HES-1 inhibits 17b-estradiol and heregulin-b1-mediated upregulation of E2F-1 Johan Hartman 1 , Patrick Mu¨ller 1 , James S Foster 2 , Jay Wimalasena 2 , Jan-A ˚ ke Gustafsson 1,3 and Anders Stro¨m* ,1 1 Center for Biotechnology, Department of BioSciences, Karolinska Institutet, Novum, S-141 57 Huddinge Sweden; 2 Department of Obstetrics and Gynecology, Graduate School of Medicine, University of Tennessee Medical Center, Knoxville, TN 37920, USA; 3 Department of Medical Nutrition, Karolinska Institutet, Novum, S-141 86 Huddinge, Sweden We have previously shown that expression of the transcription factor HES-1 is required for the growth- inhibitory effect of all-trans retinoic acid on MCF-7 cells. In this study, we have used T47D cells with tetracyclin- regulated expression of wild-type or a dominant-negative form of HES-1. Expression of HES-1 in T47D cells inhibited G 1 /S-phase transition and activation of Cdk2 elicited by estrogen. Estrogen treatment of T47D cells caused increased expression of E2F-1, and this expression was inhibited by cotreatment with all-trans retinoic acid. We show that the effect is mediated through HES-1, which directly downregulates E2F-1 expression through a CACGAG-site within the E2F-1 promoter. Furthermore, proliferation caused by heregulin-b1 treatment of T47D cells was inhibited by all-trans retinoic acid and this effect was mediated by HES-1. Interestingly, heregulin-b1- mediated upregulation of E2F-1 expression was directly inhibited by HES-1 through the same CACGAG-site as seen with estrogen-stimulated induction. In addition, we found that two important downstream target genes of estrogen and heregulin-b1 that are regulated through E2F-1, cyclin E and NPAT, were both regulated in a similar fashion by all-trans retinoic acid, and these effects were antagonized by dominant-negative HES-1. These findings establish that HES-1 inhibits both estrogen- and heregulin-b1-stimulated growth of breast cancer cells, and further suggest that growth inhibition induced in these cells by all-trans retinoic acid occurs via HES-1-mediated downregulation of E2F-1 expression. Oncogene (2004) 23, 8826–8833. doi:10.1038/sj.onc.1208139 Published online 4 October 2004 Keywords: all-trans retinoic acid; cell cycle; T47D, HES- 6; HERP; NPAT; cyclin E Introduction Growth of human breast epithelial cells is dependent on estrogen as well as epidermal growth factor receptors. Estrogens are key-players in the development of breast cancer and have been reported to take part in the initial transformation as well as to play a major role in the progression to a malignant, neoplastic state (Tsutsui et al., 1987; Bergkvist et al., 1989; Nandi et al., 1995). 17b-Estradiol (E 2 ) drives proliferation by affecting several cell-cycle factors. An important target gene induced by E 2 is the transcription factor E2F-1 (Ohtani et al., 1995). The members of the E2F-family of transcription factors (E2F-1-7) comprise key regulators of cellular proliferation, development, differentiation, and apoptosis (Muller et al., 2001). E2F-1 is the best- characterized member of the E2F-family. Overexpression of E2F-1 can drive quiescent cells into S-phase and override many different types of cell cycle arrests (DeGregori et al., 1995; Johnson et al., 1993). In nonproliferating cells, the E2F factors are stably bound to members of the Rb pocket protein family (Dyson, 1998). This association keeps the E2F in an inactive state and this complex can even function as an active repressor of transcription (Weintraub et al., 1992). In the mid-G 1 phase, during cellular proliferation, E2F-pRb complexes dissociate as a result of hyperphosphorylation by Cdks and E2F-1 subsequently acts as a transcriptional activator and induces its own transcription through binding sites within the E2F-1 promoter (Nevins, 1998). The high level of dissociated E2F-1 seen in mid-G 1 phase is essential for S-phase entry and proliferation. There is a wealth of clinical data demonstrating the importance of ErbB receptors, in particular ErbB1 (EGF-receptor) and ErbB2 (HER2/Neu), in develop- ment and malignancy of human cancer (Holbro et al., 2003). In MCF-7 cells, activation of ErbB3 by the growth factor ligand heregulin-b1 (HRG) induces ErbB2-ErbB3 heterodimerization and cellular prolifera- tion (Jhabvala-Romero et al., 2003). In cell cultures, increased ErbB2 expression or HRG treatment leads to an estrogen-independent phenotype of breast cancer cells through crosstalk between the estrogen and ErbB receptors (Pietras et al., 1995; Tang et al., 1996). Received 27 April 2004; revised 18 August 2004; accepted 18 August 2004; published online 4 October 2004 *Correspondence: A Stro¨m, Center for Biotechnology, Department of BioSciences, Karolinska Institutet, Novum, S-141 57 Huddinge, Sweden; E-mail: anders.strom@biosci.ki.se Oncogene (2004) 23, 8826–8833 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc