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J. Biol. Chem., Vol. 279, Issue 32, 34048-34061, August 6, 2004

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Molecular Characterization of the Microsomal Tamoxifen Binding Site^*

Blandine Kedjouar, Philippe de Médina, Mustapha Oulad-Abdelghani, Bruno Payré, Sandrine Silvente-Poirot, Gilles Favre, Jean-Charles Faye, and Marc Poirot

From the INSERM U 563, Centre de Physiopathologie de Toulouse Purpan, Département Innovation Thérapeutique et Oncologie Moléculaire, Institut Claudius Regaud, 20-24 rue du Pont Saint Pierre, 31052 Toulouse Cedex, France

Tamoxifen is a selective estrogen receptor modulator widely used for the prophylactic treatment of breast cancer. In addition to the estrogen receptor (ER), tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS), which is involved in ER-independent effects of tamoxifen. In the present study, we investigate the modulation of the biosynthesis of cholesterol in tumor cell lines by AEBS ligands. As a consequence of the treatment with the antitumoral drugs tamoxifen or PBPE, a selective AEBS ligand, we show that tumor cells produced a significant concentration- and time-dependent accumulation of cholesterol precursors. Sterols have been purified by HPLC and gas chromatography, and their chemical structures determined by mass spectrometric analysis. The major metabolites identified were 5-cholest-8-en-3-ol for tamoxifen treatment and 5-cholest-8-en-3-ol and cholesta-5,7-dien-3-ol, for PBPE treatment, suggesting that these AEBS ligands affect at least two enzymatic steps: the 3-hydroxysterol-⁸-⁷-isomerase and the 3-hydroxysterol-⁷-reductase. Steroidal antiestrogens such as ICI 182,780 and RU 58,668 did not affect these enzymatic steps, because they do not bind to the AEBS. Transient co-expression of human 3-hydroxysterol-⁸-⁷-isomerase and 3-hydroxysterol-⁷-reductase and immunoprecipitation experiments showed that both enzymes were required to reconstitute the AEBS in mammalian cells. Altogether, these data provide strong evidence that the AEBS is a hetero-oligomeric complex including 3-hydroxysterol-⁸-⁷-isomerase and the 3-hydroxysterol-⁷-reductase as subunits that are necessary and sufficient for tamoxifen binding in mammary cells. Furthermore, because selective AEBS ligands are antitumoral compounds, these data suggest a link between cholesterol metabolism at a post-lanosterol step and tumor growth control. These data afford both the identification of the AEBS and give new insight into a novel molecular mechanism of action for drugs of clinical value.

Received for publication, May 11, 2004 , and in revised form, June 2, 2004.

^* This work was supported by the Institut National de la Recherche Médicale, a Grant from the Association pour la Recherche sur le Cancer (ARC No. 5648), and the Caisse d'Assurance Maladie des Professions Liberales Provinces. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/ INSERM/ ULP/ Collège de France, BP 10142, 67404 Illkirch-Cedex, France.

To whom correspondence should be addressed: INSERM U 563, C. P. T. P., Département Innovation Thérapeutique et Oncologie Moléculaire, Institut Claudius Regaud, 20-24 rue du Pont Saint Pierre, 31052 Toulouse Cedex; France. Tel.: 33-5-61-42-46-48; Fax: 33-5-61-42-46-31; E-mail: poirot{at}icr.fnclcc.fr.

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