Abstract
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 5alpha-cholest-8-en-3beta-ol for tamoxifen treatment and 5alpha-cholest-8-en-3beta-ol and cholesta-5,7-dien-3beta-ol, for PBPE treatment, suggesting that these AEBS ligands affect at least two enzymatic steps: the 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-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 3beta-hydroxysterol-Delta8-Delta7-isomerase and 3beta-hydroxysterol-Delta7-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 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-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.
Highlights
Tamoxifen is a selective estrogen receptor modulator (SERM) widely used for the treatment and the prevention of breast cancer [1]
We report in this study that tamoxifen and the selective antiestrogen binding site (AEBS) ligand PBPE produced a massive accumulation of 5␣-cholest-8-en-3-ol and/or 5␣-cholesta-5,7-dien-3-ol that suggests an inhibition of the D8D7I and the 3-hydroxysterol-⌬7-reductase (DHCR7), two enzymes involved in the biosynthesis of cholesterol
Binding to the AEBS Is Not Correlated with the Inhibition of D8D7I or DHCR7 in MCF-7 Cells—First, we have shown that two ligands of the AEBS inhibited the catalytic activities of D8D7I or/and DHCR7, and second that the overexpression of D8D7I and DHCR7 resulted in the reconstitution of the [3H]tamoxifen binding site on the AEBS
Summary
We and others (5, 10 –14) have shown that AEBS ligands inhibit the growth of tumor cell lines in vitro and in vivo, demonstrating that the AEBS was involved in the mediation of the effects of these structural classes of its cognate ligands These compounds represent specific tools to study AEBS function but are anticancer drug candidates because the selective AEBS ligand DPPE (Tesmilifene) was brought up to phase II and III clinical trials for the treatment of breast and prostate cancer in association with doxorubicin [15,16,17]. Immunoprecipitation followed by tamoxifen binding to the immunoprecipitate complex showed that D8D7I and DHCR7 are associated proteins necessary for the reconstitution of the AEBS
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