SHORT REPORT CDX2 is mutated in a colorectal cancer with normal APC/b-catenin signaling Luis T da Costa 1 , Tong-Chuan He 2 , Jian Yu 1 , Andrew B Sparks 2 , Patrice J Morin 3 , Kornelia Polyak 4 , Steve Laken 2 , Bert Vogelstein 2,5 and Kenneth W Kinzler* ,2 1 Program in Human Genetics and Molecular Biology, The Johns Hopkins University, Baltimore, Maryland, MD 21231, USA; 2 Johns Hopkins Oncology Center, Baltimore, Maryland, MD 21231, USA; 3 Laboratory of Biological Chemistry, National Institute on Aging, NIH, Baltimore, Maryland MD 21224, USA; 4 Dana-Farber Cancer Institute, Boston, Massachusetts, MA 02115 USA; 5 Howard Hughes Medical Institutes, The Johns Hopkins University, Baltimore, Maryland, MD 20815, USA The majority of human colorectal cancers have elevated b-catenin/TCF regulated transcription due to either inactivating mutations of the APC tumor suppressor gene or activating mutations of b-catenin. Surprisingly, one commonly used colorectal cancer cell line was found to have intact APC and b-catenin and no demonstrable b-catenin/TCF regulated transcription. However, this line did possess a truncating mutation in one allele of CDX2, a gene whose inactivation has recently been shown to cause colon tumorigenesis in mice. Expression of CDX2 was found to be induced by restoring expression of wild type APC in a colorectal cancer cell line. These ®ndings raise the intriguing possibility that CDX2 contributes to APC's tumor suppressive eects. Keywords: CDX2; APC; colorectal cancer; mutation; regulation Mutations in the adenomatous polyposis coli (APC) gene initiate the vast majority of human colorectal tumors (Kinzler and Vogelstein, 1996) but the functional consequences of such mutations at the biochemical level were until recently poorly under- stood. An important advance was made with the discovery that the APC gene product inhibits b- catenin/TCF regulated transcription (CRT) (Korinek et al., 1997; Morin et al., 1997). This inhibition is likely mediated by the binding of b-catenin to APC (Rubinfeld et al., 1993; Su et al., 1993), which facilitates phosphorylation of b-catenin by GSK-3b (Rubinfeld et al., 1996), and leads to its degradation through ubiquitination-dependent proteolysis (Mune- mitsu et al., 1995; Aberle et al., 1997; Orford et al., 1997). APC thus eectively regulates the cellular levels of b-catenin and consequently the formation of active transcription complexes between b-catenin and Tcf-4. Consistent with this model, alterations of b-catenin that render it refractory to regulation by APC were identi®ed in a number of tumors with wild-type APC (Morin et al., 1997; Rubinfeld et al., 1997; Sparks et al., 1998), and it was shown that these b-catenin mutations as well as inactivating mutations of APC result in constitutive CRT (Morin et al., 1997). Accordingly, previous work had shown that all colorectal cancer cell lines tested had constitutive CRT (Korinek et al., 1997), suggesting that CRT deregulation is a key event in colorectal cancer initiation. The present study originated from the surprising observation that CRT is absent in RKO, a widely studied colorectal cancer cell line (Brattain et al., 1984). We subsequently found that RKO contains a mutant CDX2 gene, a gene recently implicated in intestinal tumorigenesis in mice (Chawengsaksophak et al., 1997), and that CDX2 transcription is induced by APC. These results suggest a connection between APC and CDX2 in colorectal cancers and raise the possibility that CDX2 is one of the mediators of APC's tumor-suppressing activity. b-catenin regulated transcription activity in cancer cell lines Constitutive activation of b-catenin regulated tran- scription has been reported in all colorectal cancer cell lines tested, a feature that distinguishes them from cancer cell lines derived from other tissue types (examples in Figure 1). CRT is typically measured using luciferase reporters called TOPFLASH and FOPFLASH, which contain promoters with several copies of a normal or mutant version of a TCF- response element (Korinek et al., 1997), respectively. Activation of CRT results in a high ratio of TOPFLASH to FOPFLASH luciferase activity. Surprisingly, when we measured CRT in RKO, we found that the levels of luciferase activity were similar with both TOPFLASH and FOPFLASH reporters (data not shown). However, we also observed that the levels of FOPFLASH reporter activity were signifi- cantly higher in RKO than in the other cancer cell lines tested. We were therefore unable to conclude that RKO lacked constitutive CRT activity, as it was possible that it was simply masked by high back- ground activity of the FOPFLASH control in this particular cell line. To distinguish between these two possibilities, we constructed a new pair of CRT reporter vectors (TCF-Luc and TCF*-Luc) that displayed much lower background activities in all lines tested (JY and LTC, unpublished observations). We then repeated our analysis of CRT activity in RKO using these vectors and demonstrated that RKO *Correspondence: KW Kinzler Received 1 December 1998; revised 18 March 1999; accepted 26 March 1999 Oncogene (1999) 18, 5010 ± 5014 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc