Oncogene (2003) 22, 7942–7949 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc CDX2, a homeobox transcription factor, upregulates transcription of the p21/WAF1/CIP1 gene Yun-Qing Bai1,2, Satoshi Miyake1, Takehisa Iwai2 and Yasuhito Yuasa*,1 1Department of Molecular Oncology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Tokyo 113-8519, Japan; 2Department of Surgery, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Tokyo 113-8519, Japan The CDX2 homeobox transcription factor plays key roles epithelial phenotype. Previous studies suggested that in intestinal development and homeostasis. CDX2 is CDX2 is a tumor suppressor. Firstly, heterozygous downregulated during colorectal carcinogenesis, whereas Cdx2 knockout mice develop multiple colonic polyps overexpression of CDX2 results in growth inhibition and (Chawengsaksophak et al., 1997). Secondly, the expres- differentiation of colon carcinoma and intestinal cells. sion of CDX2 is downregulated during colorectal However, the means by which CDX2 functions remain carcinogenesis (Ee et al., 1995; Mallo et al., 1997). poorly understood. p21/WAF1/CIP1 is one of the cyclin- Thirdly, CDX2 overexpression seems to inhibit growth dependent kinase inhibitors. In addition to its role in cell and/or promote differentiation of colorectal cancer cells cycle control, p21plays critical roles in differentiation and (Mallo et al., 1998) or undifferentiated intestinal tumor suppression. The overlapping in both the expression epithelial cells (Suh and Traber, 1996). Moreover, the and function of CDX2 and p21in the small intestine and ectopic expression of CDX2/Cdx2 has been reported to colon strongly suggests a link between these two genes. By be associated with intestinal metaplasia formation of the means of luciferase reporter and electrophoretic mobility stomach (Bai et al., 2002; Silberg et al., 2002; Mutoh shift assays, we show here that CDX2 transactivated and et al., 2002). Nevertheless, the molecular mechanisms physically interacted with the promoter of p21 in a p53- underlying these roles of CDX2 remain poorly under- independent manner. Moreover, overexpression of CDX2 stood. increased the mRNA expression of p21 in HT-29 colon The homeobox is a highly conserved 180-bp DNA carcinoma cells, as demonstrated by reverse transcription– sequence encoding a 60-amino-acid motif termed the polymerase chain reaction. These data suggest that p21is homeodomain. The homeodomain, with a helix–turn– a transcriptional target of CDX2. Our results may thus helix structural conformation, is the sequence-specific provide a new mechanism underlying the functions of DNA-binding domain of a family of transcriptional CDX2. regulatory proteins (McGinnis and Krumlauf, 1992). Oncogene (2003) 22, 7942–7949. doi:10.1038/sj.onc.1206634 Homeoproteins of the caudal family bind DNA via an AT-rich sequence whose consensus is A/CTTTATA/G Keywords: CDX2; transcription; p21/WAF1/CIP1 (reviewed by Freund et al., 1998). Previous studies showed that CDX2 binds cis-elements present in the gene promoters of enterocytic markers such as sucrase- isomaltase, lactase-phlorizin hydrolase, phospholipase- A, apolipoprotein B, carbonic anhydrase 1, and Introduction calbindin D9K (Freund et al., 1998). Homeobox genes such as Hox-C8 and HNF-1 have also been demon- Human CDX2 is a member of the caudal-related strated to be transcriptional targets of CDX2 (Freund homeobox gene family (McGinnis and Krumlauf, et al., 1998). In addition, expression of the gut-enriched 1992; Mallo et al., 1997). During early development in Krupel-like factor (GKLF) gene, an epithelial-specific Drosophila, caudal is involved in anterior–posterior transcription factor that functions as a suppressor of cell patterning (Macdonald and Struhl, 1986). The expres- proliferation, is dependent on CDX2 in human colon sion of the rodent Cdx2 homeobox gene is tissue specific cancer cell line RKO (Dang et al., 2001). Importantly, and present from the early embryo to the adult (James CDX2 can regulate the expression of the liver intestine- et al., 1994), and thus it is likely that Cdx2 plays roles in cadherin (LI- cadherin) gene, indicating its key role in both the establishment and maintenance of the intestinal mediating CDX2 function in intestinal cell fate determi- nation (Hinoi et al., 2002). Clearly, further investiga- tions are needed to clarify the complex network involved *Correspondence: Y Yuasa, Department of Molecular Oncology, in CDX2. Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, p21/WAF1/CIP1 (hereafter referred to as p21) was Japan; E-mail: [email protected] one of the first cyclin-dependent kinase (cdk) inhibitors Received 2 January 2003; revised 31 March 2003; accepted 7 April 2003 identified, and it can bind and inhibit G1 cyclin/Cdk CDX2 up-regulates transcription of p21 Y-Q Bai et al 7943 complexes (Harper et al., 1993; Xiong et al., 1993). p21 can induce growth suppression in vivo through cyclin D1-Cdk4 and cyclin E-Cdk2 complexes (Lin et al., 1996), and overexpression of p21 results in cell cycle arrest in G1 (Xiong et al., 1993; Luo et al., 1995) and tumor cell growth suppression (El-Deiry et al., 1993). In addition to its role in cell cycle control, p21 is also believed to inhibit DNA replication through its ability to bind proliferating cell nuclear antigen (PCNA), which is required for both replicative DNA synthesis and DNA repair. However, p21 has no inhibitory effect on the DNA repair function of PCNA (Li et al., 1994; Waga et al., 1994). Unexpectedly, p21 plays critical roles in differentiation, such as the role in control of epithelial self-renewal and commitment to differentiation (Di Cunto et al., 1998). Finally, p21 has been proved to be an unsuspected tumor suppressor, which can mediate at least some of the tumor-suppressing effects of p53 Figure 1 Effect of CDX2 on the p21 promoter. (a) Reporter (reviewed by Dotto, 2000). construct p21-Luc, in which the p21 promoter drives expression of Since firstly CDX2 and p21 are both associated with the firefly luciferase gene, was cotransfected with the expression differentiation induction and tumor growth suppression, vector pcDNA3.1CDX2 or the empty vector in different cells, as indicated. The renilla reporter plasmid (1 : 10 ratio with target and secondly their expression overlaps in the small reporter) was also included in all experiments to normalize intestine and colon (Gartel et al., 1996; Polyak et al., variation in transfection efficiency. Cells were harvested after 48 h 1996; Mallo et al., 1997; Silberg et al., 2000), a possible and assayed for luciferase activity. All values were equalized on the link between these two genes is suggested. In the present basis of the activity observed upon cotransfection with a control study, we first identified nine CDX-core binding renilla expression vector, and expressed as ratio of CDX2- expressing vector to empty vector. These results represent the sequences, TTTAT, in the sense or, ATAAA, the means and standard deviation of three independent experiments. reverse orientation (Suh et al., 1994) within the human (b) p21-Luc, p21-Luc/D1orp21-Luc/D2 were cotransfected with the p21 promoter (À2699 to þ 45 bp). By means of expression vector pcDNA3.1CDX2 or the empty vector in HT-29 luciferase reporter assay, electrophoretic mobility shift cells. p21-Luc/D1 and p21-Luc/D2 were constructed by deleting À1900 to À658 bp or À2504 to À212 bp of the p21-Luc construct, assay (EMSA) and reverse transcription–polymerase respectively, as described under ‘Materials and methods’. Numbers chain reaction (RT–PCR), we then demonstrated that on the promoter refer to positions relative to the transcription start CDX2 activated transcription of the p21 gene through site of the human p21 gene ( þ 1). Note that no CDX binding site direct physical interaction with the p21 promoter. remains upstream of position À1900 bp. The position of p21-WT, which was used as probe for subsequent EMSA, containing three clustered CDX binding sites is also indicated (À471 to À434 bp). The activity of p21-Luc was arbitrarily set at 100%. Variations in Results transfection efficiency were corrected by normalization for expres- sion of renilla luciferase. All transfections were repeated three times in duplicate Transactivation of the p21 promoter by CDX2 To determine whether or not the p21 gene is a trans- criptional target of CDX2 and, if so, whether or not p53 four cell lines described above (data not shown), is involved, SAOS-2 (p53À/À) and U-2OS (p53 þ / þ ) indicating specific transactivation by CDX2 of the p21 osteosarcoma cells were cotransfected with the p21- promoter. Since p53 is null in SAOS-2 and mutated in Luc reporter construct and expression vector pcDNA3. HT-29cells, CDX2 transactivated the p21 promoter in a 1CDX2. These two cell lines were selected because they p53-independent manner. are widely used for p53-related studies and lack To determine whether or not the activation of the p21 endogenous CDX2 expression (data not shown). Ac- promoter by CDX2 is dependent on CDX binding sites, cording to the dual-luciferase reporter assay, cotransfec- deletion mutants were made as described in ‘Materials tion of pcDNA3.1CDX2 resulted in 11.1- and 3.3-fold and methods’. As a result, deletion of putative CDX transcriptional activation of the 2744-bp p21 promoter binding sites on the 50 side from À1900 to À658 bp (p21- construct in SAOS-2 and U-2OS, respectively (Figure 1). Luc/D1) or all the binding sites (p21-Luc/D2) in the p21 Then the ability of CDX2 to activate p21 transcription promoter resulted in 35 and 79% reduction in the p21 was further studied in gastrointestinal cancer cell lines promoter activity, respectively (Figure 1b), suggesting GT3TKB and HT-29. The endogenous CDX2 expres- that these binding sites are crucial for CDX2 to sion was below the detectable level in GT3TKB (Bai transactivate the p21 promoter.