenesis. (Cancer Res 2006; 66(9): 4574-83) binding to the vitamin D receptor (VDR). VDR is down-regulated during colorectal tumorigenesis, and absence or low levels of VDR expression correlate with poor prognosis (18, 19). However, the Introduction regulatory mechanism of VDR expression is not fully understood. The transcriptional coactivator p53 binds to DNA in a The aim here was to identify and compare p53 target genes in the sequence-specific manner and induces transcription of a variety human and mouse genomes using in silico genome scanning for of genes involved in cell cycle regulation, apoptosis, inhibition of sequence conservation of potential p53 binding sites. Our results angiogenesis, and DNA repair (1, 2). Notably, the gene encoding indicate that this in silico approach is a powerful method for p53 is the most frequently mutated tumor suppressor gene in identification of p53 target genes conserved among humans and neoplasms. Consequently, identification of the transcriptional other organisms, and help determine new functions of p53 in targets of p53 is a potential key to understanding functions of tumorigenesis. Among the genes identified as a putative target of p53 and its signaling pathways in tumorigenesis, and to exploiting p53, we examined the induction of VDR by p53 family members. We their potential use as molecular targets for cancer chemothera- also examined the role of VDR in the regulation of p53 target genes. peutic drugs. Materials and Methods Construction of p53RE database and comparative analysis of p53 Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). target genes. p53REs typically consist of two copies of a 10-bp motif Requests for reprints: Takashi Tokino, Department of Molecular Biology, Cancer (RRRCWWGYYY) separated by 0 to 12 bp. We first extracted all putative p53 Research Institute, Sapporo Medical University, South 1, West 17, Chuo-ku, Sapporo binding sites in the entire human and mouse genomes. The sequence data 060-8556, Japan. Phone: 81-11-611-2111, ext. 2386; Fax: 81-11-618-3313; E-mail: were downloaded from the National Center for Biotechnology Information [email protected]. I2006 American Association for Cancer Research. (NCBI) Human Assembly 33 (April 10, 2003) and the MGSC Mouse Assembly doi:10.1158/0008-5472.CAN-05-2562 3 (February 2003) and then input to the p53RE prediction system. We set the

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. VDR Is Up-regulated by p53 parameter conditions to include the following restrictions: (i) fewer than then subjected to 2.5% Nusieve gel electrophoresis and stained with four mismatches in the 20-nucleotide consensus binding sequence, and (ii) ethidium bromide. the spacer between the 10-bp motifs has fewer than 12 bp. The output data, Real-time PCR was carried out using a CYBR Green PCR or TaqMan PCR which consisted of p53 binding site IDs, chromosomal positions, nucleotide Master Mix (Applied Biosystems, Foster City, CA) on an ABI Prism 7000. sequences, the number of mismatches, spacer length, and the 200-bp Accumulation of PCR product was measured in real time as an increase in sequences surrounding the binding sites, were stored in the p53RE fluorescent signals and analyzed using ABI Prism 7000 SDS Software database. (Applied Biosystems). Standard curves relating initial template copy We next added the gene annotation information into each of the binding number to fluorescence and amplification cycle were generated using the site entries in the p53RE database. The public database RefFlat provided by amplified PCR product as a template, and were then used to calculate the University of California Santa Cruz was used to determine the distance the mRNA copy number in each sample. The ratios of the intensities of the between the p53 binding sequences and their closest genes. This data set target genes to GAPDH signals were considered to be a relative measure of contained information from 15,824 human genes and 13,406 mouse genes; the target genes mRNA level in each specimen. The information for TaqMan the data for each gene consisted of the chromosome number and the primers/probe sets is available in Supplementary Materials and Methods. positions of the transcription start site, the first exon start position and the Northern blot analysis. Ten micrograms of total RNA were separated by first intron start position. We calculated the distance between each p53RE electrophoresis on a 1% agarose gel containing 2.2 mol/L formaldehyde. The and the corresponding transcription start site of the closest gene and integrity and equal loading of the RNA in each lane were confirmed by retrieved only the p53 binding sites located within 10 kb of the transcription ethidium bromide staining. The RNA was then transferred onto a start site. nitrocellulose transfer membrane (Protran, Schleicher and Schuell, Inc., Cell lines, cell culture, and recombinant adenovirus. The human Keene, NH) by capillary blotting in 20Â SSC; after which, hybridization was cancer cell lines used in this study were purchased from the American Type done using radiolabeled probes in 50% formamide, 5Â Denhardt solution, Culture Collection (Manassas, VA) or the Japanese Collection of Research 3Â SSC, 0.1% SDS, and 100 Ag/mL salmon sperm DNA overnight at 42jC. Bioresources (Tokyo, Japan). All cell lines were cultured under conditions After hybridization, the membranes were washed first in 1Â SSC/0.1% SDS recommended by their individual depositors. The status of the endogenous at room temperature and then in 0.25Â SSC/0.1% SDS at 60jC. The p53 expressed in these lines was wild-type for RKO and HCT116, mutant for intensities of hybridization bands were quantitated using a BAS2000 DLD-1, SW480, and MKN74, and p53-null for Saos-2 and H1299. The Bioimage Analyzer (Fuji, Tokyo, Japan). generation and purification of replication-deficient recombinant adenovi- ruses harboring p53, p73a, p73h, p63g, and the bacterial LacZ gene (Ad-p53, Ad-p73a, Ad-p73h, Ad-p63, and Ad-LacZ, respectively) were previously described (20). To examine VDR expression induced by endogenous p53, cancer cells were treated with 0.2 to 0.5 Ag/mL of Adriamycin for 12 to 24 hours and harvested. Knockdown of VDR by short hairpin RNA. Two short hairpin RNA (shRNA) sequences (sh-VDR20 and sh-VDR32) were designed using siPRECISE software (B-bridge, Inc., Sunnyvale, CA). Oligonucleotides were annealed and ligated into pFIV-H1-Puro vector (SBI, Mountain View, CA). shRNA sequences for VDR are shown in Supplementary Table S1. HCT116 cells were transfected with 2 Ag of sh-VDR20, sh-VDR32, or control pFIV-H1- Puro vector for 4 hours using Lipofectamine 2000 (Invitrogen Inc., Grand Island, NY) in 2 mL of Opti-MEM medium (Invitrogen). The culture medium was replaced with McCoy’s medium containing 1 Ag/mL puromycin and harvested after 48 hours. Chromatin immunoprecipitation. Chromatin immunoprecipitation (ChIP) assays were carried out as previously described using a ChIP assay kit (Upstate Biotechnologies, Lake Placid, NY; ref. 21). Immunoprecipitation was carried out using mouse antihuman p53 monoclonal antibody (mAb; DO-1, Santa Cruz Biotechnology, Santa Cruz, CA). The primers used were ChIP-F and ChIP-R; their sequences are shown in Supplementary Table S1. The amplified products were subjected to agarose gel electrophoresis. Luciferase assay. To construct luciferase reporter plasmids, oligonucleotide fragments corresponding to VDR-RE (wt) and VDR-RE (mut; Supplementary Table S1) were synthesized and inserted upstream of a basal SV40 promoter in the pGL3-promoter vector (Promega, Madison, WI); the resulting constructs were designated pGL3-VDR-RE (wt) and pGL3- VDR-RE (mut), respectively. H1299 cells at 50% confluence were transfected with a reporter and an effector plasmid (total, 100 ng DNA) using Lipofectin (Life Technologies, Inc., Gaithersburg, MD). After 48 hours, the cells were harvested for measurement of luciferase activity using a Luciferase Assay System (Promega). Cell extract was incubated with luciferin and light emission was measured using a Berthold Luminometer (Berthold Lumat LB9507, Belthold, Bad Wildbad, Germany). Reverse-transcription PCR. Semiquantitative reverse transcription- PCR (RT-PCR) was carried out as previously described (6). Briefly, total RNA was extracted using TRIzol (Invitrogen); after which, a 5-Ag sample was reverse transcribed using SuperscriptIII (Invitrogen). The primer sequences Figure 1. Strategy for identifying putative p53 binding sequences conserved used are shown in Supplementary Table S1. The integrity of the cDNA was between human and mouse using an in silico approach. The algorithm presented confirmed by amplifying glyceraldehyde-3-phosphate dehydrogenase here was implemented in a computer program written in the C programming (GAPDH) as previously described (21). Samples of amplified product were language. www.aacrjournals.org 4575 Cancer Res 2006; 66: (9). May 1, 2006

Figure 2. Representative p53 binding sites identified in this study and conserved across multiple species. The consensus p53 binding sequences are indicated by uppercase letters: R, purines; Y, pyrimidines; W, Aor T. Asterisks, sequences conserved across multiple species. Numbers, nucleotide position relative to the transcription start site. Numbers of mismatched nucleotides in the consensus sequence (p53), those conserved between human and mouse sequences (human-mouse), and those conserved across multiple species (human-other species) are shown below the column.

Western blot analysis. Twenty micrograms of cell lysate were subjected the gene identities in conjunction with controlled, vocabulary data mining to electrophoresis in a 10% SDS-PAGE; after which, the proteins were of literature associations, protein-protein interaction databases, and a transferred to Immobilon-Pmembranes (Millipore, Bedford, MA). The metabolism pathway database. We then combined the two pathways membranes were then blocked with 5% nonfat milk and 0.1% Tween 20 in manually by connecting the genes up-regulated by both p53 and VDR. TBS and probed with anti-VDR (Lab Vision, Fremont, CA) and antiactin mouse mAbs (Chemicon, Temecula, CA); after which, the hybridization was visualized using enhanced chemiluminescence (Amersham, Piscataway, NJ). Results Immunofluorescent staining. Cells grown on coverslips were infected In silico identification of p53 target genes. To identify novel with Ad-lacZ, Ad-p53, Ad-p63, or Ad-p73. After 48 hours, the cells were fixed with 4% paraformaldehyde and then incubated for 16 hours at 4jC with p53 target genes within the human genome, we used a anti-VDR rat mAb (Lab Vision). FITC-conjugated goat anti-rat antibody computational approach focusing on p53 binding sites (Fig. 1). (Molecular Probes, Eugene, OR) was used as the secondary antibody. Finally, The consensus p53 binding sequence consists of two copies of the nucleus was stained with 4V,6-diamidino-2-phenylindole (DAPI; Vector a 10-bp motif (RRRCWWGYYY) separated by 0 to 12 bases. Laboratories, Inc., Burlingame, CA) and the cells were examined under a Because several of the nucleotides comprising the motif are fluorescence microscope (Olympus, Tokyo, Japan). ambiguous (R, adenine or guanine; W, adenine or thymine; Y, Geneticin-resistant colony formation assay. Cells were plated in 60- cytosine or thymine), this sequence has up to 28 Â 28 (= 65,536) Â 5 mm culture dishes to a density of 1 10 per plate and incubated for 24 possible patterns, making generation of comparison patterns a hours; after which, they were transfected with pCMV-VDR or empty pCMV heavy task that requires considerable computer resources and vector (5 Ag each) using Lipofectamine Plus reagent (Invitrogen). Following takes much time to complete the general pattern matching. We transfection, cells were selected for 14 days in medium containing 0.6 mg/ mL G418 and stained with Giemsa. The colonies were then counted in therefore focused on developing a new algorithm to effectively triplicate cultures using NIH Image software. deal with the highly degenerate consensus p53 binding sequence Pathway analysis. p53 and VDR target genes were analyzed using and the variable spacer length. Instead of performing a genome- Ingenuity Pathway Analysis software (Ingenuity, Mountain View, CA). To wide pattern matching with all possible p53 patterns [patterns = infer coassociation of common p53 and VDR target genes, this method uses 28 Â 28 Â 13 (spacer) = 851,968], we broke down this consensus

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. VDR Is Up-regulated by p53 sequence into three units, two 10-bp motifs, and a spacer, Ad-p53, Ad-p63, Ad-p73a, Ad-p73h,orAd-LacZ,subsequent making it possible for us to design a strategy to pick up all semiquantitative RT-PCR showed that, of 101 genes analyzed, 32 possible 10-bp motifs and then to search for integrated p53 were up-regulated and 2 were down-regulated by introduction of binding sites with a specific spacer length. We first extracted the p53, p63, or p73 (Supplementary Table S6). Sixteen genes were not entire set of putative p53 binding sequences in the genome. The expressed at all in Saos-2 or DLD-1 cells and the expression of 51 total numbers of candidate sequences for putative p53 binding was not significantly affected. The p53 target genes with p53REs sites in the human and mouse genomes are summarized in containing two mismatches and no spacer tended to show p53 Supplementary Tables S2 and S3. If candidate p53REs are responsiveness more frequently than those with p53REs containing defined as sequences that have four or less mismatches to the three mismatches and no spacer or two mismatches and a 1-bp consensus p53 binding sequence along with a 0- to 12-bp spacer, spacer (58.8% versus 28.6%, P < 0.05). there would be 4,834,075 putative p53REs in the human genome, VDR as a direct transcriptional target of p53. Among the and a putative p53RE would be present every 620 bp, on putative p53 target genes identified by our comparative sequence average. From these, we selected candidate p53REs located within 10 kb of the transcription start site of a gene; 87,659 putative p53REs were estimated to be present in or around the Table 1. Summary of p53 and p53 family target genes known genes in the human genome. This suggests that the vast identified by in silico analysis majority of the p53 binding sequences, defined only by sequence and proximity to genes, may not be biologically functional. Gene name Fold induction To further refine the set of potentially functional p53REs, we used a comparative sequence analysis of the human and mouse p53 p63 p73a p73h genome, as the p53REs of several known p53 target genes are conserved in multiple species, including human, mouse, and rat GAS6 425.0 561.1 112.3 61.6 GEM (11), which was confirmed by our computational analysis 8.1 25.2 69.3 9.1 VDR (Supplementary Fig. S1; Supplementary Table S4). We systemati- 12.1 17.5 10.3 20.2 EFNB1 9.1 3.1 5.6 5.8 cally selected p53REs conserved in orthologous genes between SYT13* 8.9 3.3 0.8 0.6 human and mouse and eliminated repetitive elements. To search LU 5.3 3.1 31.4 14.6 for p53 binding sites that are conserved in orthologous genes HAS3 5.3 32.5 6.3 21.7 between human and mouse, we developed a simple algorithm JAG2 7.3 6.7 1.0 5.4 composed of several steps. As a first step, 7,444 genes homologous IRX5 3.9 17.0 5.9 4.5 between human and mouse were obtained from the NCBI LOXL4 2.5 17.0 11.0 15.0 database. By simple pattern matching, we compared the pair of DSIPI* 4.7 14.8 23.9 8.9 p53 binding sites located within each pair of orthologous genes. We CSDA 3.3 12.3 2.0 8.6 RABL5 then extracted 7,530 pairs of putative p53 binding sites with 3.1 10.8 3.7 3.7 MDM2 sequence similarity (z70% identity) between the orthologous 2.6 5.4 1.3 3.8 BDNF* 2.3 4.7 4.4 7.1 genes. As a second step, we eliminated the p53 binding sites that TCF12 2.2 3.4 1.8 2.1 were part of repetitive elements. The data on the 200-bp sequence ACVR2 2.2 2.2 0.7 1.0 around the p53 binding sites in the p53RE database were scanned AK1 6.4 4.4 2.2 1.7 6 using RepeatMasker Open 3.0, and any conserved segment that KCNB1 3.2 1.9 2.3 0.7 contained known human repeat motifs within the p53REs was MITF 3.2 1.3 1.9 0.5 excluded from further analysis. KIAA1536 3.8 0.1 0.1 2.4 Using this approach, we narrowed the number of candidates EDG4 1.7 128.0 6.9 5.6 for novel p53 target genes to those having conserved p53 binding LMO4 1.2 10.2 1.2 1.8 PC4 sites near their transcription start sites (Fig. 1; Supplementary 0.8 8.7 1.6 5.7 JPH2 Table S5).7 Figure 2 shows six representative p53 binding 0.6 8.1 5.2 1.4 HUS1 0.9 7.2 2.6 4.1 sequences that were predicted in this study and that are also NFE2L1* 0.6 5.0 2.9 0.5 conserved across multiple mammalian species. CLK3 1.9 4.5 0.9 3.2 Validation of p53 target genes. To assess the validity of the p53 KCNJ11 1.2 4.3 4.2 0.7 target genes predicted by the in silico analysis described above, JMJ* 0.6 3.3 2.5 1.6 their responsiveness to p53 was examined using semiquantitative ADAMTS4 1.4 3.0 5.5 1.3 RT-PCR and real-time PCR (Supplementary Fig. S2; Table 1). In 17 IL15RA* 1.0 1.2 2.8 5.9 genes, the p53 binding sites contained two mismatches in the BMP4 0.2 0.0 0.0 0.2 consensus p53 binding sequences and absence of a spacer whereas SHOX2 0.1 0.0 0.0 0.1 20 genes contained two mismatches and a 1-bp spacer. In 64 genes, Control the p53 binding sites contained three mismatches and a 0-bp spacer. When Saos-2 human osteogenic sarcoma cells, which do p21 28.8 36.7 6.8 4.6 not express endogenous p53 (22), and DLD-1 colorectal cancer GADD45A 42.8 98.2 16.8 46.5 cells, which express a mutant form of p53 (23), were infected with

*Expression of p53 and p53 family target genes identified by in silico analysis was examined by real-time PCR in Saos-2 cells or DLD-1 cells. 6 http://www.repeatmasker.org/. 7 http://www.sapmed.ac.jp/freomaru/p53.html. www.aacrjournals.org 4577 Cancer Res 2006; 66: (9). May 1, 2006

Figure 3. VDR is a direct transcriptional target of p53. A, the genomic structure of the VDR gene. Exons are shown as numbered boxes, introns as lines. B, a computational search revealed several candidate p53 binding sites in the first intron of the human and mouse VDR genes. Boxes, putative p53 binding sites; the number of nucleotides matching the consensus p53 binding sequence and the spacer length are shown above each of the potential p53 binding sites; solid boxes, binding sites that are conserved between human and mouse. The nucleotide sequences of the conserved p53 binding site (VDR-p53RE) are indicated within the dotted lines; lowercase letters, divergence from the consensus sequence. C, the conserved p53 binding sites predicted by bioinformatic analysis (VDR-p53RE) consist of five 10-bp motifs. The nucleotide sequences were compared among four mammalian species; asterisks, conserved sequences. The five 10-bp motifs in human are the most conserved to the consensus among the four species. D, ChIP analysis of the VDR-p53RE. ChIP assays were carried out with DLD-1 cells transfected with Ad-p53 or Ad-LacZ, and the chromatin was immunoprecipitated using an anti-p53 antibody (Santa Cruz Biotechnology). After extensive washing, the cross-linking was reversed and the DNAwas subjected to PCR using primers specific for the indicated conserved regions. The region around the p53RE of the p21 gene was amplified as a positive control. E, luciferase assay for VDR-p53RE. Saos-2 and H1299 cells (p53-null) were cotransfected with pGL3-VDR-RE or pGL3-p53CBS (21), together with pcDNA-p53 (p53+) or pcDNA3.1 (p53À). The relative luciferase activity was defined as the activity in the cells transfected with pGL3-VDR-RE (wt) divided by the activity in cells transfected with pGL3-VDR-RE (mut). Columns, mean of three independent experiments; bars, SD. analysis, we focused on the VDR gene, which encodes a nuclear We next carried out ChIPassays to determine whether the p53 receptor that mediates the effects of 1,25(OH)2D3 and has potential protein binds directly to the predicted p53RE in the VDR gene in tumor-suppressive activity (12). Vitamin D and its analogues cells. Immunoprecipitation of DNA-protein complexes using mouse induce cell cycle arrest and apoptosis and, potentially, could be anti-human p53 mAb was carried out on formaldehyde-cross- used for cancer prevention (12, 13). However, transcriptional linked extract of Ad-p53-infected DLD-1 cells; after which, we regulation of the VDR gene is not fully understood. Whereas our measured the abundance of the candidate sequence within the computational search revealed that its promoter and intronic immunoprecipitated complexes using PCR. The ChIP assays regions contain several potential p53 binding sites, only a single revealed that p53 protein bound reproducibly to a DNA fragment site located at position 4,695-4,704 relative to the transcription containing the candidate p53RE of the VDR gene (Fig. 3D). start site is conserved between the human and mouse genomes Furthermore, to determine whether this p53RE could be involved (Fig. 3A and B). Further examination for this region using our in transcriptional regulation, three copies of oligonucleotides p53RE prediction system revealed that it contains five copies of the containing the wild-type p53RE (VDR-RE wt) or an inactive mutant consensus 10-bp motif and that these sequences are well conserved form (VDR-RE mut) were cloned upstream of a luciferase reporter in the human, chimpanzee, mouse, rat, and dog genomes (Fig. 3C; gene; after which, the resultant constructs were transfected into Supplementary Fig. S3). From these results, we predicted this H1299 cells, with or without p53 (pcDNA-p53). Unlike the mutated region could mediate p53-dependent transactivation. sequence, wild-type VDR-RE rendered the reporter construct

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. VDR Is Up-regulated by p53 responsive to p53, leading to a 20-fold increase in luciferase activity results were confirmed by quantitative real-time PCR, which (Fig. 3E). Taken together, these findings indicate that the showed that p53 family genes induced a 4- to 10-fold increase in nucleotide sequence spanning positions 4,695-4,704 in intron 1 of the expression of VDR mRNA over that seen in cells infected the VDR gene constitutes a bona fide p53RE, and VDR is a direct with control vector (Fig. 4A, bottom). Furthermore, we confirmed transcriptional target of p53. induction of VDR mRNA in Saos-2, DLD-1, and H1299 cells by Induction of VDR by p53 family genes and genotoxic Northern blot analysis, and also induction of VDR protein in stresses. To investigate the effect of p53 on endogenous VDR Saos-2, HCT116, DLD-1, and RKO cells by Western blot analysis induction, the expression levels of VDR mRNA were examined in (Fig. 4B and C and Supplementary Fig. S4B and C). In addition, several cell lines. We found that there was significant induction immunocytochemical analysis showed that when Saos-2 cells of VDR mRNA subsequent to infection with Ad-p53 in Saos-2 and were infected with Ad-p53, a significant amount of VDR protein HCT116 cells (Fig. 4A, top) and in DLD-1 and SW480 cells accumulated in the nucleus (Fig. 4D). Similar nuclear accumu- (Supplementary Fig. S4A), but no induction in those cells lation of VDR protein was seen in Saos-2 cells infected with Ad- infected with Ad-LacZ. Interestingly, induction of VDR is more p63 and Ad-p73 but not in cells with Ad-lacZ (Fig. 4D) and in prominent in cells transfected with Ad-p63 or Ad-p73, indicating DLD-1 cells infected with Ad-p53, Ad-63, and Ad-73 (Supplemen- that p53 family members are strong inducers of VDR. These tary Fig. S4D).

Figure 4. Induction of VDR by p53 family genes. A, RT-PCR (top) and real-time PCR (bottom) analysis of VDR expression. RNAwas extracted from Saos-2 and HCT116 cells infected with Ad-lacZ, Ad-p53, Ad-p63g, Ad-p73a, or Ad-p73h, and tested for expression of VDR mRNAby RT-PCR. For real-time PCR, expression levels of VDR mRNA were normalized to those of GAPDH mRNA. Columns, mean of three independent experiments; bars, SD. B, Northern blot analysis of VDR mRNA in Saos-2 cells infected with Ad-p53, Ad-p73h, or Ad-p63 g. An ethidium-stained gel containing 28S RNA shows the amounts of mRNA loaded in each lane. C, Western blot analysis of VDR protein in Saos-2 and HCT116 cells infected with Ad-LacZ, Ad-p53, Ad-p63g, or Ad-p73h. p21 expression served as a positive control for a p53 target; actin expression was used as a loading control. D, immunofluorescence analysis of Ad-p53-induced VDR protein expression. Saos-2 cells were infected with Ad-lacZ, Ad-p53, Ad-p63, or Ad-p73h; after which, VDR was detected using an anti-VDR antibody and an FITC-conjugated secondary antibody. The nucleus was stained with DAPI. E, induction of VDR by a chemotherapeutic drug. Expression levels of VDR mRNAin response to Adriamycin( ADR) in RKO, HCT116, and U2OS cells were analyzed by RT-PCR (top) and real-time RT-PCR (bottom). RKO cells were treated with 0.2 Ag/mL Adriamycin for the indicated times (0, 12, and 24 hours). HCT116 and U2OS cells were treated with Adriamycin at the indicated concentration (0-0.5 mg/mL) for 24 hours. F, Western blot analysis of VDR and p53 proteins following treatment with Adriamycin (0-0.5 Ag/mL) for 24 hours. www.aacrjournals.org 4579 Cancer Res 2006; 66: (9). May 1, 2006

Figure 5. VDR suppresses tumor growth in a vitamin D3-dependent manner. A and B, effect of different expression levels of VDR on vitamin D3 target genes. HCT116 cells were transfected with either pCMV-Tag2 (control plasmid encoding Flag-tag) or pCMV-VDR and incubated with 0.6 mg/mL G418 in the presenceor absence of vitamin D3 (100 nmol/L). Total RNAwas prepared from HCT116 cells; after which, expression of the vitamin D3 target genes was analyzed by RT- PCR (A) or real-time RT-PCR (B). C and D, suppression of growth was evaluated by assaying geneticin-resistant colony formation. HCT116 cells were transfected with either pCMV-Tag2 (control plasmid encoding a Flag epitope) or pCMV-VDR and incubated with 0.6 mg/mL G418 in the presence or absence of vitamin D3 (100 nmol/L). After 14 days, plates were stained with Giemsa solution (C) and the colonies were counted (D). Columns, mean of three independent experiments; bars, SD.

To then determine the extent to which VDR transcription is increased in response to vitamin D3, despite the low level of VDR induced by elevation of endogenous p53, we examined the levels of expression. In contrast, genes involved in cell cycle arrest (e.g., VDR mRNA in three cell lines that have wild-type p53 (RKO, CDKN1A) showed no sensitivity to vitamin D3 unless VDR was HCT116, and U2OS cells) following treatment with Adriamycin, a overexpressed, in which case CDKN1A was up-regulated f2-fold DNA-damaging agent, which can induce endogenous p53 (Fig. 4E by vitamin D3 treatment. It thus seems that elevation of VDR and F). We found that VDR mRNA was up-regulated f3- to 4-fold expression may be necessary for the antiproliferative effect of in cells treated with Adriamycin. vitamin D3 in HCT116 cells. Consistent with this hypothesis, colony The role of VDR in the antiproliferative effect of vitamin D3. formation assays carried out in the presence and absence of The HCT116 cell line was evaluated as a suitable model system in vitamin D3 showed that HCT116 cells transfected with control which to study the function of p53 family members in inducing vector were insensitive to vitamin D3 and thus exhibited significant VDR target genes and in modulating response of the cells to colony formation, regardless of vitamin D3 treatment (Fig. 5C and vitamin D3. To investigate the biological significance of up- D). In contrast, cells expressing high levels of VDR were sensitive to regulating VDR on inhibition of cell-proliferation, HCT116 cells vitamin D3, which dramatically inhibited their colony-forming were transfected with pCMV-VDR, an expression vector containing ability (Fig. 5C and D). the full-length VDR gene for 48 hours, and high expression levels of VDR enhances the transcriptional activation of TP53 VDR mRNA were detected (Fig. 5A). Subsequently, we assessed downstream genes. We next examined whether the induction of expression of VDR-downstream genes in cells overexpressing VDR expression of VDR and its target genes by p53, p63, and p73 was and in control cells in the presence or absence of vitamin D3 enhanced by the addition of vitamin D3 in HCT116 cells. Among six (Fig. 5B). In HCT116 cells transfected with a control vector (pCMV- genes examined, three (GADD45, SPP1, and IGFBP3) were induced Tag2), expression of genes associated with mineral metabolism when Ad-p63 and vitamin D3 were added, suggesting that VDR (e.g., CYP24A1, a key gene involved in vitamin D metabolism) was may play an important role in transcriptional activation by p63

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(Fig. 6). Expression of CYP24A1 and CDH1 was induced by vitamin genome, although it is likely that most of them are nonfunctional, D3 but transfection with Ad-p53, Ad-p63, and Ad-p73 did not result at least for transactivation. In this regard, we found that in synergistic enhancement. Expression of CDKN1A was induced by comparison of the p53 binding sequences between human and Ad-p53, Ad-p63, and Ad-p73 but the effect of vitamin D3 was not mouse is highly useful for identifying functional p53REs. In several significant. To further assess the role of VDR in transactivation of known p53 target genes, the sequences surrounding the p53 p53 target genes, we examined the effects of disrupting VDR binding sites are highly conserved among different species, expression in HCT116 cells using two VDR-specific shRNA (sh- suggesting that other unknown elements (e.g., transcription factor VDR) constructs. Subsequent real-time PCR and Western blot binding sites) are necessary for p53 protein to bind to p53REs and analysis showed that VDR expression was down-regulated after function as a transcriptional coactivator. Detailed studies of puromycin selection of HCT116 cells that express sh-VDR (Fig. 7A). functional regulatory elements combined with more sophisticated These results were confirmed using two different shRNA con- comparative genomics, including comparison across multiple structs. We also found that GADD45, SPP1, and IGFBP3, but not species with varying degrees of divergence, should help in resolving CDKN1A, were suppressed by sh-VDR. The results suggest that VDR the flexible regulatory landscape of mammalian genomes. The plays a key role in the transcriptional activation of a subset of p53 reason why the sequences surrounding p53REs are conserved target genes (Fig. 7B). We next examined whether down-regulation among species remains unclear. Nevertheless, the information of VDR affects apoptosis induced by Ad-p53 and Ad-p63 (Fig. 7C). obtained in the present study should be useful not only for Down-regulation of VDR decreased the numbers of apoptotic identifying p53 target genes but also for identifying transcriptional HCT116 cells induced by Ad-p53 or Ad-p63. factors that function cooperatively with p53. Intriguingly, the The data summarized above suggest that VDR plays a novel role in sequences surrounding the functional p53RE of the VDR gene the p53 signaling pathways shown in Fig. 8. Under normal are also conserved across species (Supplementary Fig. S3). It is thus physiologic conditions, VDR induces its downstream genes associ- possible that conserved sequences close to p53REs may play a role ated with metabolic pathways, including vitamin D3–dependent in recruiting transcriptional coactivators such as p300, BRCA1, Ca2+ uptake. Under genotoxic stress, however, VDR induced by p53 PML, SWI/SNF complex, and IRF-1 to activate gene expression may serve as an effector to up-regulate expression of genes (24–28). associated with cell cycle regulation, differentiation, and apoptosis. We found that expression of the human VDR gene is directly up-regulated by p53, which confirmed an association between p53 and VDR and helps to elucidate one aspect of the biological Discussion significance of elevated VDR expression. Vitamin D analogues, In this study, we used a computational approach to predict novel such as 1,25(OH)2D3, can inhibit the growth of various types of p53 target genes through a genome-wide search for consensus p53 malignant cells, including breast, prostate, colon, skin, and brain binding sequences. Our results indicate that putative p53 binding cancer cells, as well as myeloid leukemia cells (12, 13, 15, 29). sequences are present, on average, every 620 bp in the human Moreover, 1,25(OH)2D3 shows antitumor activity in animal models

Figure 6. Transcriptional activation of VDR target genes by p53, p63g, or p73. HCT116 cells were infected with Ad-LacZ, Ad-p53, Ad-p63g, and Ad-p73h for 48 hours, then either mock treated (white column) or treated with 100 nmol/L of Vitamin D3 (gray column) for 48 hours. Total RNAwas extracted for expression analysis by RT-PCR or real-time PCR. Columns, mean of three independent experiments; bars, SD. An unpaired t test was used to determine statistically significant effects of vitamin D3 treatment. *, P < 0.05, significance of differences between mock and vitamin D3 treated cells. www.aacrjournals.org 4581 Cancer Res 2006; 66: (9). May 1, 2006

Figure 7. Disruption of VDR expression by sh-VDR. A, real-time PCR (top) and Western blot (bottom) analysis of VDR. HCT116 cells were transfected with control shRNAor VDR shRNA(sh-VDR20 or sh-VDR32), cultured with 1 Ag/mL of puromycin for 48 hours, and total RNAand cell lysates were isolated. The amount of VDR mRNAwas normalized to GAPDH.For Western blot analysis, membrane was blotted with anti-p53 and antiactin antibodies as control. B, sh-VDR suppresses expression of p53 target genes. Expression of CDKN1A, GADD45, SPP1, and IGFBP3 was determined by real-time PCR; expression levels were normalized to that of GAPDH. C, apoptosis induced by Ad-p53 or Ad-p63. HCT116 cells were infected with Ad-p53 or Ad-p63 for 48 hours and apoptotic cells were examined by flow cytometry. *, P < 0.01, apoptosis was observed in cells infected with Ad-p63 more frequently than in Ad-lacZ-infected cells.

(30) and, accordingly, clinical studies to evaluate the effect of with cell dedifferentiation and a low level of VDR protein. SNAIL vitamin D analogues in patients with colorectal cancer and other protein interacts directly with the VDR promoter, suppressing its neoplasms are under way (12, 13). The molecular mechanisms by activity and thereby abolishing induction of E-cadherin and other which 1,25(OH)2D3 suppresses cell growth involve regulation of VDR target genes by vitamin D. The fact that VDR is a target gene the cell cycle by inducing p21WAF1 and apoptosis mediated by of p53 suggests that alteration of p53 may be one of the causes of BCL-2 and inhibitor of apoptosis protein (16, 31). In addition, VDR dysregulation in cancer. vitamin D3 down-regulates the T-cell factor/h-catenin system (17). VDR pathways may partially overlap p53 pathways because VDR has been reported to be down-regulated during tumor several downstream VDR target genes are also targets of p53 (12, progression although the molecular mechanism is not fully 14, 35) and because vitamin D3 can induce cell cycle arrest, understood. Conversely, VDR is up-regulated by a variety of growth differentiation, and apoptosis (Fig. 8). Under normal physiologic factors, Sp1 (32, 33), WT1 (34), and vitamin D3, and high levels of conditions, VDR may preferentially induce target genes involved VDR expression are associated with a good prognosis in colorectal in Ca2+ uptake and cellular differentiation (e.g., CYP24A1). In the cancer (18). In addition, Palmer et al. (19) recently reported that presence of genotoxic stress however, VDR seems to induce VDR is down-regulated by the transcriptional repressor SNAIL. target genes associated with cell cycle regulation and apoptosis. They showed that high levels of SNAIL expression are associated This difference in VDR target genes under normal and stressed

Figure 8. Diagram of common p53 and VDR target genes. The pathways were drawn by Ingenuity Pathway Analysis Software. The p53 target genes (e.g., p21, IGFBP3,andGADD45a) can be activated either directly by p53 or via activation of VDR.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. VDR Is Up-regulated by p53 conditions may be partially explained by the p53-dependent up- In summary, we have identified novel p53 target genes by regulation of VDR protein expression induced by genotoxic comparative analysis of p53REs. In particular, we also showed that stress. VDR, which is a transcriptional regulator downstream of p53- VDR target genes also can be induced by stimulation of vitamin dependent cellular signaling, is a direct transcriptional target of D3 in the presence of an inactive p53 mutant or in p53-deficient p53 and plays a role in p53-mediated suppression of tumor growth. cells (35, 36). Consistent with these findings, we observed that p63 Our results indicate that this in silico approach is a powerful and p73 also induce VDR gene expression in cell lines. Although method for identification of p53 target genes conserved among both p63 and p73 can bind to p53REs (37), they specifically bind to humans and other organisms and could serve to facilitate analysis those elements containing three or four copies of the 10-bp of the function of p53 in tumorigenesis. consensus motif separated by spacer sequences (21). This is consistent with the fact that the p53RE in the VDR gene contains Acknowledgments C five copies of the 10-bp motif (Fig. 3 ). Recent studies have shown Received 7/20/2005; revised 1/31/2006; accepted 2/24/2006. that both vitamin D and p63 are associated with development and Grant support: Grants-in-Aid for Scientific Research on Priority Areas from the differentiation in several organs. Our results indicate that there is Ministry of Education, Culture, Sports, Science, and Technology (M. Toyota, K. Imai, and T. Tokino) and a grant from New Energy and Industrial Technology Development an association between p63 and VDR expression, which raises the Organization (M. Toyota). possibility that up-regulation of VDR by p63 at appropriate stages The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance in various cell types might increase their susceptibility to the with 18 U.S.C. Section 1734 solely to indicate this fact. proapoptotic activity of vitamin D metabolites. We thank Drs. Joseph F. Costello and William F. Goldman for valuable discussion.

References differentiation of the myelomonocytic cell line U937. 26. Lee D, Kim JW, Seo T, et al. SWI/SNF complex Genes Dev 1996;10:142–53. interacts with tumor suppressor p53 and is necessary 1. Levine AJ. p53, the cellular gatekeeper for growth and 15. Danielsson C, Torma H, Vahlquist A, Carlberg C. for the activation of p53-mediated transcription. J Biol division. Cell 1997;88:323–31. Positive and negative interaction of 1,25-dihydroxyvita- Chem 2002;277:22330–7. 2. Vogelstein B, Lane D, Levine AJ. Surfing the p53 min D3 and the retinoid CD437 in the induction of 27. Lill NL, Grossman SR, Ginsberg D, DeCaprio J, network. Nature 2000;408:307–10. human melanoma cell apoptosis. Int J Cancer 1999;81: Livingston DM. Binding and modulation of p53 by 3. Zhao R, Gish K, Murphy M, et al. Analysis of p53- 467–70. p300/CBPcoactivators. Nature 1997;387:823–7. regulated gene expression patterns using oligonucleo- 16. Diaz GD, Paraskeva C, Thomas MG, Binderup L, 28. Tanaka N, Ishihara M, Lamphier MS, et al. Cooper- tide arrays. Genes Dev 2000;14:981–93. Hague A. Apoptosis is induced by the active metabolite ation of the tumour suppressors IRF-1 and p53 in 4. Yu J, Zhang L, Hwang PM, et al. Identification and of vitamin D3 and its analogue EB1089 in colorectal response to DNA damage. Nature 1996;382:816–8. classification of p53-regulated genes. Proc Natl Acad Sci adenoma and carcinoma cells: possible implications for 29. Chen TC, Schwartz GG, Burnstein KL, Lokeshwar BL, U S A 1999;96:14517–22. prevention and therapy. Cancer Res 2000;60:2304–12. Holick MF. The in vitro evaluation of 25-hydroxyvitamin 5. Oda K, Arakawa H, Tanaka T, et al. p53AIP1, a 17. Palmer HG, Gonzalez-Sancho JM, Espada J, et al. D3 and 19-nor-1a,25-dihydroxyvitamin D2 as therapeu- potential mediator of p53-dependent apoptosis, and its Vitamin D(3) promotes the differentiation of colon tic agents for prostate cancer. Clin Cancer Res 2000;6: regulation by Ser-46-phosphorylated p53. Cell 2000;102: carcinoma cells by the induction of E-cadherin and the 901–8. 849–62. inhibition of h-catenin signaling. J Cell Biol 2001;154: 30. Harris DM, Go VL. Vitamin D and colon carcinogen- 6. Adachi K, Toyota M, Sasaki Y, et al. Identification of 369–87. esis. J Nutr 2004;134:3463–71S. SCN3B as a novel p53-inducible proapoptotic gene. 18. Evans SR, Nolla J, Hanfelt J, et al. Vitamin D receptor 31. Chakrabarty S, Wang H, Canaff L, et al. Calcium Oncogene 2004;23:7791–8. expression as a predictive marker of biological behavior sensing receptor in human colon carcinoma: interaction 7. Hoh J, Jin S, Parrado T, et al. The p53MH algorithm in human colorectal cancer. Clin Cancer Res 1998;4: with Ca(2+) and 1,25-dihydroxyvitamin D(3). Cancer Res and its application in detecting p53-responsive genes. 1591–5. 2005;65:493–8. Proc Natl Acad Sci U S A 2002;99:8467–72. 19. Palmer HG, Larriba MJ, Garcia JM, et al. The 32. Jehan F, DeLuca HF. The mouse vitamin D receptor 8. Dermitzakis ET, Reymond A, Lyle R, et al. Numerous transcription factor SNAIL represses vitamin D receptor is mainly expressed through an Sp1-driven promoter potentially functional but non-genic conserved sequen- expression and responsiveness in human colon cancer. in vivo. Arch Biochem Biophys 2000;377:273–83. ces on human chromosome 21. Nature 2002;420:578–82. Nat Med 2004;10:917–9. 33. Wietzke JA, Ward EC, Schneider J, Welsh J. Regula- 9. Dermitzakis ET, Reymond A, Scamuffa N, et al. 20. Ishida S, Yamashita T, Nakaya U, Tokino T. Adeno- tion of the human vitamin D3 receptor promoter in Evolutionary discrimination of mammalian conserved virus-mediated transfer of p53-related genes induces breast cancer cells is mediated through Sp1 sites. Mol non-genic sequences (CNGs). Science 2003;302:1033–5. apoptosis of human cancer cells. Jpn J Cancer Res 2000; Cell Endocrinol 2005;230:59–68. 10. Krek A, Grun D, Poy MN, et al. Combinatorial 91:174–80. 34. Maurer U, Jehan F, Englert C, et al. The Wilms’ tumor microRNA target predictions. Nat Genet 2005;37: 21. Sasaki Y, Mita H, Toyota M, et al. Identification of the gene product (WT1) modulates the response to 1,25- 495–500. interleukin 4 receptor a gene as a direct target for p73. dihydroxyvitamin D3 by induction of the vitamin D 11. el-Deiry WS, Tokino T, Velculescu VE, et al. WAF1, a Cancer Res 2003;63:8145–52. receptor. J Biol Chem 2001;276:3727–32. potential mediator of p53 tumor suppression. Cell 1993; 22. Chen X, Ko LJ, Jayaraman L, Prives C. p53 levels, 35. Palmer HG, Sanchez-Carbayo M, Ordonez-Moran P, 75:817–25. functional domains, and DNA damage determine the et al. Genetic signatures of differentiation induced by 12. Lamprecht SA, Lipkin M. Chemoprevention of colon extent of the apoptotic response of tumor cells. Genes 1a,25-dihydroxyvitamin D3 in human colon cancer cells. cancer by calcium, vitamin D and folate: molecular Dev 1996;10:2438–51. Cancer Res 2003;63:7799–806. mechanisms. Nat Rev Cancer 2003;3:601–14. 23. Hirota Y, Horiuchi T, Akahane K. p53 antisense 36. Jiang F, Li P, Fornace AJ, Jr., Nicosia SV, Bai W. G2/M 13. Kumagai T, O’Kelly J, Said JW, Koeffler HP. Vitamin oligonucleotide inhibits growth of human colon tumor arrest by 1,25-dihydroxyvitamin D3 in ovarian cancer D2 analog 19-nor-1,25-dihydroxyvitamin D2: antitumor and normal cell lines. Jpn J Cancer Res 1996;87:735–42. cells mediated through the induction of GADD45 via an activity against leukemia, myeloma, and colon cancer 24. Zhang H, Somasundaram K, Peng Y, et al. BRCA1 exonic enhancer. J Biol Chem 2003;278:48030–40. cells. J Natl Cancer Inst 2003;95:896–905. physically associates with p53 and stimulates its 37. Levrero M, De Laurenzi V, Costanzo A, et al. The 14. LiuM,LeeMH,CohenM,BommakantiM, transcriptional activity. Oncogene 1998;16:1713–21. p53/p63/p73 family of transcription factors: over- Freedman LP. Transcriptional activation of the Cdk 25. Guo A, Salomoni P, Luo J, et al. The function of PML lapping and distinct functions. J Cell Sci 2000;113: inhibitor p21 by vitamin D3 leads to the induced in p53-dependent apoptosis. Nat Cell Biol 2000;2:730–6. 1661–70.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. Comparative Genome Analysis Identifies the Vitamin D Receptor Gene as a Direct Target of p53-Mediated Transcriptional Activation

Reo Maruyama, Fumio Aoki, Minoru Toyota, et al.

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