Identification and pharmacological characterization of cholesterol-5,6-epoxide hydrolase as a target for tamoxifen and AEBS ligands

Abstract

The microsomal antiestrogen binding site (AEBS) is a high-affinity target for the antitumor drug tamoxifen and its cognate ligands that mediate breast cancer cell differentiation and apoptosis. The AEBS, a hetero-oligomeric complex composed of 3beta-hydroxysterol-Delta8-Delta7-isomerase (D8D7I) and 3beta-hydroxysterol-Delta7-reductase (DHCR7), binds different structural classes of ligands, including ring B oxysterols. These oxysterols are inhibitors of cholesterol-5,6-epoxide hydrolase (ChEH), a microsomal epoxide hydrolase that has yet to be molecularly identified. We hypothesized that the AEBS and ChEH might be related entities. We show that the substrates of ChEH, cholestan-5alpha,6alpha-epoxy-3beta-ol (alpha-CE) and cholestan-5beta,6beta-epoxy-3beta-ol (beta-CE), and its product, cholestane-3beta,5alpha,6beta-triol (CT), are competitive ligands of tamoxifen binding to the AEBS. Conversely, we show that each AEBS ligand is an inhibitor of ChEH activity, and that there is a positive correlation between these ligands' affinity for the AEBS and their potency to inhibit ChEH (r2=0.95; n=39; P<0.0001). The single expression of D8D7I or DHCR7 in COS-7 cells slightly increased ChEH activity (1.8- and 2.6-fold), whereas their coexpression fully reconstituted ChEH, suggesting that the formation of a dimer is required for ChEH activity. Similarly, the single knockdown of D8D7I or DHCR7 using siRNA partially inhibited ChEH in MCF-7 cells, whereas the knockdown of both D8D7I and DHCR7 abolished ChEH activity by 92%. Taken together, our findings strongly suggest that the AEBS carries out ChEH activity and establish that ChEH is a new target for drugs of clinical interest, polyunsaturated fatty acids and ring B oxysterols.

Fig. 1.

Competition for [³H]Tam binding to the microsomal AEBS by Tam, α-CE, β-CE, and CT. (A) Competition assays with increasing concentrations of unlabeled Tam (▼), α-CE (●), β-CE (▲), and CT (▪) were performed on rat liver microsomes using 2.5 nM [³H]Tam. (B) Scatchard plots of [³H]Tam binding to the microsomal AEBS in the absence (▼) or presence of 100 nM α-CE (○) or 200 nM α-CE (●). (C) Scatchard plots of [³H]Tam binding to the microsomal AEBS in the absence (▼) or presence of 200 nM β-CE (△) or 500 nM β-CE (▲). (D) Scatchard plots of [³H]Tam binding to the microsomal AEBS in the absence (▼) or presence of 5 μM CT (□) or 10 μM CT (▪). The lines intercept on the x-axis, indicating that α-CE, β-CE, and CT are competitive ligands of the AEBS with respect to Tam binding. Measurements were made in triplicate for at least three separate experiments. Data are presented as the mean ± SEM.

Fig. 2.

Inhibition of ChEH by Tam, PBPE, and oleic acid. The relationship between the conversion rates of α-CE to CT and inhibitor concentrations is shown using 10 and 20 μM Tam and PBPE with rat liver microsomal ChEH. Shown are double reciprocal plots of Tam (A), PBPE (B), and oleic acid (C) versus [¹⁴C]α-CE.

Fig. 3.

Correlation between affinity of AEBS ligands for the AEBS and their potency to inhibit ChEH. Graph of the pK_i for 39 compounds tested for the inhibition of [³H]Tam binding as a function of pK_i on ChEH activity. The drug numbers and the corresponding pK_i values [−log(K_i)] are listed in Table 1. Here r is the correlation coefficient between pK_i values calculated for the inhibition of Tam binding and ChEH activity. The r² value of 0.95 and significance of correlation (P < 0.0001) are given for all structural classes of compounds (n = 39).

Fig. 4.

Expression and knockdown of D8D7I and DHCR7 in mammalian cells: Impact on ChEH and AEBS activities. (A) ChEH activity of microsomal extracts from COS-7 cells transfected with control vector (mock), D8, D7, and D8 + D7. (B) Michaelis-Menten plot of velocity versus α-CE in ChEH assays from COS-7 cells transfected with mock (●) or D8 + D7 (▪). (C) Inhibition of ChEH in microsomal extracts from COS-7 cells coexpressing human recombinant D8 and D7 with increasing concentrations of clomiphene (⋄), PBPE (○), Tam (□), 4OH-tamoxifen (△), 7-ketocholestanol (▽), or 17β-estradiol (▪). (D, E) Expression of D8 and D7 in MCF-7 cells transfected with siSC scrambled, siD8, siD7, or siD8 + siD7 at the mRNA level (D) and at the protein level (E). (F) Representative TLC autoradiogram showing ChEH activity in MCF-7 cells from three independent experiments. (G) Michaelis-Menten plot of velocity versus α-CE in ChEH assays from MCF-7 cells transfected with control scrambled siRNA (siSC; ●), siD8 (○), siD7 (□), or siD8 + siD7 (▪). (I) Scatchard plots of [³H]Tam binding to microsomal AEBS from MCF-7 cells transfected with siSC (●), siD8 (○), siD7 (□), or siD8 + siD7 (▪). Measurements were made in triplicate for at least three separate experiments. Data are presented as mean ± SEM.

Fig. 5.

Functional relationship between the AEBS and ChEH. (A) The subunits of the AEBS that bind Tam (D8D7I and DHCR7) carry out the ChEH activity. (B) Ligands of the AEBS, such as cationic SERMs (Tam and raloxifene), diphenylmethane compounds (tesmilifene and PBPE), ring B oxysterols (7-ketocholesterol and 7-hydroxycholesterol) and polyunsaturated fatty acids (DHA), are inhibitors of ChEH, leading to the blockage of CT production and CE accumulation.