1a,25-Dihydroxyvitamin D 3 regulates the expression of Id1 and Id2 genes and the angiogenic phenotype of human colon carcinoma cells Nuria Isabel Fernandez-Garcia 1,2 , Hector G Palmer 3 , Marta Garcia 4 , Alicia Gonzalez-Martin 1,2 , Marcela del Rio 4 , Domingo Barettino 5 , Olga Volpert 6 , Alberto Mun˜oz 1 and Benilde Jimenez* ,1,2 1 Instituto de Investigaciones Biomedicas CSIC-UAM, Madrid, Spain; 2 Department of Biochemistry, Universidad Autonoma de Madrid, Madrid, Spain; 3 Cancer Research, London WC2A 3PX, UK; 4 Project on Damage, Repair and Tissue Engineering CIEMAT, Madrid, Spain; 5 Instituto de Biomedicina de Valencia CSIC, Valencia, Spain; 6 Northwestern University Medical School, Department of Urology, Chicago, IL, USA 1a,25-Dihydroxyvitamin D 3 (1a,25(OH) 2 D 3 ) has antitu- mor activity in addition to its classical action on calcium metabolism and bone tissue biology. It is thought to regulate the expression of multiple target genes and thus modulate processes critical for tumor growth and metastases. Here we show that 1a,25(OH) 2 D 3 differen- tially regulates the expression of Id1 and Id2 genes, members of a family of transcriptional regulators of cell proliferation and differentiation. 1a,25(OH) 2 D 3 induced epithelial differentiation in SW480-ADH human colon carcinoma cell line by promoting expression of the proteins implicated in adherent junction formation, including E-cadherin, and by inhibiting b-catenin tran- scriptional activity. 1a,25(OH) 2 D 3 activated the human Id1 gene promoter and rapidly induced Id1 RNA and protein. Ectopic overexpression of Id1 was not sufficient to induce E-cadherin, which was critical for the morpho- logical changes induced by 1a,25(OH) 2 D 3 in SW480- ADH cells. Conversely, Id2 transcription rate, RNA and protein levels were decreased by 1a,25(OH) 2 D 3 . Id2 downregulation by 1a,25(OH) 2 D 3 mediated the antipro- liferative effect of 1a,25(OH) 2 D 3 on SW480-ADH cells. In addition, we showed that 1a,25(OH) 2 D 3 changed the levels of the inducer of angiogenesis, vascular endothelial growth factor and the potent antiangiogenic factor thrombospondin-1, leading to a balanced change in the angiogenic potential of SW480-ADH human colon carcinoma cells. Oncogene (2005) 24, 6533–6544. doi:10.1038/sj.onc.1208801; published online 11 July 2005 Keywords: Id; 1a,25(OH) 2 D 3 ; colon carcinoma; angio- genesis; E-cadherin Introduction New therapeutic approaches for colon cancer rely on biological response modifiers targeting both the tumor and its microenviroment. 1a,25-Dihydroxyvitamin D 3 (1a,25(OH) 2 D 3 ) induces growth arrest and differentia- tion of colon cancer cells (Diaz et al., 2000; Palmer et al., 2001) and has antitumor, antimetastic (Evans et al., 2000; Koli and Keski-Oja, 2000) and antiangiogenic (Iseki et al., 1999) effects in vitro and in animal models of colorectal cancer. Several nonhypercalcemic 1a,25(OH) 2 D 3 derivatives have recently entered clinical trials in patients with colorectal carcinomas and other neoplasms (Gross et al., 1998; Gulliford et al., 1998; Smith et al., 1999; Dalhoff et al., 2003). Despite the strong evidence supporting 1a,25(OH) 2 D 3 antitumor activity, it is still unclear how these results could be translated into useful therapeutic regimens and which types and individual patients would benefit from 1a,25(OH) 2 D 3 -based treatments. Understanding the antitumor mechanism of 1a,25(OH) 2 D 3 combined with clinical research will contribute to this end. Studies of the past 10 years defined a central role of the Id proteins in the control of multiple cellular processes ranging from differentiation to cell prolifera- tion, apoptosis, invasion and migration (Yokota, 2001; Zebedee and Hara, 2001). The mechanism of action of Id family members includes formation of heterodimers with basic helix–loop–helix (bHLH), Ets and Pax transcription factors and direct interaction with critical targets, such as Rb. Their broad regulatory function makes Id proteins vulnerable targets in the process of malignant transformation. Members of the Id family are generally upregulated during tumorigenesis (Israel et al., 1999; Lasorella et al., 2001) and are necessary for tumor angiogenesis (Lyden et al., 1999; Benezra et al., 2001; Sikder et al., 2003). Id proteins control epithelial cell function. Moderate expression of Id1, Id2 and Id3 in the normal colonic epithelium dramatically increases in colorectal human tumors and human colon carcinoma cell lines (Wilson et al., 2001). Ids have been implicated in several pathways central to colon carcinogenesis. The great majority of colon cancers are initiated by mutation of Received 3 August 2004; revised 1 April 2005; accepted 29 April 2005; published online 11 July 2005 *Correspondence: B Jimenez, Instituto de Investigaciones Biomedicas CSIC-UAM and Department of Biochemistry, Universidad Autono- ma de Madrid, Arturo Duperier 4, 28029 Madrid, Spain; E-mail: bjimenez@iib.uam.es Oncogene (2005) 24, 6533–6544 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc