Tamoxifen (tam) is a selective estrogen receptor modulator widely used for the management of breast cancers. Beside estrogen receptors, the highest affinity target for tam is the membranous microsomal antiestrogen binding site (AEBS). We have previously shown that the AEBS results from the associationbetween two enzymes involved in the synthesis of cholesterolprecursors (the 3ß-hydroxysterol-delta-8-delta-7-isomerase andthe 3ß-hydroxysterol-delta-7-reductase) and has no affinityfor pure antiestrogens or estrogens. In order to clarify the involvement of the AEBS in the mediation of the antiproliferative action of its cognate ligands on human breast cancer cells, we have compared the effect of tam with those of a selective AEBS ligand (N-pyrrolidino-2-[4-(benzyl)-phenoxy-ethanamine-HCl) (PBPE)). We found that PBPE and tam induced a concentration-dependant growth control and a cell arrest in G0-G1 phase of the cell cycle. The growth inhibition was associated with morphological changes including an increase of the cytoplasmic volume. Tam and PBPE stimulated the induction of milk fat globule protein (MFG), milk fat membrane globule protein and lipid droplets. The ultrastructure analysis of treated cells revealed the presence of unilamellar vesicles and multilamellar bodies corresponding to the accumulation of di- and tri-glycerides as well as free and fatty acid esters of sterols respectively. Tam and PBPE stimulation of sterol accumulation is associated with the down-regulation of the HMG-CoA reductase and the inhibition of H-Ras isoprenylation. The addition of mevalonolactone inhibited the accumulation of lipid droplets suggesting that the inhibition of the isoprenoid and cholesterol biosynthesis was responsible of the lipid accumulation. Despite the inhibition of H-Ras prenylation, a 48 hours treatment of MCF-7 cells with tam and PBPE stimulated the phosphorylation of the ERK and of the P70S6 kinase. The P70S6 kinase appeared to be phosphorylated on Thr 241, Ser 424 and also on Thr 389 suggesting both the involvement of the ERK and mTOR. The phosphorylation of the P70S6 kinase lead to a stimulation of the phosphorylation of the ribosomal S6 protein which may control the expression of the MFG since rapamycine inhibits the P70S6 kinase and the expression of the MFG. Together these results indicate that tam and PBPE have a similar mechanism of action. We propose that AEBS ligands have drastic antiproliferative activity by causing these cells to undergo cell cycle arrest and differentiation. AEBS ligands such as the PBPE analogue tesmilifen is currently evaluated in Phase III clinical trials. These findings may impact the clinical use of these drugs and may improve the prophylactic use of tam for breast cancer prevention and treatment. More importantly this study demonstrates that the AEBS represent a promising target for the development of new anticancer agents.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA