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. 2024 Nov 14;7(12):3945–3954. doi: 10.1021/acsptsci.4c00469

Aptamer Proteolysis-Targeting Chimeras (PROTACs): A Novel Strategy to Combat Drug Resistance in Estrogen Receptor α-Positive Breast Cancer

Ying Feng †, Zhilin Zhang †, Haowei Zhang †, Hui Guo †, Chunyan Tan †, Naihan Xu ‡,*, Ying Tan †,*, Yuyang Jiang †
PMCID: PMC11650730  PMID: 39698261

Abstract

graphic file with name pt4c00469_0007.jpg

Breast cancer with positive expression of estrogen receptor α (ERα+) accounts for 70% of breast cancer cases, whose predominant treatment is currently endocrine therapy. The main strategy of endocrine therapy for ERα+ breast cancer is to inhibit the ERα signaling pathway and downregulate ERα levels, which often results in mutations in the ligand-binding domain (LBD) of ERα, leading to significant resistance to subsequent treatment in patients. To combat drug resistance, we first proposed a novel aptamer PROTAC strategy through specifically targeted degradation of ERα via targeting the DNA-binding domain (DBD) of ERα. We proved that this strategy is capable of targeting ERα for degradation through ubiquitination, leading to the inhibition of proliferation in ERα+ breast cancer cells and tamoxifen-resistant breast cancer cells. Furthermore, we investigated the mechanisms involved in overcoming resistance. By circumventing drug resistance associated with LBD mutations in ERα, our approach provides a promising avenue for the discovery of new therapeutic agents.

Keywords: aptamer PROTAC, drug resistance, estrogen receptor α, breast cancer

Breast cancer is a highly heterogeneous group of diseases at the molecular level, and the traditional pathological typing is no longer adapted and adequate to the understanding of the nature of the disease.1,2 Gene expression studies have identified several different subtypes of breast cancer that differ significantly in terms of the prognosis and therapeutic targets present in cancer cells. The main breast cancer classification is divided into three parts based on the expression status of three receptors: estrogen receptor (ER), progesterone receptor (PR), and HER-2/Neu receptor.3 Clinically, they are called Luminal A subtype (ER+/PR+, HER2−), Luminal B subtype (ER+/PR+, HER2+), HER2-positive (ER–/PR–/HER2+), and triple-negative (ER–/PR–/HER2−).4,5 Breast tumorigenesis is strongly dependent on the estrogen receptor binding pathway, and endocrine therapy through estrogen inhibitor and estrogen receptor antagonist therapy has been the primary and most effective agent in the treatment of estrogen-positive breast cancer for decades.6

ERα plays an important role in the pathogenesis of breast cancer, with approximately 70% of breast cancers driven by ERα.7 ERα has been studied for decades as a transcription factor capable of activating or repressing the expression of target genes upon binding to ligands. ERα is encoded by ESR1 (estrogen receptor 1) and performs its primary function while maintaining receptor-specific signaling. ERα contains six functional structural domains: the DNA-binding domain (DBD), the ligand-binding domain (LBD), the activation functional domain (AF1 in the amino-terminal structural domain and AF2 in the LBD), hinge, and F functional domain, thereby controlling transcription and hormone-dependent gene expression8· Acquired mutations in the LBD of ESR1 have been reported in the literature as a common driver of drug resistance in ERα+ breast cancer cells.9−12 ESR1 mutations are less prevalent in patients with untreated primary breast cancer and present in approximately 20% of recurrent ER+ breast cancers, usually acquired after long-term treatment with tamoxifen.13 LBD mutations, particularly those occurring at Y537 and D538 in ERα, result in a more stable agonistic conformation of the mutant receptor. This stability reduces the sensitivity of ERα to estrogen deprivation therapy, including diminished affinity between estrogen and tamoxifen, ultimately leading to tamoxifen resistance during treatment.14 Therefore, there is an urgent need to develop a new breast cancer therapy based on a different mechanism, thus addressing the problem of drug resistance during the treatment of breast cancer.

Proteolysis-targeting chimera (PROTAC) technology, first proposed in 2001 by Crews et al., is an emerging protein degradation strategy.15,16 Unlike traditional small molecule inhibitor strategies used for cancer therapy, PROTACs aim to eradicate specific proteins rather than inhibit activity.17 PROTACs are a heterotrimeric bifunctional molecule consisting of three parts: a ligand capable of binding E3 ubiquitin ligase, another ligand capable of binding to the protein of interest (POI), and a linker connecting them. PROTACs induce the formation of a “POI–PROTAC–E3 ligase” ternary complex by “hijacking” the cellular UPS system, and the POI can be ubiquitin labeled and degraded via the proteasome pathway. The first generation of PROTACs used peptide fragments to recruit the E3 ligases and POIs. As more and more small molecule ligands for E3 ligases are discovered, the type of ligands recruiting E3 ligases is gradually changing from peptides to small molecules.18,19 Numerous laboratories are currently working on developing small molecule-based PROTACs that can recruit ER proteins using small molecules that possess structural similarities to estradiol, tamoxifen, and raloxifene. On the other end of the PROTAC, a variety of E3 ligands are used for recruitment, including small molecule ligands such as murine double mimute 2 (MDM2),20 Von Hippel–Lindau (VHL),21 cereblon (CRBN),22 and cellular inhibitor of apoptosis protein 1 (cIAP1).23 In 2014, GlaxoSmithKline used estradiol or 17-methoxyestradiol as ligands for ERs by forming different substituted mimetic peptides as VHL ligands through chains of 6–17 different atomic numbers. In 2019, Wang et al. reported a PROTAC for ERα, called ERD-308. The ER was recruited by using a raloxifene analog in which the piperidine group was substituted with the N,N-diethylamino group.24 Currently, Flanagan has designed and developed ARV471,25 in which thalidomide analogs act as E3 ligase receptors and heterocyclic compounds act as linkers to connect the target protein-binding fraction to the degradation mechanism recruitment unit to induce degradation of wild-type and mutant ERs for the treatment of ER+/HER2– advanced or metastatic breast cancer.26 In early 2022, we developed an ERE-PROTAC that efficiently degrades ERα in different breast cancer cells.27,28 Recently, we also discovered an mRNA strategy for degrading ERα.28

Studies have demonstrated that aptamers exhibit significant potential in cancer therapy due to their remarkable specificity in binding, coupled with low immunogenicity and toxicity.29 Given the higher affinity and selectivity of aptamers,30 aptamer PROTACs represent a promising approach for selectively degrading target proteins.31,31 Shipeng He’s pioneering work led to the first PROTAC-aptamer conjugate, successfully degrading BRD4 without adverse effects.32 Wang Yuchun et al. developed a novel aptamer PROTAC targeting c-Myc and exhibiting antitumor effects through c-Myc degradation.33 Additionally, recent research has also focused on optimizing the structure of aptamer PROTACs34 and enhancing the overall effectiveness of this innovative approach.32,35 Based on the advantages of aptamer PROTACs, we proposed, for the first time in this study, a strategy to address drug resistance caused by genetic mutations in ERα by altering the binding domain. Herein, the aptamer PROTAC, using the aptamer as a ligand for recruiting ERα and small molecule VH032 for recruiting VHL E3 ligase, was connected by a click chemistry reaction (Figure 1). Two aptamer PROTACs were tested in wild-type and tamoxifen-resistant cells, both resulting in wild-type and mutant ER protein degradation and displaying good proliferation inhibition in tamoxifen-resistant cells, which demonstrated aptamer PROTACs as a promising strategy for addressing drug resistance during therapy.

Figure 1.

Figure 1

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Development of aptamer PROTACs. The aptamer PROTAC is synthesized via the click chemistry reaction. The aptamer recruits ERα, and VH032 recruits VHL E3 ligase. Aptamer PROTACs promote ERα degradation through interactions with ERα and VHL E3 ligase, utilizing protein ubiquitination. This diagram was created with BioRender.

Results and Discussions

Synthesis and Characterization of Aptamer PROTACs

In the design of aptamer PROTACs, two aptamer-binding protein ERα were screened by systematic evolution of ligands by exponential enrichment (SELEX) targeting ERα-positive breast cancer cell lines. These two DNA single-strand sequences were AptamerA36 (5′-CCCGGCATGGTTGCGGAGCAGGAGTATAACACTACCATTG-3′) and AptamerC37 (5′-GTCAGGTCACAGTGACCTGATCAAAGTTAATG-3′), respectively. The small molecule VH032 is a typical ligand for the VHL E3 ligase. To obtain the best degradation efficiency, the length of the linker is optimized. Different numbers of nucleic acid bases (T bases) were used as linkers to connect VH032 with AptamerA or AptamerC (Figure 2a). Aptamer PROTACs were synthesized by linking AptamerA or AptamerC modified with azide at the 5′ end of the DNA sequence to the VH032 small molecule modified with alkynyl, respectively, through a robust bioorthogonal reaction called copper-catalyzed azide–alkyne cycloaddition (CUAAC) reaction (Figure 2b). According to a previous report, in order to maximize the efficiency of the CUAAC reaction, DNA was set to react with VH032-propargyl at a concentration ratio of 1:10 for 24 h at 37 °C. Many aptamer PROTACs were synthesized with different lengths of linkers (named PTA, P3TA, P5TA, P7TA, P9TA, P3TC, P5TC, P7TC, and P9TC). As shown in Figure 2c,d, two aptamer PROTACs, P7TA and P9TC, exhibited the best degradation efficiency, which was subsequently selected for the follow-up study. The aptamer PROTACs were purified by ethanol precipitation and finally characterized by urea polyacrylamide gel electrophoresis (Figure S1), high-performance liquid chromatography (Figure S2), and liquid chromatography-tandem mass spectrometry (Figure S3).

Figure 2.

Figure 2

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Aptamer PROTAC synthesis and the length of the linker screening. (a) The aptamer sequence (blue) with different numbers of T bases (red). (b) The 5′ end of the aptamer modified by an azide group reacts with VH032 modified by an alkynyl group through the CUAAC reaction. (c–d) Degradation effects induced by aptamer PROTACs with different length linkers in MCF-7 cells, under 5 μM concentration, respectively.

Aptamer PROTACs Degrade ERα in a Concentration- and Time-Dependent Manner

Next, we evaluated the ability of P7TA and P9TC to regulate the ERα levels. After the treatment of MCF-7 cells with varying concentrations of P7TA and P9TC for 24 h, the difference in ERα expression levels in MCF-7 cells was verified by protein immunoblotting. P7TA and P9TC were found to induce the ubiquitinated degradation of ERα in MCF-7 cells in a concentration-dependent manner. As shown in Figure 3a–d, we selected P7TA at a concentration of 5 μM and P9TC at a concentration of 2.5 μM to continue the investigation of their ubiquitination degradation of ERα in MCF-7 cells in relation to time. After different time gradients of P7TA and P9TC treatment of MCF-7 cells, Figure 3e–h showed that P7TA and P9TC were able to induce the degradation of the ERα protein in MCF-7 cells in a time-dependent manner, acting after 2 h of incubation and continuing to degrade within 24 h.

Figure 3.

Figure 3

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P7TA and P9TC degrade ERα in a concentration- and time-dependent manner. (a) Changes in ERα levels after 24 h of incubation of P7TA with MCF-7 cells at different concentration gradients. (b) Quantification of the changes in ERα levels under the (a) conditions. (c) Changes in ERα levels after 24 h of incubation of P9TC with MCF-7 cells at different concentration gradients. (d) Quantification of the changes in ERα levels under the (c) conditions. (e) Changes in ERα levels after incubation with MCF-7 cells for different times under a 5 μM concentration of P7TA. (f) Quantification of the changes in ERα levels under the (e) conditions. (g). Changes in ERα levels after incubation with MCF-7 cells for different times under a 2.5 μM concentration of P9TC. (h) Quantification of the changes in ERα levels under the (g) conditions, respectively (*p < 0.05; ***p < 0.001, ****p < 0.0001).

Aptamer PROTACs Degrade ERα in a Ubiquitin-Proteasome System-Dependent Manner

The mechanism of action of PROTACs is to specifically target the POI and the E3 ligase, forming a ternary complex of “POI–PROTAC–E3 ligase,” which can then function through the ubiquitin–proteasome pathway. In order to investigate the role of P7TA and P9TC in the degradation of the ERα protein, we designed a competitive inhibition assay. After action on MCF-7 cells for 12 h, the changes of ERα in MCF-7 cells were analyzed (Figure 4). The results showed that the higher concentration of 7TA/9TC sequences or VH032 competed with P7TA/P9TC to bind with ERα or VHL, which hindered the ubiquitination process of the ERα protein and prevented the ERα protein from being degraded (Figure 4a–d). The experimental results demonstrated that aptamer PROTACs entered MCF-7 cells to form a ternary complex with ERα and VHL and then ubiquitinated and degraded ERα.

Figure 4.

Figure 4

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Ternary complex competition assay. (a) Single VH032-propargyl, or an aptamer alone, competitively inhibits ubiquitinated degradation of ERα by P7TA. (b) Quantification of the changes in ERα levels under the (a) conditions. (c) Single VH032-propargyl, or aptamer alone, competitively inhibits ubiquitinated degradation of ERα by P9TC. (d) Quantification of the changes in ERα levels under the (c) conditions. (e) Proteasome inhibitor control assay. (f) Quantification of the changes in ERα levels under the (e) conditions. (g) Chiral small molecule of VH032-propargyl was used as a negative control. (h) Quantification of the changes in ERα levels under the (g) conditions (*p < 0.05; ***p < 0.001; ****p < 0.0001).

To prove the ubiquitin–proteasome process, P7TA and P9TC were cotreated with the proteasome inhibitor MG132 in MCF-7 cells (Figure 4e). The results showed that ERα degradation occurred when P7TA (5 μM) and P9TC (2.5 μM) were added alone and was inhibited by MG132 (20 μM), indicating that the degradation of ERα was dependent on the ubiquitin–proteasome system. Since the hydroxyl group of VH032 was recruited as the key portion for binding to VHL E3 ligase, we synthesized the negative controls, SSSP7TA and SSSP9TC, by reversing the stereochemical structure of the hydroxyl group from R to S. SSSP7TA and SSSP9TC, synthesized by the same click chemistry reaction, however, failed to degrade ERα in MCF-7 cells (Figure 4g). Together, these results suggested that P7TA and P9TC degrade ERα through the ubiquitin–proteasome pathway.

Efficacy of P7TA and P9TC in MCF-7/TAM Cells

In order to verify whether P7TA and P9TC can function equally in drug-resistant cells, the MCF-7 tamoxifen-resistant strain (MCF-7/TAM) was first verified.38,39 MCF-7 and MCF-7/TAM cells were treated with different concentrations of tamoxifen for 48 h, and cell viability was measured with the CCK-8 reagent. The results showed that MCF-7 cells were significantly more sensitive to tamoxifen than MCF-7/TAM cells, indicating that MCF-7/TAM cells were resistant to tamoxifen (Figure 5a). Then, the equivalent amount of expression of ERα in MCF-7 and MCF-7/TAM cells was verified by protein immunoblotting assay and laser confocal assay (Figure 5b).

Figure 5.

Figure 5

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Efficacy of P7TA and P9TC in MCF-7/TAM cells. (a) Survival of MCF-7 and MCF-7/TAM cells tested by the CCK-8 kit after treatment with the indicated concentrations of tamoxifen. (b) The expression of ERα was observed using Western blot assays in MCF-7 and MCF-7/TAM cell lines. (c) Quantification of the changes in ERα levels under the (b) conditions, respectively. (d) P7TA and P9TC ubiquitinated degradation of ERα in MCF-7/TAM cells. (e) Quantification of the changes in ERα levels under the (d) conditions, respectively. (f) Different concentrations of P7TA, P9TC, and tamoxifen on apoptosis of MCF-7/TAM cells. (g) Effects of different concentrations of P7TA, P9TC, and tamoxifen on the MCF-7/TAM cell cycle (*p < 0.05; ***p < 0.001).

MCF-7 and MCF-7/TAM cells were treated with different concentrations of P7TA and P9TC for 24 h, respectively, and the CCK-8 reagent was used to verify the inhibitory effect on cell proliferation (Figure 5a). The experimental results showed that both P7TA and P9TC could effectively inhibit the proliferation of MCF-7 and MCF-7/TAM cells.

To verify whether P7TA and P9TC could equally effectively ubiquitinate ERα in MCF-7/TAM cell lines, concentrations of 5 and 10 μM were set and applied to MCF-7/TAM cells for 24 h. The results of protein immunoblotting experiments showed that P7TA and P9TC could effectively degrade ERα in MCF-7/TAM cells in a concentration-dependent manner (Figure 5d,e).

The proliferative mechanism of P7TA and P9TC on ERα-positive MCF-7/TAM cells was analyzed by an apoptosis assay. MCF-7/TAM cells were treated with P7TA, P9TC, and tamoxifen at concentrations of 2.5, 5, and 10 μM for 24 h, followed by AnnexinV-FITC and PI staining, and the results of apoptosis were analyzed by flow cytometry. The results showed that the proportion of apoptotic MCF-7/TAM cells increased with increasing concentrations of P7TA, P9TC, and tamoxifen compared with the blank group, indicating dose-dependent apoptosis of MCF-7/TAM cells in the presence of P7TA, P9TC, and tamoxifen. Compared with the tamoxifen group, the proportion of apoptosis in MCF-7/TAM cells slightly increased with the increase of tamoxifen concentration, indicating that P7TA and P9TC could effectively induce apoptosis in MCF-7/TAM cells and solve the problem of tamoxifen resistance in MCF-7 cells (Figure 5f).

To investigate the effects of P7TA, P9TC, and tamoxifen on the cell cycle of MCF-7/TAM cells, we designed cell cycle experiments. MCF-7/TAM cells without any drug were used as a blank control group, and MCF-7/TAM cells were treated with 2.5, 5, and 10 μM P7TA, P9TC, and tamoxifen for 24 h, followed by staining with PI. The results of cell cycle changes were analyzed by flow cytometry. The percentage of MCF-7/TAM cells stalled in the G1 phase in the blank control group was 51.82%, and the percentage of cells stalled in the S phase was 38.83%. When the concentration of P7TA and P9TC groups increased, the proportion of cells stalled in the S phase increased, indicating that P7TA and P9TC had a concentration-dependent effect on the cell cycle of MCF-7/TAM cells and were able to induce cell stalling in the S phase. When the concentration of the tamoxifen group increased, the proportion of cells in the G0/G1 phase gradually increased, indicating that tamoxifen had a concentration-dependent effect on the cell cycle of MCF-7/TAM cells and was able to induce cell arrest in the G0/G1 phase (Figure 5g). This suggests that P7TA, P9TC, and tamoxifen cause MCF-7/TAM cell cycle abnormalities by different mechanisms.

Finally, we performed a transcriptome analysis to explore the effect of P7TA and P9TC on gene expression in TAM-resistant MCF7 cells. In our analysis, we performed RNA-Seq to investigate the biological functions of P7TA and P9TC by targeting ERα. The results showed that P7TA and P9TC exhibited relatively consistent RNA profiles (Figure S6). As shown in Figure 6a, 63 common upregulated genes and 34 downregulated genes (log2(FC) > 1 and p-value < 0.05) were selected as differentially expressed genes (DEGs) for subsequent analysis. GO and KEGG gene set enrichment analyses of DEGs demonstrate diverse biological processes and signaling pathways, such as estrogen metabolic process, metabolism of xenobiotics by cytochrome P450, cellular response to copper ions, cellular response to calcium ions, cellular response to erythropoietin, steroid hormone biosynthesis, mineral absorption, etc. These processes and pathways are highly associated with estrogen’s function and the ER signaling pathway (Figure 6c). The gene enrichment network showed three important gene sets, M1 (hormone metabolic process), M2 (extracellular matrix-related gene sets), and M3 (cell response to ions). Among them, some hub genes were identified (DE genes with top connection in the network), such as CYP1A1, UGT1A1, ALDH1A3, UGT2B15, MT2A, MT1X, MT1F, and SERPPINF1 (Figure 6d). Most of the hub genes have been reported to be strongly correlated with the ERα signaling pathway in breast cancer. CYP1A1 belongs to the cytochrome P450 family, which is involved in estrogen metabolism and ERα-related breast cancer proliferation.40 Metallothionein (MT) is downregulated in breast cancer and associated with tamoxifen resistance.41ALDH1A3 is abnormally expressed in various cancers, and its expression is negatively correlated with ER status.42 SERP1NF1 (pigment epithelium-derived factor, member 1) expression is subject to regulation by estrogen and inhibits breast cancer metastasis.43 Quantitative polymerase chain reaction (Q-PCR) by two pairs of primers for each hub gene demonstrated that P7TA and P9TC treatment resulted in upregulation of CYP1A1, MT2A, ALDH1A3, and SERP1NF1 (Figure 6e,f). These results demonstrate that the ERα-targeting PROTAC induces a series of ER-related biological processes to influence breast cancer cell proliferation and apoptosis. Furthermore, transcriptome data provide new molecular mechanisms and perspectives in ER+ breast cancer therapy by targeting ERα, since there is obvious upregulation in ion-related signaling (Figure 6d, M2) such as ferroptosis and cuproptosis.

Figure 6.

Figure 6

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Transcriptomic analysis of P7TA and P9TC-treated MCF7-TAM cell lines. (a) Differentially expressed genes selected by p < 0.05 and |log2(FC)| compared with control. (b) Heatmap of DEGs with obvious clustering. (c) GO term and KEGG pathway gene enrichment analysis of DEGs. All enriched terms are selected with p < 0.05. (d) The term-gene network from enrichment analysis. The nodes indicate enrichment terms, and the node size is proportional to the number of genes related to that term. The width of the edges is proportional to the number of genes shared between the terms. Hub genes are top connections with enriched terms. (e,f) Relative mRNA expression by two paired primers for each hub gene. The p-value was calculated by two-way ANOVA (*p < 0.05; ***p < 0.001).

Conclusion

Our study introduced a novel approach by developing aptamer PROTACs to combat drug resistance in breast cancer endocrine therapy resulting from ESR1 mutations. These aptamer PROTACs effectively link the aptamer to the VHL ligand through a click chemical reaction. They recruit ERα and VHL E3 ligase, thereby promoting the ubiquitination degradation of ERα in both MCF-7 and MCF-7/TAM cells in a concentration- and time-dependent manner. Importantly, both aptamer PROTACs demonstrated the ability to induce significant abnormalities in apoptosis and cell cycle progression in MCF-7/TAM cells, surpassing tamoxifen in apoptotic activity at equivalent concentrations.

Furthermore, differential gene expression analysis validated that P7TA and P9TC exerted substantial effects on cellular activity, growth, and apoptosis, consistent with findings from flow cytometry analyses. In conclusion, our study introduces a promising strategy to address acquired drug resistance arising from mutations in target proteins, potentially paving the way for the development of innovative therapeutics for breast cancer treatment.

Methods

Synthesis of P7TA and P9TC PROTACs

The 5′ ends of aptamer P9TA (5′-CCCGGCATGGTTGCGGAGCAGGAGTATAACACTACCATTG-3') and aptamer P9TC (5′-GTCAGGTCACAGTGACCTGATCAAAGTTAATG-3′) were modified by an azide group, and VH032-propargyl (HY-126465, MedChemExpress) was modified by an alkyne. Then, PROTAC P7TA and PROTAC P9TC were synthesized through copper-catalyzed azide–alkyne cycloaddition (CUAAC), utilizing BTTAA as a ligand to stabilize monovalent copper ions. The concentration ratio of azide-modified DNA to alkyne-modified VH032 was set at 1:10. The reaction was carried out for a duration of 24 h at a temperature of 37 °C. The oligonucleotides were separated using urea DNA polyacrylamide gel electrophoresis and purified by ethanol precipitation. The purity and molecular weight were subsequently verified through HPLC and LC–MS.

Ethanol Precipitation of DNA

200 μL portion of DNA-containing reaction liquid was moved to a centrifuge tube, followed by the addition of 20 μL of 3 mol/L sodium acetate solution and 550 μL of precooled anhydrous ethanol at 4 °C. After thorough mixing, the mixture was allowed to rest overnight at −80 °C. The centrifuge tube, containing a significant amount of white precipitate, was then placed in a precooled high-speed centrifuge at 4 °C and centrifuged at 12 000 rpm for 30 min. The supernatant was discarded, and the precipitate was washed 2–3 times with precooled 75% ethanol at 4 °C, with subsequent discarding of the supernatant. Finally, the purified DNA product was obtained by drying the precipitate in a vacuum rotary evaporator for 2–4 h.

Urea–DNA Polyacrylamide Gel Electrophoresis (Urea PAGE)

Here, 15% urea PAGE was used to verify the synthesis efficiency of P7TA and P9TC. The experiment was conducted using 1× TBE buffer under 120 V for 2 h. After electrophoresis, the gel was incubated in 1× SYBR Gold dye for 15 min. Imaging was conducted using a Molecular Imager PharosFX (Bio-Rad, USA).

Protocol for Click Chemistry

The DNA sequences for this work were synthesized by Sangon Biotech (Shanghai, China). First, 2 mM VH032-propargyl and 10 μL of DMSO were added into 0.1 M potassium phosphate solution (pH 7.0). Then, 0.05 mM CuSO4 solution and 0.3 mM BTTAA were premixed and added to the reaction solution, followed by the addition of 50 μM DNA. Finally, a 2.5 mM sodium ascorbate solution was applied and gently mixed evenly at 37 °C for 24 h.

High-Performance Liquid Chromatography (HPLC)

Waters e2695 was used to separate samples by the chromatographic column Waters XBridge OST C-18 (10 × 50 mm, 2.5 μm). 0.1 M triethylamine acetate (TEAA) (0.1 M) was used as solution A, acetonitrile was used as solution B, the flow rate was 0.5 mL/min, and each sample size was 10 μL. The column temperature was maintained at 60 °C, and the detection wavelength was set to 260 nm. The gradient program was as follows: 90% A with 10% B for the initial 4 min, 65% A with 35% B for the subsequent 19 min, and 90% A with 10% B for the final 35 min. Signals were monitored and processed with Empower 3.

Liquid Chromatography–Mass Spectrometry (LC–MS)

The chromatography was conducted on a Waters Acquity OST C-18 column (2.1 × 50 mm, 1.7 μM) with 10 mM TEAA serving as solution A and acetonitrile as solution B at a flow rate of 0.5 mL/min, and each sample size was 10 μL.

Cell Culture

MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution. MCF-7/TAM cells were cultivated in Roswell Park Memorial Institute (RPMI) 1640 with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution, and treated with 1 μM tamoxifen.

CCK-8 Cell Proliferation Test

Cells were spread and adhered evenly in a 96-well plate. Subsequently, the cells were treated with varying concentrations of tamoxifen for 48 h of incubation. Afterward, a cell culture solution containing 10% Cell Counting Kit-8 reagent was added to each well and incubated for 4 h. The optical absorbance at 450 nm was then measured by using a microplate reader (Tecan Infinite).

Western Blot Assay

A certain number of cells were cultured in a 12-well plate. Afterward, specific concentrations of drugs were coincubated with the cells for a designated period. Following incubation, RIPA buffer with 1% PMSF was used to lyse the cells. Protein was measured using the Bradford method. The lysates (15 μg of protein) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 90 V for 15 min and 120 V for 40–60 min. Subsequently, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane and incubated overnight at 4 °C with anti-ERα (estrogen receptor α (D8H8) rabbit mAb, #8644s, 1:1000) and anti-β-actin antibodies (β-actin rabbit mAb, #AC026, 1:1000). The PVDF membrane was then washed with TBST containing 0.1% Tween-20. Next, the PVDF membrane was incubated at room temperature for 2 h with an anti-rabbit secondary antibody (#A-0208, 1:1000) and washed again with 0.1% Tween-20, as previously described. Finally, the ECL method was used for imaging, and the images were visualized by using a gel imaging system (Tanon 5200).

Confocal Microscopy

A proper density of cells was inoculated in confocal dishes. After the cells were attached to the wall, the cell status and density were observed again for administration operation. After washing with PBS, the cells were fixed by the addition of 4% paraformaldehyde for a period of 15 min and then washed with PBS three times. 0.25% Triton X-100 was incubated for 5 min and then washed with PBS. 1% BSA was incubated for 1 h and then washed with PBST on three occasions. ERα primary antibody (8644S; Cell Signaling Technology, 1:300) was incubated for 1.5 h and washed with PBST three times. Then, cells were incubated with the Cy5-IgG secondary antibody (no. 550083, Zen Bioscience, 1:1000) for 40 min and washed with PBST three times. Subsequently, the nuclei were stained with DAPI for 5 min and imaged by using a confocal laser scanning microscope (NIKON A1 HD25).

Apoptosis and Cell Cycle Analysis

Following various treatments, the cells were digested and subsequently harvested and rinsed with PBS. The resulting cell suspension was centrifuged at 1000 g for 5 min. For apoptosis analysis, cells were processed in accordance with the FITC Annexin V Apoptosis Detection Kit protocol (no. 40302ES20, Yeasen Biosciences, Shanghai, China). Regarding cell cycle analysis, the cells were resuspended in 1 mL of prechilled ethanol and stored at 4 °C overnight. The ethanol in the supernatant was then removed through centrifugation, and the cells were processed in accordance with the Cell Cycle and Apoptosis Analysis Kit protocol (no. 40301ES50, Yeasen Biosciences, Shanghai, China). Subsequently, the samples were examined using a flow cytometer (Beckman Coulter) and analyzed using Modfit software. The experiments were repeated thrice, with a minimum of 10 000 gated events collected and analyzed during each repetition.

RNA Sequence and Differential Expression Analysis

Total RNA was extracted from three cell samples, either with the control or treated with P7TA and P9TC. cDNA library construction and sequencing were performed by Gene Denovo Biotechnology Co. (Guangzhou, China). High-quality reads were aligned with the human reference genome using Bowtie2. Expression abundance and variations for each of the genes were normalized to fragments per kilobase of transcript per million mapped reads (FPKM) using RNA-seq by expectation maximization (RSEM). For the identification of differentially expressed genes (DEGs), we performed differential expression analysis of treated versus control samples using the “edgeR” and “DESeq2” packages in R.44,45 Common genes with the same fold change (FC) alterations (|log2(FC)| > 1, FDR < 0.05) were selected for further analysis.

Gene Set Enrichment Analysis

Gene sets from the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were enriched in DEGs. For GO terms, BP (biological process), CC (cellular component), and MF (molecular function) were enriched. The p-value was calculated based on a hypergeometric test; the p-value cutoff was set to 0.05, and the FDR (the p-value was adjusted using the “BH” method) was set to 0.1.

Real-Time PCR

Total RNA was extracted by TRIZOL (Sangon Biotech, Shanghai, China) and subjected to reverse transcription using a Toyobo Bio RT Kit (FSQ-301). Real-time PCR was performed with SYBR Green (Transgen, AQ601) on a real-time PCR (Jena qTOWER 2.2) following the manufacturer’s instructions. Relative expression values were normalized to ACTB by the ΔΔCt method.

Acknowledgments

This work was supported by the National Key R&D Program of China, Synthetic Biology Research (2019YFA0905900). We would like to express our sincerest thanks to Professor Peter E. Lobie for sharing the specific tamoxifen-resistant MCF-7 cell line with us.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.4c00469.

  • Urea polyacrylamide gel electrophoresis results (Figure S1); HPLC chromatogram of P7TA and P9TC (Figure S2); LC–MS chromatogram of P7TA and P9TC (Figure S3); synthesis of SSSP7TA and SSSP9TC (Figure S4); identification of drug-resistant cell lines (Figure S5); Q-PCR primers (Table S1); correction and PCA (principal component analysis) of RNA-Seq samples (Figure S6) (PDF)

Author Contributions

# These authors contributed equally to this work. Y.F., Z.Z., and H.Z. contributed equally to this work. Y.T. and N.X. proposed the idea. Y.F. and Z.Z. designed and synthesized the aptamer PROTACs. Y.F. and Z.Z. designed the bioexperiments. H.Z. designed the bioinformatics experiment. H.G. was mainly responsible for domesticating drug-resistant cells MCF-7/TAM. C.T. and N.X. also provided help in the cell experiments. Y.T., N.X., Y.F., Z.Z., and H.Z. wrote the manuscript.

The authors declare no competing financial interest.

Supplementary Material

pt4c00469_si_001.pdf (1.1MB, pdf)

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Supplementary Materials

pt4c00469_si_001.pdf (1.1MB, pdf)

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