In search of novel drug target sites on estrogen receptors using RNA aptamers

Abstract

Estrogen receptor α (ERα) is a well-validated drug target for a majority of breast cancers. But the target sites on this receptor are far from exhaustively defined. Almost all ER antagonists in clinical use function by binding to the ligand-binding pocket to occlude agonist access. Resistance to this type of drugs may develop over time, not caused by the change of ERα itself, but by changes in ER associated proteins. This observation is fueling the development of reagents that downregulate ER activity through novel binding sites. However, it is challenging to find general ER antagonists that act independently from other known ER ligands. In this report, we describe the utility of RNA aptamers in the search for new drug target sites on ERα. We have identified three high affinity aptamers and characterized one of them in detail. This aptamer interacted with ERα in a way not affected by the presence or absence of either the steroidal ligands or the estrogen response DNA elements, and effectively inhibited ER-mediated transcriptional activation in a breast cancer cell line. Serving as a novel drug lead, it may also be used to guide the rational chemical synthesis of small molecule drugs or to perform screens of small molecule libraries for those that are able to displace the aptamer from its binding site.

Fig. 1.

RNA aptamers and their affinity for the unliganded estrogen receptor α (apo-ERα). (A) Aptamer sequences in the randomized region and their occurrences in the final sequenced pool. The names of the individual aptamers are given to the left of each sequence. The underlined lower case letters at the 3′ end are sequences derived from the first 3 positions (AGU, underlined) of the 3′ constant region (the primer covers 22 bases of the 25-nt sequence). The frequency of occurrence of each aptamer is given on the right. The sequences of the flanking constant region are shown at the bottom. (B) Predicted secondary structures of aptamers by mfold. The sugar-phosphate backbone is represented by bold lines, while hydrogen bonds between paired bases by faint lines. Each base is represented by a dot. (C) Binding curves of the aptamers for ERα determined using filter-binding assays. The apparent K_d determined from binding curves is shown next to the title of each curve. Each experiment was repeated at least three times and the standard deviation was shown as error bars. (D) Similar mobilities of the three aptamer-ERα complexes in electrophoretic mobility shift assay (EMSA). Binding mixtures are resolved on 4.8% polyacrylamide gel (acrylamide:bis-acrylamide=37.5:1) in 1/4× TBE buffer. ERα concentrations are 0, 10, 20, and 40 nM. The asterisks signify the radiolabel. (E) Affinity of the three aptamers for apo-ERα (0, 20, and 40 nM) and apo-ERβ (0, 20, and 40 nM). Conditions are the same as in (D).

Fig. 2.

Aptamer binding in the presence or absence of other ligands. (A) A common binding site shared by the three aptamers. In this aptamer cross competition assay, radiolabeled AptER-1 RNA (0.1 nM) is mixed with 2000× or 8000× more concentrated nonradioactive competitors AptER-1, -2, -3 RNA or estrogen response elements (ERE)-50mer before addition of apo-ERα (+, 20 nM) in the binding reactions. (B) Binding assay in the presence of 17β-estradiol (E2) or hydroxy-tamoxifen (OHT). ERα (+, 40 nM) is incubated with ligands (40 nM to 5 μM of E2 or OHT) or the vehicle control (ethanol) at 37°C for 10 minutes before addition of RNA. (C) Cross competition assay with ERE. End-radiolabeled ERE-50mer at 5 nM is mixed with 100× or 400× more concentrated unlabeled competitors ERE-50mer, AptER-1, -2, -3, or an early generation pool of the in vitro selection (G1), before addition of ERα (+, 40 nM). (D) EMSA with radiolabeled AptER-1 and GST-tagged ERα–ligand-binding domain (ERα-LBD) (100 nM) or ERα (100 nM). (E) EMSA with radiolabeled AptER-1 and His₆-tagged ERα–DNA binding domain (ERα-DBD) (100 nM) or ERα (50 nM and 100 nM). The activity of the His₆-tagged DBD is verified by EMSA with radiolabeled ERE shown in the right panel.

Fig. 3.

Minimization and augmentation of AptER-1. (A) Structural analysis of 5′-[γ-³²P]-labeled AptER-1 using in-line probing and footprinting. RNA is sequenced on denaturing gel (12%, acrylamide:bis-acrylamide=19:1) after treatment as follows: ⁻OH, partial alkaline hydrolysis; NR, no reaction; T1, partial digest of denatured AptER-1 with RNase T1 (cleaves after G residues); A, V1, and T1, folded aptamer treated with RNase A, V1, and T1 in the absence (−) or presence (+) of ERα (400 nM); IP, in-line probed sample. Note that NR and IP lanes look very much alike, indicting the hyperactive regions on the folded aptamer. The RNA cleavage by different RNases and in-line probed regions are mapped on the predicted secondary structure of AptER-1. (B) Predicted secondary structures of the truncated AptER-1 and AptER-1 dimer by mfold. Sequences in the variable regions are represented as capital letters, sequences in the constant regions or additional GC pairs in the end-most stem are shown in lower case, and sequences in the three-way junction in AptER-1×2 are enclosed in a box. The nucleotide substitutions in the 65-nt-long truncation B30 are circled in AptER-1(65nt). (C) EMSA showing different binding affinity of the truncated radiolabeled B30, sB30 (65 nt in length), and AptER-1(65nt). ERα concentration is 25 nM. (D) Comparison of binding affinity of radiolabeled AptER-1(65nt) and AptER-1×2(138nt) to ERα. Left panel: shifted band is labeled with asterisk (*). Right panel: band single-astericked (*) represents protein occupying one aptamer unit; band double-astericked (**) represents protein occupying both aptamer units.

Fig. 4.

Physical association of the RNA aptamer with ERα in living cells. (A) Localization of SNAP-ERα in HeLa cells. HeLa cells are transfected with the SNAP-hERα expression vector. Live cells are labeled with SNAP-Cell TMR-Star and Hoechst 33342, and visualized under an inverted fluorescent microscope. (B) Comparable protein expression levels in the experimental and the control cells. An aliquot of the cell samples are subjected to western blotting, and the SNAP-tagged protein is detected by rabbit anti-SNAP polyclonal antibody. (C) Quantification of SNAP-hERα or SNAP pulled down by the aptamer or immunoprecipitated by anti-SNAP antibody. HeLa cells are co-transfected with the SNAP-hERα expression vector and the pSUPER/AptER-1×2 expression vector. In the control, SNAP expression vector is used to replace the SNAP-hERα expression vector. The same cell samples are used for both pull-down assay and RNA co-IP assay. The amount of RNA in association with the SNAP proteins is quantified using real-time RT-PCR and expressed as “fold of enrichment” over the amount of RNA isolated in the control. Each experiment was repeated at least three times and the standard deviation was shown as error bars.

Fig. 5.

Inhibitory effect of the aptamer to ER-mediated transcriptional activation in MCF7 cells. (A) Accumulation level in MCF7 of AptER-1×2 expressed from the pSUPER vector. Cells are harvested at several time points within 96 hours post-transfection of the pSUPER/AptER-1×2 plasmid. The level of AptER-1×2 is quantified using real-time RT-PCR and expressed as relative expression level (fold) of β-actin. (B) Dual-luciferase assays for aptamer expressed using two different vectors. MCF7 cells are co-transfected with the firefly luciferase vector (3×ERE TATA luc), Renilla luciferase vector and an aptamer expression vector of interest. “Ctrl” is a scrambled RNA expression vector used in place of the aptamer expression vectors. All experiments are carried out in triplicate and the average is used to make the plot. (C) Specificity of the inhibitory effect. 3×ERE TATA luc is replaced with firefly luciferase reporter gene 2×PRE TK luc or pGL3 luc. The pSUPER vectors are used to express aptamers or the control RNA. Each experiment was repeated at least three times and the standard deviation was shown as error bars.