Ligands of the antiestrogen-binding site induce active cell death and autophagy in human breast cancer cells through the modulation of cholesterol metabolism

Abstract

We have recently reported that cytostatic concentrations of the microsomal antiestrogen-binding site (AEBS) ligands, such as PBPE (N-pyrrolidino-(phenylmethyphenoxy)-ethanamine,HCl) and tamoxifen, induced differentiation characteristics in breast cancer cells through the accumulation of post-lanosterol intermediates of cholesterol biosynthesis. We show here that exposure of MCF-7 (human breast adenocarcinoma cell line) cells to higher concentrations of AEBS ligands triggered active cell death and macroautophagy. Apoptosis was characterized by Annexin V binding, chromatin condensation, DNA laddering and disruption of the mitochondrial functions. We determined that cell death was sterol-and reactive oxygen species-dependent and was prevented by the antioxidant vitamin E. Macroautophagy was characterized by the accumulation of autophagic vacuoles, an increase in the expression of Beclin-1 and the stimulation of autophagic flux. We established that macroautophagy was steroland Beclin-1-dependent and was associated with cell survival rather than with cytotoxicity, as blockage of macroautophagy sensitized cells to AEBS ligands. These results show that the accumulation of sterols by AEBS ligands in MCF-7 cells induces apoptosis and macroautophagy. Collectively, these data support a therapeutic potential for selective AEBS ligands in breast cancer management and shows a mechanism that explains the induction of autophagy in MCF-7 cells by tamoxifen and other selective estrogen receptor modulators. The AEBS binds selective estrogen receptor modulators (SERM) that contain a cationic aminoethoxy side chain, but has no affinity for estrogens and non-cationic antiestrogens, such as Faslodex, 668. 2 A class of derivatives of Tx has been developed that bind to the AEBS with high affinity and selectively. This family of compounds includes as lead compounds tesmilifene (DPPE, N,N 0 -diethylamino-4-(phenylmethylphenoxy)-ethanamine,HCl) 3 and PBPE. 4 These compounds have no affinity for estrogen receptors and did not modulate other known targets of Tx such as acyl-coA:cholesterol acyltransferase and protein kinase C, 5 allowing AEBS-related cellular events to be studied. Despite a lack of a clear understanding of its mechanism of action at that time, tesmilifene was evaluated for the treatment of breast and prostate cancer with some encouraging results in phase II and III clinical trials.6-8 However, a pivotal phase III trial was recently aborted because of the lack of a therapeutic outcome. This strongly reinforces the need to understand better the mechanism of action of drugs to optimize their clinical use.The AEBS has been reported to bind unsaturated fatty acids and oxysterols (7-ketocholesterol, 7-ketocholestanol and 7-hydroxycholesterol), suggesting a link between the AEBS and the metabolism of lipids and sterols. This led us to identify the AEBS as a hetero-oligomeric complex composed of two subunits: the 3b-hydroxysterol-D 7 -reductase (DHCR7) and 3b-hydroxysterol-D 8 -D 7 -isomerase (D8D7I); these two enzymes are invol...

Not on the menu

“…18,19 Puzzled by these results we decided to test the active metabolite of tamoxifen, 4-hydroxytamoxifen (OHT), which had been reported to be a somewhat weaker inducer of autophagy based on its reduced induction of Beclin-1. 20 Contrary to expectations, we consistently found OHT to be a more potent anti-prion agent than tamoxifen insofar as its ability to clear proteinase-K-resistant prion infection from cultured cells. This result suggested that autophagy was not responsible for the anti-prion effects of tamoxifen and OHT.…”

“…20 Interestingly, these closely related compounds induce the accumulation of distinct biosynthetic intermediates of cholesterol in treated cells (Fig. 2).…”

“…25 Tamoxifen/OHT treatment shares many features with U18666A treatment. Like U18666A, both tamoxifen and OHT inhibit cholesterol synthesis and perturb cholesterol trafficking 20 (Fig. 2).…”

“…To further complicate the picture, cholesterol depletion itself has also been shown to induce autophagy. 20 Thus, the major anti-prion effects of tamoxifen and OHT are likely due to their inhibition of cholesterol biosynthesis and perturbation of lipid trafficking; in contrast, minor anti-prion activity may be due to autophagy, induced as a by-product of interference with cholesterol biosynthesis. Unlike other neurodegenerative diseases of protein aggregation, prions diseases may represent a special case, responding to perturbation of cholesterol metabolism rather than to autophagy.…”

Exosomes as intercellular signalosomes and pharmacological effectors

“…AEBS is a molecular complex constituted by two cholesterogenic enzymes, the DHCR7 reductase and the D8D7I isomerase, whose association makes a third catalytic activity, namely the cholesterol epoxyde hydrolase [32]. Ligands of AEBS trigger macroautophagy in tumor cells through modulation of cholesterol metabolism [33],and a link between microautophagy of the late endosome compartment and exosome 6 production has been proposed [22], suggesting a role of cholesterol in exosome biogenesis. Noteworthy, the ATPase Vps4 which binds ECRTIII in the ESCRT machinery (see § 1.1) also interacts with oxysterol binding proteins and sterol metabolism [34].…”