Journal of Steroid Biochemistry & Molecular Biology 93 (2005) 173–182 p53-dependent inhibition of progestin-induced VEGF expression in human breast cancer cells  Yayun Liang a , Jianbo Wu a , George M. Stancel b , Salman M. Hyder a,∗ a Dalton Cardiovascular Research Center, Department of Biomedical Sciences, University of Missouri, 134 Research Park Drive, Columbia, MO 65211, USA b Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX 77030, USA Abstract VEGF, a potent angiogenic growth factor, is up-regulated in many tumors including human breast tumors and stimulates growth of vascular networks that support tumor growth and metastasis. We previously reported that natural and synthetic progestins (P) increased VEGF mRNA and protein levels in progesterone receptor (PR) containing T47-D human breast cancer cells in a PR dependent manner, but not in PR positive ZR-75 and MCF-7, or in PR negative MDA-MB-231 cells. This indicated that factors beside PR are involved in progesterone-dependent VEGF regulation. We, therefore, tested additional tumor cell lines reported to contain PR for progestin-dependent VEGF induction. Out of nine PR-positive breast tumor cell lines, progestins induced VEGF in three cell lines that lack wild-type p53 (T47-D, BT-474, and HCC-1428) but not in cell lines that contained the wild-type p53 protein. The T47-D and BT-474 cells express mutant p53, while the p53 protein is absent HCC-1428 cells. The anti-progestin RU-486 blocked progestin-dependent induction of VEGF in T47-D and BT-474 cells but not in HCC-1428 cells. However, RU-486 partially blocked medroxyprogesterone acetate-dependent induction of VEGF in HCC-1428 cells. Estrogen receptor (ER) and PR agonists and antagonists also induce VEGF in HCC-1428 cells and this effect was partially blocked by anti-estrogen ICI-182, 780. Progestin-dependent VEGF induction was completely inhibited by PRIMA-1-activated p53 in all cell-types, but progestin-dependent transcription of a progesterone-regulated minimal promoter was only partially inhibited. PRIMA-1 induced activation of p53 in tumor cell lines was confirmed with a p53-responsive p21 reporter plasmid and by detecting increased levels of p21 proteins in cell lysates. PRIMA-1 induced p53 protein in the HCC-1428 cells while levels of mutant p53 protein in T47-D and BT-474 remained unaltered. Progestin-dependent induction of VEGF was also inhibited by stable transfection of wild-type p53 in T47-D cells. These results are consistent with the hypothesis that wild-type p53 blocks progestin-dependent induction of VEGF in breast cancer cells and this may be a novel anti-angiogenic mechanism for controlling the growth of progestin-dependent tumors. © 2004 Elsevier Ltd. All rights reserved. Keywords: Angiogenesis; Breast cancer; p53; Progestin; Progesterone receptor; VEGF Abbreviations: P, progesterone; PR, progesterone receptor; E, estrogen; ER, estrogen receptor; HRT, hormone replacement therapy; MPA, medroxy progesterone acetate; VEGF, vascular endothelial growth factor; PRIMA-1, p53 reactivation induction of massive apoptosis; RT, reverse transcriptase; TBS, tris-buffered-saline  Proceedings of the 16th International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, ‘Recent Advances in Steroids Biochemistry and Molecular Biology’ (Seefeld, Tyrol, Austria, 5–8, June 2004). ∗ Corresponding author. Tel.: +1 573 882 1261; fax: +1 573 884 4232. E-mail address: hyders@missouri.edu (S.M. Hyder). 1. Introduction Tumor growth, expansion and metastasis are promoted by development of vascular networks (i.e., angiogenesis), be- cause these networks deliver essential nutrients to the grow- ing tumor [1,2]. VEGF is one of the most potent angiogenic factors known, and it is thought to act as an angiogenic switch for expansion of quiescent tumor tissues [3,4]. VEGF expres- sion can be induced by many factors including oncogenic proteins, tumor suppressor proteins including p53, hypoxia, cytokines and steroid hormones [2,5-8]. Mutation or deletion of wild-type p53 is associated with up-regulation of VEGF in breast cancer cells [9]. Breast cancer prognosis can be pre- 0960-0760/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2004.12.011